The Pennsylvania State University

The Graduate School

The Huck Institute for Life Sciences

THE IMPACT OF VITAMIN A ON THE EXPRESSION,

NUCLEAR LOCALIZATION AND TRANSCRIPTIONAL FUNCTIONS

OF INTERFERON REGULATORY FACTOR-1

A Thesis in

Integrative Biosciences

by

Xin Luo

© 2006 Xin Luo

Submitted in Partial Fulfillment of the Requirements for the Degree of

Doctor of Philosophy

May 2006

The thesis of Xin Luo has been reviewed and approved* by the following:

A. Catharine Ross Professor and Dorothy Foehr Huck Chair in Nutrition Thesis Adviser Chair of Committee

John L. Beard Professor of Nutrition Professor-in-Charge, Graduate Program in Nutrition

Andrea M. Mastro Professor of Microbiology and Cell Biology

Michael Teng Assistant Professor of Biochemistry and Molecular Biology

Keith R. Martin Assistant Professor of Nutrition

Richard J. Frisque Co-Director, Graduate Education Integrative Biosciences Graduate Program The Huck Institutes of the Life Sciences

*Signatures are on file in the Graduate School.

ABSTRACT

Vitamin A (especially all-trans-retinoic acid; atRA) is an important regulator of

immune responses, cell death and differentiation. atRA was previously shown to increase

the expression of interferon regulatory factor (IRF)-1, a transcription factor involved in cell growth and apoptosis, differentiation, and antiviral and antibacterial immune responses.

During early stages of viral infections, the level of interferon (IFN)γ is low.

Increasing the response of the cells to low-dose IFNγ potentiates the antiviral response. In

this study, I hypothesized that atRA regulates IRF-1 through multiple mechanisms, which

include 1) potentiation of IFNγ signaling, thus inducing IRF-1, 2) regulation of IRF-1

transcription and localization in the absence of IFNγ.

In the human lung epithelial cell line, A549, pretreatment with atRA followed by

low-dose IFNγ induced a faster, higher, and more stable expression of IRF-1 than IFNγ

alone. Through the function of a receptor of atRA, retinoic acid receptor-α (RARα), atRA sequentially increased surface expression of IFNγ receptor (IFNGR)-1, activation of signal transducer and activator of transcription (STAT)-1, and expression of IRF-1. atRA pretreatment also affected the transcriptional functions of IFNγ-induced IRF-1,

increasing its nuclear localization, DNA-binding activity, and the transcription of IRF-1

target genes.

The human mammary epithelial cell line, MCF10A, was used to investigate the effect of atRA on IRF-1 in the absence of IFNγ. atRA by itself rapidly induced IRF-1

iii mRNA and protein. Sequential treatments with atRA induced almost the same level of

IRF-1 as the single dose; however, more IRF-1 was concentrated in the nucleus. The

DNA-binding activity of IRF-1 and transcription of an IRF-1 target gene were also increased. In addition, sequential treatments with atRA increased the levels of RARα and its dimerization partner, retinoid X receptor-α, in the nucleus.

Therefore, atRA may increase the response of the cells to low-dose IFNγ by affecting the IFNγ signaling pathway that leads to increased expression and transcriptional functions of IRF-1. Furthermore, atRA by itself increases both expression and nuclear localization of IRF-1, which may be mechanisms by which atRA regulates the death of cancer cells. In conclusion, these results provide evidence that atRA regulates IRF-1 through multiple mechanisms.

iv TABLE OF CONTENTS

List of Figures...... vii List of Abbreviations ...... ix Acknowledgments ...... xii

CHAPTER 1 INTRODUCTION...... 1 1. Vitamin A Metabolism and Signaling...... 2 1.1. Roles of Vitamin A in the Body...... 2 1.2. Retinoid Metabolism and Signaling in the Cell...... 4 1.3. Retinoid Receptors and Signaling...... 7 1.4. Roles of Retinoids in Antiviral Responses...... 15 1.5. Roles of Retinoids in Cell Death and Differentiation ...... 21 2. Interferon Signaling Pathways ...... 26 2.1. Signaling of Type I Interferons...... 27 2.2. Signaling of Type II Interferon...... 29 2.3. Roles of Interferons in Antiviral Responses ...... 35 2.4. Roles of Interferons in Cell Death and Differentiation...... 41 3. Members of the IRF Family of Transcription Factors...... 46 3.1. IRF-1 Subfamily ...... 48 3.2. Regulation of IRF-1 Expression and Localization...... 52 3.3. Roles of IRF-1 in Antiviral Responses...... 57 3.4. Roles of IRF-1 in Cell Death and Differentiation ...... 60 3.5. IRF-3, -4, and -5 Subfamilies...... 61 4. Interactions of Vitamin A, Interferons, and IRF-1...... 65

CHAPTER 2 STATEMENT OF HYPOTHESIS...... 70

CHAPTER 3 RETINOID-MODULATED POTENTIATION OF IFNγ SIGNALING IN A549 CELLS ...... 72 1. Abstract...... 73 2. Introduction...... 74 3. Experimental Procedures...... 76 4. Results ...... 82 5. Discussion...... 101

v CHAPTER 4 RETINOID MODULATION OF IRF-1 IN THE ABSENCE OF IFNγ IN MCF10A CELLS...... 109 1. Abstract...... 110 2. Introduction...... 111 3. Experimental Procedures...... 113 4. Results ...... 118 5. Discussion...... 135

CHAPTER 5 SUPPLEMENTAL RESULTS ...... 140 1. Abstract...... 140 2. A549 Cells: Supplemental Results and Discussion ...... 142 3. MCF10A Cells: Supplemental Results and Discussion ...... 154

CHAPTER 6 DISCUSSION ...... 163 1. How atRA Regulates IFNγ Signaling ...... 163 1.1. IFNγ Receptors...... 163 1.2. STAT-1...... 167 2. How atRA Regulates IRF-1...... 168 2.1. IRF-1 Induction...... 169 2.2. IRF-1 Localization ...... 171 2.3. IRF-1 Signaling...... 175 3. Roles of RARα...... 178 3.1. The Connection between atRA and RARα...... 178 3.2. Roles of RARα in atRA-Mediated Regulation of IRF-1 ...... 180 3.3. Am580 as a Therapeutic Agent ...... 182 4. Physiological Differences between Two Cell Lines...... 182 5. Implications ...... 184 6. Future Directions ...... 185

References...... 187

vi List of Figures

Figure 1 A model of cellular events in retinoid signaling and metabolism...... 6 Figure 2 Two families of retinoid receptors...... 8 Figure 3 Common natural and synthetic retinoids...... 10 Figure 4 A model of interactions among RAR, RXR, corepressors, and coactivators...... 12 Figure 5 Effects of vitamin A on adaptive immunity...... 20 Figure 6 Regulatory inputs in TRAIL and TRAIL-receptor (DR4 and DR5) gene expression...... 23 Figure 7 Retinoid-mediated induction of apoptosis...... 25 Figure 8 A model of cellular events in STAT-1-dependent IFNγ signaling...... 30 Figure 9 A model of IFNα/β-induced antiviral responses...... 37 Figure 10 Interferon-mediated induction of apoptosis...... 44 Figure 11 IRF family of transcriptional factors...... 47 Figure 12 Domain structure of IRF-1...... 50 Figure 13 Schematic view of the IRF-1 promoter...... 53 Figure 14 Roles of IFNγ and IRF-1 in differentiation into Th1 cells...... 59 Figure 15 A model for the interaction of RA, interferons, and IRF-1 on cell-mediated and humoral immunity...... 67 Figure 16 Overnight pretreatment with atRA followed by suboptimal concentrations of IFNγ induces a faster, higher, and more stable expression of IRF-1...... 83 Figure 17 At least 4 h of pretreatment with atRA is required to sensitize A549 cells to better respond to low-dose IFNγ...... 87 Figure 18 Pretreatment with atRA increases IFNγ-induced STAT-1 tyrosine phosphorylation...... 89 Figure 19 Pretreatment with atRA increases IFNGR-1 cell-surface expression...... 91 Figure 20 Ligands for RARα, including atRA, 9cRA, and Am580, sequentially increases the levels of IFNGR-1, activated STAT-1, and IRF-1...... 93 Figure 21 Pretreatment with atRA increases nuclear expression and DNA-binding activity of IFNγ-induced IRF-1...... 97 Figure 22 atRA and IFNγ synergistically increase transcription of IRF-1 target genes...... 100

vii Figure 23 Working model of the regulation of IFNγ-induced IRF-1 by pretreatment with atRA...... 108 Figure 24 atRA induces IRF-1 in MCF10A cells...... 119 Figure 25 RARα-selective ligand, Am580, induces IRF-1...... 123 Figure 26 atRA increases nuclear localization of RARα and RXRα...... 125 Figure 27 Re-stimulation with atRA increases nuclear localization of IRF-1...... 128 Figure 28 Re-stimulation with atRA increases nuclear IRF-1 and DNA-binding activity of IRF-1...... 131 Figure 29 Re-stimulation with atRA increases transcription of OAS-2, an IRF-1 target gene...... 134 Figure 30 Working model of atRA-mediated regulation of IRF-1 expression and localization...... 139 Figure 31 atRA increases IFNγ-induced tyrosine phosphorylation of Jak-2...... 143 Figure 32 Combination of atRA and IFNγ increases de novo synthesis of IRF-7...... 145 Figure 33 Combination of atRA and IFNγ decreases cell proliferation on Day 3...... 147 Figure 34 atRA increases cell survival on Day 7...... 149 Figure 35 Effect of atRA on TNFα and IFNβ-induced responses...... 151 Figure 36 atRA differentially regulates the binding of NF-κB dimers containing p65 and p50...... 155 Figure 37 atRA does not affect the expression, phosphorylation, or DNA-binding activity of STAT-1 to induce IRF-1...... 159 Figure 38 atRA induces the death of MCF10A cells...... 162 Figure 39 RA increases Th2 response by regulating IFNGR-1 on Th1 cells...... 166 Figure 40 Nucleocytoplasmic shuttling of proteins via the RanGTP system...... 174 Figure 41 Working model of how atRA regulates IRF-1...... 177

viii List of Abbreviations

9cRA 9-cis-retinoic acid ADAR RNA-specific adenosine deaminase ANOVA analysis of variance APL acute promyelocytic leukemia atRA all-trans-retinoic acid BSA bovine serum albumin CBP CREB-binding protein CDK cyclin-dependent kinase CRABP cellular retinoic acid-binding protein CRBP cellular retinol-binding protein CREB cAMP response element-binding protein DAPI 4’,6’-diamidino-2-phenylindole DBD DNA-binding domain DC dendritic cells DMEM Dulbecco’s minimal essential medium DR direct repeats dsRNA double-stranded RNA DTT dithiothreitol EDTA ethylenediaminetetraacetic acid EMSA electrophoretic mobility shift assay FBS heat-inactivated fetal bovine serum FasL Fas ligand FITC fluorescein isothiocyanate GAPDH glyceraldehydes-3-phosphate dehydrogenase GAS gamma-interferon activated site GBP guanylate binding protein GRIM retinoid-interferon induced mortality HAT histone acetyltransferase HDAC histone deacetylase HRE hormone-specific response elements HRP horseradish peroxidase IAD IRF-association domain ICAM-1 intercellular adhesion molecule 1 LMP low molecular weight protein

ix IFN interferon IFNAR IFNα/β receptor IFNGR IFNγ receptor Ig immunoglobulin IκB inhibitor of NF-κB IL interleukin iNOS inducible nitric oxide synthase IP-10 IFN-induced protein 10 IR inverted repeat IRF-1 IFN regulatory factor-1 IRF-E IRF-1 response element ISGF-3 IFN-stimulated gene factor 3 ISRE IFN-stimulated response element Jak Janus kinase JNK Jun-N-terminal kinase LBD ligand-binding domain LPS lipopolysaccharide LRAT lecithin:retinol acyltransferase MAPK mitogen-activated protein kinase MHC major histocompatibility complex MTT 3-(4,5-dimethyltiazol-2-yl)-2,5-diphenyltetrazolium bromide NES nuclear export signal NF-κB nuclear factor κB NK natural killer cells NLS nuclear localization signal NPC nuclear pore complex OAS 2’-5’ oligoadenylate synthetase PBS phosphate-buffered saline PIAS protein inhibitor of activated STAT PIC polyinosinic:polycytidylic acid PKC protein kinase C PKR dsRNA or ssRNA-activated serine/threonine protein kinase PMSF phenylmethylsulphonyl fluoride RA retinoic acid RANTES regulated upon activation, normal T cells expressed and secreted

x RAR retinoic acid receptor RARE retinoic acid response element Rb retinoblastoma RBP retinol-binding protein RTMBE retinyl trimethoxybenzyl ether RT-PCR reverse transcription-PCR RXR retinoic X receptor RXRE retinoic X response element SDS sodium dodecyl sulfate SDS-PAGE SDS-polyacrylamide gel electrophoresis SH2 Src-homology 2 SHP SH2-containing tyrosine phosphatase SOCS-1 suppressor of cytokine signaling 1 STAT signal transducer and activator of transcription SUMO small ubiquitin-like modifier TAP transporter associated with antigen processing TESS Transcription Element Search System Th T-helper TLR toll-like receptor TNF tumor necrosis factor TR thyroid hormone receptor TR1 thioredoxin reductase-1 TRAIL TNF-related apoptosis inducing ligand VAD vitamin A deficiency VDR vitamin D receptor

xi Acknowledgments

I would like to express my sincere thanks to Dr. A. Catharine Ross, who has been extraordinary in helping me to grow into a future scientist. As a mentor, she has consistently nourished me with her broad knowledge and creative ideas in all fields of nutrition, immunology, biochemistry, molecular biology, and cell biology. Step by step, I have learned from her how to design experiments, interpret results, and most importantly, to think creatively and thoroughly as a scientist. She has also taught me how to express myself professionally and intelligently. In addition, her encouragements all through these years have reassured my passion for science and future goal of being an independent researcher. To me, Dr. Ross is not only a professor and scientist, but also a role model.

I would like to thank all other members of my thesis committee: Drs. John L.

Beard, Michael Teng, Andrea M. Mastro, and Keith R. Martin, for their input of time and suggestions, and for their encouragements during the completion of my dissertation.

Special thanks to the Department of Nutritional Sciences, which has been like a home to me for the past few years. I would also like to thank the Huck Institutes of the

Life Sciences, for providing an excellent curriculum of courses and trainings.

I would like to thank Drs. Qiuyan Chen and Reza Zolfaghari, for teaching me molecular techniques and for helping me to solve the problems I had during the experiments. I would also like to thank all other members of Ross Lab: Yifan Ma,

Christopher Cifelli, Yao Zhang, Lili Wu, Dr. Nan-qian Li, and Madeline Stull, for providing such a pleasant environment in the lab and for being my friends.

xii Finally, I would like to thank my family for all their love and care, and for always being on my side. I thank my Mom and Dad for being the best listeners when I was blue, and for traveling a long way to come visit me while I missed them. Especially, I would like to thank my Husband, for his love and support, his encouragements and valuable input during my doctoral research, and for sharing the passion for science.

xiii CHAPTER 1 INTRODUCTION

Important roles of vitamin A (retinol) and its derivatives in regulating antiviral and/or antibacterial immune responses have been studied since the 1920’s (1). Vitamin A is also implicated in the treatment of cancer, where it functions as a regulator of cell death and differentiation (2). It has also been found to synergize with interferons, which are strong mediators of immune functions (3) and cancer cell viability (4). The interferon signaling stimulates interferon regulatory factor-1 (IRF-1), an important transcription factor involved in cell growth and apoptosis, differentiation, and antiviral and antibacterial immune responses. The goal of this dissertation is to clarify the effects of an active metabolite of vitamin A, all-trans-retinoic acid, on IRF-1 gene expression and transcriptional functions, as part of the information required for establishing the interrelationships among vitamin A, interferons, and IRF-1. Current literature about the signaling and functions of each of these components is reviewed in Chapter 1.

1 1. Vitamin A Metabolism and Signaling

Vitamin A supplementation is known to provide protection from all-cause, diarrhea-specific, and measles-specific mortality in children (5). In the case of childhood morbidity, vitamin A has no effect on the prevalence of the diseases; however, it significantly reduced the frequency of severe and lethal illnesses (6). It is now commonly accepted that, improving the vitamin A intake in populations that have vitamin A deficiency (VAD) as part of public health problems can substantially enhance the innate immunity in these people by maintaining the physical and biological integrity of the epithelium as the first barrier to infection, and by increasing the interferon response to infection once the pathogen passes this barrier. Here, the metabolism, signaling, and physiological functions of vitamin A are reviewed.

1.1. Roles of Vitamin A in the Body

Dietary vitamin A is hydrolyzed by pancreatic cholesterol hydrolase (or an intestinal intrinsic hydrolase activity) and then absorbed in the small intestine (7). Dietary pro-vitamin A, β-carotene, is usually centrally cleaved by carotene 15,15’-dioxygenase and absorbed as retinal, which is later reduced to retinol by retinol dehydrogenase (8).

Retinol is esterified within the enterocyte and transported as a part of the lipid core of the chylomicron, and metabolized in the liver and other target tissues to various kinds of retinoids including retinyl esters, retinal, retinoic acid (RA), and polar metabolites of RA

(8). Esterified retinol is mainly stored in stellate cells of the liver. The transport of retinol from the liver to extrahepatic tissues and retinol’s cycling among these tissues are thought to be the major route of maintaining vitamin A homeostasis. In the circulation,

2 retinol is associated with retinol-binding protein (RBP) to form the retinol-RBP-

transthyretin complex (9). In cells of the organs, however, retinol is usually bound to cellular retinol-binding proteins (CRBPs), whereas RA is associated with cellular retinoic acid-binding proteins (CRABPs) (10). The sequestration of retinoids by these cellular binding proteins may protect them from non-specific oxidation; conversely, the damaging effect of excessive retinoids on the cell membrane can be prevented as well. Another function of CRBPs is to direct retinol for esterification (8). RA is considered the major physiologically active metabolite of retinol, although it cannot be reduced to retinal or retinol that limits the functions of RA to cell growth, differentiation, and gene expression.

Retinol or retinal, on the other hand, can also function in reproduction and the visual cycle (8).

Recent studies in our laboratory have characterized two important enzymes of vitamin A metabolism (11). One of them is lecithin:retinol acyltransferase (LRAT), which catalyzes the esterification of retinol; the other is a cytochrome P450, CYP26, which mediates the oxidation of RA to its polar metabolites (hydroxyl- or keto-

metabolites of RA). Both of these enzymes can be regulated by RA, especially in the

liver and lung of rats and mice. In fact, the regulatory function of RA in the expression of

LRAT and CYP26 has nominated RA as a signal of the body’s vitamin A adequacy.

During VAD, for example, the availability of RA is low and both LRAT and CYP26 are

downregulated, so that retinol is made available for other functional processes, such as

oxidative activation and secretion into the plasma. In case of vitamin A supplementation

or treatment with RA, on the other hand, LRAT and CYP26 levels are increased to

prevent retinol excess (11). It is particularly interesting that although LRAT expression is

3 induced by exogenous RA in both liver and lung, the response of lung LRAT to RA is

greater. This might be due to tissue-specific regulation of LRAT by RA, and/or

quantitative differences in RA concentrations within these tissues (11). Future research is

required to explain the observation, but it is by far agreeable that vitamin A metabolism

in the liver and lung is actively regulated by RA; in contrast, since LRAT is not closely

regulated by RA availability in the intestine, the absorption and homeostasis of vitamin A

in this tissue appear to be dietary vitamin A-independent (8). Moreover, a loss of LRAT

expression and subsequent retinol esterification has been reported for several cancer cell

lines and tumor tissues (11), suggesting a role of LRAT and retinoid metabolism in

preventing tumor differentiation and/or promoting apoptosis of cancer cells.

1.2. Retinoid Metabolism and Signaling in the Cell

The exploration of the cellular functions of RA emerged from the discovery of

retinoid receptors, retinoic acid receptor (RAR) and retinoid X receptor (RXR), which

will be described in detail in Section 1.3. One of the well-studied ligands of retinoid

receptors, atRA binds RAR with high affinity (Kd < 1 nM) and RXR with relatively lower

affinity. 9-cis-retinoic acid (9cRA), on the other hand, binds to RXR with high affinity

(Kd = 10 nM) while binding to RAR with a similar strength as atRA (12). Both atRA and

9cRA can enter the cell directly from the circulation and/or be synthesized from retinol and retinal in the target cell. They bind and activate RAR and RXR, which in turn translocate into the nucleus, form heterodimers (RAR/RXR; in case of atRA and/or 9cRA) or homodimers (RXR/RXR; in case of 9cRA) that interact with hormone-specific response elements (HRE)-related response elements. These elements consist of direct

4 repeats (DR) of two consensus sequences (AGG TCA), called half sites. The spacing between the two half sites is important for the specificity of these response elements. For example, strong retinoic acid response elements (RARE) are frequently observed as DR-5 or DR-2 (separated by either five or two nucleotides, respectively). People have summarized the so-called “1-2-3-4-5 rule”, where different hormone response systems contain different spacing of direct repeats (12). HRE-mediated pathways have been shown to positively modulate the expression of enzymes and receptors involved in vitamin A cellular metabolism, such as mouse RARβ, CRBP-I, CRABP-II, and rat

CRBP-II (12). As a summary, a model of retinoid signaling and metabolism in the cell is illustrated in Figure 1.

5 RBP Retinol Retinol Esters atRA

?

RDH ⊕ CRBP ? Retinol Retinal Other RA Metabolites LRAT ⊕ REH RALDH ⊕ CYP ⊕ Retinyl CRABP Esters 9cRA atRA (Storage)

? ?

NUCLEUS 9cRA ⊕ atRA RXR RAR

RARE

9cRA 9cRA RXR RXR

HRE

Protein Expression and CYTOPLASM Cellular Differentiation

Figure 1 A model of cellular events in retinoid signaling and metabolism. atRA, all-trans-retinoic acid; 9cRA, 9-cis-retinoic acid; RBP, retinol binding protein; CRBP, cellular retinol binding protein; CRABP, cellular retinoic acid binding protein; RDH, retinol dehydrogenase; RALDH, retinal dehydrogenase; REH, retinyl ester hydrolase; CYP, cytochrome P450 enzymes; LRAT, lecithin:retinol acyltransferase; RAR, retinoic acid receptor; RXR, retinoid X receptor; RARE, retinoic acid response elements; HRE, hormone-specific response elements. Enzymes or receptors that can be positively modulated by RA are labeled with ⊕’s. Processes that have not been clarified are labeled with question marks. This figure is based on references (8, 11, 12).

6 1.3. Retinoid Receptors and Signaling

In 1987, cDNA of the first member of the RAR family, human RARα, was isolated and shown to encode an RA-activated transcription factor (13, 14). This human

RAR has a DNA-binding and a ligand-binding domain structurally and functionally similar to the members of the nuclear receptor family. Thus, it was immediately hypothesized that RA could function the same way as hormones, penetrating the cell membrane due to its hydrophobic character and binding to an intracellular receptor (i.e.

RAR). The RA-bound receptor would then translocate into the nucleus and interact with

HREs in the target gene promoters and thereby elicit transcriptional activities (12).

Meanwhile, additional RAR-related genes were isolated and up to now three different

RAR subtypes (RARα, RARβ, RARγ) are known to be expressed in a variety of tissues in mammals, birds, and amphibians. These RAR subtypes are expressed in distinct patterns throughout development and in different organs, indicating that they may function differently (Figure 2A). Nevertheless, the DNA-binding and ligand-binding domains of all subtypes are highly conserved, suggesting that they may have arisen from a common ancestral RAR gene.

7

(A) Chromosome Expression

(Human) Pattern

RARα DN A atRA or 9cRA 17q21.1 widespread

RARβ 97 % 82% 3p24 muscle, prostate

RARγ 97 % 76% 12q13 skin, lung

(B) Chromosome Expression

(Human) Pattern

RXRα DNA 9cR A 9q34.3 liver, skin, lung

RXRβ 92% 88 % 6p21.3 widespread

RXRγ 95% 86 % 1q22-q23 muscle, heart

Figure 2 Two families of retinoid receptors. Different subtypes of RARs (A) and RXRs (B) are expressed in distinct organs, but their DNA-binding and ligand-binding domains are highly conserved within each family (12).

8 In 1990, a second class of retinoid-responsive transcription factors, RXRs, was discovered (15, 16). Three different subtypes of RXRs (RXRα, RXRβ, RXRγ) have been reported in mammals, birds, and amphibians (Figure 2B). Although both RARs and

RXRs are able to respond to RA, the fact that they do not elicit the closest similarity in amino acid compositions has suggested that the response to RA by these two receptors has evolved into two distinct pathways. In fact, RAR has a greater similarity to thyroid hormone receptor β than it does to RXR (12).

The RAR family is activated by both atRA and 9cRA, whereas the RXR family is activated exclusively by 9cRA. Artificial retinoids have also been synthesized to bind certain RAR or RXR specifically, such as Am580 and Ro41-5253, which activates or antagonizes RARα, respectively (Figure 3). These synthetic retinoids have been used extensively in studies of retinoid signaling within the cell. Some of them, such as Am580, are not easily catabolized as natural retinoids and can be used to prolong the activation of specific RARs and RXRs. Thus, the roles of individual retinoid receptors, which are usually complicated in the response to a pan-activator such as atRA, can be sorted out in details by using receptor-selective retinoids.

9

(A)

(B)

(C)

(D)

Figure 3 Common natural and synthetic retinoids. (A) All-trans-RA; (B) 9-cis-RA; (C) Am580, an RARα agonist; (D) Ro41-5253, an RARα antagonist.

10 The amino terminus of RAR or RXR contains a domain of transactivation

function (AF1), which is variable among different receptors. This domain is responsible

for the recognition of coregulators and/or other transcriptional factors. The DNA-binding

domain (DBD) in the center of the protein contains two zinc-finger motifs. The domain at

the carboxyl terminus, the ligand-binding domain (LBD), has a dual function of ligand

recognition and transactivation (AF2). In the absence of ligand (Figure 4A), the LBD of the receptor is bound with transcriptional corepressors, such as nuclear receptor

corepressor 1 (NCoR1) or silencing mediator for retinoid and thyroid hormone receptor

(SMRT), which recruit histone deacetylase (HDAC) that prevents the condensed

chromatin from acetylation and thus the gene from transcriptional activation. In the

presence of ligand (Figure 4B), corepressor complex dissociates from the LBD of the

receptor due to an allosteric conformational change of the LBD. The holo nuclear

receptor allows coactivators containing LXXLL motifs to interact with its LBD. Histone

acetyltransferase (HAT) is then recruited by the coactivators to initiate transcription (17,

18).

Heterodimerization of RARα/β/γ and RXR enables the recognition of RARE by

the complex. Unlike steroid receptors that bind to response elements composed of an

inverted repeat (IR) of two core hexameric motifs RGGTCA, where R is a purine,

RAREs are usually DRs of the two motifs (half sites). The most common form, DR-5, is

found in the RARβ2 and RARα2 genes. DR-1 and DR-2 have also been found (in the

CRABPII and CRBPI genes, respectively). RXR homodimers, on the other hand, prefer

DR-1, although they bind to DR-1 less efficiently than RAR/RXR heterodimers (19).

11 (A) HDAHDACC

CCoorreepprresessosorrss

AF2 AF2

RXR RAR AF1 AF1

Basal DBD DBD Transcriptional Machinery AGGTCA AGGTCA TATA

(B) Retinoids HAHATT

CCooactiactivvaattoorrss

AF2 AF2

RXR RAR AF1 AF1

Basal DBD DBD Transcriptional Machinery

AGGTCA AGGTCA TATA

Figure 4 A model of interactions among RAR, RXR, corepressors, and coactivators. AF, transactivation function; DBD, DNA-binding domain; HDAC, histone deacetylase; HAT, histone acetyltransferase.

12 Retinoid receptors localize presumably in the nucleus, although direct evidence is scarce. One report has shown that nucleocytoplasmic translocation of RARβ is facilitated by RXR in a ligand-dependent manner (20). Moreover, it has been demonstrated that downregulation of cellular kinases, such as protein kinase C (PKC), decreases RARα nuclear localization (21). Phosphorylation of RARα on Ser-369 by an overexpressed catalytic subunit of protein kinase A increases the transcriptional activity of RARα (22), potentially through increasing nuclear translocation of the receptor. Phosphorylation at

Ser-157, on the other hand, is involved in downregulating RXR/RARα heterodimerization and transcriptional activity (23). Clearly, the modification and localization of RARs, which may differ from one cell model to another, remain to be clarified. Additionally, atRA of pharmacological concentrations is suggested to act as an inhibitor that competes the binding of acidic phospholipids to PKCα, thereby preventing

PKCα activation (24, 25). Since PKCα can phosphorylate RARα, atRA may affect modification and nuclear translocation of RARα through PKCα.

The localization and/or nucleocytoplasmic shuttling of two other nuclear hormone receptors, vitamin D receptor (VDR) and thyroid hormone receptor (TR)α, have been examined. RXR promotes nuclear accumulation of VDR, which is more cytoplasmic if unliganded (26). TRα, on the other hand, shuttles between the nucleus and cytoplasm via ligand- and energy-dependent, but nuclear export receptor-independent processes (27). In addition, RXR is suggested to act as a carrier for nuclear export of orphan receptor TR3 to the mitochondria in a 9cRA-dependent manner (28, 29). As a shared

13 heterodimerization partner, RXR may regulate the nuclear/cytoplasmic localization of

RAR as well.

The degradation of RAR and RXR is mediated by the ubiquitin-proteasome pathway. RA, a ligand for RARs, induces degradation of RARα and RARγ (30, 31) and the breakdown of the heterodimerization partner, RXR (32). RA-induced degradation of

RARγ is mitogen-activated protein kinase (MAPK)-mediated phosphorylation-dependent, and is required for RARγ turnover and thereby enhancing RARγ-mediated transactivation

(33). Alternatively, atRA-induced receptor degradation may potentially relieve transcriptional repression induced by the receptor. In addition, RARα is degraded after it is phosphorylated by c-Jun N-terminal kinases (JNK), which has a high activity in lung tumors of mice carrying an activated K-ras oncogene (34). Inhibition of JNK in the tumors increases RARα expression and ligand-induced transactivation, thereby restoring the growth inhibition functions of RA. This report suggests roles of JNK and RARα in malignant transformation of cancer cells, and the potential use of JNK inhibitors as mediators to reconstruct retinoid signaling in tumors (2).

Cross-talk between retinoid receptors and other signaling molecules has been observed in a number of situations. One of them, AP-1 (c-Jun/c-Fos), has been shown to antagonize the transactivation functions of RAR/RXR heterodimers. Overexpression of any one of the three RARs substantially suppresses AP-1-induced transcription, although a direct interaction of RARs with either AP-1 or the AP-1-binding site is not identified

(35, 36). Conversely, overexpression of either c-Jun or c-Fos can inhibit the activity of

RARE (37). β-catenin, a component of the Wnt signaling pathway, cross-talks with RAR

14 in a similar fashion. β-catenin interacts directly with RAR in a RA-dependent manner,

increasing the effect of the ligand and receptor on RARE; in turn, RAR competes with

coactivators for β-catenin binding, thus decreasing the activity of the Wnt/β-catenin signaling pathway (38). Since both AP-1 and β-catenin are associated with numerous

cancers, RA-induced downregulation of these signaling pathways suggests mechanisms

by which vitamin A may affect cancer development.

Moreover, a yeast two-hybrid screening of nuclear factor (NF)-κB inhibitor IκBβ- interacting proteins revealed RXR, which binds IκBβ, a lipopolysaccharide (LPS)- induced nuclear factor that prolongs activation of NF-κB (39), in the presence of 9cRA

(40). Such interaction represses 9cRA-induced transcriptional activity of RXR in an LPS- dependent manner. Further, NF-κB components p50 and p65 bind RXR in a ligand- independent fashion in vitro, masking the DBD of RXR; conversely, RXR also inhibits

NF-κB-mediated transactivation (41). Collectively, it can be hypothesized that in the presence of 9cRA, RXR serves as a suppressor of NF-κB-mediated transactivation by binding to both the p50/p65 heterodimer and IκBβ. In addition, at least one report has shown a direct interaction between RAR and NF-κB; and they appear to reciprocally regulate each other’s transactivation functions (42).

1.4. Roles of Retinoids in Antiviral Responses

Vitamin A was recognized as “an anti-infective agent” by Green and Mellanby in

1928 (43), not long after its discovery in 1913 (44, 45). Processes important for generating antiviral responses, including hematopoiesis, maturation and activation of

15 lymphocytes, as well as antibody and cytokine production by lymphocytes, have all been shown to be vitamin A-responsive (46). Although there is a lack of direct correlation between incidence or severity of virus infection and vitamin A status in humans, animal studies have strongly suggested that when vitamin A-deficiency is present, the damage of epithelium resulting from the infection is greater, and of longer duration (46).

Diarrhea and respiratory infections, in particular, are common in vitamin A- deficient children. In the lung, vitamin A may exhibit functions in promoting normal respiratory epithelial differentiation and growth and preventing inflammation (47).

Tissues damaged by viral infections usually have an increased demand for vitamin A.

However, therapeutic effects of vitamin A supplementation have been contradictory. In a study conducted in children admitted to the hospital with non-measles pneumonia, large doses of vitamin A (200,000 IU for children > 1 year of age; 100,000 IU for infants; administered on the day of admission, followed by another dose on the second day) had no protective effect on the recovery of these children (48). In another study, children having community-acquired pneumonia were given the same doses of vitamin A on the day of admission to the hospital, followed by half doses on the next day (49).

Unfortunately, children with vitamin A supplements developed increased clinical severity compared to the placebo group. It is thus suggested that high-dose vitamin A supplements should not be used therapeutically unless there is clinical evidence of VAD or concurrent measles infection. In addition, a meta-analysis consisting of twelve large-scale field trials in seven countries revealed that although vitamin A supplementation reduced diarrhea- or measles-specific mortality in infants and small children, it had little effect on pneumonia mortality (5).

16 In contrast, positive effects of vitamin A supplementations have also been suggested. Low plasma vitamin A concentrations were shown to be associated with increased airway infections in mechanically ventilated very-low-birth-weight neonates

(50). It was suggested that vitamin A stores in the liver were depleted to compensate the increased tissue demand caused by the infection. Vitamin A was also shown to reduce radiation-induced lung inflammation in rats (51). In addition, a high level of vitamin A

(250,000 IU/kg diet) given to influenza A virus-infected BALB/c mice was shown to increase production of salivary immunoglobulin (Ig)A and a T-helper (Th)2 cytokine, interleukin (IL)-10, while decreasing the production of serum IgG and a Th1 cytokine, interferon (IFN)γ (52). As reviewed by Ross and Stephensen (46), VAD may affect host response to viral infections in many aspects, including decreasing functions of natural killer cells and cell-mediated immune responses, and severely impairing regeneration of virus-damaged epithelia. Thus, the severity of infection is usually increased in case of

VAD.

Inflammatory response initiated by infections, on the other hand, may alter normal transport and metabolism of vitamin A (46). It is suggested that the acute phase response upon virus infection would reduce plasma levels of vitamin A, primarily by reducing vitamin A hepatic mobilization, increasing retinol excretion, and decreasing vitamin A intake and/or absorption. As a result, less retinol would be available for repairing or regenerating epithelial damages caused by the infection, and the severity of the infection and the possibility of a second infection would increase. Low levels of vitamin A in the circulation, together with the onset of the infection, would lead to alterations of normal immune responses such as innate response, lymphocyte

17 proliferation, and cytokine and antibody production (46). Thus, vitamin A supplementation, especially given before the onset of virus infection, may reduce the severity of the infection by repleting vitamin A stores and availability, boosting immune responses, and repairing infection-induced epithelial damage. To dissect the effect of vitamin A in the immune system, the roles of retinoids are reviewed here regarding both innate and adaptive responses.

In the innate stage of an immune response, vitamin A acts on epithelial barriers and cells recruited to the site of infection, such as neutrophils, macrophages, and NK cells (53). VAD is associated with the loss of mucus-producing goblet cells lying on the conjunctiva of the eye, and respiratory, gastrointestinal, and urogenital tracts. The loss of mucus decreases the ability of the epithelia to resist infections. In addition, VAD can also result in squamous metaplasia in the respiratory tract, increasing the chance of repetitive infection and invasive disease. The migration and phagocytic functions of neutrophils are also diminished in VAD (54). Furthermore, VAD induces inflammation by directing macrophages producing proinflammatory cytokines to the secondary lymphoid organs; whereas the phagocytic functions of these macrophages are impaired (53). Natural killer

(NK) cells, an important population of killer cells in the early stages of viral infection, are affected by VAD as well, with both the cell number and cytotoxic activity of NK cells diminished by VAD (46).

18 The effects of retinoids on pathogen-specific (adaptive) immunity are nicely

reviewed by Stephensen (53). Briefly recited (Figure 5), VAD decreases the Th2

response by lowering the production of IgG, IgE (55), and mucosal IgA (56). The Th1

arm of the balance is somewhat impaired, too, with functions of cytotoxic CD8+ T cells

(57) and the delayed-type hypersensitivity response (58) decreased by VAD. On the other hand, the administration of RA downregulates the production of IL-12 (41) and IFNγ (59,

60), cytokines functioning in the development of Th1 response. RA may also increase the

Th2 response through affecting IL-4 production by lymph nodes (61). In addition,

vitamin A is a regulator for the growth, differentiation, and antibody production of B

cells (62-65).

19

Figure 5 Effects of vitamin A on adaptive immunity. This figure was taken from a review article by Stephensen (53). Reprinted, with permission, from the Annual Review of Nutrition, Volume 21 (c) 2001 by Annual Reviews www.annualreviews.org

20 1.5. Roles of Retinoids in Cell Death and Differentiation

Retinoids have potent effects on cell differentiation and viability. They are used

clinically for the treatment of acute promyelocytic leukemia (APL), since the most

common forms of APL are caused by chromosomal translocations resulting in two fusion

genes: X-RARα and RARα-X (where X is the alternative RARα fusion partner) (66).

The clinical effect of RA is thought to be primarily due to its ability to promote

differentiation of promyelocytic cells.

RA has a unique role of both promoting and inhibiting apoptosis. It can prevent

activation-induced apoptosis of T cells, which nominates it as a potential therapy to

prevent the loss of lymphocytes in human immunodeficiency virus-infected individuals

(67). However, in the absence of T cell activation, RA induces apoptosis of thymocytes

(68).

On the crossroad of cell death or differentiation, retinoids may exert different effects depending on the retinoid receptors that are being called upon. In the differentiation and subsequent apoptosis of the myeloid leukemia cell line HL-60, it has been well established using receptor-selective retinoids that the differentiation phase is mediated by RARα, whereas the apoptosis phase is RXR ligand-dependent (69). It appears that retinoids and their receptors work cooperatively in regulating the balance of cell death and differentiation in order to fulfill the physiological outcome that the cells need.

Another focus of this section is on the function of retinoids to fight against cancer.

Retinoids can interfere with carcinogenesis at several levels (2). First, by inhibiting the activity of AP-1 induced by growth factors, oncogenes, and/or tumor promoters, retinoids

21 can block carcinogenesis at the promotion step (the interaction between retinoid receptors and AP-1 has been reviewed earlier, in Section 1.3). Second, retinoids can induce differentiation of progenitor cells and might overcome an obstacle in the differentiation pathway, such as the case in APL. Third, retinoids can block the cell cycle at G1 phase, frequently due to downregulation of c-Myc and cyclins E, D1/D3, A and B, and upregulation of cell cycle inhibitor p21 (70). And fourth, retinoids can induce apoptosis of cancer cells through activation of death receptors and/or their ligands, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

TRAIL is a unique tumor suppressor due to its role of selectively inducing apoptosis in cancer cells, but not normal cells (71). Retinoids have been shown to activate the TRAIL promoter through RAR/RXR-mediated transcription of IRF-1 (72). It is suggested that RARβ, itself a tumor suppressor that is epigenetically silenced in many solid tumors such as breast and lung cancers, may be involved in the activation of TRAIL

(71). Retinoids can also induce the expression of TRAIL receptors, DR4 and DR5 (73,

74), which are also p53-target genes. Other inputs that regulate TRAIL and TRAIL- receptor gene expression include chemotherapy (DNA damage), interferons, tumor necrosis factor (TNF), and HDAC inhibitors (Figure 6) (75).

22

RA

β IFNs RXR RAR p53 RE ?

DR4,5

DNA STAT p53 damage TNF ?

IRF-1 STAT NFκB TRAIL IRF-E / ISRE STAT NFκB Ac Ac Ac Ac Ac Ac Ac Ac

HDAC inhibitor

Figure 6 Regulatory inputs in TRAIL and TRAIL-receptor (DR4 and DR5) gene expression. Please refer to the text for details. This figure is based on references (71, 75). TNF, tumor necrosis factor; TRAIL, TNF-related apoptosis-inducing ligand; IRF-1, interferon regulatory factor-1; STAT, signal transducer and activator of transcription; NF-κB, nuclear factor-κB; HDAC, histone deacetylase; IFNs, interferons; DR, death receptor; RE, response element.

23 In addition to their effects on TRAIL, retinoids also modulate other components

of the apoptosis pathway (Figure 7). In prevention of activation-induced apoptosis of T

cells, RA inhibits the expression of the Fas ligand (FasL) (76); on the other hand, Fas

expression and Fas/FasL-mediated apoptosis can be induced by a synthetic retinoid,

CD437, in human lung cancer cells (77). A CD437 analog, MX3350-1, is recently shown

to decrease the levels of anti-apoptotic proteins Bcl-2 and Bcl-xL, increase pro-apoptotic

Bax, and induce cytochrome c release from the mitochondria to the cytosol (78).

Furthermore, caspase-8 can be induced by N-(4-hydroxyphenyl)retinamide, a potential chemopreventive or chemotherapeutic agent (79).

24

Ligands: TRAIL or FasL

TRAIL-R Fas Death Signals

8

- DD e

spas FADD ? Mitochondria o-ca

r P

Bcl-2 ⊕ Caspase-8 Cyt c Bax Bcl-xL

e e-9 as p

Caspase spas

Caspase Caspase-9 s cascad e o-ca r o-ca r P

P

Caspase-3 Apoptosis

Figure 7 Retinoid-mediated induction of apoptosis. The thick arrows indicate changes induced by retinoids. Please refer to the text for details. This figure is based on references (2, 76-79). TRAIL, tumor necrosis factor-related apoptosis-inducing factor; TRAIL-R, TRAIL receptor or death receptor; FasL, Fas ligand; FADD, Fas-associated death domain; DD, death domain; Cyt c, cytochrome c.

25 2. Interferon Signaling Pathways

Interferons were discovered as proteins that inhibit virus replication (3). They are

induced in response to virus infection, secreted, and act on surrounding cells (infected or uninfected) to activate a global antiviral state. In addition to their antiviral effects, interferons are also essential to cell growth, differentiation, and functions, especially those of the cells in the immune system. There are two types of interferons, type I

(mainly IFNα/β) and type II (IFNγ). IFNα/β are considered primary cytokines involved

in antiviral responses, especially during early stages of virus infection (80). Although

most types of virally infected cells are capable of producing them in cell culture, the

natural IFNα/β-producing cells are precursor dentritic cells (81). IFNγ is produced only

by certain cells in the immune system, such as natural killer cells, CD4+ T cells, and

CD8+ regulatory T cells. It is a secreted glycoprotein whose active molecule is a dimer

(3). IFNγ is expressed at a low level upon early infection, and is thought to be more important during later stages of infections when acquired immunity is activated (80).

However, since IFNγ activates different pathways and induces different genes than

IFNα/β, enhancing the functions of albeit small amount of IFNγ during early stages of

infection may improve the total immune response against the infection and encourage

faster recovery.

In general, all interferon signaling pathways start with binding of interferons to

their receptors on the surface of the target cell. Interferon receptors are composed of

several subunits with single transmembrane domains. The cytoplasmic domains of

interferon receptors recruit specific protein kinases that are activated when interferons

26 bind to the extracellular domains of the receptors. The activation process involves

receptor oligomerization and tyrosine phosphorylation of the receptors. Tyrosine kinases

involved in this process also phosphorylate themselves and other proteins important for

the activation of downstream transcription factors. Transcriptional factors after activation then translocates into the nucleus, binds to corresponding cis-acting elements of target genes, and induces or suppresses transcription of these genes. Here, both Type I and Type

II interferon signaling pathways are reviewed.

2.1. Signaling of Type I Interferons

Type I interferons are comprised of 13 IFNα genes, one single IFNβ gene, and one IFNω. They all lack introns and are clustered on the short arm of chromosome 9 in the human and chromosome 4 in the mouse (81). Most human IFNα subtypes are not glycosylated and function as monomers, whereas IFNβ is glycosylated and function as a

homodimer. IFNα genes can be divided into two groups: an immediate-early gene α4,

and genes of delayed induction including α2, α5, α6, and α8 (81). It is not known why

there are multiple IFNα genes. IFNβ, on the other hand, is suggested to be required for a

fully effective antiviral response, as IFNβ-null mice are highly susceptible to virus

infection (82). IFNα/β are considered primary cytokines involved in antiviral responses,

especially during early stages of virus infection (80). IFNω is produced only in

hematopoietic cells (3).

The IFNα/β signaling pathway contains at least two receptor subunits, IFNAR-1

and IFNAR-2, and two Janus family kinases, Tyk-2 and Jak-1. IFNAR-1 (also called the

27 α chain) has the Tyk-2 binding domain and signal transducer and activator of

transcription (STAT)-2 docking site proximal to the transmembrane domain, and a

negative regulatory domain located in the distal portion of the cytoplasmic tail. Two

phosphatases, Src homology domain 2 (SH2)-containing tyrosine phosphatase (SHP)-1

and SHP-2, have been reported to interact with IFNAR-1, although only SHP-1 appears

to dephosphorylate STAT proteins. IFNAR-1 also binds protein kinase A, extracellular-

signal-regulated kinase, and phosphatidylinositol-3 kinase, but the role of these interactions is unclear (83). There are four tyrosines in the cytoplasmic domain of

IFNAR-1, all of which are possible targets of phosphorylation by Jak or other kinases.

The long form of IFNAR-2 (also called the βL chain) has docking sites for STAT-2, as

well as Jak-1 and STAT-1 interacting domains. It is considered the primary signaling

chain, as its cytoplasmic tail has seven tyrosines, and a Box 1 motif that is consensus and

important in Jak-2 binding. Tyrosine phosphorylation occurs rapidly upon binding of type

I interferons to IFNAR-1/2, and is required for full activation of STAT-2 (83). However,

the role of tyrosines in STAT-1 activation of IFNα/β signaling remains to be elucidated.

The short form of IFNAR-2 (also called the βS chain) contains two tyrosine residues, but their role is unknown (83).

Phosphorylated tyrosines of IFNARs act as docking sites for SH2 domains of

STAT proteins. Association of STAT-2 with the cytoplasmic tail of the long IFNAR-2 leads to phosphorylation of STAT-2 at Tyr-690, which serves to recruit STAT-1. STAT-1 is phosphorylated at Tyr-701 and forms heterodimers with STAT-2 by phosphotyrosine binding. The STAT-1/STAT-2 heterodimers form while bound to the IFNARs, and upon release, they interact with a small protein called p48 or IRF-9, leading to the formation of

28 a trimeric transcription complex, interferon-stimulated gene factor 3 (ISGF-3). IRF-9 acts as the DNA-binding protein and adapter for STAT-1/STAT-2 heterodimer after the

ISGF-3 complex enters the nucleus. The complex binds and activates transcription of target genes through the conserved interferon-stimulated response element (ISRE;

AGTTTNNNTTTCC) (83, 84). It is suggested that ISGF-3-mediated transcriptional activation depends primarily on STAT-2, as STAT-1 is shared by numerous signaling pathways. In fact, the carboxyl-terminus of STAT-2 (the transcriptional activation domain) has been demonstrated to interact with cAMP response element binding protein

(CREB)-binding protein (CBP)/p300, a histone acetyltransferase (HAT). Methylation of

Arg-31 of STAT-1 by protein arginine methyltransferase, on the other hand, enhances the binding activity of the whole ISGF-3 complex (83).

2.2. Signaling of Type II Interferon

Type II interferon includes IFNγ. Unlike the intronless Type I interferons, the

IFNγ gene possesses three introns and maps to the long arm of chromosome 12 in the human and chromosome 10 in the mouse (81). The IFNγ signaling pathway (Figure 8) is initiated upon binding of IFNγ homodimers to interferon receptors, IFNGR-1 (ligand- binding chain) and IFNGR-2 (signal-transducing chain).

29

IFNGR: R-2 R-1 R-1 R-2

IFNγ dimer

CYTOPLASM Ê Jak Jak Ê PKC

Ê − SH2 S STAT-1 Ê−Y440 Y440−Ê SHP

SOCS-1

PIAS Y−Ê STAT-1 STAT-1 Ê−S Ê−Y S−Ê

? NUCLEUS

Y−Ê STAT-1 STAT-1 Ê−S Ê−Y S−Ê

GAS: TTN5AA TATA

STAT-1 co-activator complex

Figure 8 A model of cellular events in STAT-1-dependent IFNγ signaling. Please refer to the text for details. This figure is based on references (85-96). IFNGR, IFNγ receptor; STAT, signal transducer and activator of transcription; GAS, γ-interferon activated site; Jak, Janus kinase; SHP, SH2-containing tyrosine phosphatase; SOCS, suppressor of cytokine signaling; PKC, protein kinase C; PIAS, protein inhibitor of activated STAT.

30 Both IFNGR chains belong to the cytokine class II receptor superfamily and are

required for initiation of IFNγ signaling, although they have evolved to reside on different chromosomes in humans (97). The extracellular portion of IFNGR-1 chain is found to be organized into two Ig-like domains, which contains multiple disulfide bonds that are required for maximal binding of the ligand (98). The receptor chain thus has to be non-reduced to be able to bind IFNγ. In addition to the cysteine residues, several other residues in the binding interface such as lysine, tryptophan and glutamate, are also shown to be important for ligand-binding. IFNGR-1 has functions in signal transduction as well.

In fact, the intracellular portion of IFNGR-1 contains a five-residue sequence

(Y440DKPH444) distal to the membrane and a four-residue segment (L266PKS269) in the proximal region that are important for STAT-1 recruitment and Jak-1 binding, respectively. The first sequence is completely conserved in human and mouse receptors.

Upon ligand-binding, residue Tyr-440 becomes phosphorylated, presumably by Jak-1 bound to membrane-proximal four-residue segment (99), and acts in recruiting STAT-1

(100). Other residues in the sequence are likely to be responsible for enhancing the interaction between STAT-1 SH2 domain and the phosphorylated Tyr-440.

IFNGR-2, on the other hand, is not phosphorylated under any circumstances; rather, it recruits Jak-2 that assists in the phosphorylation of Jak-1 and IFNGR-1 (88).

The last 49 residues of the carboxyl-terminus of this chain (cytoplasmic portion) have been shown to be required for IFNGR-1 tyrosine phosphorylation, Jak-2 binding, Jak-1 and Jak-2 phosphorylation, and STAT-1 activation. Cytoplasmic domains of IFNGR-2, like those of IFNGR-1, can be interchanged between species with no loss of biological activity, whereas the extracellular domains are species-specific (88). Jak proteins that

31 bound to cytoplasmic domains of IFNGRs are interchangeable as well; thus, they do not contribute to the ligand specificity of Jak-STAT signaling to the same degree as STAT proteins (101).

Nevertheless, upon binding of IFNγ homodimer to IFNGR-1 (Kd = 0.2 nM), the oligomerization of IFNGR-1 and IFNGR-2 leads to phosphorylation and activation of

IFNGR-1, Jak-1, Jak-2, and STAT-1 (88). Jak-1 and Jak-2 are phosphorylated at Tyr-

1022/1023 and Tyr-1007/1008, respectively. Jak kinases are responsible for STAT-1 activation, although the cause of the latter is not restricted to either Jak-1 or Jak-2 (102).

STAT-1 is phosphorylated at Tyr-701, leading to STAT-1 activation and dimerization through SH2 domains (103, 104). STAT-1 homodimer is translocated into the nucleus via the GTPase activity of Ran (105) and binding directly to importin-α5 (105, 106).

Although STAT-1 does not possess a classical nuclear localization signal (NLS), an

Arg/Lys-rich structural element has been found within the DBD of the molecule (107).

After nuclear translocation, the homodimer binds and activates transcription of target genes through the conserved γ-interferon activated site (GAS) (108). STAT-1 is possibly exported out of the nucleus upon dephosphorylation (109, 110).

STAT-1 is important for both IFNα/β and IFNγ pathways, as STAT-1-null mice developed normally but were extremely susceptible to microbial and viral infections (85).

STAT-1 positively regulates transcription of more than 200 interferon-stimulated genes

(ISG). It also negatively modulates certain genes involved in regulating the extracellular matrix, cell cycle, and thyroid-specific functions. The primary function of many genes regulated by STAT-1 is growth control. STAT-1-null mice failed to respond to IFN- induced growth arrest, and developed tumors in response to lower doses of

32 methylcholanthrene compared to normal mice (111, 112). In addition, the lack of

STAT-1 is known to affect lymphocyte survival and proliferation in mice (113). Further,

STAT-1 activation is associated with that of p21, a cell-cycle inhibitor (114).

Tyrosine phosphorylation is thought to be essential for dimerization and activation of STAT proteins, although it has been recently reported that STAT-1 may exist as stable homodimers in absence of phosphorylation (115). It is suggested that unphosphorylated STAT-1 could bind DNA constitutively but indirectly (86). STAT-1 is also phosphorylated on Ser-727 (116) by a process involving PKC (117). The mutation of this serine residue led to a 50% reduction of IFNγ-induced IRF-1 promoter activity, indicating an effect of serine phosphorylation on maximizing the transcriptional activity of STAT-1 (116). However, little evidence has involved STAT-1 serine phosphorylation in the regulation of DNA binding, nuclear translocation, or tyrosine phosphorylation of this protein (118). It is suggested that serine phosphorylation prepares STAT-1 for an enhanced transcriptional response once a second stimulus causing the phosphorylation of

Tyr-701 is received (118). A closer look identified an evolutionarily conserved P(M)SP motif in the C-terminal region of STAT-1 (118), usually targeted by proline-directed serine kinases, such as MAPKs and PKC (119).

STAT-1 also interacts with other proteins to exert its transactivation functions. In the nucleus, STAT-1 can interact with transcriptional co-activators CBP/p300 and at least one member of protein inhibitor of activated STAT (PIAS) family (85, 87). CBP/p300, as mentioned above, has HAT activity. Its association with STAT-1 links STAT-1 to the basal transcriptional machinery and requires both the carboxyl- and amino-termini of

STAT-1 (86). Interruption of this interaction by E1A protein of the adenovirus blocks

33 STAT-1 signaling and allows viral replication in the host cell (87). PIAS-1 interacts with

STAT-1 and serves as a negative regulator (120). In addition, the interaction between

STAT-1 and NF-κB has been identified on the promoters of IRF-1 (121), intercellular adhesion molecule-1 (ICAM-1) (122), inducible nitric oxide synthase (iNOS) (123), and

IFN-induced protein 10 (IP-10) (124). STAT-1 also interacts with Sp1, leading to synergized transactivation (125).

Negative modulation of the Jak-STAT pathway is also an important mechanism of regulation in IFNγ signaling. It is mediated, at least in part, by protein tyrosine phosphatases and the proteasomal degradation machinery. SHP-2, a ubiquitously expressed cytoplasmic protein tyrosine phosphatase, can dephosphorylate Jak and block

IFNγ signaling (91, 93). Interestingly, the phosphatase activity of SHP-2 also contributes to Jak stability, as phosphorylated Tyr-1007/1008 of Jak is a critical recruitment site for suppressor of cytokine signaling (SOCS)-1, which in turn causes inactivation (94) and ubiquitination/degradation (89) of Jak. In addition, both SHP (93, 96) and SOCS-1 (95,

126) contain SH2 domains that could potentially compete with the SH2 domain of

STAT-1 for binding to Tyr-440 of human IFNGR-1 (or Tyr-441 in mice), thereby interfering with STAT-1 activation. A third group of negative regulators of the Jak-STAT pathway, PIAS proteins function as E3 ligases that promote SUMO (small ubiquitin-like modifier) modification on Tyr-703 of STAT-1 and inhibit STAT-1-mediated transcriptional activation (90, 92).

Several other signaling molecules are also activated in parallel with the STAT-1- dependent pathway in response to IFNγ stimulation. These include MAPKs; the Src-

34 family kinase Fyn; adapter proteins c-Cbl, Crk and Vav; and G-protein-linked signaling molecules C3G and Ras GTPase-activating protein 1 (85).

2.3. Roles of Interferons in Antiviral Responses

Ever since their discovery in the 1950’s, interferons have been considered essential regulators of antiviral responses. Between the two types, Type I interferons have been the focus of many studies. They are known to elicit antiviral activity in target cells, and/or to induce apoptosis in virus-infected cells (127). IFNγ, on the other hand, has started to attract more attention due to its equally significant role in antiviral responses.

For example, IFNγ is essential for the responses to double-stranded RNA (dsRNA) and encephalomyocarditis virus in cells lacking a functional IFNα/β receptor (128). Here, the antiviral events initiated by each type of interferons are reviewed.

35 IFNα/β are involved in an antiviral network (Figure 9) established by multiple

cells types of the immune system, including dendritic cells (DCs), NK cells, and CD8+ T cells. Upon virus invasion, IFNα/β are produced by plasmacytoid DCs through binding of virus-activated IRF-3 and NF-κB to IFNα/β promoters. The secretion and subsequent recognition of IFNα/β by IFNARs in an autocrine and/or paracrine fashion activate the

Jak-STAT pathway, leading to de novo transcription of the IRF-7 gene, an important player of the positive-feedback loop of IFNα/β production. IRF-7, same as IRF-3, is

phosphorylated and activated by viruses. IRF-7 and IRF-3 act cooperatively on IFNα/β

promoters, thus establishing the feedback loop (127). Such an amplification mechanism

appears to be important for the massive production of IFNα/β necessary for the

generation of a global antiviral state. Indeed, IFNα/β subsequently induce IL-12 from

myeloid DCs, which promotes IFNγ production by NK cells via the signaling of STAT-4

(129). IFNα/β also induces IL-15, an inducer of IFNγ-producing CD8+ T-cell and NK populations (130). In addition, IFNα/β can increase the cytotoxicity of NK cells (129).

Collectively, IFNα/β are immediate-early responders to viruses, serving as central

players of the antiviral network during the early stages of an infection.

36

DC P IRF-3

NF-κB

IFNγ production ISRE PRD NF-κB Cytotoxicity IP-10 & others IFNβ

PRD-LE

IFNα 1

- 4 AT

- NK ST

AT ST

Survival IFNα/β IFNARs

Jak Tyk IL-12 CD8+

IL-15

1 2

- P - IFNγ production AT AT Survival ISGF-3 P ST ST PRD IRF-9 IFNα/β

ISRE IL-12 P IRF-7 ISRE ISRE DC IL-15 IRF-7

Figure 9 A model of IFNα/β-induced antiviral responses. Please refer to the text for details. This figure is based on references (127, 129, 130). DC, dendritic cell; NK, natural killer cell; ISRE, interferon-stimulated response element; PRD, positive regulatory domain; PRD-LE, PRD-like element; IFNARs, IFNα/β receptors; ISGF-3, interferon-stimulated gene factor-3.

37 IFNγ becomes important later in the response in order to prolong the global antiviral state (131). A group of IFNγ-regulated genes (Table 1-1) are involved in such an effect. They include genes in the class I antigen presentation pathway, and those possessing antiviral, antiproliferative, and/or apoptotic effects. Some genes/proteins involved in the development and trafficking of immune cells are also regulated by IFNγ.

In the antigen presentation subgroup, IFNγ has been shown to regulate the enzymatic proteasome components, or so-called low molecular weight proteins (LMP)

(132); transporter associated with antigen processing (TAP) proteins (133); major histocompatibility complex (MHC)-I heavy chain (133); and β2-microglobulin (134). The upregulation of these proteins by IFNγ increases the amount and diversity of viral particles (antigens) presented on the cell surface and assists in the stimulation of CD8+ T cells at sites of inflammation, while avoiding autoimmune responses in uninfected regions.

38 Table 1-1 IFNγ-regulated genes and their roles in antiviral responses

IFNγ-Regulated Genes IFNγ-Mediated Changes Functional Roles

β2-microglobulin ↑ Class I antigen presentation

ADAR ↑ Antiviral

B7.2 ↑ Lymphocyte activation

Caspase-1 ↑ Antiviral; Apoptotic

Fas/FasL ↑ Apoptotic

GBP ↑ Antiviral

ICAM-1 ↑ Lymphocyte trafficking

IL-12 ↑ Leukocyte development iNOS ↑ Antiviral

IP-10 ↑ Leukocyte trafficking

IRF-1 ↑ (Multiple roles)

LMP ↑ Class I antigen presentation

MCP-1 ↑ Leukocyte trafficking

MHC-I heavy chain ↑ Class I antigen presentation

MIP-1α/β ↑ Lymphocyte trafficking

OAS ↑ Antivial p21, p27 ↑ Antiproliferation

PKR ↑ Antiviral; Antiproliferation

RANTES ↑ Lymphocyte trafficking

Rb ↑ (activated Rb) Antiproliferation

TAP ↑ Class I antigen presentation

TNFα receptor ↑ (surface) Apoptotic

39 Several genes/proteins functioning in antiviral, antiproliferative, and/or apoptotic

effects are regulated by IFNγ. These include the dsRNA-regulated protein kinase (PKR),

a serine/threonine kinase that inhibits viral protein synthesis by blocking normal cellular

translation (135); dsRNA-specific adenosine deaminase (ADAR), an enzyme that edits

viral mRNA to inhibit its replication (136); guanylate-binding proteins (GBP), GTPases

with antiviral properties (137); 2'-5' oligoadenylate synthetase (OAS), which promotes

RNase L-mediated viral RNA degradation (138); iNOS (139); p21 and p27, which are

cell cycle inhibitors (140, 141); retinoblastoma (Rb), which in the presence of IFNγ

prevents progression of the cell cycle (142); caspase-1, a cysteine protease involved in

the generation of bioactive IL-1β and IL-18 (143); Fas/FasL (144); TNFα receptor on the

surface of tumor cells (145); and IRF-1 (146), which will be reviewed extensively later in

this chapter. Taken together, IFNγ, through the functions of its target genes, limits viral

replication and prevents spreading of the viruses by inducing apoptosis of the infected

cells.

IFNγ-induced genes are also involved in the development and trafficking of

immune cells. IL-12, an NK-cell activator and differentiation factor favoring the Th1

phenotype, is upregulated by IFNγ (147). Within this subgroup are also IP-10, a

chemoattractant for monocytes and T cells (148); monocyte chemoattractant protein

(MCP)-1 (149); MIG, a chemokine in T-cell trafficking (150); macrophage inflammatory

protein (MIP)-1α/β, chemoattractants for CD4+, CD8+, and memory T cells (151); regulated on activation, normal T-cell expressed and secreted (RANTES), a protein involved in the trafficking of memory CD4+ T cells and monocytes/macrophages (152);

ICAM-1 (153); and B7.2, a surface molecule on antigen presenting cells that provides a

40 co-stimulatory signal for T-cell activation (154). By inducing the genes regulating the

development and trafficking of immune cells, IFNγ can coordinate the transition from

innate immunity to adaptive immunity during an antiviral response.

2.4. Roles of Interferons in Cell Death and Differentiation

Interferons induce programmed cell death (apoptosis) for essentially two reasons

– eliminating damaged cells such as cancer cells, and preventing viral replication by

inducing apoptosis of infected cells. A number of interferon-regulated genes described

earlier that function in antiviral responses are thus important for cell death and differentiation as well. Here, the roles of interferons in the context of cell cycle control and apoptosis of cancer cells are reviewed.

Progression of the cell cycle is tightly regulated by cyclins and cyclin-dependent

kinases (CDK). Interferons have been shown to affect the cell cycle through these

proteins. They block the cell cycle at G1 phase, or lengthen G1, G2, and/or S phases

(155). Both IFNα and IFNγ can change the phosphorylation state of Rb from the inactive, hyper-phosphorylated form to the active, hypo-phosphorylated form (156). During cell cycle progression, Rb is normally hyper-phosphorylated (inactive) by CDKs at G1 phase, thus releasing E2F-1, which in turn activates G1 cyclins that are required for G1/S transition and DNA replication; IFNα or IFNγ treatment, on the other hand, stimulates a complex of E2F and hypo-phosphorylated Rb that suppresses the functions of G1 cyclins

(157, 158). Certain S-phase cyclins, such as cyclin A and associated CDK2, are also

downregulated by IFNα (159). A phosphatase important for CDK2 activation, cdc25A, is

decreased upon IFNα treatment (160). CDK inhibitors, such as p21 and p27, are also

41 targets of interferons. IFNα is shown to upregulate p27 both transcriptionally (161) and post-transcriptionally (162); whereas IFNγ-mediated STAT-1 activation can directly induce the p21 promoter (163). IFN-induced expression of p21 and p27 is followed by increased binding of these inhibitory proteins to CDKs, blocking their kinase activities

(164). Additionally, tumor suppressor p53, which controls both cell cycle arrest and apoptosis, is modulated by interferons as well. IFNα/β induces the transcription and

protein levels of p53, ensuring the abundance of the protein for activation in response to

stress signals (165).

42 In the context of the regulation of apoptosis, interferons can affect multiple components of the death pathways (Figure 10). IFNα induces Fas/CD95 expression (166), whereas IFNγ sensitizes cancer cells to Fas-induced apoptosis in vitro (167). TRAIL is also an IFN-stimulated gene. In non-hematopoietic cells, IFNβ preferentially induces

TRAIL in comparison to IFNα, resulting in induction of apoptosis (168). Additionally,

IFNγ can regulate both the mitochondria-dependent and -independent pathways of

TRAIL-induced apoptosis (169). IFNγ also induces caspase-8 in a variety of cell lines, and the induction of caspase-8 may sensitize cancer cells to TRAIL- and/or Fas ligand- mediated apoptosis (170). Furthermore, IFNα induces apoptosis through stimulating cytochrome c release and mitochondrial membrane depolarization (171). However, the role of interferons on the bcl-2 family of anti-apoptotic proteins is a little more complex.

Bcl-2 is increased by IFNα in B-chronic lymphocytic leukemia cells and protects them from apoptosis (172). In contrast, interferons downregulate bcl-xL, thus inducing a pro- apoptotic state (173). Overall, interferons appear to affect both death receptor- and mitochondria-mediated pathways initiated by a death signal, thereby increasing apoptosis of cancer or virus-infected cells.

43

TRAIL-R or Fas/CD95

Death Signals

8

- DD e

spas FADD ? Mitochondria o-ca

r P

Bcl-2 Cyt c Caspase-8 Bcl-xL Apaf-1

e e-9 as p

Caspase spas

Caspase Caspase-9 s cascad e o-ca r o-ca r P

P

Caspase-3 Apoptosis

Figure 10 Interferon-mediated induction of apoptosis. The thick arrows indicate changes induced by interferons. Please refer to the text for details. This figure is based on references (166-174). TRAIL, tumor necrosis factor- related apoptosis-inducing factor; TRAIL-R, TRAIL receptor or death receptor; FasL, Fas ligand; FADD, Fas-associated death domain; DD, death domain; Cyt c, cytochrome c.

44 Interferons also promote the differentiation of a number of different cell types.

For instance, upon virus infection, IFNα/β produced by plasmacytoid DCs can induce

CD40-activated B cells to differentiate into Ig-secreting plasma cells with the help from

IL-6 (175). IFNα/β also increase the differentiation of monocytes into DCs (176). IFNγ, on the other hand, enhances neuronal differentiation, increasing the ability of neural stem cells to repair the damage caused by traumatic brain injury or stroke (177). It can also induce the differentiation of prostatic precursor cells into hybrid epithelial-neural- endocrine cells that secrete numerous hormone and neuropeptides important for the integrity of the prostatic epithelium (178). Another important differentiation process of the immune system, the Th1-Th2 development, can be regulated by the functions of IFN- induced IRF-1; details of this topic will be reviewed in Section 3.3 of this chapter.

45 3. Members of the IRF Family of Transcription Factors

The first member of the IRF family, IRF-1, was originally identified as a mouse

nuclear factor that specifically bound to the upstream regulatory region of IFNβ gene. In

1988, Taniguchi and colleagues cloned and characterized the cDNA encoding IRF-1

(179), and a role of IRF-1 in viral infections was then suggested, as it possessed a virus-

inducible promoter (180). Eight more members of the family, IRF-2 through IRF-9, were

subsequently identified. Recently, a new member IRF-10 was discovered and its

expression is characterized by its delayed kinetics, suggesting a unique role of this

protein in later stages of antiviral defenses (181). All IRF proteins (Figure 11) are

significantly homologous in the amino-terminal region, which comprises a domain with

five tryptophan repeats necessary for the helix-turn-helix DNA-binding motif (182). The

DBD is defined by 5 tryptophan (W) residues that are each separated by 10-18 amino

acids. Most IRFs also contain an IRF-association domain (IAD) of either type 1 or type 2,

with IAD2 containing a PEST (proline-, glutamate-, serine- and threonine-rich) domain.

Some IRFs contain repression domains and NLS. For IRF-1, -3, -5 and -7,

phosphorylation may be required for activation. Currently, IRF proteins are categorized into four subfamilies [IRF-1, IRF-3, IRF-4 and IRF-5 subfamilies (181)], and they function as transcription factors in innate and adaptive immune responses, cell death, and differentiation through interactions with their own or transcription factors other than the

IRF family members.

46

* * #

* # # * #

** #

#

* *

Figure 11 IRF family of transcriptional factors. Nuclear localization signals (NLS) are noted with asterisks. The symbol “#” indicates a repression domain. The size of each IRF in number of amino acids is also indicated. This figure was reproduced from reference (183). C, carboxyl terminus; N, amino terminus; W, tryptophan; IAD, IRF-association domain; P, phosphorylation.

47 3.1. IRF-1 Subfamily

The IRF-1 subfamily includes IRF-1 and IRF-2, whose primary structures show

62% homology in the first 154 amino acids of their amino-termini (184). They bind to the same response element called IRF-E (185). However, the carboxyl-terminus of IRF-1 contains numerous acidic amino acids and serine-threonine residues that suggest a function of IRF-1 in transactivation (184, 186), whereas that of IRF-2 is relatively rich in

basic amino acids, which may be related to its role as a transcriptional repressor (186).

Both IRF-1 and IRF-2 are expressed in many cell types, and their expression levels are

usually upregulated by interferon stimulation and viral infection. Although IRF-1 protein

is very short-lived, a series of transfection experiments have revealed that it could

activate IFNα/β promoters (184, 186). In contrast, IRF-2 protein is relatively stable and it

usually acts to repress IRF-1-induced transcriptional activation (187). Another important

role of IRF-2 is oncogenicity, as it can activate a human histone H4 gene to promote

DNA replication and cell cycle progression at the G1/S phase transition (188).

48 IFNγ is one of the strongest inducers of IRF-1. Upon binding of IFNγ to its

receptor, IRF-1 is induced through activation of STAT-1 and binding of the activated

STAT-1 to the GAS site within the IRF-1 promoter. IRF-1 mRNA, however, is also

regulated by different phases of the cell cycle in the absence of any exogenous stimuli

(189). It is elevated in serum-starved cells that stay in G0 phase but lowered after serum repletion. It is also increased during S phase. It is currently unclear what mechanisms control the cell cycle-dependent regulation of IRF-1 mRNA levels.

IRF-1 protein has a very short half-life and is degraded via the ubiquitin- proteasome pathway, although a 39-residue sequence within the carboxyl-terminal region has been identified to function in stabilizing the protein (190). The primary structure of

IRF-1 (Figure 12) contains a DBD within its amino-terminus, an NLS, two transactivation domains, two dimerization domains that interact with IRF-1 itself

(forming homodimers) and IRF-8 or IRF-2 (forming heterodimers), respectively, and repression and enhancement domains (189, 191). Little is known about posttranslational modifications of IRF-1, but it has been revealed that IRF-1 can interact with a serine/threonine protein kinase, casein kinase II (CKII), which may phosphorylate IRF-1 at several potential sites including Ser-219, Thr-224 and Thr-225 that are localized in the carboxyl transactivation region and three other residues in the amino-terminus (192).

Mutations of the serine/threonine residues have been shown to significantly decrease the transactivation activity of IRF-1 (192).

49

Figure 12 Domain structure of IRF-1. The primary structure of IRF-1 contains a DNA-binding domain within its amino- terminus, a nuclear location signal (NLS), two transactivation domains (one is acidic), two dimerization domains that interact with IRF-1 itself (forming homodimers) and IRF-8 or IRF-2 (forming heterodimers), respectively, and repression and enhancement domains. This figure is based on references (189, 191). NLS, nuclear localization signal; aa, amino acids.

50 G T G T IRF-1 can bind to both ISRE (189) and IRF-E: G(A)AAA /C /CGAAA /C /C

(185). IRF-1 target genes include those functioning in cell growth arrest, apoptosis, differentiation, and antiviral and antibacterial immune responses: 1) IFNα/β, OAS, PKR and iNOS that function in antiviral or antibacterial responses and cell growth arrest; 2)

IRF-2, p21, p53, caspase-1 and caspase-7 that function in inhibiting cell proliferation and inducing apoptosis; 3) IL-12, IL-4 and IL-5 that are involved in Th1-Th2 development; 4)

TAP and LMP proteins that function in regulating MHC class I expression; 5) cathepsin

S that is a cysteine protease and acts to complete the processing of MHC class II- associated invariant chain; 6) IL-15 that induces natural killer cell differentiation; and 7) cyclooxygenase-2 that functions in regulating inflammation (189, 193).

51 3.2. Regulation of IRF-1 Expression and Localization

A more detailed understanding of IRF-1 transcription originated from analyses of

its promoter (187, 194). An NF-κB-binding site is found in the proximal region to the

transcription start site, preceded by a CCAAT box at position −97 to −91. A

GAS/κB/ISRE combined element, which has been the focus of attention, is found close

to the CCAAT box (region −130 to −110). Several more κB sites are present upstream of

this element. There is no classical RARE in the IRF-1 promoter; however, a putative but

not functional RARE has been found between −455 and −440 (195). In addition, using

the Transcription Element Search System (TESS) provided by University of

Pennsylvania (http://www.cbil.upenn.edu/tess/), we have found a cluster of three putative

RARE half sites in the region between −584 and −550. A κB site is closely associated

with them, but the functionality of these elements is yet to be studied. A schematic view

of the IRF-1 promoter is shown in Figure 13.

Many cytokines and pathogen components are IRF-1 inducers. IFNγ, one of the

strongest inducers of IRF-1, synergizes with TNFα to increase IRF-1 transcription. The

effects of IFNγ and TNFα on IRF-1 are mediated by the proximal κB site and the

GAS/κB/ISRE combined element (121), although an additional κB site has been shown

to be functional that resides between −171 and −144, upstream of the combined element

(196). It is suggested that IFNγ-induced STAT-1 binds to GAS/κB/ISRE and that TNFα

induces NF-κB binding to the proximal κB site; and the two elements work

independently from each other (122).

52

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M M R a eas S h GA r do 9 -4 -6 c nd nd A 0 0 any n 30 bi bot bi i GA r R •1 A •1 Matik • -1

Figure 13 Schematic view of the IRF-1 promoter. Representative citations are shown in the figure with brief outlines of the results. The two boxes indicate the GAS/κB/ISRE combined element and the three putative RARE half sites identified. IR, inverted repeat; FXR, farnesoid X receptor.

53 In addition, IFNα can activate IRF-1 transcription through binding of ISGF-3 to the GAS/κB/ISRE combined element (197). Other stimuli that function through this element include LPS (198), arsenic trioxide (199), IL-12 (200), and a type of interferon in ruminants, IFNτ (201). Prolactin, on the other hand, stimulates a biphasic pattern of

IRF-1 expression along the cell cycle by regulating histone H4 acetylation of the IRF-1 promoter (202). Furthermore, IRF-1 is inducible by pro-inflammatory cytokines IL-1β

(203) and IL-6 (204).

atRA activates IRF-1 gene expression in myeloid cells (205, 206). In these cells, atRA does not activate NF-κB or STAT pathways, suggesting that an alternate mechanism is involved in IRF-1 gene activation. Also, it has been shown that atRA moderately increases IRF-1 gene expression in human APL cells through the GAS motif of the IRF-1 promoter, whereas a putative RARE identified is not functional (195). In squamous carcinoma cells, RA induces IRF-1 via a STAT-1-independent, but NF-κB- dependent pathway (207). In addition, high doses of atRA (1 µM and above) have been suggested to prolong the induction of IRF-1 by acting on both STAT-1 and NF-κB pathways in cervical squamous carcinoma SiHa cells (208); however, these cells are resistant to lower concentrations of atRA (209). Recently, treatments with 9cRA of NB4

APL or SK-BR-3 breast cancer cells are shown to increase levels of IRF-1, which subsequently induces the promoter of TRAIL (72). Although RA-mediated induction of

IRF-1 has been observed in many cell models, the molecular mechanism is still not clarified.

54 IRF-1 is a transcription factor presumably localized in the nucleus; however,

subcellular localization of IRF-1 and how IRF-1 is transported in and out of the nucleus is yet to be studied. Nuclear import of proteins is mediated by the large nuclear pore complex (NPC) (210). NPC provides passive diffusion channels for proteins smaller than

50-60 kDa; but in most cases, even small proteins are imported by an energy-dependent and receptor-mediated process. The nuclear import receptors recognize the NLS sequence present in most nuclear proteins. The export of proteins out of the nucleus, on the other hand, is mediated by the recognition of the nuclear export signal (NES) by export receptors, such as CRM1 (211). A CRM1-specific inhibitor, leptomycin B, has been used extensively in studies of nuclear export. Since nuclear transport plays an important role in regulating the activity of transcriptional factors, investigations of IRF-1 subcellular localization are necessary in studies of IRF-1 transcriptional functions. We hypothesize that retinoids may affect the transport of IRF-1 into and/or out of the nucleus, in addition to the role of retinoids on IRF-1 induction. One of the retinoid receptors, RXRα, has been shown to shuttle between the nucleus and cytoplasm ligand-dependently. Importantly, it acts as a nuclear export carrier for TR3, directing this orphan receptor to the mitochondria. RXRα-mediated translocation of TR3 to the mitochondria is crucial for the effect of TR3 in inducing apoptosis (28). Thus, IRF-1 nuclear translocation may be affected by shuttling of retinoid receptors as well.

Recently, nuclear transport of two other IRF proteins, IRF-5 and IRF-3, has been investigated. IRF-5 subcellular localization is regulated by a CRM1-dependent nuclear export pathway (212). Mutation of the NES of IRF-5 results in nuclear accumulation of

this protein, indicating the possibility of CRM1-mediated regulation of IRF-5 localization.

55 The Ser/Thr residues adjacent to the NES may also contribute to the nuclear

accumulation of IRF-5, although phosphorylation of these residues does not (212). On the

other hand, the nuclear localization of IRF-3 is regulated by nuclear sequestration (213).

The NLS of IRF-3 is constitutively active, so that IRF-3 continuously shuttles between the nucleus and cytoplasm. However, the cytoplasmic IRF-3 is phosphorylated upon infection, which concentrates in the nucleus by binding to CBP/p300 proteins that stabilize nuclear retention of IRF-3.

56 3.3. Roles of IRF-1 in Antiviral Responses

The antiviral functions of IRF-1 were recognized not long after the discovery of

IRF-1, when inhibition of encephalomyocarditis virus replication by IFNα and IFNγ was

observed to be impaired in mouse cells with a null mutation in the IRF-1 gene (180).

Lately, IRF-1 is shown to be one of the key host factors that regulate intracellular

hepatitis C virus replication through modulation of interferon-stimulated antiviral

responses (214). IRF-1 has also been found to interact with a sequence downstream of the

5' long terminal repeat of human immunodeficiency virus type-1, a segment homologous

to IRF-1 binding site, ISRE (215). The importance of this interaction is yet to be

determined, but it is hypothesized that IRF-1 may inhibit replication of this virus. On the

other hand, several viruses have developed mechanisms by which they escape the

powerful antiviral functions of IRF-1 (216).

Most antiviral genes induced by interferons (Table 1-1) are in fact IRF-1 target

genes. In addition to the roles of IRF-1 in mediating the interferon response, macrophage

functions, and antigen presentation, as reviewed in Section 2.3 (Roles of Interferons in

Antiviral Responses), IRF-1 is also important for the development of B cells and CD4+ and CD8+ T cells (182). IRF-1 plays a decisive role in the differentiation of Th1 cells

(183). T cells from IRF-1−/− mice fail to mount Th1 responses and instead exclusively

undergo Th2 differentiation both in vitro (217) and in vivo (218). The Th2 responses to

infection are characterized by marked increases in the level of IgE and the ratio of IgG1 to

IgG2a. This phenotype could be resulted from both the increase of IL-4 and the decrease

of IL-12 expression. In fact, IRF-1 is able to bind to the IL-4 promoter and function as a

transcriptional repressor (219). In addition, the compromised Th1 differentiation in

57 IRF-1−/− mice was associated with impaired production of IL-12 by macrophages and hyporesponsiveness of CD4+ T cells to IL-12 (217). The level of IL-18, a cytokine activated by IRF-1-induced caspase-1 and synergizes with IL-12 in the induction of IFNγ expression, is also lowered in IRF-1−/− mice (220). Finally, IRF-1 induces iNOS that

produces nitric oxide, which is important for both the clearance of pathogens and the

differentiation of Th1 cells (221). Taken together (Figure 14), IRF-1, induced through

STAT-1 activation by IFNγ, influences the expression of IL-12, iNOS, caspase-1 and

IL-18 in antigen presenting cells (e.g. macrophages), leading to the generation of more

IFNγ by CD4+ T cells. It also downregulates the production of IL-4 by suppressing its transcription. IFNγ produced by CD4+ T cells and IL-12 produced by antigen presenting

cells act on mature NK cells, another important source of IFNγ production. Moreover,

IRF-1 upregulates the expression of IL-15 required for NK cell proliferation (222). The

overall outcome of these feedback loops is the differentiation into Th1 cells and potential

enhancement of antiviral responses.

58

IL-15

IFNγ Mature NK cell

NK precursor IFNGR IFNγ

IFNγ

IFNGR

STAT IRF-1 IL-12R GAS IRF-E STAT IL-12 IRF-1 GAS

IRF-E iNOS NO L IL-4 IRF-E Caspase-1 IL-18 APC Th cell

Figure 14 Roles of IFNγ and IRF-1 in differentiation into Th1 cells. Please refer to the text for details. This figure is reproduced from reference (183). APC, antigen presenting cells; Th, T helper; NK, natural killer; IFNGR, IFNγ receptor; STAT, signal transducer and activator of transcription; GAS, γ-interferon activated site; IRF-1, interferon regulatory factor-1; IRF-E; IRF-1 response element; iNOS, inducible nitric oxide synthase; NO, nitric oxide; IL-12R, IL-12 receptor.

59 3.4. Roles of IRF-1 in Cell Death and Differentiation

The tumor suppressor functions of IRF-1 were demonstrated in oncogenic

transformation of IRF-1−/− primary mouse embryonic fibroblasts (146). The lack of IRF-1

leads to increased cell survival after treatments with oncogenes, similar to the observation

in the p53 knockout models. Thus, a connection between IRF-1 and p53 is suggested.

Further, the loss of IRF-1 has no effect on spontaneous tumor development, but rather

exacerbates previous tumors induced by an oncogene or by a lack of p53 (223). These

results suggest that IRF-1, together with p53, is important for the induction of apoptosis

in transformed or DNA-damaged cells. The observation that IRF-1 may not affect tumor

development directly has prompted some researchers to identify it as a tumor

susceptibility gene, rather than a tumor suppressor (193). However, the IRF-1 allele of human chromosome 5 (5q31.1) is one of the most commonly deleted segments in patients with leukemia or pre-leukemic myelodysplasia (224). The loss of IRF-1 allele is also reported in esophageal and gastric cancers (225, 226). In addition, the absence of IRF-1 is suggested to be involved in human breast oncogenesis (227). Thus, it appears that IRF-1

acts as a tumor suppressor and the loss of this gene contributes to the development of

human neoplasias.

In the cell cycle control, IRF-1 has been shown to be essential for DNA damage-

induced cell cycle arrest, as it directly induces a cell cycle inhibitor, p21 (228). Induction

of apoptosis is another mechanism of how IRF-1 regulates cell death. Indeed, IRF-1 has

been shown to induce caspase-8 activation in MCF-7 breast cancer cells (229).

IRF-1 is also involved in the differentiation of a variety of different cell types. In

the isolation of cDNA clones of myeloid differentiation-related genes, IRF-1 was found

60 to be highly expressed in precursor-enriched bone marrow cells, and therefore identified

as a potent transcriptional factor in the induction of terminal myeloid differentiation (204).

IRF-1 antisense mRNA overexpression, on the other hand, blocks the differentiation of

monoblastic U937 cells into macrophages (230). Moreover, granulocytic colony-

stimulating factor-induced granulocytic differentiation into neutrophils is accelerated in

the presence of IRF-1 ectopic expression (231).

3.5. IRF-3, -4, and -5 Subfamilies

The IRF-3 subfamily includes IRF-3 and IRF-7 that are closely related to each

other in terms of their primary structures (182). IRF-3 is expressed constitutively in all

tissues and its expression is not affected by viruses or interferons (232), whereas IRF-7 is

mainly induced by interferon signaling pathways (233). IRF-3 contains an IAD, which is

usually masked and its activation requires virus-induced phosphorylation of two

interacting autoinhibitory domains (234). Upon virus infection, IRF-3 is activated by

phosphorylation; in the nucleus, it interacts with CBP/p300 coactivators, ultimately

transactivating IFNβ and IFNα4 promoters. In turn, the induced IFNβ and IFNα4 activate several IFN-stimulated genes, including IRF-7, through signaling functions of

IFNARs and ISGF-3. IRF-7, after de novo synthesis, is phosphorylated following virus infection and acts in the transcription of other type I interferons. The synthesis of IRF-7 by IFNβ and IFNα4 and the activation of IRF-3 and IRF-7 by virus-induced phosphorylation increases the production of each of the type I interferons, thereby generating the auto-amplification mechanism of the antiviral IFNα/β system (127, 235-

61 237). IRF-3 is also involved in the MyD88-independent pathway of toll-like receptor

(TLR)-4 signaling upon treatment with LPS (238).

Members of the IRF-4 subfamily, IRF-4, IRF-8, IRF-9 and IRF-10, are

genetically close to each other in that they all contain a nuclear retention signal located

within the amino-terminus of DBD (239). Expression of IRF-4 is restricted to lymphoid

lineage, and is not affected by interferons (182). IRF-4 has been shown to play an

important role in lymphocyte development, as mice lacking this protein showed impaired

activation and functions of both B and T cells (240). IRF-4 is expressed in all stages of B

cell development as well as in mature B cells. Although the lymph nodes and spleen of

IRF-4−/− mice are normal at 4-5 weeks of age, they are enlarged more than three times at

10-15 weeks compared with those of control mice. It is shown that the numbers of T cells

and B cells increase dramatically, but the maturation and activation of these cells are

impaired in the absence of IRF-4. Moreover, T cell functions, such as cytokine

production and cytotoxic activity, are also abrogated in IRF-4−/− mice. Thus, IRF-4 is essential for both immature and mature B and T cells (241). IRF-4 also mediates T cell differentiation into Th2 cells (183). IRF-4 is induced upon ligation of the T cell receptor

(242) or IL-4 receptor (243). It interacts with STAT-6 to induce the transcription of

GATA-binding protein-3, a transcriptional factor crucial for chromatin changes that stabilize the Th2 phenotype (244, 245). IRF-8, also called interferon consensus sequence binding protein, can be induced by IFNγ, but is expressed only in myeloid and lymphoid lineages (240). The DNA-binding activity of IRF-8, important for regulation of the

immune system and oncogenesis (182), is partially dependent on its interaction with

IRF-1 or IRF-2 (246). IRF-8−/− and IRF-2−/− mice are more susceptible to Listeria

62 monocytogenes infection than IRF-1−/− mice, suggesting that IRF-2 and IRF-8 are critical for IFNγ-mediated protection against this bacterium (247). IRF-9, also termed p48 or

ISGF-3γ, acts as the DNA-binding subunit of ISGF-3, a complex essential for antiviral effects of IFNα/β (248). IRF-9 interacts with STAT-2 either in the presence or absence of

IFNα stimulation; whereas its interaction with STAT-1 is weak, or happens only when a

DNA target is present (87, 249). The newly discovered IRF-10 is inducible by interferons

and concanavalin A. It is important during later stages of the immune response against

infections by regulating the expression of several IFNγ-induced genes including MHC-I

and GBP (181).

Members of the IRF-5 subfamily, IRF-5 and IRF-6, are structurally related to

each other, although their functions are still unclear (182). Expression of IRF-5 appears

to be restricted to B cells and dendritic cells, but it may be induced by type I interferons

in most cells of lymphoid origin. It is suggested that IRF-5 plays a role in innate

immunity (250, 251). In addition, IRF-5 is recently identified as another inducer of

IFNα/β genes and may function as a direct transducer of virus-mediated signaling,

similar to IRF-3 and IRF-7 (216). Little is known about IRF-6, but the cDNA sequence of

human IRF-6 has been submitted to the GenBank database (AF027292).

Interestingly, several viruses have developed mechanisms to reduce or eliminate

antiviral functions of IRFs. It has been shown that Kaposi’s sarcoma herpes virus

(KSHV), established as an important factor in pathogenesis of Kaposi’s sarcoma and

acquired immunodeficiency syndrome-associated body cavity-based lymphoma, has

incorporated several homologs of cellular IRFs into its genome (216). Three of them,

vIRF-1, vIRF-2, and vIRF-3, have been cloned and functionally characterized. vIRF-1

63 acts similarly to IRF-2 as a transcriptional repressor. It inhibits the transactivation and

HAT activity of CBP/p300 in vitro, possibly by competing with the binding of IRF-1 or

IRF-3 to the carboxyl-terminal region of p300 (216). vIRF-2 binds to NF-κB binding sites and targets IRF-1, IRF-3, p53, and PKR, molecules that may suppress KSHV life cycle and oncogenesis. However, vIRF-2 alone is not able to induce a transformed phenotype, suggesting that it does not share the oncogenic potential of vIRF-1 (216). The third viral IRF, vIRF-3, inhibits virus-mediated induction of IFNα/β by binding to IRF-3,

IRF-7, or CBP/p300 (216).

64 4. Interactions of Vitamin A, Interferons, and IRF-1

As we have reviewed, both vitamin A and interferons are recognized as potent

regulators of antiviral responses and apoptosis. Vitamin A, especially RA, acts through

retinoid signaling pathways using retinoid receptors as mediators of transcriptional activation. Interferons, on the other hand, signal through interferon signaling pathways and use IRF-1 as a primary transcriptional factor. Recently, more attention has been drawn to study the potential interactions of vitamin A, interferons, and IRF-1, as

synergistic effects of RA and interferons have been observed in both animal models and

cultured cell lines.

Using vitamin A-deficient animal models, our laboratory has observed a strong

synergistic effect between RA and polyriboinosinic:polyribocytidylic acid (PIC), an

inducer of interferons, on antigen-specific antibody production. Expression of cytokines

(e.g. IL-10 and IL-12) and transcriptional factors (e.g. STAT-1 and IRF-1) that are

associated with activation, expansion, survival, and/or signal transduction of T cells was

also increased (252-254). These results suggest that the combination of RA and PIC may

stimulate humoral and cell-mediated immune responses against infections in immuno-

compromised animals. An interaction model has also been recommended for RA,

interferons, and IRF-1 on antibody production (255). In this model, PIC binds to a

general dsRNA receptor (TLR-3) and induces the production of interferons and other

cytokines. Interferons then signal through their receptors on the surface of target cells,

sequentially recruiting Jak and STAT proteins. After activation, STAT proteins are

translocated into the nucleus and bind to response elements within promoter regions of

IFN-stimulated genes. Among these genes is IRF-1, an important mediator of cell

65 apoptosis, differentiation, and immune responses. On the other hand, RA exerts its

regulatory functions by binding to retinoid receptors in target cells and interacting with

RAREs to stimulate target-gene expression. These target genes also include IRF-1,

suggesting an interaction between retinoid and interferon signaling pathways. The

humoral and cell-mediated immunity enhanced by the combination of RA and PIC is

characterized by 1) increased production of IgG1 and IgG2a by RA and that of IgG2b and

IgM by PIC; 2) prevention of antigen-induced apoptosis and promotion of lymphocyte survival by interferons; 3) enhanced antiviral activity by interferons; and 4) stimulation of apoptosis and cell differentiation by RA (Figure 15).

66

RA PIC

dsRNA receptor

IFNα/β IFNγ

Jak-STAT pathways

RA ⊕ STAT-1

⊕ RAR IRF-1

Cell-mediated effects: Humoral Immunity:

K Antiviral activity (IFN) K IgG1 and IgG2a (RA)

K Cell Differentiation (RA) K IgG2b and IgM (PIC)

Š Stimulation of apoptosis (RA?) K Mitogenic responses (RA)

Š Prevention of antigen-induced apoptosis; promotion of B-cell and T-cell survival (IFN)

Figure 15 A model for the interaction of RA, interferons, and IRF-1 on cell- mediated and humoral immunity. Please refer to the text for details. This figure is based on reference (255). PIC, polyriboinosinic:polyribocytidylic acid; RAR, retinoic acid receptor.

67 Possible synergism between RA and IFNs is also suggested in several in vitro

models. RA and receptor-selective retinoids have been shown to synergize with

suboptimal concentrations of IFNγ in inducing nitric oxide production and the expression of iNOS in RAW 264.7 cells (256). In human monocytic THP-1 cells, RA on its own is not able to either induce or activate STAT-1, but it enhances or prolongs the expression

(mRNA and protein) and activation (Tyr-701 phosphorylation) of STAT-1 induced by

IFNγ (257). In addition, phosphorylation-deficient STAT-1 (Y701F) suppresses RA- induced morphologic differentiation and cell growth arrest at G0/G1 phase of the cell

cycle in U937 cells (258). In fact, RA is able to induce STAT-1 expression and thereby

augment IFN-induced responses in several IFN-resistant cell lines, including the breast

tumor MCF-7 (259) and promyelocytic NB4 cell lines (260, 261). On the other hand, RA

and IFNγ synergistically induce IRF-1. IRF-1 is then recruited to two response elements

(ISRE and IRF-E) of the promoter of TRAIL (Figure 6), which induces death of cancer

cells in a paracrine fashion (71, 72). However, the mechanism of how RA and IFNγ

cooperatively induce IRF-1 is unclear.

Although synergistic cell death is marked upon the combination treatment of RA

and interferons and that IRF-1 may be a regulator of this process, people have failed to

observe an RA/IFN synergy on other IFN-induced death genes, such as OAS, RNase L,

PKR, p53, Bax, or Bcl-2 (262). This led to the employment of an antisense knockout

approach in the selection of RA/IFN-regulated death genes. Theoretically, only those

cells transfected with death specific antisense mRNA can survive in the presence of both

RA and interferons. Fourteen genes were identified using this approach, named genes

associated with retinoid-interferon induced mortality (GRIMs) (263). Of these, two have

68 been investigated. GRIM-12 is identified as human thioredoxin reductase-1 (TR1), an enzyme regulating the redox status of thioredoxin, a ubiquitous highly conserved redox protein known to regulate growth, transcription, and immune responses (264). TR1 is also hypothesized to be a tumor suppressor in vivo (265). The second GRIM, GRIM-19, is part of the mitochondria respiratory chain complex I (266) and interacts and inhibits

STAT-3, an inducer of cell survival (267). In addition to IRF-1, GRIMs may be a group of tumor suppressors that mediates the pro-apoptotic effects of retinoids and interferons.

69 CHAPTER 2 STATEMENT OF HYPOTHESIS

The hypothesis of this dissertation was developed during the course of our studies regarding atRA regulation of IRF-1 in two human cell lines, A549 and MCF10A.

As reviewed in Chapter 1, both atRA and interferons induce IRF-1. In addition, synergistic actions between atRA and IFNγ on modulation of cellular functions have been reported both in vitro and in vivo. Thus, we originally hypothesized that atRA potentiates the signaling of low-dose IFNγ to increase the expression and functions of IRF-1. We used human lung epithelial A549 cells as a model of the cells at sites of early infection, where IFNγ is present, but at low levels. By increasing IRF-1 expression, atRA could sensitize these cells to better respond to low-dose IFNγ.

While the results obtained from studies of A549 cells supported the original hypothesis, we found evidence in human mammary epithelial MCF10A cells that atRA also increased IRF-1 expression in the absence of IFNγ. Therefore, the hypothesis was revised as: atRA regulates IRF-1 through multiple mechanisms that include 1) potentiation of IFNγ signaling and thereby inducing IRF-1, and 2) regulation of IRF-1 in the absence of IFNγ stimulation. Given that IRF-1 is characterized as a tumor suppressor, atRA might induce IRF-1 gene expression as a mechanism by which it prevents growth and/or induces apoptosis of cancer cells.

Later, we observed that atRA not only induced IRF-1 gene expression, but also regulated nuclear localization of IRF-1. Thus, to test multiple ways that atRA may be able to function in regulating IRF-1, we finally hypothesized that atRA, an active

70 metabolite of vitamin A, regulates IRF-1 through multiple mechanisms, which include 1) potentiation of IFNγ signaling, thus inducing IRF-1, 2) regulation of IRF-1 transcription in the absence of IFNγ stimulation, 3) regulation of IRF-1 nuclear localization, and 4) regulation of IRF-1 target genes.

Accordingly, four specific aims were designed to test the hypothesis:

1. To determine whether atRA potentiates the actions of low-dose IFNγ in

inducing the expression of IRF-1 in A549 cells by examining the effects of

atRA and receptor selective retinoids on multiple components of the IFNγ

signaling pathway, including IFNGR-1, STAT-1 and IRF-1;

2. To determine whether atRA by itself induces the expression of IRF-1 in

MCF10A cells by examining the effects of atRA and receptor selective

retinoids on IRF-1 mRNA and protein;

3. To determine whether atRA regulates IRF-1 localization in MCF10A cells by

examining the effects of atRA and Am580 on IRF-1 nuclear localization, as

assessed by confocal microscopy, Western blot, and gel shift assay; and

4. To determine whether atRA, either in the presence or absence of IFNγ,

regulates the transcription of IRF-1 target genes, including caspase-1, TRAIL,

and OAS-2.

Chapter 3 focuses on A549 cells (Aims 1 and 4) and Chapter 4 on MCF10A cells

(Aims 2, 3, and 4).

71

CHAPTER 3 RETINOID-MODULATED POTENTIATION

OF IFNγ SIGNALING IN A549 CELLS

Adapted from

Luo XM and Ross AC (2005) Physiological and receptor-selective retinoids modulate interferonγ signaling by increasing the expression, nuclear localization and functional activity of interferon regulatory factor-1. J. Biol. Chem. 280(43):36228-36236

72 1. Abstract

Synergistic actions between all-trans-retinoic acid (atRA) and interferon (IFN)γ on modulation of cellular functions have been reported both in vitro and in vivo. However, the mechanism of atRA-mediated regulation of IFNγ signaling is poorly understood. In this study, we have used the human lung epithelial cell line, A549, to examine the effect of atRA on IFNγ-induced expression of IFN regulatory factor-1 (IRF-1), an important transcription factor involved in cell growth and apoptosis, differentiation, and antiviral and antibacterial immune responses. At least 4 h of pretreatment with atRA followed by suboptimal concentrations of IFNγ induced a faster, higher, and more stable expression of

IRF-1 than IFNγ alone. Actinomycin D completely blocked the induction of IRF-1 by the combination, suggesting regulation at the transcriptional level. Further, we found that activation of signal transducer and activator of transcription-1 (STAT-1) was induced more dramatically by atRA and IFNγ than IFNγ alone. Expression of IFNγ receptor-1

(IFNGR-1) on the cell surface was also increased upon atRA pretreatment. Experiments using receptor-selective retinoids revealed that ligands for retinoic acid receptor-α

(RARα), including atRA, 9-cis-RA and Am580, sequentially increased the levels of

IFNGR-1, activated STAT-1, and IRF-1; and that an RARα antagonist was able to inhibit the effects of atRA and Am580. In addition, atRA pretreatment affected the transcriptional functions of IFNγ-induced IRF-1, increasing its nuclear localization and

DNA-binding activity, as well as the transcript levels of IRF-1 target genes. These results suggest that atRA, an RARα ligand, regulates IFNγ-induced IRF-1 by affecting multiple

73 components of IFNγ signaling pathway, from the plasma membrane to the nuclear

transcription factors.

2. Introduction

Both vitamin A (1, 46) and interferons (81) have long been recognized as potent

regulators of antibacterial and antiviral immune responses. All-trans-retinoic acid (atRA), an active metabolite of vitamin A, acts through retinoic acid receptors (RARs) to transcriptionally activate target genes (14). Interferons (IFNs), on the other hand, signal through interferon signaling pathways and use signal transducer and activator of transcription (STAT) proteins as transcription factors (108). Recently, more attention has been drawn to potential interactions between these two pathways, as synergistic effects of atRA and IFNs have been observed in both animal models (252, 253) and cultured cell lines (256, 268, 269). However, the molecular events involved in atRA-dependent regulation of IFNγ signaling, or vice versa, are poorly understood.

IFN regulatory factor-1 (IRF-1) was discovered in studies of virus-induced

IFNα/β gene regulation and IFN-mediated antiviral responses (179). IFNγ is one of the strongest inducers of IRF-1. Upon binding of IFNγ to its receptor, IRF-1 is induced

through activation of STAT-1 and binding of activated STAT-1 to the gamma-interferon

activated site (GAS) within the IRF-1 promoter. IRF-1 protein, a transcription factor

itself, has a very short half-life and is degraded via the ubiquitin-proteasome pathway

(190). IRF-1 is known to regulate cell growth and apoptosis (193, 270). The tumor

suppressor function of IRF-1 was first demonstrated in oncogenic transformation of

74 primary IRF-1−/− mouse embryonic fibroblasts (146). Recently, it was also shown that

IRF-1 reversed the transformed phenotype of tumor cells not only in vitro, but also in

vivo (271).

RA synergizes with IFNγ to increase the level of IRF-1, which subsequently

activates promoters of IRF-1 target genes, such as tumor necrosis factor-related

apoptosis-inducing ligand (TRAIL) (72). Caspase-1 [previously termed interleukin

(IL)-1β-converting enzyme, or ICE] is another target gene of IRF-1 (272), which plays a

critical role in the production of proinflammatory cytokines IL-1α, IL-1β, IL-18, and

IFNγ (273). However, the molecular basis of IRF-1 regulation by the combination is not

fully understood. atRA by itself activates IRF-1 gene expression in NB4 promyelocytic

leukemia cells (205). It induces IRF-1 through a GAS motif, but not a putative RA

response element, of the IRF-1 promoter (195), indicating a role of STAT-1 between

atRA and IRF-1. However, IRF-1 induction by atRA in squamous carcinoma cells is

STAT-1-independent (207). In cervical squamous carcinoma SiHa cells, STAT-1-

dependence was observed only when high doses of atRA (1 µM and above) were used

(208). Collectively, it is still unclear how atRA regulates IRF-1 transcription and whether

STAT-1 is involved. RARs, common regulators of atRA functions, may also play

important roles in the regulation of IRF-1.

In this study, we hypothesized that atRA increased IFNγ-induced expression and

functions of IRF-1 through, at least in part, its regulation of the key molecules of IFNγ

signaling pathways. Using a human lung epithelial cell line, A549, we have observed that overnight pretreatment with atRA increases the levels of IFNγ receptor (IFNGR)-1 on the

75 cell surface, thereby enhancing tyrosine phosphorylation (activation) of STAT-1 upon

low-dose IFNγ stimulation. Faster, higher and more stable levels of IRF-1 are then

induced by the combination of atRA and IFNγ compared to IFNγ alone. RARα mediates

the effect of atRA in increasing cell-surface IFNGR-1, activated STAT-1, and IRF-1.

atRA pretreatment also potentiates the transcriptional activity of IFNγ-induced IRF-1,

increasing its nuclear localization and DNA-binding activity. As anticipated, IFNγ-

induced caspase-1 and TRAIL is further increased by pretreatment with atRA.

Collectively, these results indicate that atRA regulates multiple components of the IFNγ

J IRF-1 pathway, from the plasma membrane to the nuclear transcription factors.

3. Experimental Procedures

Reagents, Antibodies, and Cell Culture- atRA (prepared in ethanol), 9-cis-RA

(9cRA), all-trans-retinol (atROL), retinyl trimethoxybenzyl ether (RTMBE), actinomycin

D (Act D) and cycloheximide (CHX) were obtained from Sigma-Aldrich (St Louis, MO).

Recombinant human IFNγ was obtained from PreproTech Inc. (Rocky Hill, NJ).

Receptor-selective retinoids were provided by Michael Klaus, Hoffmann-La Roche

(Nutley, NJ). They include Am580 (RARα agonist), Ro19-0645 (RARβ agonist), CD437

(RARγ agonist), Ro25-7386 (RXR pan-agonist), and Ro41-5253 (RARα antagonist).

4’,6’-diamidino-2-phenylindole (DAPI) was obtained from Molecular Probes (Eugene,

OR). IRF-1 polyclonal antibody, IFNGR-1 monoclonal antibody, and consensus IRF-1

gel shift oligonucleotides were obtained from Santa Cruz Biotechnology (Santa Cruz,

CA). IRF-1 and STAT-1 monoclonal antibodies were obtained from Transduction

76 Laboratory (Lexington, KY). Polyclonal antibody against phospho-STAT-1 at residue

Tyr-701 was obtained from Cell Signaling Technology (Beverly, MA). A549 cells were

obtained from the American Type Culture Collection (Rockville, MD) and maintained in

F-12K medium (Life Technologies, Rockville, MD) supplemented with 10% heat-

inactivated fetal bovine serum (FBS) at 37°C in a 5% CO2-air incubator. In most

experiments, the cells were plated at approximately 70% confluency, allowed to attach in

complete medium, and adjusted to low-serum medium (supplemented with 1% FBS) for

2 h before the addition of stimuli.

Preparation of Whole-Cell and Nuclear Extracts- A549 cells were lysed in RIPA

buffer [1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS)

in phosphate-buffered saline (PBS)] containing 10% (v/v) of protease inhibitor cocktail

(Roche Applied Science; Indianapolis, IN) and 1 mM sodium orthovanadate as

phosphatase inhibitor (257). Whole-cell lysates were obtained by centrifugation at 13,000

× g for 15 min at 4°C. To obtain nuclear extract, cells were homogenized in a hypotonic

buffer [10 mM HEPES (N-2-hydroxyethylpiperazine-N’-2-ethanesulfonic acid) pH 7.9,

1.5 mM MgCl2, 10 mM KCl, 0.2 mM phenylmethylsulphonyl fluoride (PMSF), 0.5 mM

DTT, 1 mM sodium orthovanadate, 0.5% nonidet P-40]. After centrifugation at 2,500 × g

at 4°C for 5 min, the supernatant (cytoplasmic fraction) was removed. Pellets were

washed once with hypotonic buffer containing no detergent, and hypertonic buffer [final

concentrations: 20 mM HEPES pH 7.9, 10% glycerol, 1.5 mM MgCl2, 400 mM KCl, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM PMSF, 0.5 mM DTT, 1 mM sodium orthovanadate] was added to extract nuclear proteins. After 30-min incubation on ice, the mixture was centrifuged at 13,000 × g for 30 min. The supernatant was then

77 collected as the nuclear extract (257). Protein concentrations of whole-cell and nuclear

extracts were determined using Bio-Rad protein assay (Hercules, CA).

Western Blot Analysis- Whole-cell lysates (25 µg) or nuclear extract (15 µg) were denatured and separated by polyacrylamide gel electrophoresis. After separation, proteins were electrophonically transferred to nitrocellulose membranes, which were then sequentially incubated in primary antibody and horseradish peroxidase (HRP)-conjugated secondary antibody (257). Detection of the HRP-conjugate was done using the ECL system (Pierce Biotechnology; Rockford, IL). For equal loading controls, the membranes were blotted with an anti-β-actin antibody (Santa Cruz Biotechnology; Santa Cruz, CA) or stained by Ponceau S.

Reversed Transcription (RT)-Polymerase Chain Reaction (PCR)- Total cellular

RNA was isolated using Qiagen RNeasy Kit (Qiagen Inc.; Valencia, CA) according to the manufacturer's instructions. Total RNA (0.5 µg) was subjected to reverse transcription, and one-tenth of the reaction mixture was used for PCR analysis. A pair of primers was designed to detect differential expression of IRF-1 mRNAs: 5’-GGC TGG

GAC ATC AAC AAG GAT G-3’ (forward) and 5’-GAG CTG CTG AGT CCA TCA

GAG AA-3’ (reverse), amplicon size 330 base pairs (bp). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as an internal control: 5’-TGA AGG TCG GAG

TCA ACG GAT TTG GT-3’ (forward) and 5’-CAT GTG GGC CAT GAG GTC CAC

CAC-3’ (reverse), amplicon size 980 bp. Primer sequences for IRF-1 target genes were:

TRAIL – 5’-TGC GTG CTG ATC GTG ATC TT-3’ (forward) and 5’-CCA ACT AAA

AAG GCC CCG AA-3’ (reverse), amplicon size 800 bp; caspase-1 – 5’-AGG ACA AAC

CGA AGG TGA TC-3’ (forward) and 5’-TGT CCT GGG AAG AGG TAG AA-3’

78 (reverse), amplicon size 390 bp. During PCR amplification, 0.5 µCi of [α-33P]dATP was

added to each reaction as described previously (257). PCR products were separated on a

5% native polyacrylamide gel. The gel was then dried and exposed to Kodak Biomax MS

film (Eastman Kodak Company; Rochester, NY). Individual bands were cut from the

dried gel and counted in 3 ml of ScintiVerse scintillation fluid (Fisher Scientific; Fair

Lawn, NJ) using a liquid scintillation counter (Beckman Instruments; Irvine, CA) to

quantify relative gene expression levels.

Flow Cytometry Analysis of IFNGR-1- Freshly harvested A549 cells were washed

twice in cold wash buffer (0.1% NaN3, 1% FBS in PBS, pH 7.2). Anti-IFNGR-1

monoclonal antibody (0.1 µg per reaction) was added at 25 µl per well to a U-bottom 96-

well plate. Washed cells (4 × 105 in 25 µl of low-serum medium) were then added to each corresponding well, and the plate was incubated for 1 h at room temperature with mixing several times during the incubation. Cells were pelleted and washed once before the addition of 50 µl wash buffer containing PBXL-3L-conjugated anti-mouse secondary antibody (5 µl per reaction; Martek Biosciences, Columbia, MD). The cells were further

incubated for 30 min at room temperature in the dark. After they were pelleted again and

washed twice in wash buffer, 200 µl of wash buffer was added to each well to resuspend

and transfer the cells to a flow tube (130 × 10 mm) containing 400 µl of fixing solution

(1% paraformaldehyde in PBS). Fixed samples were stored at 4°C in the dark before

being analyzed using a Coulter Elite ESP flow cytometer (helium-neon laser) within 3

days.

79 Immunocytochemistry Analysis- A549 cells were plated in 12-well plates at 50%

confluency and treated appropriately. At times of harvesting, cells were washed twice in

PBS and immediately fixed with 3.7% formaldehyde (w/v) in PBS for 20 min at room

temperature. They were then permeabilized with 0.2% Triton X-100 in PBS for 5 min

and the reactions were quenched with freshly prepared 0.1% sodium borohydride in PBS

for 5 min. Afterwards, the fixed and permeabilized cell monolayer was sequentially

incubated in blocking buffer containing 10% FBS, 1% BSA, 0.02% NaN3 in PBS, anti-

IRF-1 polyclonal antibody in 1% BSA in PBS, and fluorescein isothiocyanate (FITC)- labeled anti-rabbit secondary antibody in 1% BSA in PBS in the dark. After washing the monolayer twice in PBS, cells were counterstained with 1.5 µg/ml of DAPI for 5 min in the dark. Samples were visualized under Olympus IX70 inverted system fluorescence microscope equipped with appropriate filters (Hitech Instruments; Edgemont, PA).

Images were captured using SPOT camera (Diagnostic Instruments; Sterling Heights,

MI).

Electrophoretic Mobility Shift Assay (EMSA)- Nuclear extract was prepared as described earlier, aliquoted, and stored at −80°C. For each EMSA reaction, 5 µg of nuclear protein was incubated with 30,000 cpm of [γ-32P]ATP-labeled consensus IRF-1 gel shift oligonucleotide (Santa Cruz Biotechnology; Santa Cruz, CA) for 30 min on ice.

For competition or supershift assay, unlabeled IRF-1 concensus or mutant oligonucleotides (10× or 50× in excess), or anti-IRF-1 monoclonal antibody (1 µl) were incubated with nuclear extracts for 10 min on ice prior to the addition of radiolabeled oligonucleotides. Reaction mixtures were then separated on a 5% native polyacrylamide gel. After electrophoresis, the gel was dried and subjected to autoradiography (257).

80 Statistical Analysis- In each experiment, at least three replicating samples were analyzed for each treatment/time point (i.e., n = 3). In addition, most experiments were further repeated once (i.e., n = 6). Statistical analysis was performed by using

SuperANOVA software (Abacus Concepts; Berkeley, CA) for student’s t-test, one-way analysis of variance (ANOVA), two-way ANOVA, and simple regression. Three- dimensional plot and multiple regression was performed by using SigmaPlot software

(SPSS Inc.; Chicago, IL). All data, unless specified, are shown as the mean + SEM, and difference was considered statistically significant when the P-value was less than 0.05.

81 4. Results

atRA Increases IFNγ-Induced IRF-1 Protein Expression- The dose-dependent

regulation by IFNγ on IRF-1 protein in the presence or absence of atRA was investigated

by pretreating A549 cells with atRA or vehicle overnight, and then with atRA and

different concentrations of IFNγ for 4 h (Figure 16A). IRF-1 protein was induced dose-

dependently by IFNγ. atRA by itself slightly induced IRF-1 transcripts (Figure 16B),

consistent with the reported findings (205); however, more marked increase was found in

the combination between atRA and IFNγ, where atRA was able to synergize with each

concentration of IFNγ to further increase IRF-1 expression (Figure 16A). Maximal

induction of IRF-1 was observed around 2-4 U/ml of IFNγ, with similar to a ~5.5-fold

increase with IFNγ alone and ~9-fold with IFNγ-plus-atRA. Since half-maximal

induction was produced by 0.5 U/ml of IFNγ, this concentration was chosen as the suboptimal concentration of IFNγ for use in most of the subsequent experiments. These results demonstrate that pretreatment with atRA can augment the effect of low doses of

IFNγ in IRF-1 induction.

82

Figure 16 Overnight pretreatment with atRA followed by suboptimal concentrations of IFNγ induces a faster, higher, and more stable expression of IRF-1. (On the next page) A, A549 cells were pretreated with either atRA (0.1 µM) or vehicle overnight, and then with IFNγ (0, 0.03125, 0.0625, 0.125, 0.25, 0.5, 1, 2, 4, or 100 U/ml) and atRA for 4 h. Cell lysates were prepared and assayed by Western blot. Fold increases of IRF-1 protein compared to the untreated cells are shown (n = 3; except for 100 U/ml of IFNγ, n = 1). Two-way ANOVA showed significant differences for atRA (P < 0.002) and IFNγ dose (P < 0.0001). B, A549 cells were pretreated with either atRA (0.1 µM) or vehicle overnight and then with atRA and 0.5 or 2 U/ml of IFNγ for 2, 4, and 8 h. Cell lysates were prepared and assayed for mRNA and protein levels of IRF-1 by RT-PCR and Western blot, respectively. GAPDH (for RT-PCR) and β-actin (for Western blot) were used as negative controls. The Western blot membranes were also stained by Ponceau S to assure equal loading of proteins. C, Fold increases of IRF-1 protein within each IFNγ concentration group (n = 6) are shown; the asterisks indicate significant differences between atRA and vehicle groups (student’s t-test); AUC, area under the curve. D, Cells were pretreated with either atRA or vehicle overnight, and then with Actinomycin D (Act D; 5 µg/ml) for one hour before the addition of IFNγ (0.5 U/ml). IRF-1 protein at 4 h (time of maximal expression) is shown. E, A correlation test between fold increases of mRNA and protein levels of IRF-1 was performed by using simple regression (R2 = 0.89).

83

84 The kinetics of atRA and IFNγ-regulated IRF-1 were investigated by pretreating

A549 cells with atRA or vehicle overnight, and then with atRA and IFNγ (0.5 U/ml –

suboptimal or 2 U/ml – maximal) for 2, 4, and 8 h. Both mRNA (Figure 16B) and protein

(Figure 16B and 16C) results showed that IRF-1 was induced transiently by low

concentrations of IFNγ. The expression peaked at 4 h. Treatments with atRA significantly

increased IFNγ-induced production of IRF-1, with an overall increase of 72% in case of

0.5 U/ml and 35% in case of 2 U/ml of IFNγ (Figure 16C). Specifically, the combination

of atRA and IFNγ, compared to IFNγ alone, induced significantly higher levels of IRF-1

protein at 4 h for 0.5 U/ml of IFNγ, and 4 and 8 h for 2 U/ml of IFNγ. Thus, atRA

promotes an increase of IFNγ-induced IRF-1 expression that is faster, higher, and more

stable than that induced by IFNγ alone.

Induction of IRF-1 by atRA and IFNγ is Transcriptionally Regulated-

Actinomycin D (Act D; 5 µg/ml) completely blocked the protein expression of IFNγ-

induced IRF-1 either with or without atRA (Figure 16D), indicating that production of nascent transcripts is required for IRF-1 protein induction. This was confirmed by correlation analysis (Figure 16E) between IRF-1 mRNA and protein (R2 = 0.89). These results suggest that regulation of IRF-1 transcription by atRA and IFNγ is important for their modulation of IRF-1 protein.

85 Pretreatment with atRA is Required to Sensitize A549 Cells- To test whether pretreatment with atRA was required for sensitizing A549 cells to better respond to low concentrations of IFNγ, cells were pretreated with atRA for periods of 0−16 h, followed by treatments with atRA and IFNγ (0.5 U/ml) for 4 h (Figure 17). Shorter than 4 h of atRA pretreatment did not produce a significant effect; however, 4 h of atRA pretreatment was able to significantly increase IFNγ-induced IRF-1. The increase was even more marked with longer pretreatments (8−16 h). This demonstrates that at least 4 h of pretreatment with atRA is required in atRA-mediated increase of IFNγ-induced IRF-1 protein. We hypothesize that protein(s) related to IFNγ signaling, such as those important for STAT-1 activation, may be regulated during atRA pretreatment.

86

Figure 17 At least 4 h of pretreatment with atRA is required to sensitize A549 cells to better respond to low-dose IFNγ. A549 cells were either untreated, or treated with 0.5 U/ml of IFNγ only, or pretreated with atRA (0.1 µM) for 16, 12, 8, 4, 2, 1, or 0 h before they were treated with IFNγ (0.5 U/ml) and atRA for 4 h. Cell lysates were prepared and assayed by Western blot. Fold increases of IRF-1 protein compared to untreated are shown (n = 3 or 6). The asterisks on top of the bars mark significant differences among treatment groups (one-way ANOVA). NS, not significantly different from IFNγ only.

87 Pretreatment with atRA Increases IFNγ-Induced STAT-1 Tyrosine

Phosphorylation- IFNγ signals through binding of the cytokine to IFNγ receptors and

generation of a kinase cascade that phosphorylates and activates STAT-1, which in turn

transactivates the IRF-1 promoter. STAT-1 phosphorylation at residue Tyr-701 is

required for its activation and transcriptional functions. Thus, we measured tyrosine-

phosphorylated STAT-1 (pY-STAT-1) to investigate its involvement in atRA-mediated

IRF-1 induction (Figure 18A). pY-STAT-1 protein bands were enumerated by

densitometry and plotted in Figure 18B. As anticipated, neither vehicle nor atRA in the absence of IFNγ could induce pY-STAT-1. IFNγ at 0.5 U/ml, on the other hand, induced

STAT-1 phosphorylation. pY-STAT-1 was visible after only 20 min of stimulation with

IFNγ, and peaked between 1-2 h. Comparisons between any two time points tested showed significant differences except for 20 min and 8 h, indicating rapid and transient increase of pY-STAT-1. More importantly, pretreatment with atRA significantly increased IFNγ-induced pY-STAT-1; however, the effect of a signal dose of atRA at time

0 h was similar to that of IFNγ alone, confirming the requirement of atRA pretreatment.

Protein expression of total STAT-1 was not affected by any of the treatments (Figure

18A).

When compared to the expression pattern of IRF-1 mRNA or protein, induction

of pY-STAT-1 occurred earlier, suggesting that STAT-1 activation was upstream of

IRF-1 expression. Overall, these results suggest that atRA pretreatment increases IFNγ- induced IRF-1 through a STAT-1-dependent pathway.

88

Figure 18 Pretreatment with atRA increases IFNγ-induced STAT-1 tyrosine phosphorylation. A, A549 cells were pretreated with atRA (0.1 µM) or vehicle overnight and then with atRA and 0.5 U/ml of IFNγ for 20 min, 1, 2, 4, and 8 h. An additional treatment group was designed that included overnight pretreatment with the vehicle and co-treatment of atRA with IFNγ, shown in the figure with triangles (U). Cell lysates were prepared and assayed for protein levels of STAT-1 and tyrosine phosphorylated STAT-1 (pY-STAT-1) by Western blot. B, pY-STAT-1 bands were enumerated using densitometry, and fold increases of pY-STAT-1 levels compared to the one treated with IFNγ alone are shown (n = 6). pY-STAT-1 was not detectable in the absence of IFNγ. Two-way ANOVA showed atRA and time as significant factors (P < 0.0001). For 1 and 2 h, +atRA was significantly different from both −atRA and U (*; P < 0.01).

89 Expression of IFNGR-1 on the Cell Surface is Increased upon atRA Pretreatment-

IFNGR-1 is the ligand-binding chain of IFNγ receptor complex. It is also important for

Janus kinase-1 (Jak-1) binding and STAT-1 recruitment (88). Thus, if total or surface expression of IFNGR-1 is regulated by overnight pretreatment with atRA, an increase of

IFNGR-1 expression might explain the observed effects of atRA on increasing IFNγ- induced STAT-1 activation and IRF-1 mRNA and protein expression. To test this hypothesis, A549 cells were treated with vehicle or atRA overnight and harvested at time

0 h for Western blot and flow cytometry analysis (Figure 19). Although the total protein level of IFNGR-1, assessed by Western blot (Figure 19A), was not changed by atRA, multiple experiments showed consistently that atRA significantly increased the average intensity of IFNGR-1 on the cell surface (Figure 19B and 19C). The increase was modest; but taking that IFNGR-1 is the start point of IFNγ signaling, a small increase in initial signaling could be amplified by the downstream events. The increase of IFNGR-1 surface expression by atRA is parallel with our earlier observations that both STAT-1 activation and IRF-1 expression induced by IFNγ were further increased by pretreatment with atRA.

90

Figure 19 Pretreatment with atRA increases IFNGR-1 cell-surface expression. A549 cells were treated with atRA (0.1 µM) or vehicle overnight and harvested for Western blot and flow cytometry analysis. A, Western blot of IFNGR-1 on triplicate samples after overnight treatment with either vehicle or atRA. B, Cells after the treatments were stained with IFNGR-1 monoclonal antibody, followed by PBXL-3L- conjugated anti-mouse secondary (2°) antibody. The cells were analyzed by a 633-nm- excitation laser flow cytometer. The shaded peak represents an unstained sample. The histogram shown is a representative of six independent experiments. C, Fold increases of the average intensity of PBXL-3L dye (representing the surface expression of IFNGR-1) are shown (n = 6). The asterisk indicates a significant difference between the two treatments (simple t-test).

91 Ligands for RARα Sequentially Increased the Levels of IFNGR-1, Activated

STAT-1, and IRF-1- Retinoids regulate transcription of genes through binding to members of the retinoid receptor family. Western blot analysis showed that at least two retinoid receptors, RARα and RXRα, were constitutively expressed in A549 cells (Figure

20A). To sort out which retinoid receptors were involved in the effect of atRA on IFNγ signaling and IRF-1 expression, we used several receptor-selective retinoids to treat A549 cells overnight and at time 0 h. Cells were harvested after overnight pretreatment (for

IFNGR-1 analysis), or at either 1 h 40 min (approximate peaking time of pY-STAT-1), or

4 h (approximate peaking time of IRF-1 protein) after IFNγ stimulation. The results showed that 9cRA, an RARα agonist (Am580), and the combination of Am580 and a pan

RXR-agonist significantly increased cell-surface IFNGR-1, and IFNγ-induced pY-

STAT-1 and IRF-1, to an extent similar to the effect of atRA (Figure 20B and 20C).

RARα antagonist blocked the effect of atRA on pY-STAT-1 and IRF-1, and that of

Am580 on all three levels, suggesting an important role of RARα in regulating IRF-1 induction. In fact, we found that atRA increased nuclear levels of RARα (2.1-fold) and

RXRα (2.4-fold) after overnight pretreatment with atRA, indicating that these retinoid

receptors translocate into the nucleus and act to mediate the effect of atRA on IFNγ

signaling. RTMBE, a retinoid analog without known binding activity to retinoid receptors,

was used as a negative control. atROL, a precursor of atRA, did not increase either pY-

STAT-1 or IRF-1, suggesting limited conversion of atROL to atRA during overnight

pretreatment. Protein expression of total STAT-1 was not affected by any of the

treatments (data not shown).

92

Figure 20 Ligands for RARα, including atRA, 9cRA, and Am580, sequentially increases the levels of IFNGR-1, activated STAT-1, and IRF-1. (On the next page) A, Protein expression of RARα and RXRα in A549 cells after overnight treatment with vehicle or atRA (0.1 µM). B, Cells were pretreated with different retinoids (0.1 µM; except for RARα antagonist, which was 50× in excess) overnight and with 0.5 U/ml of IFNγ for 1 h 40 min or 4 h. Cell lysates were prepared and assayed for IRF-1 protein and STAT-1 tyrosine phosphorylation (pY-STAT-1) by Western blot. RTMBE was used as a negative control. RARα antagonist is shown as “6”. C, Cells were treated overnight with different retinoids at the same concentrations described above, and then stained for the determination of IFNGR-1 cell-surface expression by flow cytometry. Percentage increases of the average intensity of PBXL-3L dye compared to the vehicle are shown (n = 6). Comparisons by one-way ANOVA showed that atRA, Am580, Am580/RXR, atRA/6, and 9cRA are significantly different from either no retinoid or RTMBE (*; P < 0.05). D, Fold increases of the levels of cell-surface IFNGR-1, pY-STAT-1, and IRF-1 are plotted on a three-dimensional scale. Multiple regression was performed that suggests significant correlations among IFNGR-1, pY-STAT-1, and IRF-1 (R2 = 0.85).

93

94 To determine the correlations among the levels of IFNGR-1, pY-STAT-1 and

IRF-1, we used a multiple regression model to analyze these three variables. Retinoid- treated levels of cell-surface IFNGR-1 before IFNγ stimulation, and those of pY-STAT-1 and IRF-1 at 1 h 40 min and 4 h after IFNγ stimulation, respectively, were normalized to the vehicle-treated levels. A three-dimensional plot (Figure 20D) showed that positive regulators of all three variables (i.e. atRA, Am580, 9cRA, and the combination of Am580 and RXR-agonist) localized in the upper inner portion of the plot. In contrast, atRA or

Am580 combined with RARα antagonist was within the lower outer portion, with the vehicle and RTMBE. Multiple regression analysis showed significant correlations among

IFNGR-1, pY-STAT-1, and IRF-1 (R2 = 0.85). This suggests that atRA (together with

Am580 and 9cRA) sequentially regulates three components of IFNγ signaling pathway: cell-surface IFNGR-1, and after IFNγ is given, STAT-1 activation (maximum at 1 h 40 min) and IRF-1 expression (maximum at 4 h).

95 Pretreatment with atRA Increases Nuclear Localization and DNA-Binding

Activity of IFNγ-Induced Nuclear IRF-1- IRF-1 stimulated by 9cRA and IFNγ as part of a

coactivation complex activates the promoter of TRAIL (72). Yet, little is known about

the trafficking of IRF-1 between cytoplasm and nucleus. To investigate the effect of IFNγ

and atRA on nuclear localization of IRF-1, we performed Western blot of nuclear extracts

(Figure 21A and 21B) and immunostaining of formaldehyde-fixed cells (Figure 21C).

IFNγ by itself increased the nuclear level of IRF-1 at 4 h, which declined at 8 h, corresponding to the kinetics of IRF-1 protein expression (Figure 16C). However, the combination of atRA and IFNγ served to concentrate IRF-1 in the nucleus at both 4 and 8 h. This suggests that atRA not only plays a role in increasing IRF-1 expression, but also functions to maintain IRF-1 levels in the nucleus for a longer period of time.

DNA-binding activity of IRF-1 was also tested as indication of its transcriptional

functions. As shown in Figure 21D, IRF-1 binding to the consensus element (IRF-E) was

induced after IFNγ stimulation, and the induction was further increased by atRA

pretreatment. The IRF-1/IRF-E complex migrated so close to nonspecific bands that the

complex was indistinct; however, supershift experiments clearly identified the complex.

atRA pretreatment markedly increased the supershifted band induced by IFNγ.

Competition and specificity controls indicated that the complex was specific. Moreover,

kinetics experiments showed that atRA markedly increased IFNγ-induced IRF-1 binding

to IRF-E at 8 h (Figure 21E). This is consistent with the earlier demonstration that with

atRA pretreatment, IFNγ-induced nuclear IRF-1 stayed at a higher level for a longer

period of time (Figure 21B).

96

Figure 21 Pretreatment with atRA increases nuclear expression and DNA- binding activity of IFNγ-induced IRF-1. (On the next page) A, A549 cells were pretreated with atRA (0.1 µM) or vehicle overnight and then with IFNγ (0.5 U/ml) and atRA for 4 h or 8 h. Nuclear extracts were prepared and analyzed by Western blot. B, Fold increases of the density of nuclear bands are shown (n = 7). The asterisks on top of the bars mark significant differences between the treatments (simple t- test). C, A549 cells were pretreated with atRA (0.1 µM) or vehicle overnight and then with IFNγ (0.5 U/ml) and atRA for 4 h or 8 h. They were fixed and stained with IRF-1 polyclonal antibody, followed by a FITC-labeled secondary antibody. Cells were counterstained with DAPI (1.5 µg/ml) before visualized under a fluorescence microscope. The pictures shown are the overlays of IRF-1/FITC (green) and nuclei/DAPI (red) images. D, A549 cells were pretreated with atRA (0.1 µM) or vehicle overnight and then with IFNγ (0.5 U/ml) and atRA for 8 h. Nuclear protein extracts (NPE) were prepared and 5 µg of NPE were incubated with [γ-32P]ATP-labeled consensus IRF-1 gel shift oligonucleotide with or without unlabeled competitor DNA (wild-type/WT or mutant/MU; 10× or 50× molar excess), or were pre-incubated with IRF-1 monoclonal antibody (mAb) prior to the addition of labeled probe. Protein-DNA complexes were resolved using a 5% PAGE gel. E, A549 cells were pretreated with atRA (0.1 µM) or vehicle overnight and then with IFNγ (0.5 U/ml) and atRA for 4 h or 8 h. NPE (5 µg) were prepared and EMSA was performed.

97

98 The effect of atRA on IRF-1 binding was abolished by co-pretreatment with

RARα-antagonist (data not shown), indicating that RARα was involved in mediating the

DNA-binding activity of IRF-1. Whether this is directly related to the effect of RARα on

IRF-1 protein levels needs further investigation.

atRA and IFNγ Synergistically Increase Transcription of IRF-1 Target Genes-

Transcript levels of two IRF-1 target genes, caspase-1 (272) and TRAIL (72), were examined by RT-PCR as indicators of biological effects of atRA and IFNγ-induced IRF-1.

Both genes were induced by IFNγ and the combination of IFNγ and atRA (Figure 22A).

Specifically, atRA pretreatment synergized with low-dose IFNγ to induce caspase-1 mRNA to more than 20-fold compared to the untreated cells at 24 h, whereas IFNγ alone induced only 12-fold (Figure 22B). TRAIL mRNA was induced about 3-fold by the combination. These results support the concept that pretreatment with atRA increases the transcriptional functions of IFNγ-induced IRF-1.

99

Figure 22 atRA and IFNγ synergistically increase transcription of IRF-1 target genes. A, A549 cells were pretreated with atRA (0.1 µM) or vehicle overnight and then with IFNγ (0.5 U/ml) and atRA for 24 or 48 h. Total RNA was isolated and transcript levels of caspase-1, TRAIL, and GAPDH was measured by RT-PCR. B, Radioactivity of caspase-1 bands was counted as described in Experimental Procedures. Fold increases of caspase-1 mRNA are shown (n = 3). The asterisks on top of the bars mark significant differences between the treatments (simple t-test).

100 5. Discussion

Vitamin A is important in the maintenance of normal immune functions. This is supported by studies of vitamin A deficiency, where disease risk is increased (274).

However, therapeutic effects of vitamin A supplementation vary, depending on the types of illness and the populations studied (5). In addition, vitamin A has failed to affect the prevalence of illness, even in the presence of a large impact on mortality. In contrast, studies on vitamin A and morbidity have consistently showed an important effect of this nutrient on the severity of illness. In one study monitoring 1455 young children weekly for a year (6), vitamin A supplemented children had less frequent vomiting and anorexia, although the prevalence of diarrhea or acute respiratory infections was not changed. The supplementation also led to fewer clinic attendances, hospital admissions, and death.

Thus, vitamin A appears to reduce the severity of infection, possibly by enhancing functions of innate and cell-mediated immune responses, and helping to repair virus- damaged epithelia (46).

Our present study was designed to study how atRA, an active metabolite of vitamin A, increases IFNγ-induced response as part of innate immunity. Type I and II

IFNs were discovered as proteins that inhibit virus replication (3). They are induced in response to virus infection, secreted, and function to activate a global antiviral state. Type

II IFN (IFNγ) is generally expressed at a low level in the early stages of an infection (80); thus, enhancing the functions of albeit small amounts of IFNγ during early infection may potentially improve the overall IFN response and encourage faster recovery.

IFNγ signaling pathway is initiated upon binding of IFNγ to IFNGR-1. The extracellular portion of IFNGR-1 chain contains multiple disulfide bonds that are

101 required for maximal binding of the ligand. The intracellular portion of IFNGR-1

contains a tyrosine residue that becomes phosphorylated upon ligand binding and acts in

STAT-1 recruitment (88). STAT-1 is phosphorylated at Tyr-701, which is required for

the activation and dimerization of STAT-1 through Src-homology 2 domains (275). The

activated STAT-1 homodimer binds and activates transcription of target genes through the conserved response element GAS (276). STAT-1 is also phosphorylated on Ser-727 in order to exert maximal transcriptional activity (116).

In the present study, we found that atRA of a sub-pharmacologic concentration

(0.1 µM) increased the effect of low-dose IFNγ (0.5 U/ml) on IRF-1 induction by about

2-fold, potentially enhancing the antiviral functions of IFNγ. IRF-1, a target of IFNγ signaling, was originally identified as a mouse nuclear factor that specifically bound to the upstream regulatory region of IFNβ gene. In 1988, Taniguchi and colleagues cloned and characterized the cDNA encoding IRF-1 (179), and a role of IRF-1 in viral infections was then suggested, as it possessed a virus-inducible promoter (180).

In previous studies, Matikainen et al. (205) showed that atRA (1 µM) rapidly upregulated IRF-1 in promyelocytic leukemia NB4 cells, inducing the mRNA level to nearly 8-fold within 3 h of treatment. There was also a 3-fold increase of IRF-1 protein levels by atRA (1 or 100 µM) in cervical squamous carcinoma SiHa cells (208). In our studies of lung epithelial A549 cells, we observed minimal effect of atRA (0.1 µM) by itself after 16 h (overnight) treatment or longer, probably due to the transient action of

lower-dose atRA on IRF-1 induction (208). However, atRA at this concentration significantly potentiated the effect of low-dose IFNγ on IRF-1 induction (Figure 16).

102 atRA pretreatment of a certain length of time (4 h or longer) was required for sensitizing these cells to better respond to IFNγ (Figure 17). We also used a protein synthesis inhibitor, cycloheximide (CHX; 10 µg/ml), together with atRA pretreatment, to determine whether the increase of IRF-1 transcription was blocked by inhibiting protein synthesis (data not shown). The result showed that both IFNγ- and IFNγ-plus-RA- induced transcription was blocked by CHX, indicating the need of certain protein(s) in both processes. Such proteins may regulate the migration of IFNGR-1 from the cytoplasm to the cell surface. Yet, whether or not these proteins are targets of atRA pretreatment requires further investigation. Nevertheless, our study has demonstrated that treatment with atRA increases the levels of cell-surface IFNGR-1 (Figure 19). This is supported by reports that upregulation of type I IFN receptors by retinoids is associated with increased signaling of IFNα/β (277, 278).

STAT-1 is tyrosine phosphorylated and activated upon IFNγ stimulation (104). atRA was observed in our model to enhance tyrosine phosphorylation (activation) of

STAT-1 induced by low-dose IFNγ, without increasing STAT-1 protein levels (Figure

18). This effect of atRA required pretreatment, suggesting that the increase of STAT-1 activation is due to the increase of cell-surface IFNGR-1 during atRA pretreatment. In comparison, the rapid effect of atRA on IRF-1 induction may not involve STAT-1 functions (205, 207), despite a report that atRA induces IRF-1 through a GAS motif of the IRF-1 promoter (195). However, the present study indicates that STAT-1 activation is required in the regulation of IFNγ-induced IRF-1 by atRA.

The use of receptor-selective retinoids in the present study revealed strong correlations among the levels of IFNGR-1, activated STAT-1, and IRF-1 (Figure 20),

103 indicating that atRA and RARα together influenced several components of IFNγ signaling pathway. Our results identified RARα ligands as important regulators of IFNγ- induced IRF-1. atRA has been shown to enhance transactivation of STAT-1 in the presence of RARα in acute promyelocytic leukemia (APL) cells (260). However, it was unknown if RARα mediated other components of IFNγ signaling. In the present study, atRA also increased nuclear levels of RARα, which, together with RXRα, may mediate the regulatory effect of atRA on multiple components of the IFNγ signaling pathway.

How atRA and RARα function in this context requires further investigation. We hypothesize that during the pretreatment period of 4 h or longer, atRA and RARα regulate the synthesis or posttranslational modification of proteins involved in surface expression of IFNGR-1, tyrosine phosphorylation of STAT-1, and/or transcription of

IRF-1.

atRA as a pan agonist for all RARs may have increased IFNGR-1 surface expression via receptors other than RARα, as the antagonist specific for RARα did not block the effect of atRA on IFNGR-1. This is supported by the observation that RARβ agonist also increased cell-surface IFNGR-1 (data not shown). Hence, additional mechanisms of atRA action might affect IFNGR-1, but not pY-STAT-1 or IRF-1.

Nevertheless, our current findings suggest an essential role of RARα in mediating the effect of atRA on IFNγ signaling.

atRA pretreatment also potentiated the transcriptional activity of IFNγ-induced

IRF-1, increasing its nuclear localization and DNA-binding activity (Figure 21). Notably, pretreatment with atRA maintained higher IRF-1 levels in the nucleus for a longer period

104 of time, with a nearly 4-fold difference between IFNγ-alone and atRA-plus-IFNγ at 8 h.

This is possibly due to an unknown role of atRA in facilitating nuclear translocation of

IRF-1 and/or preventing shuttling of the protein out of the nucleus. Reports are scarce on

this subject; however, a retinoid-mediated increase of nuclear localization of calcyclin binding protein was observed during neuronal differentiation (279). Another report has shown that nucleocytoplasmic translocation of RARβ is facilitated by RXR in a ligand- dependent manner (20). Our results are consistent with these findings, where atRA facilitated or maintained the nuclear localization of RARα, RXRα, and IRF-1 and may

thereby prolong the transactivation of IRF-1.

IFNγ-induced caspase-1, an enzyme important for the production of IFNγ during

an innate immune response, was further increased by pretreatment with atRA (Figure 22).

Caspase-1 processes the precursors of IL-1β (280) and IL-18 (273) to their biologically

active forms. IL-1β and IL-18, alone or in synergy with IL-12, increases

lipopolysaccharide (LPS)-induced IFNγ production by natural killer cells, evidenced by

marked reduction of LPS-induced serum IFNγ titers in caspase-1−/− mice (273) and

IL-12-deficient mice (281). It is very likely that IFNγ (low-dose)-induced caspase-1,

which can be enhanced by atRA pretreatment, feeds forward IFNγ signaling during early

stages of an infection.

TRAIL, another target gene of IRF-1, induces apoptosis selectively in cancer cells,

sparing normal cells that are generally TRAIL-insensitive (282). IFNγ-9cRA cotreatment

synergistically induces TRAIL mRNA levels due to sustained occupancy of IRF-1 on the

TRAIL promoter (72), supporting the use of combination therapies between IFNs and

105 retinoids in cancer treatments. The induction of TRAIL could also be important in the cellular response to infection, but has received little attention in this context. Our data indicate that the cell’s retinoid status can affect the induction of TRAIL by low doses of

IFNγ (Figure 22), suggesting another mechanism by which retinoids may improve the host’s response to infectious disease.

106 In summary, the present study has detailed the regulating roles of atRA in IFNγ signaling and IRF-1 induction. Using RARα as a mediator, atRA pretreatment sensitizes

A549 cells to better respond to low-dose IFNγ, increasing cell-surface IFNGR-1, STAT-1 activation, levels of IRF-1 mRNA and protein, and transcriptional functions of IRF-1

(Figure 23). Could these results in a model system of the cellular response to low-dose

IFNγ be relevant to human studies of vitamin A status and infectious disease? In epidemiological studies conducted in populations where vitamin A deficiency remains a public health problem [see WHO review (5)], supplementation with vitamin A has been shown to reduce the severity of infectious diseases (6), and to significantly reduce all- cause mortality rates in children and pregnant women (283, 284). It is possible that vitamin A, through conversion to physiological levels of atRA, results in the generation of an “active atRA state”, which serves to improve the integrity of epithelial tissue barriers as a first line of host defense, and to enhance the initial response to viral and bacterial infections through potentiation of the response to low levels of IFNγ, such as are likely to be present in the early stages of an infection. A heightened host response to IFNγ, produced when the “active atRA state” is adequate, may provide an important advantage in quickly responding to viral and bacterial agents, and could be part of the mechanism whereby vitamin A has been shown to be effective in reducing morbidity and mortality in human populations.

107

Figure 23 Working model of the regulation of IFNγ-induced IRF-1 by pretreatment with atRA. In A549 cells, atRA pretreatment of 4 h or longer facilitates the shuttling of RARα and its dimerization partner, RXRα, from the cytoplasm to the nucleus in a ligand-dependent manner (1). This enhances the transactivation activity of RARα/RXRα heterodimer, leading to yet to be characterized changes that prepare the cells for increased response to low-dose IFNγ. Via the actions of RARα, atRA increases the levels of IFNGR-1 on the cell surface (2), enhancing tyrosine phosphorylation (activation) of STAT-1 upon IFNγ stimulation (3). IRF-1, a target gene of STAT-1, is then induced (4) and translated (5); its nuclear localization and DNA-binding activity (6), and target gene (caspase-1 and TRAIL) transcription (7), are also increased.

108

CHAPTER 4 RETINOID MODULATION OF IRF-1 IN

THE ABSENCE OF IFNγ IN MCF10A CELLS

Adapted from

Luo XM and Ross AC (2005) Dual regulatory actions of retinoids in IRF-1 gene expression and nuclear localization. (manuscript in preparation)

109 1. Abstract

Retinoids have been shown to induce interferon regulatory factor-1 (IRF-1), an

important transcription factor involved in cell growth regulation and immune responses.

However, it is unclear how retinoids affect the localization of IRF-1, in addition to the

effect on its expression. In this study, we have used the human mammary epithelial cell

line, MCF10A, to examine the effect of all-trans-retinoic acid (atRA) on IRF-1 gene expression and nuclear localization, as part of mechanisms by which retinoids modulate transcriptional functions of IRF-1. IRF-1 mRNA and proteins were induced rapidly by atRA in MCF10A cells. The protein level peaked at 8 h with maximal induction of more than 30-fold, and declined afterwards. A second dose of atRA, given 16 h after the first one, re-stimulated mRNA and protein levels of IRF-1; however, it did not increase the maximum level. Am580, a retinoic acid receptor (RAR)α-selective ligand, induced IRF-1

in a similar fashion; whereas an RARα antagonist inhibited the effect of atRA and

Am580. In addition, atRA and Am580 increased the nuclear level of RARα. Retinoid X receptor (RXR)α, on the other hand, localized to the nucleus upon second exposure to

atRA. More importantly, although re-stimulation with atRA or Am580 induced almost

the same level of IRF-1 expression as the first dose, more IRF-1 was concentrated in the

nucleus after re-stimulation, indicating that the retinoids can affect nuclear localization of

IRF-1. The increase in nuclear IRF-1 was accompanied by enhanced DNA-binding activity of IRF-1 to its concensus binding element and elevated expression of an IRF-1 target gene, 2',5'-oligoadenylate synthetase-2 (OAS-2). These results suggest that atRA may affect both expression of IRF-1 and its nuclear localization, to increase its signaling

capacity.

110 2. Introduction

Retinoids are potent regulators of immune responses (46) as well as cell growth

and differentiation (2). They induce gene transactivation through ligation to RAR and

RXR proteins, which form heterodimers and bind to the retinoic acid response element

(RARE) in the promoter region of target genes (18). In the presence of ligand, the

receptor proteins are capable of recruiting coactivators and histone acetyltransferase

required for initiation of transcription. atRA, one of the most studied retinoids, is

implicated in the induction of many genes via interaction with RARα, β, or γ. One of the

genes upregulated by atRA, interferon regulatory factor-1 (IRF-1) (205), may be an

important mediator of retinoid functions in enhancing immune responses and promoting

cancer prevention.

IRF-1 was discovered in studies of virus-induced IFNα/β gene regulation (179).

The antiviral functions of IRF-1 were then recognized, when inhibition of

encephalomyocarditis virus replication by interferons (IFNs) was observed to be impaired

in cells from mice with a null mutation in the IRF-1 gene (180). In addition to its

functions on antiviral responses, IRF-1 is known to regulate cell growth and apoptosis

(193, 270). It is also a tumor suppressor, as demonstrated in oncogenic transformation of

primary IRF-1−/− mouse embryonic fibroblasts (146). However, the transformed phenotype of tumor cells can be reversed by exogenous IRF-1 not only in vitro, but also in vivo (271).

In previous studies, atRA has been shown to synergize with IFNγ to increase the level of IRF-1 (285). However, atRA by itself can also induce IRF-1, as shown in

111 promyelocytic leukemia NB4 cells bearing a natural mutation of RARα, where atRA (1

µM) rapidly upregulated IRF-1 mRNA nearly 8-fold (205). A 3-fold increase of IRF-1

protein levels by atRA (1 or 100 µM) was also found in cervical squamous carcinoma

SiHa cells (208). A number of studies have investigated the mechanisms of atRA-

mediated in activation of IRF-1 promoter (195, 207, 208), where a functional RARE is

yet to be identified. However, it has not been examined whether atRA can regulate

subcellular localization of IRF-1, in addition to the effect of this retinoid on IRF-1

expression. Nuclear localization of IRF-1, a transcription factor, is very likely to be

essential for the transactivation of its immune function- and/or apoptosis-related target

genes.

In this study, we hypothesized that atRA modulates both gene expression and

nuclear localization of IRF-1. Using a human mammary epithelial cell line, MCF10A, we

examined the effects of sequential treatments of atRA on IRF-1 expression, localization, and DNA-binding activity. MCF10A is a non-tumorigenic mammary epithelial cell line that has the characteristics of normal breast epithelium (286). However, it is readily transformed; and the breast cancer progression in MCF10A cells is associated with alterations in retinoid receptors, which can be reversed by atRA (287). Thus, we chose these cells as a susceptible cancer model. We found that atRA induced rapid expression of IRF-1 mRNA and protein. Although re-exposure to atRA stimulated similar levels of

IRF-1 as the first dose, it significantly increased nuclear IRF-1. DNA-binding activity of nuclear IRF-1 and transcription of IRF-1 target gene, OAS-2, were also increased by sequential treatments with atRA. An RARα-specific ligand, Am580 affected IRF-1 in a similar fashion to atRA, indicating the involvement of RARα ligation in regulating IRF-1

112 gene expression and nuclear localization. Further, we found that subcellular distribution

of RARα and RXRα were regulated by atRA and Am580, which may contribute to the

effects of the retinoids on IRF-1. Together, it is suggested that retinoids, via functions of

RAR/RXR, may modulate IRF-1 expression, localization, and transcriptional functions,

which may function as part of the mechanism by which retinoids can prevent breast

cancer progression.

3. Experimental Procedures

Reagents, Antibodies, and Cell Culture- atRA (prepared in ethanol and stored at

−20°C), 9-cis-RA (9cRA), retinyl trimethoxybenzyl ether (RTMBE) and actinomycin D

(AD) were obtained from Sigma-Aldrich (St Louis, MO). Receptor-selective retinoids

were provided by Michael Klaus, Hoffmann-La Roche (Nutley, NJ). They include

Am580 (RARα agonist) and Ro41-5253 (RARα antagonist). IRF-1 polyclonal antibody

and concensus gel shift oligonucleotide were obtained from Santa Cruz Biotechnology

(Santa Cruz, CA). IRF-1 monoclonal antibody was obtained from Transduction

Laboratory (Lexington, KY). Alexa Fluor 568-conjugated anti-rabbit IgG and To-Pro.3 iodide were obtained from Molecular Probes (Eugene, OR). MCF10A cells were maintained in DMEM/F-12 medium (GIBCO/Invitrogen, Carlsbad, CA) supplemented with 5% heat-inactivated horse serum (GIBCO), 10 µg/ml human insulin (Sigma), 10 ng/ml epidermal growth factor (Invitrogen), 100 ng/ml cholera toxin (Sigma), and 0.5

µg/ml hydrocortisone (Sigma) at 37°C in a 5% CO2-air incubator. In most experiments,

the cells were plated at approximately 70% confluency, allowed to attach in complete

113 medium, and adjusted to low-serum medium (supplemented with 0.5% horse serum) for

16-24 h before the addition of stimuli. For immunofluorescence experiments, the cells were plated at 20% confluency and grown overnight before use.

Preparation of Whole-Cell and Nuclear Extracts- For extraction of whole-cell lysates, MCF10A cells were lysed in RIPA buffer [1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS) in phosphate-buffered saline (PBS)] containing 10% (v/v) of protease inhibitor cocktail (Roche Applied Science; Indianapolis,

IN) and 1 mM sodium orthovanadate as phosphatase inhibitor (257). Whole-cell lysates were obtained by centrifugation at 13,000 × g for 15 min at 4°C. To obtain nuclear extract, cells were homogenized in a hypotonic buffer [10 mM HEPES (N-2- hydroxyethylpiperazine-N’-2-ethanesulfonic acid) pH 7.9, 1.5 mM MgCl2, 10 mM KCl,

0.2 mM phenylmethylsulphonyl fluoride (PMSF), 0.5 mM DTT, 1 mM sodium orthovanadate, 0.5% nonidet P-40]. After centrifugation at 2,500 × g at 4°C for 5 min, the supernatant (cytoplasmic fraction) was removed. Pellets were washed once with hypotonic buffer containing no detergent, and hypertonic buffer [final concentrations: 20 mM HEPES pH 7.9, 10% glycerol, 1.5 mM MgCl2, 400 mM KCl, 0.2 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM PMSF, 0.5 mM DTT, 1 mM sodium orthovanadate] was added to extract nuclear proteins. After 30-min incubation on ice, the mixture was centrifuged at 13,000 × g for 30 min. The supernatant was then collected as the nuclear extract (257). Protein concentrations of whole-cell and nuclear extracts were determined using Bio-Rad protein assay (Hercules, CA).

Western Blot Analysis- Whole-cell lysates (25 µg) or nuclear extract (5 µg) were denatured and separated by polyacrylamide gel electrophoresis. After separation, proteins

114 were electrophonically transferred to nitrocellulose membranes, which were then

sequentially incubated in primary antibody and horseradish peroxidase (HRP)-conjugated

secondary antibody (257). Detection of the HRP-conjugate was done using the ECL system (Pierce Biotechnology; Rockford, IL). For equal loading controls, the membranes were blotted with β-actin antibody (Santa Cruz Biotechnology; Santa Cruz, CA) for comparing whole-cell proteins or histones (H1 and core proteins) antibody (CHEMICON

International, Inc.; Temecula, CA) for nuclear proteins.

Reversed Transcription (RT)-Polymerase Chain Reaction (PCR)- Total cellular

RNA was isolated using Qiagen RNeasy Kit (Qiagen Inc.; Valencia, CA) according to the manufacturer's instructions. Total RNA (0.5 µg) was subjected to reverse transcription, and one-tenth of the reaction mixture was used for PCR analysis. A pair of primers was designed to detect differential expression of IRF-1 mRNAs: 5’-GGC TGG

GAC ATC AAC AAG GAT G-3’ (forward) and 5’-GAG CTG CTG AGT CCA TCA

GAG AA-3’ (reverse), amplicon size 330 base pairs (bp). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was employed as an internal control: 5’-TGA AGG TCG GAG

TCA ACG GAT TTG GT-3’ (forward) and 5’-CAT GTG GGC CAT GAG GTC CAC

CAC-3’ (reverse), amplicon size 980 bp. Primer sequences for IRF-1 target gene 2'-5' oligoadenylate synthetase-2 (OAS-2) were: 5’-CCA GGA GAA GCT GTG TAT CT-3’

(forward) and 5’-GTC TTC AGA GCT GTG CCT TT-3’ (reverse), amplicon size 440 bp.

During PCR amplification, 0.5 µCi of [α-33P]dATP was added to each reaction as described previously (257). PCR products were separated on a 5% native polyacrylamide gel. The gel was then dried and exposed to Kodak Biomax MS film (Eastman Kodak

Company; Rochester, NY).

115 Confocal Microscopy- MCF10A cells were plated in Lab-Tek 2-well chambered

coverglasses (Nalge Nunc International; Rochester, NY) at 20% confluency. After the

treatments, cells were washed twice in PBS and immediately fixed with 3.7%

formaldehyde (w/v) in PBS for 20 min at room temperature. They were then

permeabilized with 0.2% Triton X-100 in PBS for 5 min and the reactions were quenched

with freshly prepared 0.1% sodium borohydride in PBS for 5 min. Afterwards, the fixed

and permeabilized cell monolayer was sequentially incubated in blocking buffer

containing 10% FBS, 1% BSA, 0.02% NaN3 in PBS, anti-IRF-1 polyclonal antibody in

1% BSA in PBS, and Alexa Fluor 568-labeled anti-rabbit secondary antibody and

To-Pro.3 iodide in 1% BSA in PBS in the dark. After washing the monolayer twice in

PBS, a drop of SlowFade® Gold antifade reagent (Invitrogen; Eugene, OR) and a coverslip were mounted onto the cells. Samples were visualized under the Olympus

Fluoview 300 Confocal Laser Scanning Microscope (Olympus America Inc.; Melville,

NY). Images were analyzed by the Fluoview software. To quantify nuclear fluorescence,

a small circle (5 µm in diameter) was applied to a total of 100 nuclei (excluding the

nucleoli) in each treatment; the fluorescence intensity in the circle was then read by the

software and analyzed as an index of nuclear protein expression.

Electrophoretic Mobility Shift Assay (EMSA)- Nuclear extract was prepared as

described earlier, aliquoted, and stored at −80°C. For each EMSA reaction, 5 µg of

nuclear protein was incubated with 15,000 cpm of [γ-32P]ATP-labeled consensus IRF-1 gel shift oligonucleotide (Santa Cruz Biotechnology; Santa Cruz, CA) for 30 min on ice.

For competition or supershift assay, unlabeled IRF-1 concensus or mutant oligonucleotides (50×), or anti-IRF-1 monoclonal antibody (1 µl) were incubated with

116 nuclear extracts for 10 min on ice prior to the addition of radiolabeled oligonucleotides.

Reaction mixtures were then separated on a 5% native polyacrylamide gel. After electrophoresis, the gel was dried and subjected to autoradiography (257).

Statistical Analysis- Statistical analysis was performed by using SuperANOVA software (Abacus Concepts; Berkeley, CA) for one-way analysis of variance (ANOVA), two-way ANOVA, and simple regression. All data, unless specified, are shown as the mean + SEM, and difference was considered statistically significant when the P-value was less than 0.05.

117 4. Results

atRA Induces IRF-1 in MCF10A Cells- atRA alone induced IRF-1 in MCF10A cells. A titration analysis identified an optimal concentration of 0.1 µM of atRA (Figure

24A), which was used in the subsequent experiments. IRF-1 protein was increased within

2 h of atRA stimulation, reached a maximum at 8 h, and declined afterwards (Figure 24B).

The maximal induction was over 30-fold; and the level of IRF-1 protein remained

elevated over initial level 16 h after treatment. The increase of protein expression was

accompanied by that of IRF-1 mRNA (Figure 24C), which started to rise within 1 h after atRA treatment and was increased about 3-fold from 4-12 h. The atRA-mediated induction of IRF-1 transcript level was blocked by the transcription inhibitor actinomycin

D, indicating that atRA may activate IRF-1 transcription. As anticipated, the IRF-1 message level fluctuated in cells treated with vehicle, consistent with the literature that

IRF-1 is regulated by progression of the cell cycle (189). Despite this fluctuation, atRA significantly induced IRF-1 at each of the time points between 2-12 h. These results indicate that atRA could rapidly induce both IRF-1 mRNA and protein expression in

MCF10A cells.

118

Figure 24 atRA induces IRF-1 in MCF10A cells. (On the next page) A, MCF10A cells were treated with vehicle or atRA (1, 10, 100, or 1000 nM) overnight, and then lysed and assayed by Western blot for IRF-1 expression. The membranes were also blotted for β-actin to assure equal loading of proteins (data now shown). Fold increases of IRF-1 protein over the vehicle-treated level are shown (n = 3). atRA-treated cells had significantly higher levels of IRF-1 protein as determined by one-way ANOVA (*). B, Cells were treated with vehicle or atRA (0.1 µM), cultured for 2-16 h, and then lysed and assayed by Western blot. Fold increases of IRF-1 protein compared to the untreated level at time 0 h are shown (n = 3). Two-way ANOVA showed atRA and time as significant factors. For all time points, +atRA was significantly different from the vehicle (*). C, Cells were treated with vehicle or atRA (0.1 µM) for 1-12 h and RT-PCR was performed to assess the mRNA levels of IRF-1 and GAPDH. In some experiments, the cells were pretreated with Actinomycin D (AD; 5 µg/ml) 1 h prior to the addition of atRA. After normalization by GAPDH mRNA, fold increases of IRF-1 mRNA compared to that of vehicle-treated cells at 0 h are shown (n = 3). Two-way ANOVA showed treatment and time as significant factors. The asterisks indicate significant differences between +atRA and vehicle groups. D, Ccells were treated with atRA (0.1 µM) overnight (o.n.), followed by a second dose of the same concentration at time 0 h, shown in the figure as “++atRA”. They were then cultured for 2, 4, or 8 h, and lysed and assayed by Western blot for IRF-1 expression. Fold increases of IRF-1 protein compared to the vehicle at time 0 h are shown (n = 4). Two-way ANOVA showed atRA and time as significant factors. For 2, 4, and 8 h, ++atRA was significantly different from the vehicle (*). E, Cells were treated the same as in D. RT-PCR was performed to assess the mRNA levels of IRF-1 and GAPDH. After normalization by GAPDH mRNA, fold increases of IRF-1 mRNA compared to that of vehicle-treated cells at 0 h are shown (n = 3). Two-way ANOVA showed treatment and time as significant factors. The asterisks indicate significant differences between ++atRA and vehicle groups. F, Regression analysis showed significant correlation between fold increases of IRF-1 mRNA and protein, regardless of +atRA or ++atRA.

119

120 IRF-1 is a transcriptional factor with a half-life of less an hour (288). Because the increase of IRF-1 protein in MCF10A cells was transient, with a marked reduction after it peaked at 8 h, and considering that atRA is readily metabolized, we next tested the effect of re-exposing the cells to a second dose of atRA, of the same concentration, 16 h after the first dose. The re-exposure induced a second wave of IRF-1 protein (Figure 24D). At

2 and 8 h after the second dose, the atRA-treated cells had significantly higher levels of

IRF-1 protein as compared to the residual level of the first dose (16 h after application).

Similarly, IRF-1 mRNA was increased after re-exposure (Figure 24E), and the level of mRNA was highly correlated with IRF-1 protein (Figure 24F). Notably however, the second dose of atRA did not further increase the maximal induction of either IRF-1 mRNA or protein as compared to the first dose, indicating that previous exposure to atRA may not “prime” or potentiate the response of IRF-1 to this retinoid.

121 IRF-1 is Induced by the RARα-Selective Ligand, Am580- atRA is a pan agonist of all RARs; thus, we asked which receptor family members may mediate the effect of atRA on IRF-1 induction in MCF10A cells. Am580, a ligand specific for RARα, increased

IRF-1 protein expression (Figure 25), indicating that RARα may mediate the effect of atRA on IRF-1 induction. This was confirmed by experiments using co-treatments between the agonists (atRA or Am580) and an RARα antagonist (Figure 25), where the increase of IRF-1 level was either partially (in the case of atRA) or completely (in the case of Am580) blocked by the antagonist. However, the Am580-mediated induction was relatively smaller than that of atRA. Since the same dosage (0.1 µM) was given, which is higher than the Kd values of atRA and Am580 for RARα [0.2 nM (289) and 36 nM (290), respectively], it is unlikely that the differences in IRF-1 induction are due to different receptor affinities. On the other hand, ligands for RARβ and RARγ also induced IRF-1.

The fold increases of IRF-1 protein were 12.4 ± 1.0 for RARβ and 11.8 ± 1.4 for RARγ agonists, comparable to 15.5 ± 1.5 for Am580 (Figure 25). In addition, 9-cis-RA (9cRA) also induced IRF-1; since additional ligation with RXR did not further increase the effects of RAR agonists (data not shown), 9cRA may act through RARs, but not RXRs.

The negative control, RTMBE, a retinoid analog without known binding activity to retinoid receptors (291), did not significantly increase IRF-1 protein level. In summary, it is likely that atRA increases IRF-1 protein though each of RARα, RARβ, and RARγ.

122

Figure 25 RARα-selective ligand, Am580, induces IRF-1. MCF10A cells were treated with vehicle, atRA (0.1 µM) or Am580 (0.1 µM) overnight (o.n.), followed by a 2nd dose of different retinoids of the same concentration (except for RARα antagonist “6”, which was 5 µM) and further incubation of 6 h. Cells were lysed and assayed by Western blot for IRF-1 protein expression. Fold increases of IRF-1 protein compared to the vehicle at time 0 h are shown (n = 3 or 6). One-way ANOVA was performed and outcome is as following. Compared to vehicle, atRA induced significantly higher level of IRF-1 after o.n. treatment, whereas Am580 did not. When vehicle was the 1st dose (black bars), atRA, atRA/6, Am580, and 9cRA as the 2nd dose induced significantly higher levels of IRF-1 protein. When atRA or Am580 was the 1st dose (darker or lighter gray bars, respectively), re-stimulation with the same retinoid further increased IRF-1 level significantly. The inhibitory effects of 6 were significant in all cases (*), with the percentages of reduction indicated in the figure.

123 atRA Increases Nuclear Location of RARα and RXRα- Since each of the ligands

for RARα (atRA, Am580, and 9cRA) induced IRF-1, we next tested the expression and/or localization of RARα and its dimerization partner, RXRα. The basal expression of

RARα in MCF10A cells is low and mostly within the nucleus. The initial dose of atRA

increased the nuclear intensity of RARα, and the intensity was similar after the cells were

exposed to a second dose of atRA (Figure 26A). Co-incubation of the initial dose of atRA

with RARα antagonist reduced the overall fluorescence; however, RARα still resided in

the nucleus. In contrast, the RARα antagonist blocked the effect of the second dose of

atRA, with RARα showing a diffuse localization over the nucleus and the cytoplasm,

indicating that antagonism may affect the localization of this receptor. Although Am580

slightly increased both nuclear and cytoplasmic RARα, while the antagonist partially

decreased the effect of Am580 on the overall expression of RARα, Am580 did not alter

the relative distribution of RARα between the nucleus and the cytoplasm. Together, these

results indicate that while both atRA and Am580 induce RARα expression, atRA may

have an additional effect on the localization of RARα.

124 RXRα showed a different distribution pattern in untreated cells. RXRα was

localized mostly in the nucleus; however, a cluster of fluorescence is clearly visible

adjacent to the nucleus (Figure 26B). Stimulation of the cells with the first dose of atRA

increased the fluorescence intensity in the cytoplasm, including the cluster close to the

nucleus. Interestingly, nuclear and perinuclear staining of RXRα were enhanced after the

cells were treated with two sequential doses of atRA. The second dose of atRA may have

assisted in the transport of RXRα from the cytoplasmic cluster to the nucleus. The identity of the cluster and the role of atRA on RXRα translocation require further investigation. Nevertheless, atRA regulated the expression and/or localization of RARα and RXRα.

Figure 26 atRA increases nuclear localization of RARα and RXRα. (On the next page) MCF10A cells were treated with one dose (overnight) or two doses (overnight, and then for 3 h) of vehicle, atRA (0.1 µM), or Am580 (0.1 µM). In some experiments, RARα antagonist (“6”; 5 µM) was given with the retinoids (in the case of a single dose, or “+”), or with the second dose (in the case of two doses, or “++”). Cells were fixed, permeabilized, and stained with primary (1°) and secondary (2°) antibodies as described in Experimental Procedures. Representative confocal images from at least ten microscopic fields are shown, with red fluorescence indicating RARα (A) or RXRα (B).

125

126 Re-stimulation with atRA Increases Nuclear Localization of IRF-1- Whereas the

experiments shown in Figure 24 showed that re-stimulation with atRA induces a second

wave of IRF-1 mRNA and protein without elevating the maximal levels, it was unknown

whether atRA alters the localization of IRF-1. As shown in Fig. 27A, the initial dose

increased mostly the cytoplasmic level of IRF-1. In contrast, IRF-1 fluorescence was

concentrated in the nucleus by sequential doses of atRA. Quantification of nuclear IRF-1

fluorescence revealed modest increases with the first dose of atRA at both 4 and 8 h; however, the second dose markedly elevated these levels (Figure 27B). Co-incubation with the RARα antagonist partially decreased the effects of atRA, indicating a role of

RARα in regulating IRF-1 localization. Similar results were obtained using Am580 as the stimulus, although a significant difference between the first and second exposures was observed only at 4 h. Collectively, these results suggest a novel role of atRA in regulating

IRF-1 localization.

127

Figure 27 Re-stimulation with atRA increases nuclear localization of IRF-1. (On the next page) A, MCF10A cells were treated with vehicle or atRA (0.1 µM) overnight, followed by a dose of atRA and incubation for 4 h, shown in the figure as “+atRA” and “++atRA”, respectively. Primary (1°) and secondary (2°) antibodies were used as described in Experimental Procedures and confocal microscopy was performed. Overlays of Alexa Fluor 568 (IRF-1; red) and To-Pro.3 (nuclei; blue) are shown. B, Cells were treated with vehicle, atRA (0.1 µM) or Am580 (0.1 µM) overnight, followed by a 2nd dose of different retinoids of the same concentration (except for RARα antagonist “6”, which was 5 µM) and further incubation of 4 h (upper panel) or 8 h (lower panel). Confocal microscopic images were quantified by the Fluoview software, and averages of IRF-1 fluorescence from a hundred nuclei per treatment were plotted. One way ANOVA was performed. For each time point, the asterisks indicate significant differences compared to the level caused by two doses of vehicle. In addition, four groups of results were color-coded, and different letters of matching colors with the bars indicate significant differences within the group.

128

129 Re-stimulation with atRA Increases Nuclear IRF-1 and DNA-Binding Activity of

IRF-1- atRA-mediated nuclear localization of IRF-1 was confirmed by Western blot of

nuclear protein extracts and EMSA experiments. The initial exposure of MCF10A cells

to atRA increased both IRF-1 nuclear expression and DNA-binding activity; however, a

second dose of atRA further increased both levels (Figure 28A). In addition, the increase

of nuclear IRF-1 was highly correlated with that of IRF-1 DNA-binding activity.

Sequential doses of Am580 functioned similarly to atRA, although the peak of Am580-

induced IRF-1 nuclear expression and DNA-binding activity was later than after atRA,

with higher responses at 8 h (Figure 28A). Unlike atRA that is unstable and readily

degraded in solutions, Am580 is very stable (292) and the lack of its catabolism may contribute to sustained effects of this compound. As expected, the RARα antagonist

blocked the effects of atRA and Am580 on DNA-binding activity of IRF-1 (Figure 28B).

These results agreed very well with those obtained by immunofluorescence staining,

indicating that sequential doses of atRA or Am580 increased IRF-1 protein in the nucleus

and its DNA-binding activity.

130

Figure 28 Re-stimulation with atRA increases nuclear IRF-1 and DNA-binding activity of IRF-1. (On the next page) MCF10A cells were treated with vehicle, atRA (0.1 µM) or Am580 (0.1 µM) overnight, followed by a 2nd dose of different retinoids of the same concentration (except for RARα antagonist “6”, which was 5 µM) and further incubation of 4 or 8 h. A, Western blot of nuclear protein extracts (NPE) and EMSA were performed as described in Experimental Procedures. For EMSA, NPE were incubated with an anti-IRF-1 monoclonal antibody prior to the addition of [γ-32P]ATP-labeled IRF-1 consensus oligonucleotide. Protein- DNA complexes were resolved using a 5% PAGE gel. Only the supershifted bands are shown. B, NPE were incubated with an anti-IRF-1 monoclonal antibody prior to the addition of [γ-32P]ATP-labeled IRF-1/IRF-E consensus oligonucleotide with or without unlabeled competitor DNA (wild-type/WT or mutant/MU; 50× molar excess). Protein- DNA complexes were resolved using a 5% PAGE gel. The arrow indicates complexes supershifted by IRF-1 antibody, with the 4-h supershifted bands exposed longer and shown as well. Original IRF-1/IRF-E-containing complexes are pointed out by a short segment. A nonspecific band is also shown (*).

131

132 Re-stimulation with atRA Increases Transcription of IRF-1 Target Gene OAS-2-

As part of the evidence that retinoid-induced nuclear localization of IRF-1 could be related to increased functions of this transcription factor, we next tested the expression of an IRF-1 target gene, OAS-2. Sequential doses of atRA or Am580 significantly enhanced

OAS-2 mRNA. The effect of Am580 on OAS-2 was completely inhibited by RARα- antagonist, indicating a role of RARα in regulating the transcriptional function of IRF-1.

Since the RARα antagonist partially downregulated the effect of atRA, other factors

(such as RARβ or RARγ) may also confer the ability of atRA to increase IRF-1-mediated gene transactivation.

133

Figure 29 Re-stimulation with atRA increases transcription of OAS-2, an IRF-1 target gene. MCF10A cells were treated with vehicle, atRA (0.1 µM) or Am580 (0.1 µM) overnight, followed by a 2nd dose of different retinoids of the same concentration (except for RARα antagonist “6”, which was 5 µM) and further incubation of 8 h. A, Total RNA was isolated and transcript levels of OAS-2 and GAPDH was measured by RT-PCR. B, Fold increases of OAS-2 mRNA after normalization are shown (n = 3 or 6). One way ANOVA was performed. The asterisks indicate significant differences compared to the level caused by two doses of vehicle. In addition, two groups of results were coded as black or gray, with regular or capitalized letters indicating significant differences, respectively.

134 5. Discussion

IRF-1 is regulated by a number of factors (189). Previously, we showed that atRA

increases IFNγ-induced expression of IRF-1 by affecting multiple components of IFNγ

signaling pathway (285). We next focused on atRA and investigated whether it

independently induces IRF-1. In MCF10A cells, atRA alone increased IRF-1 expression,

consistent with the observations in a few other cell lines (195, 205, 207).

The present study was conducted to study whether atRA, in addition to its role in

inducing IRF-1, modulates IRF-1 localization as one way of regulating transcriptional

activity of IRF-1. IRF-1 has been shown to primarily reside in the cytoplasm in a number of breast cancer cell lines, while IFNγ simulation induces its nuclear localization (293).

How IFNγ exerts such an effect and whether atRA could function similarly are unknown.

In the present study, we found that although an initial exposure of cells to atRA increased

mostly cytoplasmic IRF-1 (Figure 27A), re-stimulation of MCF10A cells with a second dose of atRA increased the nuclear localization of IRF-1 (Figures 27 and 28). The

increase of nuclear level of IRF-1 was apparently due to accumulation in the nucleus

rather than an increase of whole-cell expression, since the second exposure to atRA

induced a similar level of IRF-1 as the first dose (Figure 24). These results of these

experiments suggest a novel role of atRA in nucleocytoplasmic transport of IRF-1.

Nuclear import of proteins is thought to be mediated by the large nuclear pore

complex (NPC) (210). NPC provides passive diffusion channels for proteins smaller than

50-60 kDa but in most cases, even small proteins are imported by an energy-dependent

and receptor-mediated process. The nuclear import receptors recognize the nuclear

localization signal (NLS) present in most nuclear proteins. The export of proteins out of

135 the nucleus, on the other hand, is mediated by recognition of the nuclear export signal

(NES) by export receptors, such as CRM1 (211).

IRF-1 possesses a putative NLS (191), whereas no NES has been identified.

IRF-1 can be serine phosphorylated (192); although a direct correlation between IRF-1 phosphorylation and transcriptional activity is yet to be established. In the present study, we have observed that atRA increases the content of IRF-1 in the nucleus. The mechanism of atRA-mediated regulation of IRF-1 nuclear transport is unclear; however, we hypothesize that atRA may function through the shuttling of retinoid receptors (RAR and RXR). One of them, RXRα, has been shown to shuttle between the nucleus and the cytoplasm ligand-dependently (28). RXRα has been shown to act as a nuclear export

carrier for TR3, directing this orphan receptor to the mitochondria, a process crucial for

the effect of TR3 in inducing apoptosis (28). In our studies that tracked the localization of

RXRα in MCF10A cells, the sequential exposure of cells to atRA induced the

translocation of RXRα from a cytoplasmic cluster to the nucleus (Figure 26B). Thus, we

hypothesize that atRA-mediated relocation of RXRα in MCF10A cells may contribute to

the movement of IRF-1, either through a direct interaction between the two proteins, or

by other more complex processes. Since atRA is solely a ligand for RARs, but not for

RXRα, we speculate that increased nuclear localization of RARα, observed after atRA

treatment (Figure 26A), may assist in nuclear sequestration of RXRα. Furthermore,

nuclear localization of both IRF-1 and RXRα was increased by sequential doses of atRA,

but not by one dose, indicating that previous exposure to atRA may be required. The

mechanism and significance of this effect need further investigation; however, it is

136 hypothesized that the initial dose of atRA may affect the production and/or activation of a yet to be identified transporter, which mediates the nuclear import, export, and/or nuclear retention of IRF-1 when re-exposed to atRA. Nuclear localization of IRF-1, as visualized by confocal microscopy, was confirmed by observations that both nuclear expression and

DNA-binding activity of IRF-1 were increased by sequential treatments with atRA

(Figure 28).

Recently, IRF-5 subcellular localization is reported to be regulated by a CRM1- dependent nuclear export pathway (212). Mutation of the NES of IRF-5 results in nuclear accumulation of this protein, potentially increasing transactivation functions of IRF-5

(294). The importance of nuclear import and sequestration in transcriptional regulation has also been reported. For instance, nuclear localization of IRF-3 appears to be initiated by its NLS (213), which is constitutively active and shuttles IRF-3 between the nucleus and the cytoplasm; upon virus infection, IRF-3 is serine phosphorylated and bound to

CREB-binding protein or p300 that stabilizes nuclear retention of IRF-3. How IRF-1 subcellular localization is regulated requires further investigation.

Increased nuclear localization could potentially enhance the transcriptional functions of IRF-1, inducing genes important for strengthening immune responses and/or promoting cancer prevention. atRA-induced nuclear IRF-1 was transcriptional active, as transcription of IRF-1 target gene OAS-2 was enhanced (Figure 29). OAS-2 is a known

IFN-stimulated gene that promotes RNase L-mediated immune response (138) and tumor suppression (295). The tumor suppressor functions of RNase L can also amplify the apoptotic signals generated by another IRF-1 target gene, tumor necrosis factor-related apoptosis inducing ligand (TRAIL) (296). Thus, atRA-induced IRF-1 nuclear localization

137 and transcriptional activity may be important for the generation of a multi-component

network containing OAS-2, RNase L, TRAIL, and other IRF-1-responsive genes which

function in concert to induce apoptosis of cancer cells. Similar effects were found in the

case of Am580, an atRA analog specific for RARα, suggesting possible implication of

this synthetic, yet more stable, retinoid in therapeutic practices.

In summary, the present study has provided evidence for dual regulatory roles of

atRA on IRF-1 expression and nuclear localization. Using RARα and RXRα as mediators, atRA can induce both IRF-1 mRNA and protein. In addition, sequential treatments of atRA can also increase IRF-1 nuclear localization, DNA-binding activity, and transcription of IRF-1 target gene, OAS-2 (Figure 30). Such effects may not be seen in a chronic exposure model, as IRF-1 expression and nuclear localization may already be

increased by repeated dosing of atRA. However, our sequential study has revealed the

possibility of separate mechanisms of atRA actions on IRF-1. In the regulation of IRF-1

gene expression, atRA facilitates the shuttling of RARα/RXRα heterodimer from the

cytoplasm to the nucleus, leading to yet to be characterized changes that activate the

IRF-1 promoter. Sequential treatments of atRA, on the other hand, increase IRF-1 nuclear

localization by altering the functions of transport proteins responsible for nuclear

translocation and/or sequestration of IRF-1. Together, the dual functions of atRA can lead

to increased DNA-binding activity of IRF-1 and elevated transcription of IRF-1 target

genes.

138

Figure 30 Working model of atRA-mediated regulation of IRF-1 expression and localization. In MCF10A cells, either the first (1°) or second (2°) dose of atRA facilitates the shuttling of RARα and its dimerization partner, RXRα, from the cytoplasm to the nucleus in a ligand-dependent manner (1). This enhances the transactivation activity of RARα/RXRα heterodimer, leading to yet to be characterized changes that activate the IRF-1 promoter. IRF-1 is thus induced (2) and translated (3). Sequential doses (1° + 2°) of atRA increases nuclear translocation of IRF-1, a process possibly assisted by the shuttling of RARα/RXRα (4). Increased nuclear localization and DNA-binding activity of IRF-1 is followed by augmented transcription of IRF-1 target gene, OAS-2 (5).

139 CHAPTER 5 SUPPLEMENTAL RESULTS

1. Abstract

In our studies of A549 cells, we have shown that atRA potentiates IFNγ signaling, thus inducing IRF-1 expression and transcriptional functions. Here, additional results are presented. Consistent with the effect of atRA on IFNγ-induced STAT-1 activation, atRA increased phosphorylation of IFNγ-induced Jak-2, the kinase upstream of STAT-1. In addition, the combination between atRA and IFNγ induced IRF-7 at 72 h, a protein required to be synthesized de novo during antiviral responses. In terms of cell growth and/or apoptosis, the combination decreased the proliferation and number of cells after 3 days of incubation. However, it also decreased the apoptosis of these cells. Interestingly, atRA increased cell survival after 7 days of incubation, suggesting that it may regulate the balance between cell growth and apoptosis. In addition, atRA increased IRF-1 protein induced by two other proinflammatory cytokines, IFNβ and TNFα, indicating that atRA may affect proteins shared in the regulation of inflammatory responses. Preliminary microarray results revealed a number of genes up- or downregulated in the presence of atRA, as compared to IFNγ alone.

In MCF10A cells, we found that atRA by itself induced the expression, nuclear localization, and transcriptional functions of IRF-1 in an RARα-dependent fashion. As supplemental results, we present here that atRA affected the DNA-binding activity of

NF-κB complexes, increasing the ratio of NF-κB activation complex (p65:p50 heterodimer) to repression complex (p50:p50 homodimer) within 30 min of treatment,

140 which may potentially lead to activation of IRF-1 transcription. In contrast, the expression, phosphorylation, or DNA-binding activity of STAT-1 was not affected by atRA. These results suggest that atRA-mediated induction of IRF-1 is RARα- and

NF-κB-dependent, but STAT-1-independent, indicating a distinct role of atRA that parallels interferon signaling to IRF-1. In addition, apoptosis of MCF10A cells was increased by short-time treatments with atRA.

141 2. A549 Cells: Supplemental Results and Discussion

atRA Increases IFNγ-Induced Tyrosine Phosphorylation of Jak-2- Jak kinases are responsible for activation of STAT-1. To investigate the role of Jak kinases in the atRA- mediated increase of STAT-1 activation, we assessed the expression and tyrosine phosphorylation of Jak-2, which has been shown to exert activities in A549 cells (297,

298). The results (Figure 31) showed that 0.5 U/ml of IFNγ increased Jak-2 expression and phosphorylation. Overnight pretreatment with atRA further increased the levels of tyrosine-phosphorylated Jak-2 induced by IFNγ, consistent with observations that atRA increased cell-surface IFNGR-1 and potentiated the effect of IFNγ on STAT-1 tyrosine phosphorylation (Figure 18). Like IFNγ-induced STAT-1 phosphorylation, tyrosine- phosphorylated Jak-2 was not further increased by co-treatment with atRA, especially at

4 and 8 h (Figure 31B). Interestingly, while protein expression of Jak-2 was increased by

IFNγ at 2 and 4 h, pretreatment with atRA prolonged this increase until 8 h (Figure 31A), suggesting a role of atRA in regulating the expression and/or degradation of Jak-2 protein.

Overall, these results suggest that Jak-2 activation may be related to the regulatory effect of atRA on IFNγ-induced STAT-1 tyrosine phosphorylation.

142

Figure 31 atRA increases IFNγ-induced tyrosine phosphorylation of Jak-2. A, A549 cells were pretreated with atRA (0.1 µM) or vehicle overnight and then treated with atRA and 0.5 U/ml of IFNγ for 2, 4, and 8 h. An additional treatment group was also designed that included overnight pretreatment with vehicle and co-treatment of atRA with IFNγ, shown in the figure with triangles (U). Cell lysate was prepared and assayed for levels of Jak-2 and tyrosine-phosphorylated Jak-2 (pY-Jak-2) by using Western blot. B, Fold increases of pY-Jak-2 were enumerated by densitometry (n = 1).

143 atRA and IFNγ Combination Increases de novo Synthesis of IRF-7- During the early stages of an antiviral response, IRF-1 signaling initiated by IFNs induces de novo synthesis of IRF-7, which is activated by virus and positively feeds back to produce more interferons (127). Thus, we examined the effects of atRA and IFNγ on this IRF-1 target gene. The result (Figure 32) showed that low-dose IFNγ by itself did not significantly induce IRF-7 mRNA, whereas the combination between atRA pretreatment and IFNγ significantly increased the transcript level of IRF-7 at 72 h. The increase was relatively modest at this time point; however, once the IFNs/IRF-1/IRF-7 positive feedback loop is triggered by IRF-7 induction, the robust IFN-mediated antiviral responses would be generated in a timely fashion. Thus, pretreatment with atRA may potentiate the effect of low-dose IFNγ to induce de novo synthesis of IRF-7 and thereby encourage a more rapid response against infections.

144

Figure 32 Combination of atRA and IFNγ increases de novo synthesis of IRF-7. A549 cells were pretreated with atRA (0.1 µM) or vehicle overnight, and then stimulated with IFNγ (0.5 U/ml) and another dose of the retinoid. Cells were harvested 72 h after IFNγ stimulation and tested for IRF-7 transcript levels. Fold increases of IRF-7 mRNA compared to that of the vehicle-treated level are shown (n = 4). Student t-test showed significant difference between the combination treatment and IFNγ alone (*).

145 atRA and IFNγ Combination Decreases Cell Proliferation on Day 3- Since both atRA and IFNγ are regulators of cell growth and apoptosis, we examined the viability of

A549 cells in response to atRA and IFNγ treatments. Using MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, which is based on the cleavage of yellow tetrazolium salt MTT into purple formazan by an enzyme of the respiratory chain of the mitochondria, we found that the combination of atRA and IFNγ significantly decreased the number of metabolically active cells on Day 3, whereas either atRA or IFNγ alone was not effective (Figure 33A). Consistently, the combination

treatment significantly decreased the proliferative index (299) calculated after flow

cytometry analysis of CFSE (carboxyfluorescein diacetate, succinimidyl ester)-stained

proliferating cells (Figure 33B). Thus, atRA and IFNγ cooperatively inhibited

proliferation of A549 cells on Day 3.

However, either the combination of atRA and IFNγ, or IFNγ alone, decreased cell

apoptosis, as assessed by Annexin V staining (300) that detects phosphatidylserine on the

outer membrane leaflet of apoptotic cells (Figure 33C). The atRA and IFNγ combination

decreased both apoptosis and cell proliferation, resulting in an overall outcome of

decreased number of cells (Figure 33A). On the other hand, IFNγ alone decreased

apoptosis without changing proliferation, leading to a slight increase of cell viability after

the treatment.

Collectively, these results showed that atRA and IFNγ act cooperatively to

decrease the proliferation of A549 cells. IRF-1, the anti-proliferative protein induced by the combination, may mediate such an effect.

146

Figure 33 Combination of atRA and IFNγ decreases cell proliferation on Day 3. A, A549 cells were plated at a density of 1,500 cells per cm2, and then pretreated and treated with atRA (0.1 µM) and IFNγ (0.5 U/ml) as described before, for 3 days. MTT assay coupled with a standard curve was performed to determine the number of metabolically active cells. Student t-test showed significant difference, as indicated by an asterisk (n = 3). B, CFSE-stained A549 cells were plated at a density of 1,500 cells per cm2 and treated. Flow cytometry analysis was performed and proliferative index was calculated. One-way ANOVA was then performed (n = 4). C, Cells were plated at a density of 60,000 cells per cm2 and treated. Annexin V (AV)-FITC and propidium iodide (PI) staining was performed, followed by flow cytometry analysis. Numbers of AV + PI − cells were plotted and one-way ANOVA was then performed (n = 3).

147 atRA Increases Cell Survival on Day 7- Since the combination of atRA and IFNγ decreased apoptosis of A549 cells on Day 3, we examined whether the survival of these cells could be affected by the treatments. atRA, either by itself or in combination with

IFNγ, significantly increased cell survival, measured by MTT assay, on Day 7 (Figure

34A). Both vehicle and IFNγ treatment resulted in low numbers of metabolically active cells, whereas atRA and the combination of atRA and IFNγ had a lot more cells (Figure

34B), indicating that atRA may have protected the cells from serum deprivation-induced death. IFNγ at a high concentration (500 U/ml) had a small protective effect (data not shown); however, this high dosage may not be physiologically relevant. These results suggest that atRA may affect cell survival in addition to its effect on apoptosis. Whether atRA acts through inducing growth-related genes or simply as regulator of the nutritional status of the cell requires further investigation.

148

Figure 34 atRA increases cell survival on Day 7. A, A549 cells were plated at a density of 1,500 cells per cm2, and then pretreated and treated with atRA (0.1 µM) and IFNγ (0.5 U/ml) as described before, for 7 days. atRA was re-administered every 2 days. MTT assay coupled with a standard curve was performed to determine the number of metabolically active cells. Log10 transformed data were plotted and one-way ANOVA was performed. B, Microscopic images of formazan- containing A549 cells are shown (10× magnification), taken 7 days after the treatments.

149 atRA Increases IRF-1 Protein Induced by IFNβ and TNFα- To determine whether the potentiation effect of atRA was specific for IFNγ signaling, we investigated the IRF-1 response of A549 cells to two other proinflammatory cytokines, IFNβ and TNFα. The results showed that atRA increased IRF-1 protein levels induced by IFNβ, TNFα, or both

(Figure 35), indicating that the potentiation effect of atRA may be general during an inflammatory response. atRA also slightly increased IFNβ-induced IRF-1 mRNA and transcription of IRF-1 target gene, OAS-2. TNFα-induced mRNA levels of IRF-1 and

OAS-2, on the other hand, were not further increased by atRA pretreatment. However,

TNFα itself increased all these levels, and the combination of TNFα and IFNβ was not

different from TNFα alone, suggesting that TNFα may be a more powerful stimulator of

IRF-1 response in these cells. Nevertheless, atRA sensitizes the cells to better respond to

each of IFNγ, IFNβ, and TNFα, increasing IRF-1 proteins induced by these cytokines. It

is possible that atRA affects a common regulator upstream of the signaling pathways

initiated by the cytokines. For instance, atRA may activate an unidentified carrier protein

responsible for the translocation of cytokine receptors from the cytoplasm to the cell

surface. Physiologically, atRA pretreatment may generate an “atRA state” that sensitizes

the cells for enhanced response to proinflammatory cytokines.

150

Figure 35 Effect of atRA on TNFα and IFNβ-induced responses. A549 cells were pretreated with atRA overnight, and then treated with atRA again and TNFα or IFNβ or both for indicated periods of time. IRF-1 mRNA and protein levels, and mRNA level of OAS-2 were assayed.

151 Comparison Between atRA and IFNγ Combination and IFNγ Alone Reveals

Genes that are Differentially Regulated- To identify candidate genes regulated by atRA

and IFNγ in A549 cells, preliminary Affymetrix microarray analysis was performed. The

cells were pretreated with vehicle or atRA (0.1 µM) and then treated with the

combination of atRA and IFNγ (0.5 U/ml) at 3 h, a time point before IRF-1 protein

reached maximal level (Figure 16). Total cellular RNA was isolated from approximately

6 × 105 cells using Qiagen RNeasy Mini Kit. For biotin-labeled target synthesis, reactions

were performed using standard Affymetrix protocols (Affymetrix, Santa Clara, CA).

After the synthesis of double-stranded cDNA and biotin-labeled cRNA, and subsequent fragmentation of cRNA, hybridization reactions of fragmented cRNA on the Affymetrix

GeneChip® Human Genome U133 Set were performed by Dr. Craig Praul at the DNA

Microarray Facility of Penn State University, University Park. The microarray data were

analyzed by Affymetrix GeneChip® Operating Software version 1.1.1.

Out of ~33,000 genes on the Human Genome U133 Set, only a small number of genes were differentially regulated by the combination of atRA and IFNγ, compared to

IFNγ alone. The combination upregulated 20 genes, and downregulated 78 genes, by two fold or greater. As anticipated, the induction of IRF-1 upon atRA and IFNγ combination treatment was higher than the response to IFNγ alone. One of IRF-1 target genes, low molecular weight protein (LMP)-7, was strongly upregulated (15 times greater than IFNγ alone). LMP-7 is a proteasomal subunit involved in the MHC class I presentation pathway (301); thus, the increased transcription of LMP-7 by atRA and IFNγ may assist in MHC-I-mediated presentation of exogenous antigens, such as viral particles. In

152 addition, an atRA-target cytochrome P450 gene, CYP26 (302), was upregulated 6.5-fold.

On the other hand, the housekeeping genes, GAPDH, β-actin, and ISGF-3 (STAT-1), were not changed by either treatments.

Other genes that were differentially regulated by the combination of atRA and

IFNγ, versus IFNγ alone, include RanGTP activating protein-1 (GAP-1) and RanGTP- binding protein (RanBP), which decreased upon the combination treatment. The protein products of these two genes are important for active shuttling of proteins (cargos) into and out of the nucleus. Thus, downregulation of these proteins by the combination of atRA and IFNγ may potentially slow down nuclear export and/or prolong nuclear retention of some proteins, such as IRF-1, whose nuclear localization is important for transactivation of genes involved in antiviral responses or cell apoptosis. In addition, a protein tyrosine phosphatase was downregulated 4-fold, indicating that the presence of atRA may potentially prohibit dephosphorylation of proteins important in IFNγ signaling, such as tyrosine phosphorylated STAT-1. Moreover, the combination of atRA and IFNγ increased transcription of CD14 antigen, which is part of the LPS receptor complex comprised of CD14, TLR-4 and MD-2. Thus, the sensitivity of the cells to bacterial component LPS may be enhanced.

To summarize, the preliminary Affymetrix microarray analysis revealed a number of genes differentially regulated by IFNγ alone and the combination of atRA and IFNγ.

However, the analysis did not provide changes, other than the regulation of IRF-1, which we wanted to follow up.

153 3. MCF10A Cells: Supplemental Results and Discussion

atRA Induces Binding of NF-κB Activation Complex to DNA- Whereas atRA rapidly induced IRF-1 mRNA and protein in MCF10A cells, as shown in Figure 24, it was unclear how atRA functioned to increase IRF-1 transcription. The promoter of IRF-1 possesses multiple NF-κB binding elements (Figure 13), and at least one of them is functional (121). However, current literature has been controversial on whether NF-κB mediates the effect of atRA on IRF-1, with atRA (1 µM) decreasing NF-κB binding to

DNA at 2-24 h after the treatment in myeloid cells (205) or increasing the binding at 1-6 h in squamous carcinoma cells (207). Since the effect of atRA on NF-κB may be cell type-specific, we tested the DNA-binding activity of NF-κB in atRA-treated MCF10A

cells. The result showed that atRA (0.1 µM) treatment for 30 min increased the intensities

of three NF-κB-related complexes (C1, C2, and C3) compared to vehicle; each of them

could be competed by an excessive amount of unlabeled wild-type NF-κB

oligonucleotide (Figure 36A).

154

Figure 36 atRA differentially regulates the binding of NF-κB dimers containing p65 and p50. (On the next page) A, MCF10A cells were treated with vehicle or atRA (0.5 µM) for 30 min and NPE were prepared for EMSA experiments. NPE were incubated with [γ-32P]ATP-labeled NF-κB consensus oligonucleotide with or without unlabeled competitor DNA (WT or MU). Protein-DNA complexes were separated on a 5% PAGE gel and three NF-κB-related complexes (C1, C2, and C3) were identified. B, NPE were incubated with anti-p50 or p65 antibodies prior to the addition of radiolabeled NF-κB consensus oligonucleotide. Protein-DNA complexes were separated on a 6% PAGE gel. Three supershifted NF-κB dimers (p65:p50, p50:p50, and p65:p65) were identified and indicated in the figure. C and D, MCF10A cells were treated with vehicle or atRA (0.5 µM) for 0.5-22 h as indicated in the figure. NPE were prepared and incubated with anti-p50 or p65 antibodies prior to the addition of radiolabeled NF-κB consensus oligonucleotide. Protein-DNA complexes were separated on a 6% PAGE gel. The supershifted NF-κB dimers identified in B were numerated by densitometry. Relative intensities of the dimers compared to the vehicle-treated level at 30 min were plotted (n = 2). C- Ratio of p65:p50 to p50:50. D- p65:p65.

155

156 NF-κB proteins are comprised of homo- or heterodimers of Rel proteins (303). To

determine which dimers were regulated by atRA in MCF10A cells, we used antibodies

against three common Rel proteins in mammalian cells, p50, p65 and c-Rel, in the EMSA

experiments. While anti-c-Rel did not affect any of the complexes (data not shown), C1

was supershifted by each of the p50 and p65 antibodies (Figure 36B). Further, it was

identified that C1 contained at least three dimers, p65:p65, p65:p50, and p50:p50, as

indicated in the figure. On the other hand, C2 and C3 may contain other proteins so that

they were not affected by either p50 or p65 antibody.

Both p65:p65 and p65:p50 dimers act as activators of NF-κB-mediated

transcription. However, since p50 does not possess a transactivation domain, as present in p65, the p50:p50 homodimer functions as a repressor (303). Thus, we quantified the two bands supershifted by the p50 antibody and calculated the ratio of p65:p50 to p50:p50 as an index of transactivation activity. As shown in Figure 36C, atRA increased the ratio at

30 min and 1 h, indicating that an activating NF-κB complex may bind to DNA preferentially. Although the effect of atRA on NF-κB-mediated transactivation was transient, with the ratio back to the control level (vehicle-treated) at 2 h, it may be in time for inducing IRF-1, whose mRNA level started to increase within 1 h of atRA treatment

(Figure 24). The p65:p65 dimer, on the other hand, was not changed by atRA between

0.5 and 2 h (Figure 36D). Whether or not the atRA-regulated NF-κB proteins can bind to the IRF-1 promoter requires further investigation. Interestingly, both the ratio of p65:p50 to p50:p50 and the level of p65:p65 was decreased at 16 and 22 h after atRA treatment, indicating that the repressor functions of NF-κB may dominate at these time points, and/or the DNA-binding activity of NF-κB is downregulated. NF-κB components, along

157 with their inhibitors, IκB proteins, shuttle dynamically between the nucleus and the cytoplasm (304, 305). Future research is necessary to investigate whether atRA exerts a complex, yet interesting, role in the nucleocytoplasmic shuttling of NF-κB.

atRA-Mediated Induction of IRF-1 is STAT-1-Independent- Since the IRF-1 promoter contains a GAS element and that atRA increases IFNγ-induced STAT-1 activation in A549 cells, we examined whether atRA could induce IRF-1 through the functions of STAT-1 in MCF10A cells. Unlike in A549 cells, where atRA and IFNγ synergistically induced IRF-1 protein, these two stimuli acted additively to increase

IRF-1 in MCF10A cells (Figure 37A), suggesting that atRA and IFNγ may affect IRF-1 transcription through different mechanisms. Further, we found that atRA pretreatment did not potentiate IFNγ-induced tyrosine phosphorylation of STAT-1; but rather, it decreased

STAT-1 tyrosine phosphorylation at 2 h, indicating that the increase in IRF-1 protein by the combination treatment of atRA and IFNγ was not due to an increase in STAT-1 activation (Figure 37B). In addition, none of the receptor selective retinoids affected

STAT-1 tyrosine phosphorylation (Figure 37C). Since serine phosphorylation is required for transcriptional functions of STAT-1, we also tested serine-phosphorylated STAT-1.

Serine-727 of STAT-1 is constitutively phosphorylated in MCF10A cells, and neither one dose (Figure 37D) nor sequential doses (Figure 37E) of atRA altered the level of serine phosphorylation. Finally, the DNA-binding activity of STAT-1 to a consensus GAS element was also unaffected after atRA treatment (Figure 37F). These results suggest that atRA-mediated induction of IRF-1 in MCF10A cells is STAT-1-independent.

158

Figure 37 atRA does not affect the expression, phosphorylation, or DNA- binding activity of STAT-1 to induce IRF-1. (On the next page) A, MCF10A cells were pretreated with atRA (0.1 µM) overnight, and then treated with atRA and IFNγ (0.5 U/ml) for 2, 4, and 8 h. Cell lysates were prepared and assayed for IRF-1 protein by Western blot. The membranes were also stained by Ponceau S to assure equal loading of proteins. B, Cells were pretreated with atRA (0.1 µM) overnight, and then treated with atRA and IFNγ (0.5 U/ml) for indicated times. Western blots of STAT-1 and pY-STAT-1 are shown. Positive (IFNα-treated HeLa) and negative (untreated HeLa) control lysates were included to test the specificity of pY-STAT-1 antibody. C, Cells were treated with different retinoids (0.1 µM; except for RARα antagonist, which was 50× in excess) for 6 h. Cell lysates were prepared and assayed for STAT-1 and pY- STAT-1 by Western blot. RTMBE was used as a negative control. RARα antagonist is shown as “6”. D, Cells were treated with vehicle or atRA (0.1 µM), cultured for 2-16 h, and then lysed and assayed by Western blot for pS-STAT-1. E, Cells were pretreated with atRA (0.1 µM) overnight, and then treated with a second dose for 2, 4, and 8 h. Cell lysates were prepared and assayed for pS-STAT-1 by Western blot. F, Cells were treated with vehicle or atRA (0.5 µM) for 30 min and NPE were prepared for EMSA experiments. NPE were incubated with [γ-32P]ATP-labeled GAS/ISRE consensus oligonucleotide with or without unlabeled competitor DNA (WT or MU). Protein-DNA complexes were separated on a 5% PAGE gel and two STAT-1-related complexes (C4 and C5) were identified.

159

160 atRA Induces Death of MCF10A Cells- Since atRA induced IRF-1 in MCF10A

cells, we tested whether atRA could affect the viability of these cells by using Annexin V

(AV) and propidium iodide (PI) staining. Whereas overnight treatment with atRA did not

affect either PI+ (necrotic or late apoptotic) or PI−AV+ (early apoptotic) cells, both one dose and sequential doses of atRA increased percentages of PI+ cells at 8 h after the second dose of atRA (or 24 h after the first dose) compared to the vehicle treatment

(Figure 38). However, two doses of atRA did not induce more death of MCF10A cells than a single dose of atRA. This could be due to the effects of different doses of atRA to induce IRF-1 to similar levels at this time point (Figure 24). Since sequential additions of atRA increased both expression and nuclear localization of IRF-1 (Figure 27), we hypothesize that they are more effective in inducing the death of MCF10A cells than a single dose of atRA, after longer periods of incubation. This hypothesis is supported by the literature that repetitive exposures of MCF10A cells to atRA induce the death of these cells (306). The percentages of early apoptotic (PI−AV+) cells, on the other hand, were

not changed by atRA during the time the experiments were performed.

161

Figure 38 atRA induces the death of MCF10A cells. MCF10A cells were treated with vehicle or atRA (0.1 µM) overnight, followed by a second dose at time 0 h. After they were harvested, the cells were stained with AV-FITC and PI, and then analyzed by flow cytometry. Percentages of PI+ and PI−AV+ cells are shown (n = 3). The asterisk indicates significant differences among the vehicle and atRA treatments; whereas “+atRA” and “++atRA” are not significantly different.

162 CHAPTER 6 DISCUSSION

1. How atRA Regulates IFNγ Signaling

Synergy between retinoids and IFNγ has been observed both in vitro and in vivo.

The molecular mechanism of the synergy, however, is not completely clear. Current literature focuses on STAT-1, a major player in the IFNγ signaling pathway, whose IFNγ- induced phosphorylation or DNA-binding activity could be increased by atRA. In some cases, atRA elevates the expression level of STAT-1 prepared to be stimulated by IFNγ.

Other components of the IFNγ signaling pathway, on the other hand, have not been examined in detail. Our studies of the pathway have revealed an effect of atRA pretreatment on cell-surface expression of IFNGR-1, a possible event upstream of the effect of atRA on IFNγ induced tyrosine phosphorylation of STAT-1. Other molecular events that may affect STAT-1 activation, in addition to IFNGR-1 expression on the cell surface, are also discussed.

1.1. IFNγ Receptors

IFNγ signal transduction is initiated through the binding of IFNγ homodimer to the IFNGRs on the cell surface. Thus, how the expression and/or localization of IFNGRs are regulated can contribute to the functions of IFNγ in the immune system. This is evidenced by the observations that patients carrying IFNGR-1 mutations are more susceptible to mycobacterial infections (307), and that the IFNγ-induced development of

163 CD8+ T cell responses during an acute viral infection is completely abrogated in mice adoptively transferred with IFNGR-deficient T cells (308).

Recent literature has suggested a possible role of IFNGRs in regulating the balance of T helper (Th) responses. A lack of IFNGR-2 expression is found in Th1 cells, which may be related to the effect of IFNγ in inhibiting the proliferation of Th2, but not

Th1 cells (309). In addition, IFNGR-1 and IFNGR-2 colocalize with T-cell receptor

(TCR) in the immunological synapse established between naïve Th0 cells and mature dendritic cells (310), possibly due to the need of proximity between the cytokine receptors (expressed on the surface of Th0 cells) and their ligand, IFNγ (secreted by dendritic and other cells). The colocalization of IFNGRs with TCR is hypothesized to be responsible for the previously characterized TCR signaling-induced transient activation of STAT-5 tyrosine, STAT-1 serine phosphorylation and subsequent downregulation of

IL-4 receptor signaling (311), preferentially driving the Th response toward Th1 differentiation (310). Taken together, IFNGR-1 and IFNGR-2 crosstalk with TCR in the early stages of Th differentiation, suppressing the signaling of IL-4, a Th2 cytokine, and thus contributing to the Th1 lineage commitment. After that, the expression of IFNGR-2 is downregulated in mature Th1 cells, preventing the anti-proliferative effect of IFNγ signaling on these cells. Furthermore, the disappearance of colocalization of IFNGR and

TCR in mature Th cells (310) corresponds nicely with the notion that Th1 cells could escape IFNγ-induced growth arrest and/or apoptosis (309).

In our study, the expression of IFNGR-1 on the surface of A549 cells was found to be upregulated by overnight treatment with atRA (Figure 19). This is particularly interesting in that atRA (or vitamin A), a hypothesized Th2 inducer, may affect the

164 balance between Th1 and Th2 responses through regulating IFNGR-1. If the surface expression of IFNGR-1 on Th1 cells is increased by atRA, the same as on A549 cells, an increase of IFNγ signaling and subsequent inhibition of Th1 cell proliferation may, at least in part, account for the effect of atRA in driving the balance towards Th2 response.

A hypothesized model is illustrated in Figure 39.

An additional role of IFNGR-1 has been proposed by Johnson and colleagues

(312-314) that involves the adaptor function of IFNGR-1 to connect IFNγ, which contains a NLS, and the NLS-less STAT-1. Upon ligation of IFNγ to IFNGR-1 and subsequent recruitment of STAT-1 to the cytoplasmic domain of IFNGR-1, the trimeric complex of IFNγ/IFNGR-1/STAT-1 is endocytosed in caveolae-like microdomains (315), followed by the interaction of the complex with nuclear importer proteins via the NLS of

IFNγ and nuclear translocation of IFNγ, IFNGR-1, and STAT-1 (314). IFNGR-1 is thus a carrier for STAT-1’s nuclear import. Whether or not RA is involved in this process is not yet established.

165 A Th1 Cell IFNγ NK or Th1 Cells TCR/IFNGR-Mediated Survival Transient Downregulation of IL-4 Signaling IFNGR IFNGR

IFNγ TCR

Mature Naïve Dendritic Cell Th0 Cell IFNGR IL-4

Apoptosis

Th2 Cell B Th1 Cell RA

Apoptosis

IFNGR TCR

IFNγ

Mature Naïve ? Dendritic Cell Th0 Cell IFNGR IL-4

Survival

Th2 Cell

Figure 39 RA increases Th2 response by regulating IFNGR-1 on Th1 cells.

166 1.2. STAT-1

STAT-1 is a common regulator in both type I and type II interferon signaling

pathways. Type I interferons stimulate interaction of STAT-1, STAT-2, and p48 (IRF-9)

which as a complex (ISGF-3) binds to ISRE of the target promoter. Type II interferon or

IFNγ activates tyrosine phosphorylation and homo-dimerization of STAT-1 that binds to

GAS. STAT-1-deficient mice show a complete lack of responsiveness to both types of

interferons; however, they respond normally to several other cytokines that induce and

activate STAT-1 in vitro (111), indicating an essential role of STAT-1 basal expression in

interferon signaling pathways.

atRA has been shown to induce STAT-1 expression in many cell lines. In human

breast cancer MCF7 cells, atRA increases STAT-1 proteins and thus tyrosine

phosphorylated STAT-1, which sensitizes these cells for an increased response to interferon stimulation (259, 316). The effect of atRA on STAT-1 is mediated by RARβ, an RAR subtype that is usually downregulated in breast cancer cells, but is inducible by atRA (316). atRA also increases STAT-1 expression and activation in many monocytic cell models, leading to elevated levels of STAT-1-mediated transactivation both in the presence and absence of interferons (257, 258, 260, 261, 317, 318). In fact, a functional

RARE is present in the mouse STAT-1 promoter, which is preferentially responsive to

RARβ/RXRα heterodimer in vivo (319). Both tyrosine phosphorylation (258, 260) and

serine phosphorylation (318) of STAT-1 are upregulated by atRA.

In our study of lung epithelial A549 cells, atRA by itself did not increase either

expression or tyrosine phosphorylation of STAT-1. However, pretreatment with atRA

potentiated the activation of STAT-1 induced by low-dose IFNγ (Figure 18). The lack of

167 STAT-1 phosphorylation after atRA treatment is probably due to an effect of atRA on

preventing dephosphorylation rather than inducing tyrosine phosphorylation. Our

preliminary observation that atRA decreased the transcript level of a protein tyrosine

phosphatase supports this hypothesis. By inducing and/or activating proteins that prevent

dephosphorylation of STAT-1 during the pretreatment, atRA could prolong the activation

signal transduced by IFNγ stimulation.

The PIAS (protein inhibitor of activated STAT) family of proteins is involved in

downregulation of STAT-1 signaling. PIAS-1 interacts with STAT-1 and inhibits the

DNA-binding activity of activated STAT-1 (120). The activity of PIAS-1 depends on the

binding affinity of STAT-1 to its target promoter; if the STAT-1/DNA interaction is

weak, such as in the transcription of IP-10, PIAS strongly inhibits STAT-1 binding to

DNA (320). Possessing an LXXLL motif, PIAS proteins may act as corepressors (321). It is likely that atRA may affect PIAS by RARs or RXRs, which via ligand-binding domains might interact with the LXXLL motif of PIAS, thus relieving the repression on

STAT-1 transactivation. Interactions between RAR/RXR and the motif have been suggested (17); but whether or not the retinoid receptor could bind PIAS is unknown, and may be an interesting angle in investigating the effect of atRA on STAT-1 signaling.

2. How atRA Regulates IRF-1

Downstream of IFNγ-induced Jak-STAT signaling is the production of a

transcription factor, IRF-1. IRF-1 is essential for most, if not all, responses mediated by

IFNγ, evidenced by a complete abolishment of interferon responses to infections in mice

lacking the wild-type IRF-1 gene. The phenomenon in IRF-1-deficient mice parallels that

168 of STAT-1 knockouts, indicating a close relationship between these two transcription

factors. However, IRF-1 could be induced by stimuli that bypass STAT-1 as well. For

instance, both bacterial wall component LPS and proinflammatory cytokine TNFα increase IRF-1 transcription though NF-κB, not STAT-1. In fact, distinct mechanisms of

IRF-1 induction are found depending on the sources of stimuli. One of the stimuli, atRA, may regulate IRF-1 both though common factors (e.g. STAT-1) and in a unique fashion

(e.g. though interactions with RAR/RXR). Our present studies have revealed effects of

atRA in many aspects of IRF-1, including its mRNA and protein levels, localization,

DNA-binding activity, and potential to transcribe IRF-1 target genes. Overall, atRA

regulates the signaling of IRF-1, by which it exerts functions in antiviral immunity and/or prevention of cancer growth.

2.1. IRF-1 Induction

IRF-1 is known to function in regulation of host defense (182) and cell death

(193). atRA activates IRF-1 gene expression in myeloid cells (205, 206). In these cells, atRA did not activate NF-κB or STAT pathways, suggesting that an alternate mechanism

is involved in IRF-1 gene activation. Also, atRA moderately increases IRF-1 gene

expression in human APL cells through the GAS motif of the IRF-1 promoter, whereas a putative RARE was not functional (195). In squamous carcinoma cells, RA induces

IRF-1 via a STAT-1-independent, but NF-κB-dependent pathway (207). In addition, high doses of atRA (1 µM and above) prolong the induction of IRF-1 by acting on both

STAT-1 and NF-κB pathways in cervical squamous carcinoma SiHa cells (208); however, these cells are resistant to lower concentrations of atRA (209). In our studies of MCF10A

169 cells, atRA stimulated a 30-fold induction of IRF-1 protein (Figure 24). Preliminary

investigation of the mechanism revealed an effect of atRA on activation of NF-κB within

30 min of incubation (Figure 36), whereas neither STAT-1 expression nor its tyrosine or

serine phosphorylation was affected (Figure 37). In contrast, A549 cells did not

significantly respond to atRA alone (Figure 16). Thus, although RA-mediated induction

of IRF-1 has been repetitively observed, the molecular mechanism is still not clarified,

which might be dependent upon the responsiveness of different cell lines to the retinoid.

Indeed, a detailed investigation of the response of IRF-1 to atRA in different cell

models could be correlated to (1) expression pattern of retinoid receptors, and/or (2) the cell’s response to atRA-induced cell death and differentiation. In most breast cancer cell lines, for example, RARβ is down-regulated, whereas RARα, RARγ, and RXRs are variably expressed. In addition, altered localization of RXRα in the nucleus is correlated

with the loss of retinoid responsiveness (322). In our studies, MCF10A cells responded to

each of RARα, RARβ, and RARγ agonists (Figure 25 and related text), indicating that

they may be physiologically normal as mammary epithelial cells. Nuclear localization of

both RARα and RXRα were also regulated by atRA. Correspondingly, atRA strongly induces IRF-1 in MCF10A cells (Figure 24) and growth of these cells is inhibited by

retinoids after long periods of incubation (306). In contrast, lung carcinoma A549 cells are RA-resistant (323) and did not respond to RARβ or RARγ agonists to increase IRF-1

expression (Figure 20). These observations suggest the importance of retinoid receptors

in atRA-mediated IRF-1 induction.

To investigate the effect of RAR/RXR on IRF-1 transcription, a logical step is to

look for the presence of RARE in the IRF-1 promoter. Using the Transcription Element

170 Search System (TESS) (http://www.cbil.upenn.edu/tess/), we have found a cluster of three putative RARE half sites in the region between −584 and −550 (Figure 13). These half sites are adjacent to a κB site and an IRF-E; if all of these response elements are functional, RAR/RXR, NF-κB, and IRF-1/IRF-2 may cooperatively activate the IRF-1 promoter. In fact, a multi-component transactivation complex may be present about 420 base pairs downstream of this promoter segment, where a functional GAS/κB/ISRE combined element has been identified. This combined element is proximal to the transcription initiation site, potentially bringing the −584 to −550 region close to the basal transcription machinery by DNA-looping, as observed for spaced double RXREs, which loop the DNA by binding to RXR tetramers (324). This hypothesis is supported be the report that RXR may interact with STAT-1 through VDR (325). Alternatively,

RAR/RXR, NF-κB, and IRF-1/IRF-2 may be involved in the formation of an enhanceosome at the distal segment of the IRF-1 promoter, mimicking the multi- component complex that binds to an enhancer of the IFNβ promoter (326). We have started to clone the promoter of IRF-1; future studies on transactivation activities of different regions of this promoter and the interactions between the promoter and

RAR/RXR, NF-κB, STAT-1, IRFs may help to further explain how IRF-1 transcription is regulated by atRA.

2.2. IRF-1 Localization

Like other proteins, IRF-1 should be translated outside the nucleus; thus, translocation of this protein back into the nucleus may be an important regulatory step of

171 IRF-1’s transcriptional activity. IFNγ induces nuclear localization of IRF-1 (293). We

observed a similar effect of sequential treatments with atRA in MCF10A cells, which increased IRF-1 nuclear levels in addition to the effects on whole-cell expression of

IRF-1 (Figure 27). It is likely that atRA affects IRF-1 nuclear localization through increasing the import, decreasing the export, or retaining the protein within the nucleus.

In addition, although atRA itself did not significantly induce IRF-1 protein or nuclear localization in A549 cells, it potentiated the effects of IFNγ on these processes. atRA pretreatment increased nuclear IRF-1 at 4 and 8 h after IFNγ stimulation, evidenced by both Western blots of nuclear protein extracts and fluorescence microscopic experiments, which was apparently distinct from the effects of IFNγ alone. A detailed discussion of the effect of atRA on IRF-1 nuclear localization in MCF10A cells can be found in Chapter

IV. Here, another possible mechanism of how atRA regulates nucleocytoplasmic transport of IRF-1 is hypothesized.

172 Transport of proteins between the cell nucleus and the cytoplasm is thought to be mediated by the RanGTP gradient (210). The RanGTP level is higher in the nucleus than the cytoplasm, and the gradient is maintained by the cytoplasmic RanGTP activating protein (GAP) that converts the bound GTP to GDP, and the nuclear RanGTP exchange factor (GEF) that regenerates RanGTP (Figure 40). Protein transported by this system, or so-called cargo, are shuttled by the movement of RanGTP associated with importin or exportin proteins. How the RanGTP gradient is initially established is unknown; however, its functionality is required for nucleocytoplasmic shuttling.

Our preliminary microarray results showed that atRA decreased the transcript levels of GAP and another protein closely associated with GAP, RanBP. If the observation can be confirmed by further studies, we may be able to provide evidence that atRA decreases GAP and RanBP in order to interfere with the RanGTP gradient, thus maintaining the proteins already in the nucleus, and/or to prevent the release of cargos exported from the nucleus, a process that requires the functions of GAP and RanBP. By slowing down the active transport of transcription factors (e.g., IRF-1) across the nuclear membrane, atRA may sustain the transactivation functions of these proteins.

173

Cytoplasm Nucleus (low RanGTP) (high RanGTP)

Cargo

Cargo Imp Imp

Import Cargo

Imp Imp RanGTP RanGTP

GAP RanGDP RanGDP GEF

RanGTP RanGTP Cargo Exp Exp Cargo

Export Cargo Cargo Exp Exp

Figure 40 Nucleocytoplasmic shuttling of proteins via the RanGTP system. Imp, importin; Exp, exportin; GAP, RanGTP activating protein; GEF, RanGTP exchange factor. This figure is based on reference (210).

174 2.3. IRF-1 Signaling

Nuclear IRF-1 induced by atRA alone or in combination with IFNγ was

transcriptional active, as evidenced by increased DNA-binding activity of IRF-1 and

elevated transcription of IRF-1 target genes. We found a correlation between nuclear

expression/localization of IRF-1 and its DNA-binding activity (by EMSA experiments),

suggesting that this transcription factor, once within the nucleus, may be always ready for

binding. To date, no evidence has been reported that IRF-1 must be modified (e.g.

phosphorylated) in order to transactivate its target genes. Confocal microscopic images of

MCF10A cells (Figure 27) showed a punctuate distribution of IRF-1 in the nucleus

(excluding nucleoli). We hypothesize that these IRF-1 speckles may coincide with the

sites of new transcription. Further studies are necessary to verify this hypothesis. In

addition, chromatin immunoprecipitation (ChIP) assays, though not included in our study,

may provide direct evidence of IRF-1/DNA interactions within the cell. Nevertheless, our results support the possibility that the increase in IRF-1 binding to DNA is due to that of

IRF-1 nuclear localization, which is facilitated by atRA.

To examine the effect atRA on IRF-1-induced transcription, we tested the

expression of a number of IRF-1 target genes including caspase-1, TRAIL, and OAS-2.

In A549 cells, we observed a strong potentiation of atRA on IFNγ-induced transcription

of caspase-1 and TRAIL (Figure 22). Caspase-1 is an enzyme that processes IL-1β and

IL-18 to their active forms, which in turn increase the production of IFNγ and positively

feedback IFNγ signaling. TRAIL, on the other hand, is a death ligand that induces

apoptosis of cancer cells, but not normal cells. Interestingly, we found an effect of atRA on IFNγ-induced TRAIL in lung carcinoma A549 cells; however, the non-tumorigenic

175 mammary epithelial cells MCF10A did not respond to atRA with increased expression of

TRAIL (data not shown). This could be due to the specificity of TRAIL to induce

apoptosis selectively in cancer cells. However, the elevation of TRAIL did not correlate

with the increase of IRF-1 protein – for example, atRA strongly induced IRF-1 in

MCF10A cells, but did not affect TRAIL – suggesting that other factors may contribute

to the selectivity of TRAIL-induced apoptosis.

OAS-2 was modestly increased by atRA treatments in MCF10A cells (Figure 29).

OAS-2 is a known IFN-stimulated gene that promotes RNase L-mediated antiviral

response (138) and tumor suppression (295). RNase L is an endoribonuclease that cleaves

both viral and cellular RNA species, leading to viral clearance and cell apoptosis,

respectively. It also amplifies apoptotic signals generated by TRAIL (296). Collectively,

atRA-mediated nuclear localization and possibly enhanced transcriptional activity of

IRF-1 may be important for establishing a multi-component network containing at least

OAS-2, RNase L and TRAIL, which mediate orchestrally the effects of atRA and IRF-1 on antiviral responses and/or apoptosis of cancer cells. It is not clear why atRA, though induced high levels of IRF-1, did not strongly affect OAS-2 transcription. It is possible that additional signal(s) are required for IRF-1-induced transactivation.

In summary, atRA appears to have multiple regulatory roles on IRF-1, including its transcription (expression) and localization. A working model of how atRA affects

IRF-1 is shown in Figure 41.

176

IFNγ

(1) IFNγ atRA + RAR/RXR signaling

(3) (2)

IRF-1 mRNA IRF-1 protein Nuclear IRF-1

IRF-1-induced transactivation

Figure 41 Working model of how atRA regulates IRF-1. Through functions of RAR/RXR, atRA potentiates IFNγ-induced IRF-1 transcription by regulating the IFNγ signaling pathway (1). atRA also affects IRF-1 transcription in the absence of IFNγ (2). After transcription and translation, IRF-1 protein localizes to the nucleus, a process facilitated by atRA (3). Nuclear localization of IRF-1 further leads to transcription of IRF-1 target genes.

177 3. Roles of RARα

Retinoids function mostly through ligation to retinoid receptors. atRA is pan-RAR agonist; thus, we used receptor-selective retinoids in our studies to identify the receptor subtype mediating the effects of atRA. Although RARβ- or RARγ-agonist may be functional in some cases (e.g. IRF-1 induction in MCF10A cells), we have repeatedly observed that RARα-specific agonist, Am580, to act similarly to atRA and that the

RARα-antagonist could at least partially inhibit the effects of atRA or Am580. Therefore, it would be interesting to discuss the possible roles of RARα in mediating the effects of atRA on IRF-1.

3.1. The Connection between atRA and RARα

Crystal structure of atRA binding to the ligand-binding domain of RARα has confirmed the close connection between the two molecules (327). However, it is still controversial whether atRA binds to RARα in the cytoplasm and thus inducing nuclear translocation of the receptor, or it binds RARα that is already present on the promoter of the target gene. Although not providing direct evidences, our results support the first statement. In both A549 and MCF10A cells, atRA induced nuclear levels of RARα, either by increasing the overall expression level or by inducing nuclear translocation.

Am580 induced a small but obvious increase of nuclear RARα (Figure 26), confirming the hypothesis that retinoid-receptor ligation facilitates nuclear translocation of the complex. The RARα antagonist partially blocked Am580-mediated increase of nuclear

RARα, indicating that antagonism may induce a conformation of RARα not favoring

178 nuclear translocation. On the other hand, atRA increased both expression and nuclear localization of RARα. A single treatment of atRA (0.1 µM) for 3 h induced whole-cell

RARα markedly (Figure 26), although the RARα promoter does not possess a functional

RARE. atRA might increase RARα protein expression by inhibiting its degradation

through the ubiquitin proteasome pathway (328). Although conflicting results have been

reported on whether atRA induces or prevents degradation of RARα, it is possible that

the effects of atRA on RARα are cell type-specific; in MCF10A cells, atRA may

primarily prevent RARα degradation, thereby increasing the whole-cell level of RARα.

In addition, atRA may also affect nuclear translocation of the receptor though ligation.

This is supported by the use of RARα antagonist, which inhibited not only the overall

expression of RARα increased by atRA, but also nuclear localization of RARα induced

by sequential doses of atRA (Figure 26). In cells treated with “++atRA/6”, RARα

appeared to be equally distributed between the nucleus and cytoplasm. Together, these

results suggest that atRA and Am580 may increase nuclear levels of RARα, possibly

through enhancing translocation of the receptor into the nucleus.

The shuttling of RARα across the nuclear membrane may be facilitated by

dimerization with RXRα. In A549 cells, nuclear RXRα was found to be elevated almost

the same folds as RARα. In fact, RXRα may serve as a carrier protein for many of its

dimerization partners, including RARα, VDR, and TR3. We found RXRα to localize to a

perinuclear cluster in untreated MCF10A cells (Figure 26). A signal dose of atRA may

have induced the brightness of the cluster (possibly the Golgi apparatus) and cytoplasmic

expression of RXRα, but it did not increase the nuclear level, confirming that atRA is not

179 a natural ligand for RXRα. In contrast, sequential exposures to atRA induced significant

increase of nuclear RXRα, accompanied by disappearance of the perinuclear cluster.

Although the pattern of atRA-regulated RXRα localization is distinct from that of RARα,

RXRα may facilitate nuclear translocation of RARα, or sequestrate RARα within the

nucleus, at least after sequential treatments with atRA.

3.2. Roles of RARα in atRA-Mediated Regulation of IRF-1

As mentioned earlier, RARα may be related to all known functions of atRA on

IRF-1. First, RARα ligands atRA, 9cRA, and Am580 sequentially increased cell-surface

IFNGR-1, STAT-1 tyrosine phosphorylation, and IRF-1 induction (Figure 20). The effects of atRA and Am580 on STAT-1 and IRF-1 were completely inhibited by incubation with an RARα antagonist, indicating an important role of RARα in these

processes. How RARα mediates the effects of atRA on IFNγ signaling is unknown.

However, since pretreatment with atRA was required for increased response to IFNγ, we

hypothesize that atRA and RARα may assist in the production and/or activation of

proteins regulating the IFNγ signaling pathway. For instance, liganded RARα may be required for the functions of a protein transporter that translocates IFNGR-1 to the cell membrane. In addition, RARα may be able to suppress the transcription of a protein

tyrosine phosphatase, as seen in our preliminary microarray results, so that STAT-1

dephosphorylation could be prevented. The dependence of IRF-1 induction on RARα is

probably due to the effect of this receptor on STAT-1 tyrosine phosphorylation.

180 Second, RARα ligands directly induced IRF-1 protein levels in MCF10A cells

(Figure 25). RARα antagonist blocked the effect of either one or two doses of Am580, suggesting a direct association between RARα and IRF-1 transcription. Although a classical RARE (DR-5) is not present in the IRF-1 5’ promoter region, we hypothesize that RARα may interact with the promoter as an enhancer of transcription. An indirect interaction between RARα and DNA is also possible. In this case, RARα may be involved in a multi-component complex containing transcription factors known to bind the promoter of IRF-1, such as STAT-1 and NF-κB.

Third, RARα ligands increased nuclear localization of IRF-1 (Figure 27).

Although the effect of Am580 on nuclear IRF-1 was weak compared to atRA and that combination with the RARα antagonist only partially decreased the IRF-1 fluorescence within the nucleus, the involvement of RARα in the regulation of IRF-1 localization was significant. The shuttling of RARα between the nucleus and cytoplasm may facilitate the trafficking of IRF-1 into the nucleus. On the other hand, RXRα is translocated from the perinuclear cluster to the nucleus after sequential treatments with atRA. The double treatment increased significantly higher levels of nuclear IRF-1 than a single dose of atRA, coinciding with the response of RXRα to the treatments. Thus, we hypothesize that

RXRα may function as a primary chaperone of IRF-1 nuclear translocation; whereas

RARα may help to sequestrate IRF-1 within the nucleus after it is translocated.

181 3.3. Am580 as a Therapeutic Agent

When investigating the effects of atRA and Am580 on IRF-1 localization and

DNA-binding activity (Figure 28), we observed that these two retinoids had different

kinetics of regulation on IRF-1. Compared to atRA which had a greater effect at 4 h,

Am580 modestly increased nuclear localization and DNA-binding activity of IRF-1 at

this time point; whereas at 8 h, the effect of Am580 is greater. This could be due to the

difference between the stabilities of atRA and Am580. atRA is light sensitive, unstable in

solution, and readily catabolized by cytochrome P450 enzymes; whereas Am580 is a

stable synthetic retinoid. Thus, while atRA may be deprived after a period of incubation

with the cells, the effect of Am580 can be persistent, thus making it a potential

therapeutic agent in treatments that require continuous supplies of RAR-α agonizing

retinoids. The stability of Am580 may not help to lower the dosage of the retinoid need

for the treatment, however, because it has a higher Kd value than atRA. Thus, teratogenic effects exist for both atRA and Am580 at high doses.

4. Physiological Differences between Two Cell Lines

In our study of the effects of atRA on IRF-1, we have used two cell lines – the

human lung epithelial carcinoma cell line A549, and the human mammary epithelial cell

line MCF10A. These two cell lines are physiologically different, and they respond to

atRA and IFNγ differently.

The A549 cell line was initiated in 1973 through explant culture of lung

carcinoma tissue from a 58-year-old Caucasian male (329). These cells have properties of type II alveolar epithelial cells, including the ability to synthesize surfactant (330). The

182 MCF10A cell line, on the other hand, is a non-tumorigenic epithelial cell line produced

by long term culture in serum free medium with low Ca2+ concentration (286). MCF10A cells were derived from adherent cells in the population. They exhibit three-dimensional growth in collagen, and show no signs of terminal differentiation.

Morphologically, the two cell lines are similar. Both of them are adherent and their growth can be contact inhibited. However, MCF10A cells require supplementation of the growth medium with insulin, epidermal growth factor, cholera toxin, and

hydrocortisone, indicating that they are non-tumorigenic and need additional growth

factors to survive in culture. A549 cells, on the other hand, are carcinoma cells. Thus, the

degrees of transformation of these two cell lines are different, possibly leading to distinct

patterns of RAR/RXR expression and regulation. MCF10A, like normal epithelial cells,

could respond to ligands for all types of RARs; whereas A549 cells were sensitive to

RARα only in most cases. The lack of RARβ and RARγ functions in A549 cells may

contribute to the minimal induction of IRF-1 stimulated by atRA alone.

In addition, since the cell lines are derived from different human tissues, their

response to atRA and IFNγ could be different. For instance, the lung A549 cells came

from alveolar epithelium that may have been constantly exposed to respiratory pathogens

and thereby, interferons; so that their IFNγ system (e.g. translocation of IFNGR-1 to the

cell surface) is active. Although the level of IFNGR-1 on the surface of MCF10A cells

was not tested, we speculate that it is not regulated by atRA, so that the synergy between

atRA and IFNγ on IRF-1 was not observed. However, the mammary MCF10A cells

contain an assortment of functional retinoid receptors, which may mediate the strong

effect of atRA itself on IRF-1. In addition, different redox conditions in the lung and

183 mammary tissues may contribute to the differences between A549 and MCF10A cells in

their responses to atRA treatment, as hypoxia has been shown to interact with IRF-1

induction pathways (331).

Nevertheless, the discrete responsiveness of these two cell lines to atRA has

helped us to identify multiple roles of atRA in the regulation of IRF-1 expression,

localization, and transcriptional activity.

5. Implications

We have used atRA at a concentration of 0.1 µM (or 100 pmol/mL) in our studies

of A549 and MCF10A cells. This concentration is likely achievable in vivo, as it is

comparable to physiological levels of atRA. atRA is present in the plasma and tissues at

concentrations of 4~14 pmol/mL and 40~580 pmol/g, respectively (332). Following a 45 mg/m2 oral dose of atRA, a pharmacological dose usually used in the treatment of APL,

peak plasma concentrations on the first day of treatment range from 0.1 to 8 µM, with a

median peak concentration of approximately 1 µM (333). Thus, the concentration of atRA that we have used is far below the plasma level induced pharmacologically.

Maintaining an optimal level of vitamin A in the body, as achieved by vitamin A supplementation in populations having vitamin A deficiency as a public health problem, is the key for improving the immune response against infections and/or preventing cancer progression.

Our study has also provided part of an explanation how vitamin A could decrease the severity of the disease after virus infections. During early stages of an infection, IFNγ

184 is usually low; whereas the antiviral functions of IFNγ are required for facilitated viral

clearance and faster recovery from the infection. atRA pretreatment, or an optimal level

of vitamin A in the body beforehand, enhances the response of immune cells to low

levels of IFNγ during early infection. The enhanced signaling of low-dose IFNγ leads to

increased production of casapse-1, an enzyme that process IL-1β and IL-18 in order to

stimulate robust production of IFNγ. Thus, a positive feedback loop is established,

generating a global antiviral state. atRA pretreatment may initiate an earlier onset of the

antiviral state, thus facilitating virus clearance and reducing the severity of the infection.

Finally, we hypothesize IRF-1 as a common mediator of atRA functions. By

increasing the expression and/or transcriptional activity of IRF-1, atRA (or vitamin A)

could be potentially beneficial for both antiviral immunity and inducing cell death and

differentiation in cancer cells.

6. Future Directions

As shown by our laboratory and others, atRA regulates IRF-1 either in the

presence or absence of IFNγ in vitro. However, despite a study by DeCicco et al. (253)

that showed an increase of IRF-1 in rats treated with atRA and PIC, an interferon and

cytokine inducer, the relationship between atRA and IRF-1 is not yet established in vivo.

It would be interesting to investigate in animal models the regulation of IRF-1 by atRA and whether IRF-1, the same as in vitro, could mediate the effects of vitamin A on immune responses, such as the Th1-Th2 balance and antiviral immunity. It would be also

185 interesting to examine the involvement of tumor models (e.g. lung and breast cancers) that are responsive to retinoid treatments.

Also, to further investigate the molecular mechanisms of atRA-mediated regulation of IRF-1 transcription, the promoter of IRF-1 could be dissected to identify the segments regulated by atRA. The three RARE half sites are possible candidates of atRA targets. Other transcription factors, such as NF-κB and STAT-1, may act cooperatively with RAR/RXR on IRF-1 promoter; thus, the interactions among these molecules would be also interesting to study. In addition, it is still unclear how atRA regulates nuclear translocation of IRF-1 in the molecular level. Investigations of NPC, importins/exportins, and the RanGTP system would help to clarify the mechanism(s).

To test our working hypothesis that IRF-1 is a common mediator of vitamin A functions, antisense knockdown of IRF-1 in vitro and/or site-specific knockout of the gene in vivo would be interesting experiments to perform. If IRF-1 is essential for the functional processes stimulated by vitamin A, the absence of this gene could potentially downregulate the effects of vitamin A. Likewise, it would be also interesting to target

RARα, and investigate if the presence of this receptor is required for the expression and functionality of IRF-1.

Finally, it would be interesting to test if other micronutrients, which may also improve the immune system and/or prevent cancer progression, regulate IRF-1. It has been shown that Hippophae rhamnoides (a rich source of vitamins A, C, and E, and microelements such as sulfur, selenium, zinc, and copper) increases the DNA-binding activity of IRF-1 (334). It is possible that the antioxidant properties of these nutrients contribute to increased functions of IRF-1 and thus chemoprevention.

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216 Vita

XIN M. LUO

EDUCATION Ph.D., Integrative Biosciences (Nutritional Biochemistry) May 2006 The Pennsylvania State University, University Park, PA M.S., Nutritional Sciences Aug 2001 The Pennsylvania State University, University Park, PA B.S., Biochemistry and Molecular Biology Jul 1998 Beijing University, Beijing, China RESEARCH EXPERIENCE Graduate Research Assistant Aug 2001 – Dec 2005 Graduate Program in Integrative Biosciences & Department of Nutritional Sciences The Pennsylvania State University, University Park Graduate Research Assistant Aug 1999 – Aug 2001 Department of Nutritional Sciences, The Pennsylvania State University, University Park Undergraduate Research Assistant Sept 1997 – Jul 1998 Department of Biochemistry and Molecular Biology, Beijing University, Beijing, China RESEARCH PUBLICATIONS Luo XM, Ross AC. Dual regulatory actions of retinoids in IRF-1 gene expression and nuclear localization. (manuscript in preparation) Luo XM, Ross AC. Physiological and receptor-selective retinoids modulate interferonγ signaling by increasing the expression, nuclear localization and signaling of interferon regulatory factor-1. Journal of Biological Chemistry (in press) Luo XM, Fosmire GJ, Leach RM Jr. (2002) Chicken keel cartilage as a source of chondroitin sulfate. Poultry Science 81(7):1086-1089. TEACHING EXPERIENCE Instructor Jun 2005 – Aug 2005 Nutrition 100 (Contemporary Nutrition Concerns) Instructor Jan 2003 – May 2003 BMB 212 (Introduction to Biochemistry Laboratory) Teaching Assistant Aug 2002 – Dec 2002 BIOL 472 (Animal Physiology) AWARDS Life Science Consortium Fellowship 2001 – 2005 Integrative Biosciences Scholarship 2001 Young Eagle Scholarship 1998 Advanced Science Honor Program Fellowship 1994 – 1998 PROFESSIONAL MEMBERSHIP American Society for Nutritional Sciences (ASNS) American Association of Immunologists (AAI)