Apocarotenoids Modulate Retinoid Receptors

Home , Apocarotenal, Apocarotenoid, Tetraterpene, Violaxanthin

APOCAROTENOIDS MODULATE RETINOID RECEPTORS

Dissertation

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Abdulkerim EROĞLU

The Ohio State Biochemistry Program

The Ohio State University

2012

Dissertation Committee:

Earl H. HARRISON, Ph.D., Advisor

Robert W. CURLEY, Ph.D.

Ouliana ZIOUZENKOVA, Ph.D.

Jeanette W. MARKETON, Ph.D.

Abdulkerim EROĞLU

2012

Abstract

β-carotene (BC) is the major dietary source of provitamin A. Central cleavage of BC catalyzed by β-carotene oxygenase 1 yields two molecules of retinaldehyde. Subsequent oxidation produces all-trans-retinoic acid (ATRA) which functions as a ligand for a family of nuclear transcription factors, the retinoic acid receptors (RARs). Eccentric cleavage of BC at non-central double bonds is catalyzed by other enzymes and can also occur non-enzymatically. The products of these reactions are β-apocarotenals and β- apocarotenones, whose biological functions in mammals are unknown. We used reporter gene assays to show that none of the β-apocarotenoids significantly activated RARs and

RXRα. β-Apo-13-carotenone was found to antagonize the activation of RXRα by 9-cis- retinoic acid and was effective at concentrations as low as 1 nM. Molecular modeling studies revealed that β-apo-13-carotenone makes molecular interactions like an antagonist of RXRα. The results suggest a possible function of BACs on RXRα signaling. Moreover, β-apo-14’-carotenal, β-apo-14’-carotenoic acid, and β-apo-13- carotenone antagonized ATRA-induced transactivation of RARs. Competitive radioligand binding assays demonstrated that these putative RAR antagonists compete directly with retinoic acid for high affinity binding to purified receptors. Molecular modeling studies confirmed that β-apo-13-carotenone can directly interact with the ligand binding site of the retinoid receptors. β-Apo-13-carotenone and the β-apo-14’- carotenoids inhibited ATRA-induced expression of retinoid responsive genes in Hep G2 ii cells. Finally, we developed an LC/MS method and found 3-5 nM β-apo-13-carotenone was present in human plasma. These findings suggest that β-apocarotenoids function as naturally-occurring retinoid antagonists. We have also tested apo-lycopenoids that have a structural resemblance to β-apo-13-carotenone to see if they exert a similar action as β- apo-13-carotenone in modulating retinoid receptor activation. We found that apo-13- lycopenone was able to block ATRA induced expression of RARβ and CYP26A1 like the action of β-apo-13-carotenone. This suggests that the ionone ring may not be prerequisite for β-apo-13-carotenone’s binding to RARs. The antagonism of retinoid signaling by these metabolites may have implications for the activities of dietary β- carotene as a provitamin A and as a modulator of risk for cardiovascular disease and cancer.

iii

Dedication

This document is dedicated to my parents and my wife.

Acknowledgements

I am greatly indebted to people who have helped me throughout my graduate studies. It is almost impossible to individually thank everyone who provided assistance or encouragement during my studies. I want to give my sincere thanks to all the people who are not mentioned here. I am eternally grateful for my advisor, Dr. Earl Howard Harrison, who generously gave me the opportunity to study in his laboratory and supported me through out my Ph.D. I appreciate his patience and guidance in teaching me directly en route to becoming an independent scientist and in correcting my poor English writing. Dr. Robert W. Curley, Jr. helped me a lot to learn analytical chemistry techniques and forge my ideas and to test them in his laboratory. I especially enjoyed many hours I spent working with him and his crew. Both Harrison lab and Curley lab are such great places to work and I am indebted to all the members of these labs, past and present. I have benefited from the advice of many of them. I thank Dr. Jian Sun, Mrs. Shiva Raghuvanshi, Dr. Matt Feshman, Mr. Carlo Sena and other members of our lab for their assistance. I also thank Dr. Curley lab members, Dr. Suresh Narayanasamy and Damian Hruszkewycz who contributed significantly to the analytical experiments in synthesis of β-apocarotenoids and apo-lycopenoids. Without their work, the studies that presented here would not be complete. I also thank Dr. Steven J. Schwartz and his lab members for their contribution to chapter 3. They analyzed the existence of β-apo-13-carotenone in human plasma samples. I am sincerely grateful to Dr. Ouliana Ziouzenkova and Dr. Jeanette Webster Marketon for their invaluable suggestions and guidance.

I am grateful to this country for opening its doors to me as a visiting research scholar back in 2005. I am eternally grateful to my parents Kadir and Kezzi Eroğlu for whom none of this would be possible and to my sister Yasemin Eroğlu and my brother Suleyman Eroğlu who are friends as well as siblings. I am also so grateful for my wife Arife Eroğlu for cheering me up and for her devoted support, love, inspiration and wise counsel.

Vita

2005……………B.S. Molecular Biology and Genetics, Istanbul Halic University

2009……………Virginia Vivian Research Award

2010……………American Society for Biochemistry and Molecular Biology (ASBMB)

Graduate/Postdoctoral Travel Award to Attend Experimental Biology

Meeting 2010 in Conjunction with ASBMB Annual Meeting

2011……………American Society for Nutrition (ASN) Travel Stipend to Attend

New Developments in Carotenoid Research an International Conference

2011……………Poster Competition Winner (1st place) at New Developments in

Carotenoid Research an International Conference

2011…………....Outstanding Poster Award at the 2011 Carotenoids Research Interaction

Group (CARIG) Meeting

2007-2012...... Graduate Research Associate, Department of Human Nutrition,

The Ohio State University

Publications

1. Eroglu, A., Hruszkewycz, D.P., Curley, R.W. & Harrison, E.H. The eccentric cleavage product of β-carotene, β-apo-13-carotenone, functions as an antagonist of RXRα. Archives of Biochemistry and Biophysics 504, 11-16 (2010).

2. Eroglu, A. et al. Naturally-occurring eccentric cleavage products of provitamin A carotene β-carotene function as antagonists of retinoic acid receptors. Journal of Biological Chemistry 287, 15886-15995 (2012).

Fields of Study

Major Field: Biochemistry vii

Table of Contents

Abstract...... ii

Dedication...... iv

Acknowledgement ...... v

Vita...... vii

Table of Contents………………………………………………………………………..viii

List of Abbreviations ...... xi

List of Tables ...... xiv

List of Figures...... xv

Chapter 1: Literature review

1.1 Introduction...... 1

1.2 Absorption, Metabolism, and Transport of β-carotene and Vitamin A…………….....2

1.3 Cleavage and Oxidation of Carotenoids to Apocarotenoids…………………………..8

1.4 Occurrence and Functions of Apo-lycopenoids and β-Apocarotenoids, in Foods, and in Mammalian Tissues and Plasma………………………………………………………12

1.5 Retinoic Acid Receptors and Retinoid X Receptors...... 24

1.6 Figures...... 30

viii

Chapter 2: The eccentric cleavage product of β-carotene, β-apo-13-carotenone, functions as an antagonist of RXRα

Abstract...... 37

2.1 Introduction ...... 37

2.2 Materials and Methods...... 39

2.3 Results and Discussion ...... 44

2.4 Implications...... 48

2.5 Figures and Tables ...... 49

Chapter 3: Naturally-occurring eccentric cleavage products of provitamin A carotene β- carotene function as antagonists of retinoic acid receptors

Abstract...... 56

3.1 Introduction ...... 57

3.2 Experimental Procedures ...... 58

3.3 Results and Discussion ...... 66

3.4 Conclusions and Implications...... 72

3.5 Figures...... 74

Chapter 4: Apo-lycopenoids modulate transcription of RARE-mediated genes

Abstract...... 88

4.1 Introduction ...... 89

4.2 Hypothesis ...... 91

4.3 Materials and Methods...... 91

4.4 Results and Discussion ...... 93

4.5 Figures...... 95

Bibliography ...... 102

List of Abbreviations

β-carotene ...... BC

Scavenger receptor class B, type 1 ...... SRBI

Cluster determinant 36...... CD36

Niemann-Pick C1-Like 1 protein ...... NPC1L1

Lecithin:retinol acyltransferase...... LRAT

Acyl:CoA retinol acyltransferase...... ARAT

Cellular retinol-binding protein 1 ...... CRBPI

Cellular retinol-binding protein 2 ...... CRBPII

Cellular retinoic acid binding protein ...... CRABP

Very low density lipoprotein ...... VLDL

Low density lipoprotein ...... LDL

Retinol binding protein ...... RBP

Transthyretin...... TTR

Retinal pigment epithelium……………………………………………………………RPE

Stimulated by retinoic acid gene 6 protein homolog ...... STRA6

β-apocarotenoid ...... BAC

xi all-trans-retinoic acid ...... ATRA

9-cis-retinoic acid…………………………………………………………………9-cis-RA

Alcohol dehydrogenase...... ADH

Retinol dehydrogenase ...... RDH

Retinal dehydrogenases ...... RALDH

Short-chain dehydrogenase/reductase ...... SDR

Dehydrogenase/reductase SDR family member 9 ...... DHRS9

Abscisic acid ...... ABA viviparous 14...... vp14 retinal pigment epithelium protein of 65 kDa ...... RPE65

β-carotene-15,15’-monooxygenase 1 (β-carotene-oxygenase 1) ...... BCO1

β-carotene 9’-10’-dioxygenase (β-carotene-oxygenase 2) ...... BCO2

Retinoic Acid Receptor...... RAR

Retinoid X Receptor ...... RXR

Intestine Specific homeoboX...... ISX

Gap junction communication ...... GJC acyclo-retinoic acid ...... ACR antioxidant response element...... ARE nuclear factor E2-related factor 2 ...... Nrf2

Sirtuin 1 ...... SIRT1

Thin layer chromatography...... TLC

High-performance liquid chromatography ...... HPLC

xii

Liquid chromatography-mass spectrometry ...... LCMS

Alpha-Tocopherol, Beta-Carotene Cancer Prevention ...... ATBC

Beta Carotene and Retinol Efficacy Trial...... CARET human pregnane receptor ...... PXR peroxisome proliferator-activated receptor...... PPAR ligand-independent activation function ...... AF1 ligand-dependent transcriptional activation function ...... AF2

DNA binding domain ...... DBD hormone response element...... HRE retinoic acid response element...... RARE retinoid X response element ...... RXRE ligand-binding domain...... LBD

Direct repeat...... DR

Homeobox...... Hox hepatocyte nuclear factor ...... Hnf steroid receptor coactivator...... SRC histone acetyltransferase ...... HAT nuclear receptor corepressors...... NCoR histone deacetylases...... HDAC silencing mediator of retinoid and thyroid hormone receptor ...... SMRT cytochrome 450, family 26, subfamily a, polypeptide 1...... CYP26A1

xiii

List of Tables

Chapter 2: The eccentric cleavage product of β-carotene, β-apo-13-carotenone, functions as an antagonist of RXRα

2.1 Inhibition of 9-cis-retinoic acid induced transactivation of RXRα by β-apocarotenoids...... 51

xiv

List of Figures

Chapter 1: Literature Review

1.1 Structures of major carotenoids found in human plasma…………………………….30

1.2 General view of the metabolism of natural retinoids and of β-carotene, from their presence in the intestinal lumen to their actions at the target cell level………………… 31

1.3 Retinoid metabolism………………………………………………….……………...32

1.4 Structural and functional domains of RXRα and ligand domain binding (LBD) structure…………………………………………………………………………………..33

1.5 The 1-to-5 Rule...... 34

1.6 The mechanism of nuclear receptor i.e., RXR activation upon ligand binding……...35

Chapter 2: The eccentric cleavage product of β-carotene, β-apo-13-carotenone, functions as an antagonist of RXRα

2.1 The cleavage products of β-carotene………………………………………………...49

2.2 The antagonistic effect of β-apo-13-carotenone on RXRα...... ……………………...52

2.3 Docking of β-apo-13-carotenone with the RXRα tetramer………………………….53

2.4 Docking of β-apo-13-carotenone with the RXRα dimer…………………………….54

Chapter 3: Naturally-occurring eccentric cleavage products of provitamin A carotene β- carotene function as antagonists of retinoic acid receptors

3.1 The cleavage products of β-carotene...... 74

3.2 Chemical synthesis of all possible β-apocarotenoids....……………………………..76

3.3 β-Apocarotenoids do not transactivate retinoic acid receptors……………………....78

3.4 β-Apocarotenoids antagonize ATRA-induced transactivation of retinoic acid receptors………………………………………………………………………………… 80

3.5 β-Apo-13-Carotenone is a potent antagonist of retinoic acid receptor-mediated induction of reporter gene expression and blocks all-trans retinoic acid (ATRA) induction of endogenous gene expression………………………………………………………….82

3.6 β-Apo-13-Carotenone is a high affinity ligand for purified retinoic acid receptors and fits into the ligand binding site…………………………………………………………..84

3.7 β-Apo-13-Carotenone is a high affinity ligand for purified RXRα...... 86

3.8 Analysis of β-apo-13-carotenone in human plasma by HPLC/MS....……………….87

Chapter 4: Apo-lycopenoids may modulate transcription of RARE-mediated genes

4.1 Structures of lycopene….……………………………………………………...…….95

4.2 Suggested metabolic pathway for lycopene cleavage by BCO2....………………… 96

4.3 Apo-lycopenoids that has structural similarity to β-apo-13-carotenone…………….97

4.4 Methods used to synthesize geranic acid and apo-11-lycopenoic acid.……………..98

4.5 Citral, geranic acid, and pseudoionone modulate mRNA levels of RAR-target genes

(RARβ and CYP26A1).…………………………………………………………………99

xvi

4.6 Apo-11-Lycopenals may down-regulate ATRA-induced gene expression of RARβ and CYP26A……………………………………………………………………………100

4.7 Apo-13-Lycopenone blocked ATRA-induced RARβ and CYP26A1 at similar extent as β-apo-13-carotenone…..……………………………………………………………..101

xvii

CHAPTER 1: LITERATURE REVIEW

1.1 INTRODUCTION

Carotenoids are polyisoprenoids and over 700 are found in nature (1-5). Carotenes are synthesized biochemically from eight isoprene (C5H8) units. Oxygenated carotenoids are called xanthophylls and non-oxygenated, hydrocarbon carotenoids, are called carotenes

(See Fig. 1.1). The characteristic feature of the carotenoids is the polyene chain (long conjugated double bond system) that allows them to absorb light between 450-570 nM.

Chlorophyll also absorbs light in this range and carotenoids can serve as accessory pigments enhancing light harvesting in photosynthesis. One of the other functions of carotenoids is their ability to function as antioxidants. Their electron-rich, conjugated double bond makes them reactive against oxidizing free radicals. Some carotenoids can be a source of vitamin A activity i.e., provitamin A carotenoids. In 1930, Moore demonstrated that orally fed carotene was converted into the colorless form of vitamin A in the liver of rats (6). The Swiss organic chemist Karrer elucidated the structures of β- carotene and vitamin A (7).

1 Carotenoids occur naturally in fruits and vegetables and they are synthesized in plants and microorganisms from acetyl-coenzyme A by a series of condensation reactions. The first C40 carotenoid in the biosynthetic pathway is phytoene. Phytoene is dehydrogenated to other acyclic carotenoids including lycopene and ultimately cyclized to carotenes (8).

β-Carotene, α-carotene, β-cryptoxanthin, lycopene, and lutein are the primary carotenoids found in human plasma (See Fig. 1.1). Among them β-carotene, α-carotene, and β-cryptoxanthin are provitamin A carotenoids, the others are non-provitamin A carotenoids; lycopene is the acyclic carotenoid and lutein has two hydroxlated β-ionone rings. In order to exhibit a provitamin A activity, the carotenoid molecule must have at least one unsubstituted β-ionone ring and the correct number and position of methyl groups in the polyene chain (9). Hence, α-carotene, and β-cryptoxanthin show 30 to 50% of provitamin A activity (10,11) and 9-cis and 13-cis isomers of β-carotene less than 10%

(12) of the provitamin A activity of all-trans-β-carotene.

1.2 ABSORPTION, METABOLISM, AND TRANSPORT OF β-CAROTENE AND

VITAMIN A

Provitamin A carotenoids are partly converted to vitamin A (as retinyl esters), in the intestinal mucosa. In the enterocytes, both carotenoids and retinyl esters are incorporated into chylomicrons and secreted into lymph for delivery to the blood (13). Following consumption of carotenoid containing foods, carotenoids are released from their food matrix and they are incorporated into mixed micelles consisting of lipids and bile components (14). The intestinal absorption of carotenoids occurs via passive diffusion or 2 through facilitated transport via scavenger receptor class B, type 1 (SR-B1), and perhaps cluster determinant 36 (CD36) and Niemann-Pick C1-Like 1 protein (NPC1L1) (15-18).

In humans, some of the absorbed β-carotene is cleaved oxidatively by β,β-carotene-

15,15′-monooxygenase 1 (BCMO1) into two molecules of all-trans-retinal (RAL), that can be either further oxidized irreversibly to all-trans-retinoic acid (ATRA) or reduced reversibly to all-trans-retinol (ROL) (14). The intact, absorbed β-carotene molecule can accumulate in humans in the blood and tissues, which also occurs in other mammals such as horses and ferrets (19). In rodents and chickens, it has been reported that almost all of the β-carotene present in foods is converted to vitamin A (20).

After being absorbed, vitamin A (retinol), either preformed or resulting from cleavage of

β-carotene, undergoes esterification with long-chain fatty acids, particularly with palmitic acid (21, 22). The enzymes responsible for esterification are lecithin:retinol acyltransferase (LRAT) (See Fig. 1.2) (23), which utilizes the acyl group at the sn-1 position of phosphatidylcholine as a source of fatty acids, and acyl:CoA retinol acyltransferase (ARAT), which utilizes fatty acyl-CoAs (24, 25). ROL in its free form may cause deleterious effects on cells. To prevent these deleterious effects, retinol binds with high affinity to specific cytoplasmic proteins, the cellular retinol-binding proteins,

CRBPI and CRBPII (26). CRBPII plays primary roles in the regulation of ROL absorption into enterocytes and its intracellular metabolism, also acting on the reaction of retinaldehyde reduction to retinol (27-29). CRBPII bound to ROL directs the RAL more specifically toward microsomal reductase, thus preventing its free access to cytosol reductases (29). 3 Inside the enterocytes, CRBPII-bound ROL undergoes esterification specifically by the enzyme LRAT. However, when present in high intracellular concentrations, vitamin A is esterified by the enzyme ARAT (30). Retinyl esters formed in the intestine are also incorporated into chylomicrons for absorption into the lymph. The carotenoids present in the chylomicron remnants may be converted to retinoids in the liver or be incorporated into very low density lipoproteins (VLDL) and thus be again transported to peripheral cells (31). Low density lipoprotein (LDL) is the major carrier of β-carotene in plasma

(32).

It has been observed that the liver is the major site of β-carotene accumulation in the organism when diets supplemented with this carotenoid are administered to various species such as ferrets, rats, and chickens (20, 33). Liver is also the major site of vitamin

A storage in the animal. Stellate cells are the major cellular site of vitamin A storage in the liver, containing 80 to 90% of total hepatic retinol, which is present in the form of retinyl esters (34). Stellate cells also have large quantities of CRBPs and cellular retinoic acid binding protein (CRABP), as well as enzymes capable of synthesizing (LRAT and

ARAT) and hydrolyzing retinyl ester (35, 36). As also observed in the small intestine, under physiological conditions, ROL bound to CRBP is esterified in the liver preferentially by the enzyme LRAT. When present in high concentrations, ROL can also be esterified by the enzyme ARAT. Thus, the two enzymes may be involved in the esterification of hepatic ROL depending on the concentrations of the latter and whether or not it is bound to CRBP (30, 37). For ROL to be released from stellate cells into the

4 circulation, the stored retinyl esters must first be hydrolyzed. ROL then associates with the plasma retinol binding protein (RBP) and is secreted from the liver.

The retinol-RBP complex (holo RBP) in the blood associates with the thyroxine binding protein, transthyretin (TTR). The retinol:RBP:TTR complex circulates in plasma and delivers vitamin A to peripheral cells.

Free vitamin A can diffuse through membranes yet nearly all vitamin A bound in the retinol:RBP:TTR complex cannot freely pass through cell membranes. Also even some molecules that can diffuse through still need membrane transporters to facilitate their transport. The existence of an RBP receptor that mediates cellular vitamin A uptake was studied comprehensively (38-48). There is a specific cell surface receptor for RBP on the retinal pigment epithelium (RPE) and on intestinal epithelial cells (38-41), the placenta

(42-45), choroid plexus (43, 46), testis (43, 47), and macrophages (48). The cell surface

RBP receptor specifically binds to RBP and mediates vitamin A uptake from vitamin A- loaded RBP (holo-RBP) (40-42, 45, 47, 48).

Recently, stimulated by retinoic acid gene 6 protein homolog (STRA6) was identified as the RBP receptor that is a widely expressed, multitransmembrane domain protein (49). It mediates cellular uptake of vitamin A by removing retinol from the RBP complex and transporting it across the plasma membrane, where it can be metabolized. Additionally,

STRA6 was found to localize to the cellular locations expected of the RBP receptor in tissues (49). 5 1.2.a - Formation and metabolism of all-trans-retinoic acid (ATRA)

After 1987, with the important discovery of the existence of nuclear retinoic acid receptors (50, 51) researchers started to devote more time to studies related to the mechanisms involved in the regulation of the intracellular concentrations of ATRA.

Conversion of ROL to ATRA initially involves the oxidation to retinal and subsequent oxidation of the retinal to ATRA. In this respect, the enzymes alcohol dehydrogenase

(ADH) and retinol dehydrogenase (RDH) have been reported to act in the formation of retinal (52). Retinal dehydrogenases (RALDH) catalyze the oxidation of retinal to retinoic acid. Collectively, all these enzymes including ADHs, RDHs, and RALDHs are involved in the intercorversion of various isomers of ROL, RAL, and ATRA.

ADH, a group of cytosolic enyzmes, catalyzes the oxidation of variety of substrates reversibly including all-trans-retinol and its isomers including 9-cis and 13-cis (53).

While ADH1, ADH2, and ADH4 are involved in oxidation of all-trans and 9-cis-retinol;

ADH7 uses only all-trans-retinol as substrate; they all use NAD as cofactor. In contrast, retinol dehydrogenase is present both in the cytosol and in the microsome fraction, where it catalyzes the oxidation of retinol to retinal (54).

RDH enzymes are microsomal members of short-chain dehydrogenase/reductase (SDR) family. The RDH isoenzyme present in the cytosol has little affinity for the substrate and is inhibited by apo-CRBP. In contrast, the microsomal isoenzyme has high affinity for retinol in its free or CRBP-bound forms, and is not inhibited by the latter. Currently, there are three RDHs that seem physiologically relevant in humans i.e., RDH10, RDH16, and 6 dehydrogenase/reductase SDR family member 9 (DHRS9) involved in converting ROL into RAL (55). A retinal-CRBP complex, then migrates to the cytosol, where the enzyme retinal dehydrogenase is also present and can oxidize it to retinoic acid.

There are at least three retinal dehydrogenases (RALDHs) involved in the catalysis of dehydrogenation of RAL to ATRA. RALDH1, RALDH2, RALDH3 are encoded by

Aldh1a1, Aldh1a2, and Aldh1a3 in humans. They recognize as a substrate the RAL generated by the enzyme retinol dehydrogenase (See Fig. 1.3) (56). Both RALDH1 and

RALDH2 display widespread tissue expression (57-59) and both recognize CRBP1 as a substrate yet RALDH1 is inhibited by apo-CRBP1 (55) but not RALDH2 (60). Another

RALDH identified i.e., RALDH4 recognizes only 9-cis-retinal as substrate (61). More studies needed to be done for understanding the physiological role of RALDH4.

In situations of relative vitamin A deficiency, there is an increase in apo-CRBP concentration (53). This can stimulate the hydrolysis of stored retinyl esters and inhibit

LRAT, but not microsomal retinol dehydrogenase, thus permitting the continuous production of retinoic acid under these conditions (62).

Several isoenzymes of the cytochrome P450 system can also participate in the catabolism of ATRA, producing polar compounds such as 4-OH- and 18-OH-retinoic acids, which will later be excreted by the cell (63-65). CYP26A1 (cytochrome 450, family 26, subfamily a, polypeptide 1) was identified and shown to be essential for the generation of

4-hydoxy-retinoic acid and 4-oxo-retinoic acid, as well as other hydroxylated retinoid 7 derivatives (66, 67). Cells of different tissues such as brain, ovary, testicle, uterus, kidney, and liver contain a cytosol protein capable of binding to retinoic acid (CRABP) whose real function is still unclear (68). This protein mainly has binding affinity for all- trans-retinoic acid but also binds to its 9-cis and 13-cis isomers and to its polar metabolite, 4-hydoxy-retinoic acid (69, 70). Furthermore, another protein with affinity for

ATRA has been reported, the cellular retinoic acid-binding protein II (CRABPII), which has properties similar to those of CRABP. This protein is detected during embryogenesis, being restricted to the skin in adults (71). CRABP-II is responsible for shuttling of ATRA from cytosol to the nucleus. However, CRABP can also direct this retinoid to the cytochrome P450 system. On this basis, it has been suggested that this protein participates in the homeostasis of retinoic acid inside the cell, sequestering it and limiting its distribution and biological effects (68, 72)

1.3 CLEAVAGE & OXIDATION OF CAROTENOIDS TO APOCAROTENOIDS

Apocarotenoids are molecules resulting from the oxidative cleavage of double bonds in the carotenoid molecule. These apocarotenoids are formed by chemical reactions in foods that contain carotenoids or by enzymatic cleavage of intact carotenoids. To date, a plethora of apocarotenoids has been identified in both plants and animals. In plants, abscisic acid (ABA) is the physiologically important phytohormone derived from 9-cis- violoxanthin and 9-cis-neoxanthin (73). Another example of plant apocarotenoids are strigolactones that can serve as signaling molecules and as shoot branching regulators

(74-76). In animals, the best-known apocarotenoids are vitamin A and its derivatives

(retinoids). The first study that a carotenoid is the precursor of vitamin A came from 8 Moore (6) where he described conversion of β-carotene to vitamin A in the small intestine of the rat. This was the first evidence that a carotenoid is the precursor of retinoids. Then, the structure of β-carotene was elucidated and the central cleavage mechanism at the central carbon double bond (15,15′) of β-carotene for its conversion to vitamin A was proposed. (7). Goodman & Huang (77) and Olson & Hayaishi (78) characterized the respective enzymatic activity in cell-free homogenates from rat small intestine but the enzyme was never purified.

1.3.a - Enzymatic cleavage of carotenoids

The molecular characterization of a carotenoid-cleaving enzyme was achieved first in plants (73) by analyzing the ABA-deficient maize mutant, vp14 (viviparous 14). ABA is a plant growth regulator involved in the induction of seed dormancy and in adaptation to various stresses, such as drought (79). Vp14 protein does cleave 9-cis-neoxanthin and 9- cis-violaxanthin to form xanthoxin and a C25 by-product in ABA synthesis pathway (73).

The product of this cleavage reaction is cis-xanthoxin, which is readily converted to

ABA. The molecular identification of vp14 led to the findings of related carotenoid cleavage enzymes in other organisms - in bacteria (80), fungi (81), and animals (82, 83).

These are non-heme iron oxygenases containing a ferrous iron as an essential cofactor for the reaction as well as four conserved histidines and a glutamate residue. There are three different family members of these enzymes encoded in mammals. First, the retinal pigment epithelium protein of 65 kDa (RPE65) was identified (84) which catalyzes the concerted hydrolysis of all-trans-retinyl palmitate to 11-cis-retinol. The other two

9 members incorporate molecular oxygen into their substrates, and catalyze the oxidative cleavage of double bonds of the polyene chain of carotenoids.

Mammalian β-β-carotene-15,15’-monooxygenase 1 (BCMO1) uses mostly provitamin A carotenoids as substrates, and it cleaves them at the central 15,15’ double bond to yield retinal. It has been reported that BCMO1 only catalyzes the cleavage of β-carotene, α- carotene, and β-cryptoxanthin and requires substrates having at least one non-substituted

β-ionone ring (85).

The other carotenoid-cleaving oxygenase catalyzes the eccentric (asymmetric) cleavage of β-carotene and is known as β-carotene-9’-10’-dioxygenase (BCDO2). This reaction results in the formation of one molecule of β-apo-10’-carotenal, and one molecule of β- ionone (86). Later on, it was found that this enzyme could also catalyze the eccentric cleavage of xanthophylls such as zeaxanthin and lutein (87, 88), and acyclic carotenoid, lycopene (89). The cellular localization of these two enzymes is different from each other; while central cleavage enzyme, BCMO1, is a cytosolic protein (85), eccentric cleavage enzyme, BCDO2, is localized to mitochondria (87).

The mechanism of the central cleavage is still unclear, earlier study described that it acts as a monoxygenase (90) yet more recently it was shown as a dioxygenase reaction (91).

For the sake of clarification, we use β-carotene-oxygenase 1 (BCO1) for BCMO1 and β- carotene-oxygenase 2 (BCO2) for BCDO2. BCO1 has been cloned from the Drosophila

10 melanogaster (83), chicken (82), mouse (92-94), and human (95). BCO2 has been identified and cloned from mice, humans, and zebrafish (86).

First, it was thought that the eccentric cleavage of β-carotene was an alternative pathway to produce retinoids and vitamin A (96). However, BCO1 knockout mice (BCO1-/-) that are fed β-carotene become vitamin A deficient despite the expression of BCO2 suggesting that BCO1 is the primary enzyme for retinoid production and BCO2 is responsible for different physiological functions (97). Also, it was seen that while both

BCO1 and BCO2 are expressed in human tissues in kidney tubules, exocrine pancreas,

Leydig and Sertoli cells in the testis, small intestinal and stomach mucosa, adrenal gland, and epithelium in the eye; only BCO2 is expressed in cardiac and skeletal muscle, prostate and endometrial connective tissue, and the endocrine pancreas suggesting that

BCO2 has a function independent of vitamin A production (98).

1.3.b - Negative Feedback Regulation of β-carotene conversion and intestinal absorption

Vitamin A deficiency resulted in enhanced BCO1 activity in rats (99). The promoter of mouse BCO1 gene contains a direct repeat of retinoic acid response element, suggesting its regulation by all-trans-retinoic acid (ATRA) signaling via retinoic acid receptors

(RARs) at the transcriptional level (100). ATRA activates the ISX (Intestine Specific homeoboX), an intestine-specific transcription factor that was found to regulate BCO1 and SR-BI; expression in the intestine (101, 102). ISX in turn inhibits the expression of

BCO1 and SR-BI, as a result intestinal β-carotene absorption and β-carotene conversion 11 to retinoids would be inhibited (103), explaining the mechanism of negative feedback regulation of intestinal β-carotene absorption and conversion.

1.4 OCCURRENCE AND FUNCTIONS OF APO-LYCOPENOIDS AND β-

APOCAROTENOIDS, IN FOODS, AND IN MAMMALIAN TISSUES AND

PLASMA

1.4.a - The Occurrence of Apo-lycopenoids

The occurrence of apo-lycopenoids such as apo-6′-lycopenal and apo-8′-lycopenal in tomato extracts and tomato paste (104) and in raw tomatoes (105) was reported some time ago. Recently, in addition to apo-6′-, and 8’-lycopenal, apo-10'-, 12'-, and 14'- lycopenals were found in both raw and processed food products (106). Also, in the same study all of these apo-lycopenals (apo-6′-, 8′-, 10'-, 12'-, and 14'-) were detected in human plasma. Whether they originated from enzymatic cleavage of lycopene or from consumption of apo-lycopenal containing fruits and vegetables is not clear, though.

Another study revealed the presence of apo-8’-lycopenal and apo-10’-lycopenal in the liver of 14C-lycopene-fed rats, suggesting that and these are putative cleavage products of the BCO2 (107). Apo-10’-lycopenal was not detected in the lung tissues of lycopene- supplemented ferrets, but the reduction product of this apo-lycopenoid, apo-10’- lycopenol was observed (89).

12 1.4.b - The Biological Activities of Apo-lycopenoids

Lycopene oxidation products, apo-lycopenoids, may be responsible for some of the biological activities attributed to lycopene (108, 109). There are number of reports indicating that apo-lycopenoids are biologically active compounds both in vitro and in vivo.

Gap junctions, considered to contain cell-to-cell channels, allow adjacent cells to exchange nutrients, waste products, and information. Gap junction communication (GJC) plays a role in the control of cell growth via differentiation, proliferation, and apoptosis

(110). The central cleavage of lycopene yields acyclo-retinoic acid (ACR), the linear analogue of ATRA, which was shown to enhance GJC although at supra-physiological concentrations (50 µM) in human fetal skin fibroblasts (111). ACR was also found to reduce cell viability by inducing apoptosis in human prostate cancer cells, yet not at define physiological concentrations (112). In another cell-based study, this metabolite was shown to inhibit cell growth in human mammary MCF-7 cells; in the same study it was observed that ACR activates RARE but not as well ATRA activating RARE though.

(113).

The eccentric cleavage of lycopene catalyzed by BCO2 yields apo-10’-lycopenal that can be either further oxidized into apo-10’-lycopenoic acid or reduced to apo-10’ lycopenol depending on whether NAD+ or NADH is present as a cofactor (71). Apo-10’-lycopenoic acid is implicated as the biologically active apolycopenoid particularly in lung carcinogenesis both in vitro and in vivo by inducing phase II enzymes (114); a process is 13 mediated through antioxidant response elements (ARE) and activation of ARE transcription factor Nrf2 (nuclear factor E2-related factor 2).

It is known that BCO2 is highly expressed in adipose tissue (80); it was hypothesized that apolycopenoids that are synthesized in adipose tissue should up-regulate RARs thereby displaying an impact on adipose tissue (115). In this study, they found that apo-10’- lycopenoic acid induced RAR activation both in vitro and in vivo and this apo-lycopenoid displayed a similar effect as ATRA in adipocyte tissue biology by showing anti- inflammatory response in adipose tissue yet it does not have any effect on adipogenesis

(115).

Sirtuin 1 (SIRT1) is a key regulator of lipid metabolism (116) which is involved in attenuation of adipogenesis and inducing fat mobilization (117, 118). In a most recent study, it was reported that apo-10’-lycopenoic-acid led to the increase in SIRT1 enzyme activity by treatment with this metabolite in ob/ob mice resulting in prevention of fatty liver (119).

1.4.c - The Occurrence of β-apocarotenoids

In 1970s, Gunguly and co-workers (120, 121) reported studies regarding the existence and possible biological activities of β-apocarotenoids (BACs). In one of them, β-apo-8’-,

10’-, and 12’-carotenals were tested in the curative-growth assay in rats. The assay was performed in which the total gain in weight of vitamin A-deficient rats receiving β- carotene, β-apo-8’-, 10’-, and 12’-carotenal were assessed and the bio-potencies of these 14 BACs were calculated against β-carotene taken as 100%, on a molar basis. The biopotencies found were 72 +/- 3.7, 78 +/- 2.8, 72 +/- 5.1 for β-apo-8’-carotenal, β-apo-

10’-carotenal, β-apo-12’-carotenal respectively (120). In the same study, they demonstrated that β-apo-carotenals can be oxidized to the corresponding β-apocarotenoic acids in mitochondrial and microsomal fractions of the homogenates of rat and chicken liver and that addition of either NAD+ or NADP+ produced an increase in the aldehyde oxidase activity in both species (120). Another study by the same group showed that retinal, β-apo-8'-, 10'- and 12'-carotenal were isolated from the intestine of chickens fed

β-carotene and they were identified on the basis of thin-layer chromatography and spectroscopic analysis (121).

The formation of β-apocarotenoids can occur enyzmatically as it was shown in the study that when human, monkey, ferret and rat tissue whole tissue homogenates. These homogenates were incubated with β-carotene in the presence of NAD and dithiothreitol and significant amounts of β-apo-8’-, 10’-, 12’-carotenal, as well as retinal and retinoic acid were found (122). There were no β-apocarotenals or retinoids detected in the control incubations without tissue homogenates. Moreover, the amounts of β-apo-carotenals and retinoids formed were markedly reduced when NADH, or when dithiothreitol and cofactors were deleted from the incubation and replaced by NAD. Both β-apocarotenals and retinoid production were inhibited completely by adding disulfiram, an inhibitor of sulfydryl containing enzymes (122).

15 Olson and co-workers detected β-apocarotenoids in vitamin A-deficient rats but they were present in <5% of the retinoids and only in intestinal preparations from β-carotene metabolism (123). Collectively, all these studies mentioned above suggesting that β- apocarotenals are formed as products of β-carotene oxidation in vivo.

BACs can also be formed via autoxidation, thermal degradation of β-carotene, or during food processing. The spontaneous autoxidation of toluene solutions of β-carotene (200

µM) with 100% oxygen at 60°C for 120 min. yielded a homologous series of carbonyl cleavage products of β-carotene including retinal, β-apo-13-carotenone, β-apo-14’- carotenal, β-apo-12’-carotenal, and β-apo-10’-carotenal as they were separated by reverse-phase HPLC, and individual peaks were characterized with an on-line diode array detector (124).

In a simulated deodorization using purified palm oil and β-carotene (BC) in which they were separated, purified and the individual compounds in fractions were isolated using a silicic acid column, β-apo-13-Carotenone, β-apo-14’-carotenal, and all-trans-retinal were identified by infrared spectroscopy and mass spectrometry (125). In another food related study, thermal degradation (various heat treatments) of β-carotene yielded β-apo-8′- carotenal, β-apo-10′-carotenal β-apo-12′-carotenal, β-apo-14′-carotenal and these oxidation products were found to be 5% of initial BC (126).

16 β-apocarotenoids (BACs) can directly come from our diet as it was shown in a number of studies. Processed mango juice, acerola juice and dried apricots were analyzed by means of analytical methods using chromatographic methods such as thin layer chromatography

(TLC), high-performance liquid chromatography (HPLC) and visible absorption spectra obtained by a photodiode array detector, and mass spectra. These processed foods were tested for the identification of β-apocarotenals including β-apo-8’, 10’-, 12’-, and 14’- carotenal derived from β-carotene. None of these oxidation products were detected in acerola juice, or dried apricots and only β-apo-12’-carotenal was found in mango juice

(127).

Our laboratory and our collaborators reported the existence of BACs in recently published studies. The levels of β-apo-13-carotenone (C13 Ketone), and β-apocarotenals including β-apo-8’-, 10’-, 12’-, and 14’-carotenal in melons (a cantaloupe and a greenhouse grown Orange Dew) were measured by utilizing liquid chromatography mass spectrometry (LC/MS). β-Apo-8′-, 12′, and 14′-carotenals and β-apo-13-carotenone were detected in these two different types of melons (128).

More recently, our collaborators developed sensitive HPLC/MS procedure for detection of BACs in human plasma. The specificity and sensitivity was assured by multiple reaction monitoring. Human plasma samples from six free-living individuals were analyzed and the plasma concentration of β-apo-13-caroteneone was found to be 3.8 +/-

0.6 nM (101).

17 Additionally, BACs can arise from β-carotene metabolism as demonstrated in a number of studies. In one study, β-apo-8’-carotenal was detected in the plasma of a healthy man followed by ingestion of a small oral tracer dose of [14C]-β-carotene (130). Our group undertook the study in which the concentrations of β-carotene, retinal, β-apo-8′-, 10′-,

12′- and 14′-carotenal were measured both in serum and liver of wild type and BCO1-/- mice that were on a β-carotene containing diet. It was found that while β-apo-10’-, and

12’-carotenals were detected in serum and livers from wild type and BCO1 knock out mice, only β-apo-12’-carotenal levels were found to be significantly higher in the liver of knock out mice compared to wild type, suggesting that BCO2 was possibly up-regulated in the BCO1 deficient mice (131).

1.4.d - The Biological Activities of β-apocarotenoids

β-Carotene is one of the substances most extensively used in chemoprevention clinical trials against various types of cancers (132). Chemoprevention can be defined as the application of natural or synthetic molecules to prevent, inhibit or reverse the carcinogenic machinery (133). Epidemiologic studies demonstrated that individuals eating more fruits and vegetables that are rich in carotenoids and people having higher serum β-carotene levels have a lower risk of cancer, particularly lung cancer (134, 135).

Randomized intervention trials test dietary interventions through randomized experimental designs to prevent most of the biases that can be seen in observational studies. Two large randomized intervention trials of β-carotene supplementation were

18 designed to test whether β-carotene could reduce the risk of lung cancer: the Alpha-

Tocopherol, Beta-Carotene Cancer Prevention (ATBC) Study and the Beta Carotene and

Retinol Efficacy Trial (CARET).

In 1994, ATBC reported its findings on more than 29000 participants of a double-blind, placebo-controlled trial on the prevention of lung cancer and other cancers by supplementation with micronutrients (136). The results for β-carotene that was given 20 mg in one capsule taken daily for five to eight years were not expected because they provided no evidence for benefit in the prevention of lung cancer in older male cigarette smokers and rather there were more incidents of lung cancer diagnosed in those receiving

β-carotene supplements.

In the CARET study, β-carotene (30 mg) and retinyl palmitate 25000 IU (13 664 retinol equivalents) were given to more than 18000 men and women with a history of smoking or asbestos exposure (137). Among them 388 developed lung cancer, with a 28% increase in lung cancer incidence that received the β-carotene–retinyl palmitate combination daily for an average of 4 years compared with participants who received placebo. This study was halted ahead of schedule for exactly the same reasons.

The reason for the adverse effects of β-carotene observed in the CARET and ATBC trials may be the fact that the subject group in these trials was restricted to smokers. It is possible that β-carotene could be easily oxidized under the conditions of smoking and asbestos exposure, also the doses given in these trials were supra-physiological levels (20 19 mg in ATBC, and 30 mg in CARET vs. the physiological concentration of β-carotene is 6 mg).

The biological activities of oxidation products of β-carotene (eccentric cleavage products of β-carotene) were examined in a number of studies based on considering the biological properties of the parental compound i.e., β-carotene. In one study, the interaction between benzo[a]pyrene (an important carcinogen found in cigarette smoke) and β- carotene or β-apo-14’-carotenoic acid in normal human bronchial epithelial (NHBE) cells was examined (138). Also, they tested whether benzo[a]pyrene, β-carotene or β-apo-14’- carotenoic acid affected the expression levels of RARβ in NHBE cells. The found that while a carcinogen benzo[a]pyrene treatment led to the down-regulation of RARβ, β- carotene or β-apo-14’-carotenoic acid treatment doubled the levels of RARβ.

Additionally, they observed that down-regulated RARβ by carcinogen was completely reversed by treatment of β-carotene or β-apo-14’-carotenoic acid. Also, they demonstrated that β-apo-14’-carotenoic acid can transactivate RARβ2 but that was ten to hundred-fold less than activity seen by a physiological agonist i.e., ATRA. However, this transactivation seen by β-apo-14’-carotenoic acid was claimed to be due to its metabolism to ATRA. They tested another β-apocarotenoid, β-apo-13-carotenone, in the same study and this compound did not display a significant transcriptional activity of

RARβ.

20 It is known that ATRA does exhibit anticancer activities by repressing activator protein-1

(AP-1) (139). β-Apocarotenoic acids including β-apo-8’-, 10’-, 12’- and 14’-carotenoic acid were tested their ability to inhibit breast tumor cell growth in both estrogen receptor positive (MCF-7 cells) and negative (Hs578T and MDA-MB-231) human breast cancer cells which express RARs (140). It was observed that after 2-6 days of treatment none of the β-apocarotenoic acids and ATRA at various concentrations (10 nM, 100 nM and 1

µM) induced cell growth inhibition but 9-days of treatment with 1 µM β-apo-12′-CA, and

β-apo-14’-carotenoic acid displayed half of the inhibition elicited by 1 µM ATRA in

MCF-7 cells on the basis of live cell calculations. Additionally, 1 µM β-apo-14’- carotenoic acid exerted the growth inhibitory response in Hs578T cells. The induced growth inhibitory effect of β-apocarotenoids was associated with repression of AP-1 activity similar to the action of ATRA but not dependent on RAR proteins. They concluded that these β-apocarotenoic acids display anti-proliferative effects through anti-

AP-1 activity and these compounds are biologically active in breast tumor cells (140).

Acute myeloid leukemia occurs as a result of the propagation of a clone of cells that have a block in their differentiation pathway to functional mature granulocytes or monocytes

(141). The human cell line HL-60 was developed (142) and it has been widely used in order to examine the compounds that induce maturation of these cells. ATRA inhibits proliferation of HL-60 cells and induces them to differentiate into morphologically functional mature granulocytes (143, 144). β-carotene and some of its metabolites were tested in promyelocytic leukemia cell lines such as HL-60 cells and U937 cells for their

21 ability to function as biologically active compounds on myeloid differentiation. β-

Carotene inhibited proliferation and differentiation of HL60 cells (145) and U937 cells

(146). β-Carotene cleavage products such as β-apo-14’-carotenoic acid were found to stimulate the differentiation of U937 leukemia cells (147) and β-apo-12’-carotenoic acid was observed as a biologically active compound and it was capable of inhibiting the proliferation of HL-60 cells (148).

In addition to these ascribed anti-carcinogenic properties of the parent compound i.e., β- carotene, it was shown that β-carotene can be an activator of the human pregnane receptor (PXR) (149). PXR, a member of the nuclear receptor family of ligand-activated transcription factors, is involved in xenobiotic detoxification to prevent accumulation of toxic substances in liver (150). It was tested whether β-carotene cleavage products are biologically active in terms of eliciting PXR activity.

β-Apocarotenals including β-apo-8’-, and 12’-carotenal, β-carotene, and a known PXR agonist i.e., rifampicin, were tested in reporter assays using Hep G2 liver cells. While β- carotene itself elicited a 5-6 fold reporter gene activity increase, β-apocarotenals exerted less or no activation compared to 7-fold induction by rifampicin (151).

Whether β-apocarotenals are biologically active compounds was also challenged by

Ziouzenkova et al. (152). They tested whether eccentric cleavage products of β-carotene such as β-apo-8’- 12’- and 14’-carotenals display any biological activity and whether

22 they have any effect on gene expression. Based on ATRA being a suppressor in adipocyte differentiation via peroxisome proliferator-activated receptor gamma (PPARγ)

(153), they tested whether β-apocarotenals displayed any activity on adipocyte differentiation assays using 3TC3-L1 cells. They found that only β-apo-14’-carotenal decreased adipogenesis (lipid accumulation) in a concentration-dependent manner. Next, they tested whether this suppression of adipogenesis is through association with RARs and found that β-apo-14’-carotenal can be a weak partial agonist suggesting on a RARα- independent mechanism leading to β-apo-14’-carotenal’s repression of adipogenesis.

Since adipogenesis is also regulated by activation of retinoid X receptor (RXR) and

RXR-PPAR or RXR partnering with other nuclear recetors (154), they tested RXRα activation in the presence of a synthetic agonist of RXR and β-apo-8’-, 12’- and 14’- carotenals in human bovine cells. Only β-apo-14’-carotenal effectively inhibited agonist- induced RXRα activation with inhibition constant (Ki ) value of 500 nM. In addition,

RXR partner nuclear receptors were tested including PPARα, PPARδ, and PPARγ. It was found that β-apo-14’-carotenal decreased agonist-induced PPARα and PPARγ activation very effectively, and PPARδ modestly. Also, they found that β-apo-14’-carotenal displays a inflammatory response via PPARα inhibition in vivo in wild type mice and this response was not observed in PPARα deficient mice. Collectively, this study suggested that β-apo-14’-carotenal did block RXR, PPARα, and PPARγ activation both in vitro and in vivo (152).

23 In plants, the biological activities of β-apocarotenoids are well established and detailed.

β-Apo-13-carotenone is a biologically active compound blocking the growth of root hairs in plants (155). β-Ionone exerts a number of biological activities such as a pollinator attractant, fruit or vegetable flavor (156). Along with β-ionone, β-cyclocitral (157) was shown to contribute to the flavor and/or aroma of various foods including tomato (158).

The existence of β-apocarotenoids is well established yet there is limited knowledge on biological activity and biochemical characterizations of these metabolites in higher organisms.

We hypothesize that these metabolites modulate the transcription of nuclear receptors such as RARs and RXRs since they are analogs of physiological ligand for RARs, e.g,

ATRA and the putative ligand for RXRs e.g., 9-cis-RA. Our goal is to fill the gaps in the knowledge on the biochemistry and the molecular biology of β-apocarotenoids in mammals in a comprehensive manner.

1.5 - RETINOIC ACID RECEPTORS AND RETINOID X RECEPTORS

All-trans-retinoic acid (ATRA) and some synthetic retinoids act through ligand-regulated trans-acting transcription factors that bind to cis-acting DNA regulatory elements in the promoter regions of target genes. Retinoic acid receptors (RARs) are members of the steroid/thyroid hormone family of nuclear receptor that consist of RARα (NR1B1),

RARβ (NR1B2) and RARγ (NR1B3) encoded by separate genes (159, 160) that form

24 heterodimers with retinoid X receptors (RXRs). Each receptor subtype has also been demonstrated to have at least two isoforms due to differential splicing and promoter usage. All-trans-retinoic acid (ATRA) is the physiological ligand of RARs and upon binding to receptor, ATRA signaling occurs which leads to the transcription of target genes involved in various cellular events such as cellular differentiation, proliferation, and apoptosis (161). In the absence of ATRA, transcriptional silencing (repression) does occur at un-liganded RARs.

RARα is widely expressed in numerous tissues and cell lines (50, 51); RARβ is expressed in a variety of epithelial cells (162), and expression of RARγ is restricted to skin (163). RXRs including RXRα (NR2B1), RXRβ (NR2B2), and RXRγ (NR2B3) are expressed in almost every tissue of the body: RXRα is the major subtype in skin and expressed in liver, lung, muscle, kidney, and intestine; RXRβ is expressed ubiquitously; and expression of RXRγ was detected in brain, cardiac, and skeletal muscle (164).

ATRA is the major biologically active retinoid by virtue of activating the RARs globally thereby exerting pleiotropic events in cell growth and differentiation in embryonic development (ATRA as morphogen) and adult physiology (165, 166).

Nuclear receptors including RARs, and RXRs comprise modular structural/functional domains denoted A to F (See Fig. 1.4.a) (167) with different regions corresponding to autonomous functional domains that can be interchanged between related receptors without loss of function. The N-terminal A/B region contains a ligand-independent activation function (AF-1). The DNA binding domain (DBD) is the most conserved 25 domain of nuclear receptors, and it is responsible of recognition of specific DNA sequences called hormone response elements: retinoic acid response elements (RAREs) or retinoid X response elements (RXREs). The sequence-specific DNA recognition is achieved through two zinc-finger motifs of the DBD.

The E region contains the moderately conserved and largest domain e.g., ligand-binding domain (LBD). It consists of a ligand-binding pocket, a dimerization surface and a ligand-dependent transcriptional activation function (AF-2). The short D region (linker domain) is not well conserved among the different receptors and serves as a hinge between the DBD and the LBD, allowing rotation of the DBD. The C-terminal F domain is present in RARs but not in RXRs and has no clear function yet. The canonical retinoid signaling (RAR-mediated regulation of transcription with the LBD as the key player) relies on binding to specific sequence elements (response elements) on DNA located in the promoters of target genes.

1.5.a - Hormone Response Elements (HRE)

Like other nuclear receptors, retinoid receptors regulate transcription by binding to specific DNA sequences in target genes known as hormone response elements or HREs.

The analysis of a large number of naturally occurring as well as synthetic HREs showed that a sequence of 6 base pairs represents the core recognition motif. Although some monomeric receptors can bind to a single hexameric motif, most receptors bind as homo- or heterodimers to HREs composed typically of two core hexameric motifs. For dimeric

26 HREs, the half-sites can be configured as palindromes (Pal), inverted palindromes (IPs), or direct repeats (DRs).

RARs and RXRs recognize response elements composed of two direct repeats of the consensus sequence 5’-AGGTCA-3’ separated by one to five base pairs known as the 1-5 rule (See Fig. 1.5) (168).

In the case of RAR-RXR heterodimers, the response element can be distinguished as either DR1 or DR5, and DR5 is the preferred HRE for the RARs (169). RAREs were found in the promoters of a large number of ATRA target genes implicated in a wide variety of functions. A DR1 element has been found in the rat CRBPII gene promoter

(170). DR2 elements were identified in the CRBPI and CRABPII gene promoters (171,

172). The classical DR5 elements are found in the promoters of the RARβ2 gene itself

(173) of the CYP26A1 (cytochrome 450, family 26, subfamily a, polypeptide 1) gene

(174) and of several Homeobox (Hox) and hepatocyte nuclear factor (HNF) genes (175,

176). For RXR-RXR homodimers, the response element is identified as DR1 (177).

1.5.b - The structure of Ligand Binding Domain (LBD)

The LBDs are formed by 12 conserved α-helical regions (H1 to H12) and a β-turn

(situated between H5 and H6). The LBD contains the ligand-binding pocket (LBP), the main dimerization domain and the ligand-dependent activation function-2 (AF-2). LBP consists of hydrophobic residues. It is involved in selectivity of ligand binding, maximizing hydrophobic contacts and its shape matches the volume of the ligand (178- 27 180). The C-terminal helix 12 or H12 (AF-2) is the key mediator between active

(recruitment of coactivators) and inactive configuration (presence of corepressors) of nuclear receptors.

Upon agonist binding to receptor, there is a conformational change (See Fig. 1.4.b/c. and

Fig. 1.6.) in H12 that induces an α-strand to β-helix transition leading to H11 formation, which in turn stimulates release of corepressors and H12 repositions itself where it can contact co-activators through their LXXLL motifs (181, 182) where L represents leucine and X represents any amino acid (183-186).

The ligands are stabilized through extensive van der Waals contacts, and ionic, hydrogen bonds involving their carboxylate moiety, a conserved arginine in H5, and water molecules (187).

1.5.c - Coactivators

Coactivators are proteins that cooperate with nuclear hormone receptors to activate transcription. The three p160 members of steroid receptor coactivators (SRCs) are the main coactivators: SRC1, SRC2, and SRC3 and these contain three copies of a highly- conserved NR box (LXXLL) motif which interact with H12 of nuclear receptors (188-

190). SRC coactivators have the ability to acetylate core histones in nucleosomes via recruiting histone acetyltransferases (HATs) leading to relaxation of the chromatin structure and providing a more accessible DNA for transcription initiation (191, 192).

28 1.5.d - Corepressors

Corepressors are proteins that cooperate with nuclear hormone receptors to repress transcription. These proteins tie up histone deacetylase-containing complexes to promoters of target genes. The two main types are nuclear receptor corepressors (NCoRs) and silencing mediator of retinoid and thyroid hormone receptors (SMRTs). These proteins oppose the action of coactivators and several have been identified related to retinoid signaling (193-197). They inhibit transcription of target genes by binding to the

LBD of nuclear receptors, such as RAR and RXR (198). The binding of corepressors leads to change in the AF2 domain of receptor and recruits a large protein complex containing histone deacetylases (HDAC) to a target gene (199-201). HDAC recruitment results in target gene silencing. The HDAC activity leads local histone deacetylation and this hypoacetylated chromatin is more condensed and restricts access of transcription factors and the general transcription machinery to the DNA template (202, 203). In addition to that, where the coactivator complex contacts and stabilizes the preinitiation complex, SMRT and N-CoR make inhibitory contacts that interfere with transcriptional initiation (204-207).

29 1.6 FIGURES b-CAROTENE: FRIEND OR FOE? 165

FIG. 1. Structures of various carotenoids found in human serum.

adrenal tissue containedFigure more 1.1 ofStructures the 9-cis andof major 13-cis carotenoidsisomers tion(carotenes, of two and molecules xanthopylls) of retinal found (Devery in and Milborrow, of b-carotene. They also found numerous isomers of lyco- 1994; Nagao et al., 1996), at least in in vitro studies. pene in serum and tissue,human which plasma. are not necessarily of dietary In addition to this pathway, whereby retinal is either oxi- origin. Thus, there is isomerization of dietary carotenoids dized to retinoic acid or reduced to retinol, there is another and accumulation of these isomers in vivo. However, we metabolic pathway in which b-carotene is metabolized to a know very little about the different actions of these isomers, series of shortened carbonyls known as apocarotenals. These with the exception of the 9-cis isomer of b-carotene, which molecules then undergo oxidation to apocarotenoic acids, can serve as a precursor for the all-trans- and 9-cis-isomers and, by a mitochondrial b-oxidation-like pathway, form reti- of both retinoic acid (Wang et al., 1994) and retinal (Nagao noic acid (Wang and Krinsky, 1997) (Fig. 4). As yet, we and Olson, 1994). The importance of these observations lies know very little about the biological activity of these shorter- in the fact that 9-cis-retinoic acid is the speciﬁc ligand for chain b-carotene derivatives. the RXR nuclear receptor (Heyman et al., 1992; Levin et The next question that we would like to address is whether al., 1992). there is any link between carotenoid ingestion and the risk We also know that b-carotene is an important precursor of cancer in humans. This question was stimulated by Peto of vitamin A in humans and may account for 35–50% of et al. (1981), who ﬁrst suggested that a protective association dietary vitamin A. There are two pathways for the metabo- might exist between dietary b-carotene and a reduced risk lism of b-carotene (Fig. 4). The central cleavage pathway30 of human cancer. There have been many studies that suggest involves the direct attack of a dioxygenase-like enzyme on that b-carotene and other carotenoids might decrease the the central, 15,15؅-double bond with the subsequent forma- risk of cancer in animals or act as inhibitory compounds with respect to the proliferation of tumor cell lines (Krinsky, 1994). Epidemiological data have strongly supported a role for dietary carotenoids as protective agents with respect to the risk of developing a variety of tumors. In a survey on the relationship between food intake and cancer, Block et al.

FIG. 2. The primary actions of carotenoids. FIG. 3. The quenching of singlet oxygen by carotenoids.

AID FAAT 2387 / 6k24$$$182 12-02-97 08:03:28 ftoxas

Figure 1.2 General view of the metabolism of natural retinoids and of β-carotene, from their presence in the intestinal lumen to their actions at the target cell level

(23). (β-Carotene, βC; retinyl ester, RE; lecithin:retinol acyltransferase, LRAT; cellular retinol-binding protein typeII, CRBPII; retinyl ester hydrolase, REH; retinol binding protein, RBP; transthyretin, TTR; retinoic acid receptor, RAR; retinoid X receptor,

RXR).

Figure 1.3 Retinoid Metabolism (56).

Figure 1.4 Structural and Functional domains of RXRα and ligand domain binding

(LBD) structure (167) a. Schematic representation of a nuclear receptor: variable N- terminal region (A/B), a conserved DNA-binding domain (DBD) or region C, a linker region D, and a conserved E region that contains the LBD and they may also contain a

COOH-terminal region (F) of unknown function. b. un-bound RXRα: Human RXRα

LBD without a bound ligand as found in Protein Data Bank (PDB) crystal structure of the apo-RXRα LBD homodimer 1 LBD. c. agonist-bound-RXRα: Human RXRα LBD complexed with transcriptional agonist SR11237 (BMS649, with carbon atoms in gray) as found in PDB crystal structure 1MVC. Protein backbones are shown in ribbon format.

Figure 1.5 The 1-to-5 Rule (168). Direct repeats of the recognition motif (AGGTCA) separated by one to five base pairs

Fig. 1.6 The mechanism of nuclear receptor i.e., RXR activation upon ligand binding (182) In the unliganded RXR LBD (left panel), structural elements that are important for activation function 2 (AF2) are represented as colored helices (H3, blue;

H4, green; H11/H12, red). Upon agonist such as 9-cis-RA binding, helix H12 folds into active site where it interacts with LXXLL motifs (orange helix) of coactivators.

CHAPTER 2 (*): THE ECCENTRIC CLEAVAGE PRODUCT OF β-CAROTENE,

β-APO-13-CAROTENONE, FUNCTIONS AS AN ANTAGONIST OF RXRα

* Eroglu, A., Hruszkewycz, D.P., Curley, R.W. & Harrison, E.H. The eccentric cleavage product of β-carotene, β-apo-13-carotenone, functions as an antagonist of RXRα. Archives of Biochemistry and Biophysics 504, 11-16 (2010).

36 ABSTRACT

In this study, we investigated the effects of eccentric cleavage products of β-carotene, i.e.

β-apocarotenoids (BACs), on retinoid X receptor alpha (RXRα) signaling.

Transactivation assays were performed to test whether BACs activate or antagonize

RXRα. Reporter gene constructs (RXRE-Luc, pRL-tk) and RXRα were transfected into

Cos-1 cells and used to perform these assays. None of the BACs tested activated RXRα.

Among the compounds tested, β-apo-13-carotenone was found to antagonize the activation of RXRα by 9-cis-retinoic acid and was effective at concentrations as low as

1 nM. Molecular modeling studies revealed that β-apo-13-carotenone makes molecular interactions like an antagonist of RXRα. The results suggest a possible function of BACs on RXRα signaling.

2.1 INTRODUCTION

Carotenoids are C40 polyisoprenoids that are biosynthesized from eight isoprene units followed by cyclization, isomerization, and oxidation (208). Approximately 600 carotenoids have been characterized in nature (209). Among them, 50-60 display provitamin A activity (210, 14). In order to exhibit a provitamin A activity, the carotenoid molecule must have at least one unsubstituted β-ionone ring and the correct number and position of methyl groups in the polyene chain (9). Two pathways have been described for the cleavage of BC in mammals (211). β,β-Carotene-15,15′-oxygenase

(BCO1) catalyzes the central cleavage of BC to yield retinal (the initial product of symmetric or central cleavage of BC). BCO1 enzymes from fruit fly (83), chicken (82),

37 mouse (92) and human (95) have been cloned and biochemically characterized. The second pathway of BC metabolism is the eccentric cleavage which occurs at double bonds other than the central 15,15’ double bond of the polyene chain of BC to produce β- apocarotenals and β-apocarotenones. Little is known about the biological function of β- apocarotenoids (BACs) in higher animals.

The retinoid X receptor (RXR) is a member of the nuclear receptor superfamily of ligand dependent transcription factors and consists of three distinct subtypes (α, β, and γ) (212-

214). RXRs are nuclear receptor proteins which can modulate the transcriptional activity of target genes by binding as RXR heterodimeric complexes or RXR homodimers to gene promoters. The first identified RXR, referred to as RXRα, was initially described as an orphan receptor (215). 9-cis-Retinoic acid (9-cis-RA), a stereoisomer of all-trans-retinoic acid (ATRA) is a high-affinity ligand for RXRα, as well as for the two additional related subtypes, RXRβ and RXRγ, that were later identified (215-221). RXRs form heterodimers with a number of orphan and nuclear hormone receptors including retinoic acid receptors (RARs), thyroid receptor (TR), vitamin D receptors (VDRs), peroxisome proliferator-activated receptors (PPARs), liver X receptors (LXRs), farnesoid X receptors

(FXRs), and pregnane X receptors (PXRs) (217, 222-224).

RXRs can also form homodimers in vitro, indicating the existence of a RXR-specific signaling (225, 226). It was observed that liver-specific inactivation of RXR in mice was associated with abnormalities in major metabolic pathways which supports the pleiotropic role of this receptor (227). RXR-selective synthetic retinoids, i.e. rexinoids, 38 are valuable in elucidating the role of RXRs. One particular rexinoid, LGD 1069, is currently used for the treatment of refractory advanced-stage cutaneous T-cell lymphoma

(228-233).

The purpose of the present study was to test BACs as potential ligands for RXRα. We utilized a transient cotransfection (receptor/reporter) assay to screen these compounds as agonists or antagonists of RXRα. Our data indicate that β-apo-13-carotenone acts as a potent antagonist of activated (liganded) RXRα. Molecular modeling studies suggest that

β-apo-13-carotenone makes molecular interactions which are similar to those made by

ATRA bound as an antagonist to RXRα.

2.2 MATERIALS & METHODS

2.2.a - The preparation of β-apocarotenoids

β-Cyclocitral and β-ionone were purchased (Sigma-Aldrich; Milwaukee, WI, USA) and purified (preparative TLC) prior to use. β-apo-8’-carotenal (Sigma-Aldrich) and β-apo-

12’-carotenal (CaroteNature; Lupsingen, Switzerland) were purchased and used as obtained. β-Cyclogeranic acid was prepared by air oxidation of β-cyclocitral and crystallization of the product. In the other instances where a β-apocarotenal precursor was purchased, the aldehyde group was converted to its methyl ester (KCN/acetic acid/MnO2/methanol) according to the procedure of Corey and co-workers (234), followed by saponification to the acid (β-apo-12’- and β-apo-8’carotenoic acid). For preparation of the remaining β-apocarotenoids, the ethyl ester of β-ionylideneacetic acid

39 was prepared (as an 80:20 trans/cis isomer mixture about the newly formed double bond) by the Wadsworth-Emmons reaction of β-ionone with triethylphosphonoacetate. In addition to saponifying this ester to β-ionylideneacetic acid, reduction to the alcohol

(DIBAL-H) followed by allylic oxidation (MnO2/dichloromethane) produced β- ionylideneacetaldehyde (BIA).

BIA was converted to apo-13-carotenone (C13 ketone) by Wittig reaction with 2- oxopropyltriphenylphoshonium chloride (235). Prior to any conversions of BIA, its cis/trans isomers were separated by column chromatography.

Wadsworth-Emmons reaction of retinaldehyde with triethylphosphonoacetate provided essentially isomerically pure β-apo-14’-carotenoic acid after saponification. Two stage reduction-oxidation (DIBAL-H; MnO2/dichloromethane) provided β-apo-14’-carotenal.

Wadsworth-Emmons reaction as for the conversion of retinal to ethyl-β-apo-14’- carotenoic acid converted β-apo-12’-carotenal to β-apo-10’-carotenoic acid after saponification. All reactions and compound handling were performed under gold fluorescent lights in oven dried glassware under a dry argon atmosphere.

All products were purified by appropriate column or preparative thin layer chromatography and analyzed by HPLC for purity (Model 127 pump and 166 detector,

Beckman Instruments, San Ramon, CA, USA; Metachem Polaris 5 um C18, 250 x 4.6 mm column, Varian Inc., Palo Alto, CA, USA). Chemical and structural characterization was carried out using ultraviolet spectrophotometry (Beckman DU-40 40 spectrophotometer), NMR spectroscopy (Bruker DRX400; Billerica, MA, USA) and electrospray mass spectrometry (Micromass QTOF; Milford, MA, USA).

2.2.b - Cell Culture

Cos-1 cells (ATCC CRL-1650 from the American Type Culture Collection, Manassas,

VA) were cultured in Dulbecco's modified Eagle medium (Sigma), supplemented with

10% fetal bovine serum (FBS), 100 U/ml penicillin, 100 mg/ml streptomycin, and 0.2 ml

/100 ml fungizone (antifungal reagent). The cells were maintained in an incubator at a high humidity, 5% CO2, and 37°C.

2.2.c - Plasmid Constructs

The control reporter plasmid pRL-TK was obtained from Promega (Madison, WI, USA).

It contains the herpes simplex virus thymidine kinase (HSV-TK) promoter region upstream of cDNA encoding the native Renilla (Renilla reniformis) luciferase enzyme

(236). This plasmid was used as an internal control in cotransfection experiments to normalize for transfection efficiency. The pRXRE-tk-Luc and pSG5-RXRα expression vectors were kind gifts from Dr. Noa Noy (Case Western Reserve University School of

Medicine, Cleveland, OH, USA). The experimental reporter plasmid pRXRE-tk-Luc contains five tandem repeats of a 35-bp sequence (DR-1) from the promoter of the mouse

CRBP-II gene (225).

41 2.2.d - RXRα Transactivation Assay

Cos-1 cells were plated at 1.5 x 105 cells/35-mm2 tissue-culture plate in DMEM with

10% FBS. The cells were grown overnight at 37°C with 5% CO2. The next day, the cells were transfected in serum-free medium with three plasmids mixed in the following amounts per well, 0.05 µg of pRL-TK, 2 µg of pRXRE-luciferase, 2.5 µg of pSG5-RXRα in triplicates using Lipofectamine 2000 (Invitrogen) according to the manufacturer's protocol. Following transfection, the plates were incubated at 37°C in 5 % CO2 for 4 h.

The medium was then changed to complete DMEM. Note that complete DMEM contains

10% charcoal stripped FBS instead of 10% FBS. Charcoal stripped FBS has been treated with activated carbon to adsorb lipophilic compounds including retinoids.

Twenty hours after transfection, cells were treated with test compounds that were dissolved in ethanol or 0.1% ethanol alone for an additional 24 h. Cells were washed once with PBS and lysed by incubation with 500 µl passive lysis buffer (Promega) for 15 min at room temperature. A 20-µl aliquot of cell lysate was then assayed for luciferase activities using a GloMax 96 Microplate Luminometer (Promega) and the Dual

Luciferase Reporter (DLR) assay system (Promega), according to the supplier's recommendations. For each experiment, the firefly luciferase activity (experimental reporter) was normalized to Renilla luciferase (control reporter) activity. The change in normalized firefly luciferase activity was calculated relative to that for cells that were transfected with vehicle (ethanol), which was set as 1. Fold Activation = Average

(Firefly/Renilla) from test compound / Average (Firefly/Renilla) from vehicle (Ethanol treated cells). 42 2.2.e - Molecular Modeling and Docking

Structures of proteins and their co-crystallized ligands were recalled from the protein data bank and were displayed, along with test ligands that were built and manipulated, using

Sybyl 7.1 (Tripos, Inc.; St. Louis, MO). The RXRα which binds all-trans-retinoic acid in an antagonist tetrameric form (237) is available as PDB entry 1G5Y, while the dimeric

RXRα which binds 9-cis-retinoic acid in the agonist form (238) is recalled from PDB entry 1FBY.

Computational docking of the C13 ketone (β-apo-13-carotenone) to these proteins was conducted using Surflex-Dock v. 2.11 (239) which is available from Tripos, Inc. Surflex-

Dock allows ring and acyclic structure flexibility and all-atom optimization during determination of the docked ligand poses and uses specific information about molecular interactions between known ligands and the target protein to guide the search process for docked poses.

Thus, in the case of both protein structures, ligand was extracted from the protein crystal structure and re-docked to the protein to determine if the program successfully finds the original docked pose of the crystallized ligand. Subsequently, the C13 ketone, built to resemble the native bound ligand was then docked to the protein structure and inspected for the quality of the match relative to the ligand in the crystal structure.

43 2.3 RESULTS & DISCUSSION

2.3.a - The eccentric cleavage products of β-carotene: β-apocarotenoids

β-Carotene, the most potent dietary provitamin A carotenoid, can be metabolized in mammals via two enzymatic pathways. BCO1 catalyzes the cleavage of the 15,15’ double bond resulting in two retinaldehyde molecules, and the eccentric cleavage takes place at double bonds other than the central 15,15’ double bond of the polyene chain of

β-carotene to produce β-apocarotenals and β-apocarotenones, i.e. β-apocarotenoids

(BACs) with different chain lengths (See Fig. 2.1).

Presumably the aldehydes can be oxidized to the corresponding carboxylic acids or reduced to the alcohols. β-apo-10’-Carotenal and β-ionone were shown to be products of

β,β-carotene-9′,10′-oxygenase (BCO2) (86). β-apo-13-Carotenone and β-apo-14'- carotenal were identified as enzymatic cleavage products of β-carotene in homogenates of intestinal mucosa of rat (240) but the enzyme responsible was not determined. Another study using tissue homogenates from human, monkey, ferret and rat which were incubated with β-carotene found β-apo-8'-, 10'-, and 12'-carotenals, retinal, and all-trans- retinoic acid (122). More recently, β-apo-8'-carotenal was detected in plasma after digestion of β-carotene in a healthy human subject (130).

There is no doubt that the formation of BACs occurs; however, it is not known whether the products of this pathway are biologically active. We have tested all possible eccentric

44 cleavage products of β-carotene (except the 10’-aldehyde) for their ability to transactivate

RXRα.

2.3.b - Effects of β-apocarotenoids on transactivation of RXRα

The biological properties of the BACs were characterized using a cell-based receptor/reporter cotransfection (transactivation) assay (225, 241, 50) in which the full length RXRα in a eukaryotic expression vector was co-transfected with two reporter plasmids, a firefly luciferase reporter (experimental reporter) containing the RXRE from

CRBPII and renilla luciferase (control reporter) which serves an internal control to normalize for transfection efficiency. Initially, assays using 9-cis-RA for transactivation of RXRα were used to ensure that the assay was reproducible.

Dose response experiments employed a range of 10-5 M (10 µΜ) to 10-10 M (0.1 nΜ) 9- cis-RA. As expected, the receptor was activated with increasing concentrations of 9-cis-

RA (data not shown) with half maximal activation observed at 10-8 M. Maximal fold induction at 10-5 M 9-cis-RA varied from 2.2 - 5.1 in individual experiments. We next screened all BACs at 1 µΜ + 10 µΜ for activation of RXRα. None of the BACs activated RXRα (data not shown).

2.3.c - Antagonist effects of β-apocarotenoids on activated (liganded) RXRα

Next, we tested whether any of the BACs could act as antagonists of 9-cis-RA activation in RXRα. We screened the BACs in our assays to test antagonistic behavior by

45 incubating 9-cis-RA alone or equimolar mixtures of 9-cis-RA and the test compound.

The results shown in Table 1 are expressed as a percent of the activation observed with 9- cis-RA alone that is set to 100%. The maximal, normalized fold induction for 9-cis-RA alone varied from 2.2 to 3.8 (2.9 average). The data shown in Table 1 are a compilation of multiple experiments in each of which the compounds used were tested in triplicate wells. β-apo-13-Carotenone was the most potent antagonist of the compounds tested under the conditions of these experiments.

Following the screening experiments shown in Table 2.1, we tested more completely the effects of β-apo-13-carotenone on 9-cis-RA-induced gene expression. A classic antagonist is defined as a compound that shifts the concentration-response curve of an agonist to the right (242, 243).

To characterize the interaction between both 9-cis-RA and β-apo-13-carotenone, a cumulative concentration-response curve of the agonist was established in the absence or presence of increasing fixed concentrations of the antagonist. As shown in Figure 3.2, increasing concentrations of β-apo-13-carotenone shifted the curves to the right in a concentration-dependent manner. Importantly, concentrations as low as 1 nM shifted the

9-cis-RA dose response curve.

46 2.3.d - Molecular Modeling Experiments to identify interaction between β-apo-13- carotenone and RXRα

Computational docking experiments were performed to determine if the antagonistic activity observed for β-apo-13-carotenone might be due to direct association with the ligand binding site of RXRα. ATRA has been crystallized with a tetrameric form of

RXRα that antagonizes RXR signaling (237). Shown in Figure 3 is the ligand binding domain of this tetramer (in green) with bound ATRA (in white).

When ATRA is removed from the protein and redocked using Surflex-Dock, the rebound

ATRA assumes the same position as in the crystal structure (not shown). More importantly, when β-apo-13-carotenone is constructed using the bound ATRA as template, its docked structure with RXRα (shown in yellow) is virtually coincident with the bound ATRA. Alternatively, 9-cis-RA binds as an agonist to a dimeric form of RXRα but in a twisted, high energy conformation (238) we find to be around 70 kcal/mol above a calculated low energy ligand conformation.

Shown in Figure 4 is a simplified view of efforts to dock β-apo-13-carotenone with this agonist-bound form of RXRα. In white is the structure of the bound 9-cis-RA crystallized in the RXRα ligand binding site. In yellow is the best pose found by Surflex-Dock in attempts to dock β-apo-13-carotenone into this binding site when it was built in a conformation resembling that of the 9-cis-RA.

47 The RXR protein structure has been removed to facilitate observing the very different position β-apo-13-carotenone assumes in the docking experiment. It should be noted that a similar result was obtained if the β-apo-13-carotenone conformation from Figure 3 was docked (not shown) or even when redocking of the extracted 9-cis-RA was attempted

(not shown), perhaps reflecting the effects of the highly strained structure of the bound 9- cis-RA. Regardless, the results of these modeling experiments suggest that β-apo-13- carotenone should be capable of functioning as an antagonist to the RXRα protein.

2.4 - IMPLICATIONS

The biological activities of BACs are well understood in plant biology but there is limited information regarding their role in mammals. β-Ionone is a pollinator attractant and β- cyclocitral and β-ionone contribute to fruit or vegetable flavor (156, 157). β-apo-13-

Carotenone blocks the growth of root hairs by interfering with PIN2-mediated auxin transport (155). Also, this compound is believed to be the precursor of the well-known fungal pheromone trisporic acid (244). Previous work by others has demonstrated that β- apo-14’-carotenal can function to inhibit RXRα at 10 µM concentrations (152). The results reported here suggest that β-apo-13-carotenone, the other product of the oxidative cleavage of β-carotene at the 13,14 double bond may affect gene expression mediated by

RXRs in mammals. The pathways for the production of these cleavage products are not fully known and may involve enzymatic or non-enzymatic processes. These compounds are also likely present in the plant-derived foods consumed by mammals.

48 2.5 FIGURES & TABLES

Figure 2.1 The cleavage products of β-carotene. β-carotene can be cleaved either symmetrically (denoted as “e” cleavage) by β,β-carotene-15,15′-oxygenase (BCO1) yielding all-trans-retinaldehyde that can be further oxidized to all-trans-retinoic acid

(ATRA) by retinal dehydrogenases. The second pathway of β-carotene cleavage is called the eccentric cleavage (denoted as “a”, “b”, “c”, “d” cleavages) and it occurs at double bonds other than the central 15,15′ double bond of the polyene chain of β-carotene to produce β-apo-carotenals and β-apo-carotenones with different chain lengths.

50 Compound % Activation of 9-cis-RA alone β-Cyclocitral (“a” cleavage) 83 β-Cyclogeranic acid (“a” cleavage) 93 β-Ionone (“b” cleavage) 83, 106 β-Ionylideneacetaldehyde (“c” cleavage) 87, 98 β-Ionylideneacetic acid (“c” cleavage) 99 β-Apo-13-carotenone (“d” cleavage) 7, 9 Retinaldehyde (“e” cleavage) 31, 78 β-Apo-14′-carotenal (“d” cleavage) 75, 75 β-Apo-14′-carotenoic acid (“d” cleavage) 87 β-Apo-12′-carotenal (“c” cleavage) 69, 114 β-Apo-12′-carotenoic acid (“c” cleavage) 96 β-Apo-8′-carotenal (“a” cleavage) 73, 78 β-Apo-8′-carotenoic acid (“a” cleavage) 73, 84, 86

Table 2.1 Inhibition of 9-cis-retinoic acid induced transactivation of RXRα by β- apocarotenoids. Transactivation assays were carried out in the presence of 10 µM 9-cis-

RA and 10 µM of the indicated β-apocarotenoids in triplicate wells. Results are expressed as the percent activation observed with 9-cis-RA alone that is defined as 100%. For most compounds two or more independent experiments were conducted.

Figure 2.2 The antagonistic effect of β-apo-13-carotenone on RXRα. Dose response curve of 9-cis-RA alone (♦), and increasing concentrations of β-apo-13-carotenone: 10−9

M (●), 10−8 M (×), 10−7 M (∆), and 10−6 M (■). The fold induction of 10−5 M 9-cis-RA

(the absolute fold induction of this point was 2.29 over the vehicle treated cells) was set to 100% and the other experimental points were calculated relative to this.

Figure 2.3 Docking of β-apo-13-carotenone with the RXRα tetramer. Shown is the structure of the ligand-binding domain of the tetrameric RXRα (in green) from PDB entry 1G5Y with: (a) the bound crystallized all-trans-retinoic acid (ATRA) isomer (in white) and the Surflex-Dock docked structure of β-apo-13-carotenone (yellow) when built in a conformation resembling that of the bound ATRA; (b) same as in (a) with the protein helix 3 removed to facilitate visualization of the quality of docking in the binding site; (c) as in (a) with the RXR protein removed to show details of the similarity of the crystallized ATRA ligand and the docked pose of β-apo-13-carotenone.

Figure 2.4 Docking of β-apo-13-carotenone with the RXRα dimer. Shown is the structure of 9-cis-retinoic acid (9-cis-RA) (in white) crystallized in the binding site of the

RXRα from PDB entry 1FBY, along with the docked structure of β-apo-13-carotenone

(in yellow) in the ligand binding site after its construction in a conformation that resembles that of the bound 9-cis-RA (the protein structure has been removed to facilitate appreciation of the significant difference between the bound and docked structures).

CHAPTER 3 (*): NATURALLY-OCCURRING ECCENTRIC CLEAVAGE

PRODUCTS OF PROVITAMIN A β-CAROTENE FUNCTION AS

ANTAGONISTS OF RETINOIC ACID RECEPTORS

* Eroglu, A. et al. Naturally-occurring eccentric cleavage products of provitamin A carotene β-carotene function as antagonists of retinoic acid receptors. Journal of Biological Chemistry 287, 15886-15995 (2012).

55 ABSTRACT

Importantly however, β-apo-14’-carotenal, β-apo-14’-carotenoic acid, and β-apo-13- carotenone antagonized ATRA-induced transactivation of RARs. Competitive radioligand binding assays demonstrated that these putative RAR antagonists compete directly with retinoic acid for high affinity binding to purified receptors. Molecular modeling studies confirmed that β-apo-13-carotenone can directly interact with the ligand binding site of the retinoid receptors. β-Apo-13-carotenone and the β-apo-14’- carotenoids inhibited ATRA-induced expression of retinoid responsive genes in Hep G2 cells. Finally, we developed an LC/MS method and found 3-5 nM β-apo-13-carotenone was present in human plasma. These findings suggest that β-apocarotenoids function as naturally-occurring retinoid antagonists. The antagonism of retinoid signaling by these metabolites may have implications for the activities of dietary β-carotene as a provitamin

A and as a modulator of risk for cardiovascular disease and cancer.

56 3.1. INTRODUCTION

Capability for the synthesis of compounds with vitamin A activity is limited to plants and microorganisms (245). It has been known since the 1930’s that cleavage of the central double bond of β-carotene by vertebrates gives rise to retinaldehyde (vitamin A aldehyde) (6, 7, 94) which can subsequently be reduced to retinol (vitamin A alcohol) or oxidized to all-trans-retinoic acid (vitamin A acid) (55). In mammals, ATRA functions as a hormone agonist for the retinoic acid receptor family of nuclear transcription factors and directly activates several hundred genes which contain retinoic acid response elements (RAREs) in their promoters (51, 159); it is this global effect on the regulation of gene transcription that renders vitamin A essential for embryonic development, growth, and differentiation in mammals, including humans. β-Carotene is cleaved eccentrically at double bonds other than the central one to yield β-apocarotenals and β-apocarotenones

(86, 122, 240), molecules that have been detected in foods (128) and in the blood of both humans (130) and animals (131), but whose function in these is unknown. Here we show that some of these compounds (particularly β-apo-14’-carotenal, β-apo-14’-carotenoic acid, and β-apo-13-carotenone) function as antagonists of retinoic acid receptors α, β, and γ and block the ATRA-induced activation of endogenous genes that contain RAREs in their promoters. Moreover, these molecules directly compete for ATRA binding to all receptor subtypes and in the case of β-apo-13-carotenone, the binding affinity is in the nanomolar range and comparable to ATRA itself.

57 Thus, depending on the extent of oxidative cleavage at its various double bonds, dietary

β-carotene can produce differing proportions of both agonists and antagonists of retinoic acid receptors. This Janus face may account for the unexpected and negative effects of large doses of β-carotene used in human clinical trials.

3.2 EXPERIMENTAL PROCEDURES

Cell lines – Cos-1 cells from ATCC (Rockville, MD) were cultured in DMEM supplemented with 10% FBS. Hep G2 cells were cultured in MEM supplemented with

10% FBS. Cells were maintained at 37°C with 5% CO2 in air.

Characterization of β-apocarotenoids – Compounds were characterized by a mix of 1H

(400 MHz) and 13C (100 MHz unless noted) NMR spectroscopy, UV and mass (ESI) spectrometry and HPLC analysis (Polaris C18 analytical column with 1 ml/min methanol:water of appropriate ratio, all compounds were determined to be at least 94% pure). Essential compound data follows and procedures for their synthesis are provided below in Results and Discussion.

β-Cyclocitral: HRMS calculated for C10H16O (M+Na) 175.1099, observed 175.1101.

1 β-Cyclogeranic acid: H NMR (CD3COCD3) δ 1.14 (6H, s), 1.45-1.49 (2H, m), 1.66-

1.71 (2H, m), 2.01-2.04 (2H, m), 2.08 (3H, m); UV (ethanol) λmax 295 nm (ε 7,720);

HRMS calculated for C10H16O2 (M+Na) 191.1048, observed 191.1041.

58 β-Ionone: HRMS calculated for C10H16O (M+H) 193.1592, observed 193.1579.

1 β-Ionylideneacetaldehyde: H NMR (CDCl3) δ 1.05 (6H, s), 1.46-1.55 (2H, m), 1.60-

1.67 (2H, m), 1.74 (3H, s), 2.03-2.10 (2H, m), 2.39 (3H, s), 5.96 (1H, d, J = 8 Hz), 6.23

(1H, d, J = 16 Hz), 6.74 (1H, d, J = 16 Hz), 10.14 (1H, d, J = 8 Hz); UV (ethanol) λmax

272 nm (ε 14,800); HRMS calculated for C12H22O (M+H) 219.1749, observed 219.1737.

1 β-Ionylideneacetic acid: H NMR (CDCl3) δ 1.07 (6H, s), 1.50-1.53 (2H, m), 1.63-1.69

(2H, m), 1.75 (3H, s), 2.06-2.09 (2H, m), 2.36 (3H, s), 5.85 (1H, s), 6.23 (1H, d, J = 16.2

Hz), 6.67 (1H, d, J = 16.2 Hz), 10.14 (1H, d, J = 8 Hz); UV (ethanol) λmax 296 nm (ε

20,000); HRMS calculated for C12H22O2 (M+H) 235.1698, observed 235.1688.

1 β-apo-13-Carotenone: H NMR (CDCl3) δ 0.98 (6H, s), 1.41-1.44 (2H, m), 1.54-1.60

(2H, m), 1.67 (3H, s), 1.99-2.00 (2H, m), 2.01 (3H, s), 2.25 (3H, s), 6.08-6.15 (3H, m),

13 6.37 (1H, d, J = 16 Hz), 7.53 (1H, dd, J = 15, 11.9 Hz); C NMR (CDCl3; 75 MHz) δ

13.10, 19.14, 21.72, 27.63, 28.93, 33.14, 34.25, 39.59, 127.67, 129.27, 130.98, 131.27,

136.68, 137.48, 139.30, 145.55, 198.48; UV (ethanol) λmax 341 nm (ε 25,300); HRMS calculated for C18H26O (M+Na) 281.1881, observed 281.1859.

1 β-apo-14’-Carotenal: H NMR (CDCl3) δ 1.00 (6H, s), 1.40-1.44 (2H, m), 1.54-1.60

(2H, m), 1.70 (3H, s), 1.93-2.04 (2H, m), 2.00 (3H, s), 2.07 (3H, s), 6.10-6.44 (6H, m),

6.90 (1H, dd, J = 14.9, 11.5 Hz), 7.50 (1H, dd, J = 14.9, 11.5 Hz), 9.58 (1H, d, J = 7.9

13 Hz); C NMR (CDCl3) δ 13.42, 19.26, 21.61, 27.57, 29.19, 33.55, 34.46, 40.10, 127.59,

59 128.06, 130.12, 130.95, 133.89, 136.31, 136.63, 137.43, 137.63, 137.99, 143.71, 190.39;

UV (ethanol) λmax 402 nm (ε 55,000); HRMS calculated for C22H30O (M+Na) 333.2194, observed 333.2190.

1 β-apo-14’-Carotenoic acid: H NMR (CDCl3) δ 1.05 (6H, s), 1.46-1.54 (2H, m), 1.61-

1.71 (2H, m), 1.77 (3H, s), 2.06 (3H, s), 2.06-2.10 (2H, m), 2.13 (3H, s), 5.92 (1H, d, J =

15.0 Hz), 6.13-6.32 (4H, m), 6.39 (1H, d, J = 15.1 Hz), 6.90 (1H, dd, J = 15.0, 11.5 Hz),

13 7.88 (1H, dd, J = 15.1, 11.5 Hz); C NMR (CDCl3) δ 13.65, 19.63, 22.19, 29.38, 33.53,

34.68, 40.01, 119.46, 128.58, 129.57, 130.37, 136.13, 137.81, 138.16, 139.23, 143.19,

146.14, 173.21 UV (ethanol) λmax 378 nm (ε 52,200); HRMS calculated for C22H30O2

(M+Na) 349.2143, Observed 349.2136.

1 β-apo-12’-Carotenal: H NMR (CDCl3) δ 1.00 (6H, s), 1.45-1.43 (2H, m), 1.65-1.57

(4H, m), 1.69 (3H, s), 1.85 (3H, s), 1.99 (3H, s), 2.02 (3H, s), 6.14 (3H, m), 6.19 (1H, dd,

J = 13.8, 14.2 Hz), 6.36 (1H, d, J = 11.9 Hz), 6.68 (1H, d, J = 11.8 Hz), 6.77 (1H, dd, J =

14.2, 13.8 Hz), 6.92 (1H, dd, J = 13.8, 11.6 Hz), 7.00 (1H, d, J = 11.6 Hz), 9.42 (1H, s);

13 C NMR CDCl3) δ 10.12, 12.74,13.27, 20.24, 22.18, 25.49, 29.38, 33.53,34.68, 40.02,

127.31, 127.63, 127.66, 130.47, 130.81, 136.31, 136.62, 137.53, 137.67, 137.89, 138.22,

141.38,149.47,194.38; UV (methanol) λmax 426 nm (ε 75,600 ); HRMS calculated for

C25H34O (M+H) 351.2682, observed 351.2689.

1 β-apo-12’-Carotenoic acid: H NMR (CDCl3) δ 1.05 (6H, s), 1.47-1.50 (2H, m), 1.60-

1.65 (2H, m), 1.74 (3H, s), 2.00-2.03 (11H, m), 6.14-6.22 (3H, m), 6.27 (1H, d, J = 12.2

60 Hz), 6.36 (1H, d, J = 14.9 Hz), 6.52 (1H, dd, J = 13.7, 12.4 Hz), 6.74 (1H, dd, J = 14.9,

13 11.5 Hz), 6.93 (1H, d, J = 12.2 Hz), 7.43 (1H, d, J = 11.5 Hz); C NMR (CDCl3) δ

12.84, 13.25, 19.65, 22.19, 29.38, 33.53, 34.68, 40.03, 125.53, 127.51, 127.94, 128.04,

130.11, 130.86, 131.39, 136.92, 137.38, 137.98, 138.24, 141.09, 141.37, 173.92; UV

(ethanol) λmax 407 nm (ε 67,000); HRMS calculated for C25H34O2 (M+Na) 389.2457, observed 389.2463.

1 β-apo-10’-Carotenal: H NMR (CD3COCD3) δ 1.01 (6H, s), 1.46-1.43 (2H, m), 1.62-

1.58 (2H, m), 1.68 (3H, s), 1.96 (6H, s), 1.87 (3H, s), 2.02 (2H, m), 6.09 (1H dd, J = 9.4,

7.7 Hz), 6.19 (2H, m), 6.35 (2H, m), 6.74 (2H, m), 6.86 (1H, dd, J = 11.5, 11.5 Hz), 7.27

13 (1H, d, J = 7.6 Hz), 7.33-7.30 (2H, m), 9.55 (1H, d, J = 7.7 Hz); C NMR (CD3COCD3)

δ 11.95, 12.08, 21.22, 28.54, 28.89, 29.08, 29.27, 29.47, 29.66, 32.86, 39.57, 126.56,

126.85, 127.04, 127.30, 128.15, 129.08, 131.14, 132.00, 135.32, 137.11, 137.98, 140.97,

156.16, 192.91; UV (hexane) λmax 438 nm (ε 73,100 ); HRMS calculated for C27H36O

(M+Na) 399.2664, observed 399.2664.

1 β-apo-10’-Carotenoic acid: H NMR (CDCl3) δ 1.00 (6H, s), 1.42-1.45 (2H, m), 1.56-

1.62 (2H, m), 1.69 (3H, s), 1.91 (3H, s), 1.98-2.00 (8H, m), 5.82 (1H, d, J = 15.3 Hz),

6.09-6.19 (3H, m), 6.36 (1H, d, J = 14.9 Hz), 6.22 (1H, d, J = 11.7 Hz), 6.31 (1H, d, J =

14.9 Hz), 6.53 (1H, dd, J = 13.2, 11.9, Hz), 6.70 (1H, dd, J = 12.4, 11.7 Hz), 6.78 (1H, d,

13 J = 12.4 Hz) 7.40 (1H d, J = 15.3 Hz); C NMR (CDCl3) δ 12.98, 13.24, 13.39, 18.81,

19.66, 22.20, 29.39, 31.36, 33.54, 34.69, 40.03, 115.64, 126.99, 127.74, 128.74, 128.94,

130.05, 130.99, 131.97, 133.67, 134.94, 137.13, 137.55, 138.03, 138.26, 139.88, 140.89,

61 151.46, 173.20; UV (ethanol) λmax 407 nm (ε 67,000). HRMS calculated for C27H36O2

(M+Na) 415.2613, observed 415.2608.

1 β-apo-8’-Carotenoic acid: H NMR (CDCl3) δ 1.07 (6H, s), 1.49-1.52 (2H, m), 1.60-

1.69 (2H, m), 1.76 (3H, s), 1.94 (3H, s), 2.02-2.07 (2H, m), 2.02 (3H, s), 2.04 (3H, s),

6.15-6.23 (3H, m), 6.31 (1H, d, J = 11.6 Hz), 6.40 (1H, dd, J = 14.8 Hz), 6.49 (1H, d, J =

11.6 Hz) 6.64-6.84 (5H, m), 6.98 (1H, dd, J = 10.7 Hz); UV (ethanol) λmax 441 nm (ε

108,700); HRMS calculated for C30H40O2 (M+Na) 455.2926, observed 455.2944.

Retinoids and other materials – All-trans-retinoic acid, retinal, retinyl acetate, and 13- cis-retinoic acid were from Sigma (St. Louis, MO). 9-cis-RA was obtained from Enzo

Life Sciences. RARβ/γ selective antagonist CD 2665 was from Tocris Bioscience

(Ellisville, MO). All-trans-[3H]RA (50.8 Ci/mmol), and 9-cis-[3H]RA (52.9 Ci/mmol) were purchased from American Radiolabeled Chemicals (St. Louis, MO), and Perkin-

Elmer Corp (Boston, MA) respectively. Recombinant proteins including RARα, RARβ,

RARγ, and RXRα were from Active Motif (Carlsbad, CA). Probes and primers for

TaqMan assays were from Applied Biosystems (Carlsbad, CA).

Transactivation assays – Cos-1 cells were transfected with plasmids in serum-free medium using Lipofectamine 2000 reagent (Invitrogen). Reporter vectors used were renilla luciferase under the control of the thymidine kinase promoter and firefly luciferase under the control of a retinoic acid response element (RARE). Plasmids with cDNAs for retinoic acid receptors α, β, and γ were cotransfected in individual experiments. Four 62 hours after transfection, cells were treated with test compounds that were dissolved in ethanol or with 0.1% ethanol alone for an additional 24 h. Cell lysates were then assayed for luciferase activities using a GloMax 96 Microplate Luminometer (Promega) and the

Dual Luciferase Reporter (DLR) assay system (Promega). For each experiment, the firefly luciferase activity (experimental reporter) was normalized to renilla luciferase

(control reporter) activity. The change in normalized firefly luciferase activity was calculated relative to that for cells that were transfected with vehicle (ethanol), which was set as 1. In some experiments we used the human RARγ stably-transfected reporter cell system from Indigo Biosciences, Inc. (State College, PA) according to the supplier’s directions. These cells show a much more robust response of ATRA-induced reporter gene expression than the transiently transfected cells.

Ligand-Binding Assays – Purified human recombinant retinoid receptors were used at 50 fmol with a non-retinoid-binding protein (keyhole limpet hemocyanin) to maintain ~0.1 mg protein/mL to prevent loss of receptor protein and ligand during the course of binding experiments. For binding analyses, proteins and ligands were incubated at 4°C for 16h.

For equilibrium saturation binding assays, proteins were incubated with various concentrations of all-trans-[3H]RA for RARs and 9-cis-[3H]RA for RXRα and in the absence or presence of a 100-fold excess of unlabeled compound to determine nonspecific binding. Specific binding was determined by a hydroxyapatite-binding assay

(246). The quantity of labeled compound bound was determined by liquid scintillation counting. For competitive binding assays, a constant amount of all-trans-[3H]RA (5 nM) for RARs or 9-cis-[3H]RA (10 nM) for RXRα was incubated with test compounds (from 63 -6 -10 10 M to 10 M). Apparent dissociation constants (Kd) and inhibition constants (Ki) were determined using GraphPad Prism Version 5.0 with non-linear regression.

Gene expression Assays - Hep G2 cells were treated with test compounds for 4 h and total RNA was isolated from each well and subjected to reverse transcription.

Quantitative PCR was carried out using TaqMan chemistry for human CYP26A1

(Hs01084852_g1), and human RARβ (Hs00977137_m1)) as target genes and human

GAPDH as a housekeeping gene. The comparative Ct method (ΔΔCt) was used to quantify the results obtained by real-time RT-PCR.

Molecular modeling – Structures were displayed and modeled on a Silicon Graphics O2 running Sybyl v.7.1 (Tripos). Structures were minimized using Sybyl’s Maximin method and docked to the RARβ ligand binding domain crystal structure (PDB entry: 1xap) using the available FlexX routine (247).

Quantitative HPLC/MS analysis of β-apo-13-carotenone in human plasma – Blood plasma was purchased from Innovative Research (Novi, MI). Fresh blood plasma was prepared from six individuals over the course of two weeks, shipped on dry ice to Ohio

State University, and stored at -80°C before extraction. For each individual, 1 mL of plasma was split among 5 glass tubes (200 µL per tube). Ethanol (1 mL) was added to each tube, and the samples were sonicated using an Ultrasonic Dismembrator (Model

150E, Fisher Scientific). Hexanes (10 mL) were added to each tube and again the mixture was sonicated. The tubes were centrifuged for 5 min at 300g to facilitate phase 64 separation. The upper organic layers from each vial were pooled into a single vial, and the extraction with hexanes repeated a second time. The pooled extracts were dried under nitrogen gas. The samples were redissolved in 200 µL 1:1 methyl t-butyl ether/methanol and analyzed using HPLC-MS/MS. HPLC was conducted on a C30 column (4.6x150mm, s5, YMC) in reversed phase with (A) methanol/0.1% formic acid (80/20 v/v) and (B) methanol/0.1% formic acid/methyl t-butyl ether (20/2/78 v/v/v) as mobile phase solvents at 1.8 mL/min and 35 °C (Agilent 1200 SL, Agilent Technologies, Santa Clara, CA).

Eluent was introduced to a triple quadrupole mass spectrometer (QTrap 5500, AB Sciex,

Concord, Canada) via an atmospheric pressure chemical ionization probe operated in positive ion mode. Three MS/MS transitions monitored for β-apo-13-carotenone were m/z 259>175, 119 and 69 at collision energies of 21, 31, and 27 eV respectively with

150ms dwell times and using nitrogen as CAD gas. Other MS parameters included: curtain gas 10, heated nebulizer temperature 425 °C, nebulizer current 5 µA, declustering potential 60, entrance potential 10, and exit potential 11. Calibrating solutions of β-apo-

13-carotenone were prepared in 1:1 methyl t-butyl ether/methanol with concentrations based on an extinction of 25,300 at 368 nm in ethanol. The dried residue of serum extract was redissolved in 100 µL of methyl t-butyl ether then 100 µL of methanol added with mixing before centrifuging prior to injection (10 µL). For quantitation, the m/z 259>175 transition was used as it had superior signal:noise and the other two transitions were used for qualitative purposes to confirm peak identity. The limit of detection for β-apo-13- carotenone was 280 pM. We have found that stock solutions of β-apo-13-carotenone in ethanol are stable for 2.5 years at -30ºC. The compound is stable in plasmas frozen at -80

C for periods of at least two weeks. 65 3.3 RESULTS & DISCUSSION

Synthesis of β-Apocarotenoids – Although the occurrence and biological activity of selected β-apocarotenoids in mammals has been reported in various systems, progress has been hampered because many of the compounds are not available commercially. In order to comprehensively assess the activity of β-apocarotenoids we first undertook to purify or synthesize all of the possible eccentric cleavage products of β-carotene (See Fig. 3.1).

Retinal, retinoic acid, and β-apo-8’-carotenal (Fluka) were used as obtained. β-

Cyclocitral and β-ionone (both Sigma-Aldrich) were purified by preparative TLC prior to use. Eleven of the β-apocarotenoids were synthesized by standard organic chemical transformations including oxidation of aldehydes to acids (234), Wadsworth–Emmons and Wittig homologation, and reduction of esters to acids followed by oxidation to aldehydes (See Fig. 3.2).

β-Apocarotenoids antagonize retinoic acid-induced expression of reporter and endogenous retinoic acid-responsive genes – We first screened all of the compounds for their potential to activate RARα, RARβ, and RARγ, using Cos-1 cells transiently transfected with cDNAs for the individual RARs and with an RARE-luciferase reporter.

None of the compounds was as effective as the pan-agonist, ATRA, in activating the

RARs and indeed most showed no agonist activity at all (See Fig. 3.3). The slight activity of some of the longer chain β-apocarotenoids at high concentration is consistent with previous reports (138, 152, 248). We then used the same transactivation assay to screen all of the compounds for their potential to antagonize the ATRA-induced activation of the

66 individual RARs by treating the cells with maximally effective doses of ATRA and equimolar concentrations of the β-apocarotenoids (See Fig. 3.4).

While the shorter products of the eccentric cleavage of β-carotene had little or no effect on ATRA-induced transactivation, β-apo-10’-carotenoic acid and β-apo-12’-carotenoic acid both led to 40-50% inhibition of ATRA-induced activation of all three RAR isoforms. Even more striking inhibition was observed for the products of the “d” cleavage

(See Fig. 3.1) of β-carotene (viz., β-apo-14’-carotenal, β-apo-14’-carotenoic acid, and β- apo-13-carotenone), with the greatest inhibitory activity being displayed by β-apo-13- carotenone. Thus, it appeared that these five β-apocarotenoids could be functioning as

RAR antagonists.

In order to characterize the antagonist function β-apo-13-carotenone more quantitatively we used stably-transfected RARγ reporter cells. Cells were treated with ATRA in a concentration range of 0.5 nM to 3 µM in the absence or presence of fixed concentrations

(1, 10 or 100 nM) of β-apo-13-carotenone (See Fig. 3.5a). We observed a progressive shift in the ATRA dose-response curve with increasing concentrations of β-apo-13- carotenone in the nanomolar range. Higher concentrations of ATRA were able to overcome inhibition by β-apo-13-carotenone, suggesting direct competition between the two compounds for binding. This suggestion is supported by the results of competitive radioligand binding assays and molecular modeling discussed below.

67 We then asked whether β-apo-13-carotenone and the β-apo-14’-carotenoids would antagonize the ATRA-induced transcription of endogenous genes. For these experiments, we treated Hep G2 cells in culture with ATRA for 4 hours and measured the mRNA levels for RARβ and cytochrome P450-26A1 (CYP26A1). Both of these genes have canonical RAREs in their promoters and their transcription is directly upregulated by

ATRA treatment (174, 249).

As shown in Fig. 3.5b, treatment with 10 nM ATRA led to 9-fold induction in RARβ mRNA levels and ~20-fold increases on CYP26A1 mRNA levels. Treatment with 10 nM

BC, β-apo-13-carotenone, or other β-apocarotenoids expectedly did not markedly induce expression of either gene (data not shown). However, co-treatment with ATRA and β- apo-13-carotenone or the β-apo-14’-carotenoids led to marked inhibition of the ATRA- induced gene expression (See Fig. 3.5b). Importantly, the inhibition by β-apo-13- carotenone was greater than that for β-apo-14’-carotenal or β-apo-14’-carotenoic acid.

This is in keeping with the greater affinity of -apo-13-carotenone for the RARs than that of the β-aop-14’-carotenoids. Co-treatment of the cells with ATRA and the parent compound (BC) or with a β-apocarotenoid that does not antagonize ATRA-induced transactivation (viz. β-ionylideneacetic acid) had no effect on ATRA-induced gene expression in HepG2 cells.

β-Apocarotenoids bind to the ligand binding site of RARs with high affinity – Retinoic acid receptors (like other type II nuclear receptors) function in the regulation of

68 endogenous gene expression by binding as heterodimers with retinoid X receptors (159,

250). The heterodimers bind to specific response elements (RAREs and RXREs) in the promoter regions of genes via their respective DNA-binding domains. In the unliganded state the transcription factor complex binds to nuclear co-repressors and transcription is repressed. Binding of ATRA (or other agonist) to the ligand-binding domain of RAR induces a conformational change in the RAR (at helix 12) and this leads to co-repressors dissociating from the receptor and the unmasking of a co-activator binding site (250). In the case of RAR-RXR heterodimer the binding of an agonist to RXR alone does not lead to activation, but the binding of an RXR agonist in concert with an RAR agonist leads to supraactivation of transcription (250).

We wanted to know whether the β-apocarotenoids that demonstrated an antagonist

“activity” in the cell-based assays did so by directly binding to the RAR ligand-binding domain and competing for ATRA binding. Thus, we conducted radioligand binding assays using purified recombinant RARα, RARβ, and RARγ and tritium-labeled ATRA in the presence of increasing concentrations of unlabeled ATRA (as a positive control),

CD 2665 (a synthetic antagonist of RARβ/γ known to bind to the ligand binding site), retinyl acetate (a retinoid that does not bind and used as a negative control), and the selected β-apocarotenoids (See Fig. 3.6a). For all three receptors the three β- apocarotenoids with the highest antagonist activity competed for ATRA binding. In fact,

β-apo-13-carotenone displayed the same affinity for the RARs as ATRA itself (i.e., 2-6 nM) (See Fig. 3.6b). The affinity of binding for β-apo-14’-carotenal and β-apo-14’-

69 carotenoic acid to RARs was in the 15-60 nM range, while those of β-apo-10’- and β- apo-12’-carotenoic acids were greater than 300 nM.

The high affinity binding of β-apo-13-carotenone to the ligand binding site of RARβ was also demonstrated by the results of molecular modeling studies. We displayed the crystal structure of RARβ with the retinoid agonist TTNPB in the binding site (PDB entry:1xap),

“extracted” the ligand computationally, and then attempted to dock both ATRA and β- apo-13-carotenone into the binding site using FlexX (247). Both molecules docked smoothly (See Fig. 3.6c left); indeed the RMSD for β-apo-13-carotenone was very slightly better than for ATRA. Moreover, the crystal conformation of TTNPB and the energy-minimized and then docked conformations of ATRA and β-apo-13-carotenone had nearly identical RMSD values (See Fig. 3.6c right).

The studies reported here demonstrate that the products of the oxidative eccentric cleavage of β-carotene at the C13-C14 double bond yields products that are antagonists of RARs and that the most active molecule in this regard is β-apo-13-carotenone. We previously showed that this compound was also the most effective β-apocarotenoid in inhibiting the transactivation of RXRα by its agonist, 9-cis-retinoic acid, and molecular modeling studies demonstrated that it could potentially bind to the transcriptionally silent tetramer of RXRα (251). Therefore, we tested the three β-apocarotenoids resulting from cleavage of the C13-C14 double bond of β-carotene for their binding to purified recombinant RXRα (See Fig. 3.7). The β-apo-13-carotenone competed for 9-cis-retinoic

70 acid binding with an affinity (7-8 nM) identical to 9-cis-retinoic acid itself. The affinities of β-apo-14’-carotenal and β-apo-14’-carotenoic acid for RXRα were greater than 250 nM in keeping with their lack of effect on inhibiting RXR transactivation. Given that a number of nuclear receptors form heterodimers with RXR and that ligand binding (either agonist or antagonist) to the RXR leads to modulation of the transcriptional activity of the heterodiomers (250), these eccentric cleavage products of β-carotene could have complex global effects on gene expression.

The most potent β-apocarotenoid antagonist of RARs, β-apo-13-carotenone, is found in human plasma at concentrations that are biologically significant – Although the mechanisms responsible for the formation of the eccentric cleavage products of β- carotene in mammals are not fully known, it is clear that some of the long-chain β- apocarotenals (e.g., 8’, 10’, 12’, 14’) are found in the plasma of humans (130) and experimental animals (131) and that these are increased under conditions of oxidative stress and high dietary doses of β-carotene (252). We have also found that all of these β- apocarotenals and, specifically, β-apo-13-carotenone are present in fresh cantaloupe and orange-fleshed melons (128); thus these compounds may be absorbed directly from the diet.

In order to further establish the relevance of β-apo-13-carotenone’s potent antagonist activity on retinoid receptors, we developed sensitive HPLC/MS procedures for its detection in human plasma. Figure 3.8 shows the analysis of human plasma and authentic

71 standards by HPLC/MS. We used multiple reaction monitoring to insure the specificity and sensitivity of the assay. We then analyzed the plasmas of six free-living individuals and found the plasma concentration of β-apo-13-carotenone to be 3.8 +/- 0.6 nM.

Importantly, this is in the range of normal concentrations of retinoic acid in plasma and approximately the same as the binding constant of the compound for the retinoid receptors. This would suggest that β-apo-13-carotenone can function at physiological concentrations as an endogenous modulator of retinoid signaling in humans.

3.4 CONCLUSIONS AND IMPLICATIONS

Our results demonstrate that β-carotene can generate both RAR agonists (ATRA) and

RAR antagonists (β-apo-14’-carotenal and β-apo-13-carotenone) depending on the extent of cleavage at the central C15-C15’ double bond or the C13-14 double bond, respectively. These findings may have implications for the unexpected and negative effects of high doses of β-carotene in human clinical trials of cancer prevention (253). An example is the now-famous CARET trial which, based on observational epidemiology, explored whether supplemental β-carotene would decrease incidence of lung cancer in a highly susceptible population, namely smokers and asbestos workers (137, 254).

Surprisingly, the supplemented subjects had a higher incidence of disease and the trial had to be halted early. It was apparent that the doses of β-carotene used in the trial (30 mg/day) were much higher than the range of normal dietary intakes associated with a decreased risk of disease in the observational studies (253).

72 The possible mechanisms involved were explored in elegant studies employing a novel animal model, the smoking ferret (252, 255). These studies revealed that under conditions of high dietary β-carotene and the oxidative stress of smoking there was a clear increase in preneoplastic lung cancer lesions in the animals. The authors concluded that oxidative stress led to increased eccentric cleavage of β-carotene and that the mixture of cleavage products led to disruption of retinoid-signaling via indirect mechanisms. The present work demonstrates that specific β-apocarotenoids exert an anti-vitamin A activity by directly interacting with RARs as high-affinity antagonists. Our analyses of both β- carotene-containing animal diets and fruits containing β-carotene suggest that any dietary source of β-carotene also contains β-apocarotenoids. It may also be useful to consider these findings in attempts to alleviate vitamin A deficiency in humans through the biofortification of crops with high levels of β-carotene.

73 3.5 FIGURES

Figure 3.1 β-Apocarotenoids. Structures of the β-apocaroteniods synthesized (indicated by [S]), purified and characterized for this study. R = CHO in the carotenals and R =

COOH in the carotenoic acids.

75 Figure 3.2 Chemical synthesis of all possible β-apocarotenoids. Reagents and conditions used in the synthesis of the various compounds are shown in lower case roman numerals and yields are shown in parentheses. i) 5N KOH/EtOH (+/- benzene), rt, 12 h

(99%, BIAA; 96%, 12’-CA; 92%, 10’-CA; 94%, 14’-CA); ii) LAH, THF, rt, 45 min (for compound 2, 9); DIBAL-H, CH2Cl2, rt, 30 min (for compound 6); iii) MnO2, Celite,

CH2Cl2, rt, 4 h (73%, BIA; 36%, 10’-AL; 6%, 14’-AL); iv)

(triphenylphosphoranylidene)-2-propanone, toluene, reflux, 12 h (61%); v) NaH, dialdehyde shown, CH2Cl2, 0 °C to rt, 48 h (59%); vi) KCN, CH3COOH, MnO2, MeOH, rt, 90 h (21%, 4; 2%; 10); vii) NaH, triethylphosphonoacetate, THF, 0 °C to rt, 48 h

(83%, 5; 94%, 1; 74%, 8); viii) O2, CH2Cl2, 48 h (quant.).

77 Figure 3.3 β-Apocarotenoids do not transactivate retinoic acid receptors. Histograms of activation of RARE-reporter genes in cells transfected with retinoic acid receptors α

(left), β (middle) and γ (right). Normalized fold-activation relative to vehicle-treated cells is shown for all-trans-retinoic acid (left most bar in each histogram) or the β- apocarotenoids resulting from cleavage at the “a”, “b”, “c”, or “d” sites from top to bottom, respectively. Compounds were tested individually at 10-5 M. n = 3 to 6; mean +/- s.d. Compound abbreviations are given in Fig. 3.1.

78 10 RARα 10 RARβ 10 RARγ

8 8 8 BCA BCA BCL BCL 8'-AL 8'-AL 8'-CA 8'-CA BCA BCA 8'-AL 8'-AL BCL BCL 8'-CA 8'-CA 8'-AL 8'-AL 8'-CA 8'-CA BCA BCA 6 BCL 6 6

4 4 4

FOLD ACTIVATION ACTIVATION FOLD 2 2 2

0 0 0

RARα RARβ RARγ 6 6 6 BI 10'-AL 10'-AL 10'-CA 10'-CA BI 10'-CA 10'-CA 4 10'-AL 4 4 BI 10'-CA 10'-CA 10'-AL 10'-AL

2 2 2 FOLD ACTIVATION ACTIVATION FOLD ACTIVATION FOLD FOLD ACTIVATION ACTIVATION FOLD

0 0 0

8 8 8 RARα RARβ RARγ 6 6 6 BIAA BIAA BIA BIA 12'-CA 12'-CA 12'-AL 12'-AL BIA BIA BIAA BIAA 12'-CA 12'-CA 12'-AL 12'-AL BIAA BIAA BIA BIA 12'-AL 12'-AL 12'-CA 12'-CA 4 4 4

2 2 2 FOLD ACTIVATION ACTIVATION FOLD

0 0 0

10 RARα 10 RARβ 10 RARγ

8 8 8

6 6 6 14'-AL 14'-AL C13 Ketone C13 Ketone 14'-CA 14'-CA 14'-AL 14'-AL 14'-CA 14'-CA 14'-AL 14'-AL 4 4 C13 Ketone 4 14'-CA

2 2 2 FOLD ACTIVATION ACTIVATION FOLD

0 0 0

79 Figure 3.4 β-Apocarotenoids antagonize ATRA-induced transactivation of retinoic acid receptors. Histograms of activation of RARE-reporter genes in cells transfected with retinoic acid receptors α (left), β (middle) and γ (right). Percent of maximal activation of cells treated with 10-5 M ATRA alone (left most bar in each histogram) or co-treated with 10-5 M ATRA and 10-5 M of the β-apocarotenoids resulting from cleavage at the “a”, “b”, “c”, or “d” sites are shown in a, b, c, and d, respectively. n = 3 to 6; mean

+/- s.d. Compound abbreviations are given in Fig. 3.1.

80 RARα RARβ RARγ BCL BCL BCL BCL BCA BCA BCA BCL BCL 8'-AL 8'-AL 8'-AL 8'-AL BCA BCA 8'-CA 8'-CA 8'-CA 8'-CA 8'-CA 100 8'-AL 100 100

80 80 80

60 60 60

40 40 40

20 20 20 % Activation of ATRA alone ATRA Activation of % 0 0 0

RARγ I RARβ B RARα BI 100 100 BI 10'-AL 10'-AL 10'-AL 10'-AL 10'-AL 10'-CA 10'-CA 10'-CA 100 10'-CA

80 80 80

60 60 60

40 40 40

Activation of ATRA alone ATRA Activation of 20 20

% 20

0 0 0 RARα RARβ RARγ BIA BIA BIA BIA BIA BIA BIAA BIAA 100 BIAA 100 100 BIAA 12'-AL 12'-AL 12'-AL 12'-AL 12'-CA 12'-CA 12'-CA 12'-CA 12'-CA 12'-CA 12'-AL 12'-AL

80 80 80

60 60 60

40 40 40

20 20 20 % Activation of ATRA alone ATRA Activation of % 0 0 0 RARα RARβ RARγ

100 100 100

80 80 80 14'-CA 14'-CA 14'-CA 14'-CA 14'-AL 14'-AL 14'-AL 14'-AL 14'-AL 14'-AL 14'-CA 14'-CA C13 Ketone C13 Ketone C13 Ketone 60 60 60

40 40 40

20 20 20 % Activation of ATRA alone ATRA Activation of %

0 0 0 81 Figure 3.5 β-Apo-13-Carotenone is a potent antagonist of retinoic acid receptor- mediated induction of reporter gene expression and blocks all-trans-retinoic acid

(ATRA) induction of endogenous gene expression. a, Dose response curves for transactivation of RARγ (left-upper panel) by ATRA in the absence () or presence of 1 nM (green ▲), 10 nM (), or 100 nM (blue ▲) C13 ketone. Points shown are the means of 6 determinations for ATRA alone or 3 determinations for each of the curves with C13 ketone. Variations about the means were generally less than 10% except at very low concentrations of ATRA. b, Induction of expression of mRNAs for RARβ (left-lower panel) or cytochrome P450, 26A1 (CYP26A1) (right-lower panel) by 10 nM ATRA treatment alone or by co-treatment with ATRA and the test compounds at 10 nM including b-carotene (BC), b-ionylideneacetic acid (BIAA), β-apo-14’-carotenal (14’-

AL), β-apo-14’-carotenoic acid (14’-CA), and β-apo-13-carotenone (C13 ketone). mRNA levels were quantified by RT-PCR and are shown as the fold induction compared to vehicle treated cells. n = 3; mean +/- s.d.

RARγ 100

25 Percent Response

0 -1 0 1 2 3 [log ATRA] nM ATRA + 100 nM C13 Ketone + 10 nM C13 Ketone + 1 nM C13 Ketone

b 12

24 ATRA b ATRA ATRA ATRA + BIAA + BIAA ATRA ATRA + BC ATRA ) - mRNA ) - mRNA

9 + BC ATRA ATRA + BIAA + BIAA ATRA β 18 RAR ATRA + 14'-AL + 14'-AL ATRA

6 12 + 14'-AL ATRA ATRA + 14'-CA + 14'-CA ATRA ATRA + 14'-CA + 14'-CA ATRA

3 6 ATRA + C13 Ketone ATRA Expression Fold Change ( ATRA + C13 Ketone ATRA Expression Fold Change (CYP26A1) - mRNA Expression Fold Change (CYP26A1) - mRNA

0 0

83 Figure 3.6 β-Apo-13-Carotenone is a high affinity ligand for purified retinoic acid receptors and fits into the ligand binding site. a, Competitive displacement of 5 nM tritiated ATRA from purified RAR proteins by unlabeled ATRA () as a positive control,

C13 ketone (▲), 14’-CA (+), 14’-AL (), and 13-cis-retinoic acid () as a negative control for RARα (left) experiment, CD 2665 (), retinyl acetate () as a negative control for RARβ (middle) and RARγ (right) experiments. Points shown are means of n=3 with a variance of less than 10%. b, Binding affinities (in nM) of β-apocarotenoids to RARs calculated from the data shown in Fig. 3.6a and additional experiments with β- apo-12’- and β-apo-10’-carotenoic acids. For ATRA and the C13 ketone variance shown is for three independent experiments. c, Molecular modeling of the docking of ATRA

(red) and β-apo-13-carotenone (purple) into the ligand binding site (protein backbone in green) of RARβ (PDB entry:1xap)(left). On the right is shown the energy minimized then docked conformations of ATRA (red) and β-apo-13-carotenone (purple) overlaid onto the conformation of the agonist TTNPB (white) as observed in the X-ray structure.

84 Figure 3.6a

RARα RARβ RARγ 100 100 100

80 80 80

60 60 60 H] ATRA bound ATRA H] 3

% [ 40 40 40

20 20 20

0 0 0 -11 -10 -9 -8 -7 -6 -5 -11 -10 -9 -8 -7 -6 -5 -11 -10 -9 -8 -7 -6 -5 log [M] log [M] log [M] Figure 3.6b Binding Affinity – Ki (nM)

β-Apocarotenoids RARα RARβ RARγ ATRA 3 ± 1 4 ± 2 3 ± 1 β-Apo-13-carotenone 5 ± 1 4 ± 2 4 ± 1 β-Apo-14'-carotenal 25 15 52 β-Apo-14'-carotenoic acid 34 25 58 β-Apo-12'-carotenoic acid 306 313 284 β-Apo-10'-carotenoic acid 378 507 351

Figure 3.6c

a RXRα 100

60 H] 9cisRA bound H] 9cisRA 3 40 % [

0 -11 -10 -9 -8 -7 -6 -5 log [M]

Ki (nM)

β-Apocarotenoids RXRα 9-cis-RA 7 β-Apo-13-carotenone 8 β-Apo-14'-carotenal 245 β-Apo-14'-carotenoic acid 738

Figure 3.7 β-Apo-13-Carotenone is a high affinity ligand for purified retinoid X receptor alpha. a, Competitive displacement of 10 nM tritiated 9-cis-RA from purified

RXRα protein by unlabeled 9-cis-RA () as a positive control, C13 ketone (▲), 14’-AL

(), 14’-CA (+), and retinyl acetate () as a negative control. Points shown are means of n=3 with a variance of less than 10%. b, Binding affinities of β-apocarotenoids to RXRα calculated from the data shown in Fig. 3.7a.

Figure 3.8 Analysis of β-apo-13-carotenone in human plasma by HPLC/MS.

Multiple reaction monitoring (MRM) chromatogram of β-apo-13-carotenone in blood plasma (top) and a standard (bottom) as analyzed by atmospheric pressure chemical ionization in positive mode after C30 HPLC. The MRM was composed of three transitions – m/z 259.2>175.1 (blue), 119.1 (red) and 69.0 (green) and the matching elution time and relative intensities of the transitions confirm the peak identity.

CHAPTER 4: APO-LYCOPENOIDS MODULATE TRANSCRIPTION OF RARE-

MEDIATED GENES (UNPUBLISHED WORK)

ABSTRACT

Lycopene, an acyclic carotenoid can be oxidatively cleaved at its polyene chain C=C double bonds to yield apo-lycopenoids. These compounds were found in vivo and in foods such as in tomato paste. We hypothesize that shorter cleavage products of lycopene that have structural resemblance to β-apo-13-carotenone may exhibit similar responses in a retinoid-mediated system. First we have synthesized the structural analogs of β-apo-13- carotenone. Then, we selected retinoic acid receptor (RAR) mediated genes such as

RARβ and CYP26A1 that can be induced by all-trans-retinoic acid (ATRA), the physiological ligand of RARs. Citral, geranic acid, pseudoionone, apo-11-lycopenal, apo-

11-lycopenoic acid, and apo-13-lycopenone were tested for their ability to modulate transcription of RARβ and CYP26A1. We found that apo-13-lycopenone which has the same molecular weight as β-apo-13-carotenone but lacks the β-ionone ring was able to inhibit ATRA induced RARβ, and CYP26A1 in a similar manner as β-apo-13- carotenone. This suggests that the ionone ring may not be required for β-apo-13- carotenone’s accommodation on RARs. More studies need to be done to characterize the interaction between the antagonist ketone and RARs.

88 4.1 INTRODUCTION

Lycopene, an acyclic hydrocarbon carotenoid, is one of more than 600 carotenoids synthesized by plants and photosynthetic microorganism (8). It is a tetraterpene hydrocarbon containing 11 conjugated and 2 non-conjugated double bonds with 40 carbon atoms and 56 hydrogen atoms (C40H56) and a molecular mass of 536 (See Fig.

4.1). Lycopene is a lipophilic hydrocarbon, soluble in non-polar solvents like chloroform, ether, and benzene but not in polar protic solvents such as methanol or ethanol. Lycopene lacks the β-ionone ring at its terminal ends unlike β-carotene hence it is a non provitamin A carotenoid. Although it is not a pro-vitamin A carotenoid, lycopene is a potent antioxidant allowing it to prevent the accumulation of reactive oxygen species by virtue of having conjugated double bonds.

A growing body of research suggests that intake of lycopene-containing foods, as well as blood lycopene concentrations, are inversely related to incidence of cardiovascular disease and prostate cancer (256, 257). Also, in vitro studies demonstrated that this carotenoid inhibited neoplastic cell growth in lung (258, 259), prostate (260, 261), and human promyelocytic cells (145, 262).

Lycopene is the most abundant carotenoid in tomatoes (Lycopersicon esculentum) and tomato sauce and ketchup are concentrated sources of lycopene (33–68 mg/100g) compared to unprocessed tomatoes (263). Other sources of dietary lycopene include watermelon, pink grapefruit, apricots, pink guava and papaya (264-266).

89 Lycopene is tightly bound to macromolecules within the food matrix and its bioavailability from food is relatively poor (267). There are a number of factors determining the bioavailability and absorption of lycopene such as isomeric form, food processing, co-ingestion of dietary fat or presence of other carotenoids in the diet. The cis form of lycopene found in processed tomato products was found to be more bioavailable than its trans form found in fresh tomatoes (268, 269). Food processing e.g., cooking or heating may improve lycopene bioavailability by breaking down cell walls in which the bonding forces between lycopene and tissue matrix weakened, thereby making lycopene more accessible and enhancing cis isomerization (265). Ingestion of dietary fat (265) improves lycopene absorption and bioavailability. Lycopene in food is found primarily in the trans form yet more than 50 % of the lycopene present in the human serum and tissues is cis isomers (270).

Followed by intestinal absorption, lycopene is incorporated into chylomicrons and released into the lymphatic system for transport to the liver (271). In the plasma, lycopene is transported in lipoproteins including low-density lipoprotein (LDL) and very low-density lipoprotein (VLDL) and distributed to multiple organs such as testes, adrenal glands, and liver.

It has been shown that lycopene can be metabolized by β-carotene-9',10'-oxygenase

(BCO2) (89). This enzyme is responsible for the eccentric oxidative cleavage of cis- lycopene isomers (5-cis and 13-cis) to form apo-10’-lycopenal (See Fig. 4.2) (19) that may be further oxidized to apo-10’-lycopenoic acid or reduced to apo-10’-lycopenol by 90 dehydrogenases using NADH or NAD+ as cofactors. In the same study, all-trans- lycopene supplementation in ferrets resulted in the formation of apo-10'-lycopenol in the lung (89). In another study, apo-8′-lycopenal and apo-12′-lycopenal have been found in the liver of rats that were fed with lycopene-enriched diets (107). In a recent study, apo-

6′-, apo-8′-, apo-10′-, apo-12′-, apo-14′- and apo-15′-lycopenals were detected in the plasma of humans who had consumed tomato paste (106). Yet, in this study the detected apo-lycopenals were also found in tomato paste so it is not clear whether circulating apo- lycopenoids found originated from human lycopene metabolism or directly absorbed from the diet.

4.2 HYPOTHESIS

We demonstrated that β-apo-13-carotenone served as an antagonist of RXRα (chapter 2) and RARs (chapter 3). We hypothesize that apo-lycopenoids that have a structural resemblance to β-apo-13-carotenone (See Fig. 4.3) may exert a similar function in modulating retinoid receptor activation.

4.3 MATERIALS & METHODS

4.3.a. Synthesis of apo-lycopenoids

Dr. Robert W. Curley’s laboratory used analytical methods to synthesize apo-lycopenoids of interest including pseudoionone, citral, geranic acid, apo-11-lycopenal, apo-11- lycopenoic acid, and apo-13-lycopenone.

91 The smallest lycopenoic acid of these 6 apo-lycopenoids, geranic acid, was synthesized from citral via a two-step oxidative process. The longer precursor, ethyl apo-11- lycopenoate, was synthesized from citral using a Wadsworth-Emmons reaction and from this molecule apo-11-lycopenoic acid was obtained (See Fig. 4.4). All reactions and compound handling were performed under gold fluorescent lights in oven dried glassware under a dry argon atmosphere. All products were purified by appropriate column or preparative thin layer chromatography and analyzed by HPLC for purity

(Model 127 pump and 166 detector, Beckman Instruments, San Ramon, CA, USA;

Metachem Polaris 5 um C18, 250 x 4.6 mm column, Varian Inc., Palo Alto, CA, USA).

Chemical and structural characterization was carried out using ultraviolet spectrophotometry (Beckman DU-40 spectrophotometer), NMR spectroscopy (Bruker

DRX400; Billerica, MA, USA) and electrospray mass spectrometry (Micromass QTOF;

Milford, MA, USA).

4.3.b. Effects of apo-lycopenoids on retinoic acid-induced gene expression in the Hep

G2 cells

Hep G2 (liver hepatocellular carcinoma) cells were cultured in Eagle's Minimum

Essential Medium (MEM) supplemented with 10% Fetal Bovine Serum (FBS). Cells were maintained at 37°C in incubators with 5% CO2 in air atmosphere. Hep G2 cells were treated with apo-lycopenoids dissolved in ethanol for 16 hours.

92 Cells were harvested at 0 h and 16 h after treatment. Total RNAs were isolated from cell pellets using a NucleoSpin RNA II (Macherey Nagel) and subjected to reverse transcription using a High Capacity RNA-to-cDNA kit (Applied Biosystems).

Quantitative PCR (qPCR) was carried out using TaqMan chemistry for human CYP26A1

(Hs01084852_g1), and human RARβ (Hs00977137_m1) as target genes and human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (HsNM_002046.3) as a housekeeping gene. Probes and primers for TaqMan assays i.e., RARβ, CYP26A1, and

GAPDH were obtained from Applied Biosystems. The Applied Biosystems 7900HT

Real-Time PCR System was used at The Ohio State University Comprehensive Cancer

Center nucleic acid shared resource for qPCR experiments. The comparative Ct method

(ΔΔCt) was used to quantify the results obtained by the real-time reverse transcription polymerase chain reaction (RT-qPCR).

4.4 Results & Discussion

We have tested the ability of apo-lycopenoids of interest to promote gene expression of

ATRA inducible genes such as CYP26A1 and RARβ. Hep G2 cells received only test apo-lycopenoids. Also, we tested their effects on ATRA-induced expression of these genes. In this case, cells were treated with both inducer i.e. ATRA, and a test apo- lycopenoid, simultaneously. At 0 h and 16 h, cells were harvested and RNAs were collected. First, short apo-lycopenoids i.e., pseudoionone, citral, cyclogeranic acid, were tested in gene expression assays. We found that none of these compounds stimulated the transcription of these target genes. Additionally, these shorter apo-lycopenoids did not affect ATRA-induced expression of RARβ and CYP26A1 (See Fig. 4.5). 93 Then, we proceeded with the apo-11-lycopenal (11-AL) and apo-11-lycopenoic acid (11-

CA) pair. The compounds alone did not induce significant gene expression. However, apo-11-lycopenoic acid was found to display some antagonist activity of ATRA-induced

RARβ and CYP26A1 (See Fig. 4.6). ATRA itself induced gene expression of RARβ at

25.7 fold change and co-treatment of equimolar (10 nM) 11-CA and ATRA led to the reduction of ATRA’s fold induction by about 30% from 25.7 to 17.8. In the case of

CYP26A1, the reduction of ATRA-induced expression is less effective; 11-CA blocked only 1/5th of the ATRA’s induction from 155.2 to 123.2 (See Fig. 4.6).

Finally, we tested apo-13-lycopenone. Apo-13-lycopene has the same molecular weight as β-apo-13-carotenone, the difference is the apo-lycopenoid analog does not contain a cyclic β-ionone ring. As expected this apo-lycopenoid did not activate CYP26A1 and

RARβ gene expression. However, this compound did inhibit ATRA-stimulated gene expression of both target genes but not to quite the same extent as β-apo-13-carotenone.

For ATRA induced CYP26A1 expression the lycopenoid was nearly as effective as the β- apocarotenoid. This suggests that the β-ionone ring may not be prerequisite in order for the ketone apocarotenoid to function as antagonists of retinoid target genes. (See Fig.

4.7). Apo-13-lycopenone has the same number of double bonds i.e., three conjugated double bonds and one non-conjugated double bond as β-apo-13-carotenone. More studies need to be done to exploit the mechanism of antagonist action of apo-13-lycopenone

94 4.5 FIGURES

Figure 4.1 Structures of lycopene

Figure 4.2 Suggested metabolic pathway for lycopene cleavage by BCO2 (22)

Fig. 4.3 Apo-lycopenoids that have structural similarity to β-apo-13-carotenone.

Fig. 4.4 Methods used to synthesize geranic acid and apo-11-lycopenoic acid: Shown are the synthetic reaction pathways for the two lycopenoic acids of interest. (3) Geranic acid was obtained from (2) methyl geranoate and (5) apo-11-lycopenoic acid was synthesized from (4) ethyl apo-11-lycopenoate. Both ester intermediates were made from

(1) citral.

98 50

45 ATRA

300 ATRA ATRA nM nM nM ATRA

40 ATRA + 10 nm ATRA nM nM

250 acid + 10 acid + 10 + 10

35 + 10 ATRA Citral Geranic Pseudoionone + 10 nM Citral Geranic Pseduoionone

ATRA nM nM nM 30 10 nM nM nM nM

) - mRNA ) - mRNA 200 10 10 10 β 10 10 23.1 10 10 152.0

RAR 21.9 25 155.0 147.1 153.5 22.0 20.7 150 20

15 Acid 100

Acid

Expression Fold Change ( Expression Expression Fold Change (CYP26A1) - mRNA Fold Change (CYP26A1) - mRNA Expression Citral Geranic Pseduoionone 10 nM nM nM Citral Geranic Pseduoionone

10 10 10 50 nM nM nM

5 1.9 10 10 10 1.7 1.1 1.2 2.5 2.4 0 0 Fig. 4.5 Citral, geranic acid, and pseudoionone modulate mRNA levels of RAR- target genes (RARβ, and CYP26A1). Induction of expression of mRNAs for RARβ (on left) or cytochrome P45026A1 (CYP26A1) (on right) by test apo-lycopenoids including citral, geranic acid, and pseudoionone treatment alone or by co-treatment with ATRA and test apo-lycopenoid. mRNA levels were quantified by RT-PCR and are shown as the fold induction compared to vehicle treated cells. n = 3; mean +/- s.d.

99 100 300

90 ATRA 250 ATRA 80 nM nM ATRA 70 nM 11'-AL + 10 11’-CA + 10 200 10 nM ATRA nM ATRA

) - mRNA ) - mRNA 60 10 10 β nM

nM 155.2 143.2

50 150 123.2 11'-AL + 10 ATRA 11'-CA + 10 nM nM 40 nM 0 10 1

25.7 100 30

Expression Fold Change (RAR Expression 24.0 Expression Fold Change (CYP26A1) - mRNA Fold Change (CYP26A1) - mRNA Expression 10 11’-AL 11’-CA 11'-AL 11'-CA 17.8

20 nM nM nM nM 50 10 10 10 10 10 4.5 2.1 11.9 3.5 0 0 Fig. 4.6 Apo-11-Lycopenals may down-regulate ATRA-induced gene expression of

RARβ, and CYP26A1. Induction of expression of mRNAs for RARβ (on left) or

CYP26A1 (on right) by test apo-lycopenoids including apo-11-lycopenal (11-AL) and apo-11’-lycopenoic acid (11-CA) or by co-treatment with ATRA and test apo- lycopenoid. mRNA levels were quantified by RT-PCR and are shown as the fold induction compared to vehicle treated cells. n = 3; mean +/- s.d.

100 50 250

40 200 ATRA ATRA nM

35 nM 10

30 ATRA ATRA 135.2 ATRA

) - mRNA ) - mRNA 150 nM β nM nM 10 25

19.5 ATRA nM Apo-13-Lycopenone + 10 20 C13 ON + 10 nM

nM 100 + 10 10 carotenone 15 12.9 Apo-13-Lycopenone + 10 10 -Apo-13- nM nM C13 ON β Apo-13-Lycopneone

10 7.9 -Apo-13-carotenone Apo-13-Lycopneone β

50 10 10

nM nM

Expression Fold Change (RAR Expression 29.4 nM 10 nM Expression Fold Change (CYP26A1) - mRNA Fold Change (CYP26A1) - mRNA Expression 10 10

10 18.1 5 1.7 1.3 1.9 3.2 0 0

Fig. 4.7 Apo-13-Lycopenone blocked ATRA-induced RARβ and CYP26A1 at similar extent as β-apo-13-carotenone. Induction of expression of mRNAs for RARβ

(on left) or CYP26A1 (on right) by apo-13-lycopenone, and β-apo-13-carotenone (C13

ON) or by co-treatment with ATRA and test apocarotenoids. mRNA levels were quantified by RT-PCR and are shown as the fold induction compared to vehicle treated cells. n = 3; mean +/- s.d.

101

BIBLIOGRAPHY

1. Britton, G. Carotenoids Vol.4:Carotenoids-Natural functions. Birkhäuser, Basel 4, p. 189 (2008).

2. Lu, S. & Li, L. Carotenoid metabolism: biosynthesis, regulation, and beyond. J. Integr. Plant Biol. 50, 778-785 (2008).

3. Maresca, J.A., Graham, J.E. & Bryant, A.D. The biochemical basis for structural diversity in the carotenoids of chlorophototrophic bacteria. Photosynth. Res. 97, 121-140 (2008).

4. Takaichi, S. & Mochimaru, M. Carotenoids and carotenogenesis in cyanobacteria: unique ketocarotenoids and carotenoid glycosides. Cell. Mol. Life Sci. 64, 2607- 2619 (2007).

5. Ye, Z.W., Jiang, J.G. & Wu, G.H. Biosynthesis and regulation of carotenoids in Dunaliella: progresses and prospects. Biotechnol. Adv. 26, 352-360 (2008).

6. Moore, T. Vitamin A and carotene: The conversion of carotene to vitamin A in vivo. Biochemistry Journal 24, 692-702 (1930).

7. Karrer, P., Helfenstein, A., Wehrli, H. & Wettstein, A. Über die Konstitution des Lycopins und Carotins. Helv. Chim. Acta 13, 1084 (1930).

8. Straub, O. et al. Key to Carotenoids, 2nd ed., Birkhauser Verlag, Basel 296 (1987).

102 9. Wirtz, G.M. et al. The substrate specificity of β,β-carotene 15,15’- monooxygenase. Helv. Chim. Acta 84, 2301-2315 (2001).

10. Bauernfield, J.C. Carotenoids as colorants and vitamin A precursors. Technological and nutritional applications. Academic Press, New York (1981).

11. Van Vliet, T., Van Schaik, F., Schreurs, W. H. P. & Van den Berg, H. In vitro measurement of β-carotene cleavage activity methodological considerations and the effect of other carotenoids on β-carotene cleavage. Intl. J. Vit. Nutr. Res. 66, 77-85 (1996).

12. Nagao, A., During, A., Hoshino, C, Terao, T. & Olson, J.A. Stoichiometric conversion of all-trans beta-carotene to retinal by pig intestinal extract. Archives of Biochemistry and Biophysics 328, 57-63 (1996).

13. Olson, J.A. Provitamin A function of carotenoids: the conversion of β-carotene into vitamin A. Journal of Nutrition 119, 105-108 (1989).

14. Parker, R.S. Absorption, metabolism, and transport of carotenoids. FASEB Journal 10, 542-551 (1996).

15. Moussa, M. et al. Lycopene absorption in human intestinal cells and in mice involves scavenger receptor class B type I but not niemann-pick C1-like. Journal of Nutrition 138, 1432-1436 (2008).

16. Van Bennekum, A. et al. Class B scavenger receptor-mediated intestinal absorption of dietary β-carotene and cholesterol. Biochemistry, 44, 4517-4525 (2005).

17. Yonekura, L., & Nagao, A. Intestinal absorption of dietary carotenoids. Molecular Nutrition and Food Research, 51, 107-115 (2007).

18. Harrison, E.H. Mechanisms involved in the intestinal absorption of dietary vitamin A and provitamin A carotenoids. Biochimica et Biophysica Acta 1821, 70-77 (2012).

103 19. Bondi, A. & Sklan, D. Vitamin A and carotene in animal nutrition. Prog. Food Nutr. Sci. 8, 165-191 (1984).

20. Mercado, J.D.R., Holmgren, S.C., Fox, J.G. & Russel, R.M. Dietary β-carotene absorption and metabolism in ferrets and rats. Journal of Nutrition 119, 665-668 (1989).

21. Erlanson, C. & Borgstom, B. Identity of vitamin A esterase activity of rat pancreatic juice. Biochimica et Biophysica Acta 167, 629-631 (1968).

22. Dew, S.E. & Ong, D.E. Specificity of the retinol transporter of the rat small intestine brush border. Biochemistry 33, 12340-12345 (1994).

23. Elaine, R.S. & Fernando, S.M. Natural retinoids and β-carotene: from food to their actions on gene expression. The Journal of Nutritional Biochemistry 9, 446- 456 (1998).

24. MacDonald, P.N. & Ong, D.E. Evidence for a lecithin-retinol acyltransferase activity in the rat small intestine. Journal of Biological Chemistry 263, 12478- 12482 (1988).

25. Helgerud, P., Petersen, L.B. & Norum, K.R. Retinol esterification by microssomes from the mucosa of human small intestine: evidence for acyl- coenzym A retinol acyltransferase activity. J. Clin. Invest. 71, 747-753 (1983).

26. Ong, D.E. Cellular transport and metabolism of vitamin A: roles of the cellular retinoid-binding proteins. Nutr. Rev. 52, 24-31 (1994).

27. Wolf, G. The intracellular vitamin A-binding proteins: an overview of their functions. Nutr. Rev. 49, 1-11 (1991).

28. Kakkad, B.P. & Ong D.E. Reduction of retinaldehyde bound to cellular retinol- binding protein (type II) by microsomes from rat small intestine. Journal of Biological Chemistry 263, 12916-12919 (1988).

104 29. Napoli, J.L. Retinoic acid biosynthesis and metabolism. FASEB Journal 10, 993- 1001 (1996).

30. Blomhoff, R., Green, M.H., Green J.B., Berg, T. & Norum, K.R. Vitamin A metabolism: new perspectives on absorption, transport and storage. Physiological Reviews 71, 951-990 (1991).

31. Blomhoff, R. Transport and metabolism of vitamin A. Nutr. Rev. 52, 13-23 (1994).

32. Clevidence, B.A. & Bieri, J.C. Association of carotenoids with human plasma lipoproteins. Methods Enzymol. 214, 33-46 (1993).

33. Levin, G., Amotz, A.B. & Mokady, S. Liver accumulation of soluble all-trans or 9-cis β-carotene in rats and chicks. Comp. Biochem. Physiol. 107A, 203–207 (1994).

34. Harrison, E.H., Gad, M.Z. & Ross, A.C. Hepatic uptake and metabolism of chylomicron retynil esters: probable role of plasma membrane and endosomal retinyl ester hydrolases. Journal of Lipid Research 36, 1498-1506 (1995).

35. Yamada, M. et al. Biochemical characteristics of isolated rat liver stellate cells. Hepatology 7, 1224-1229 (1987).

36. Yumoto, S., Ueno K., Mori, S., Takebayashi N. & Handa S. Morphological and biochemical analyses of lipid granules isolated from fat-storing cells in rat liver. Biomed. Res. 2, 147-160 (1988).

37. Yost, R.W., Harrison, E.H. & Ross, A.C. Esterification by rat liver microsomes of retinol bound to cellular retinol-binding protein. Journal of Biological Chemistry 263, 18693-18701 (1988).

38. Heller, J. Interactions of plasma retinol-binding protein with its receptor. Specific binding of bovine and human retinol-binding protein to pigment epithelium cells from bovine eyes. Journal of Biological Chemistry 250, 3613-3619 (1975).

105 39. Bok D. & Heller J. Transport of retinol from the blood to the retina: an autoradiographic study of the pigment epithelial cell surface receptor for plasma retinol-binding protein. Exp. Eye Res. 22, 395-402 (1976).

40. Rask, L. & Peterson P.A. In vitro uptake of vitamin A from the retinol-binding plasma protein to mucosal epithelial cells from the monkey's small intestine. Journal of Biological Chemistry 251, 6360-6366 (1976).

41. Chen C.C. & Heller, J. Uptake of retinol and retinoic acid from serum retinol- binding protein by retinal pigment epithelial cells. Journal of Biological Chemistry 252, 5216-5221 (1977).

42. Sivaprasadarao, A. & Findlay, J.B. The mechanism of uptake of retinol by plasma-membrane vesicles. Biochemistry Journal 255, 571-579 (1988).

43. Smeland et al. Tissue distribution of the receptor for plasma retinol-binding protein. Biochemistry Journal 305, 419-424 (1995).

44. Sivaprasadarao, A. & Findlay, J.B. Structure-function studies on human retinol- binding protein using site-directed mutagenesis. Biochemistry Journal 300, 437- 442 (1994).

45. Sundaram M., Sivaprasadarao, A., DeSousa M.M. & Findlay, J.B. The transfer of retinol from serum retinol-binding protein to cellular retinol-binding protein is mediated by a membrane receptor. Journal of Biological Chemistry 273, 3336- 3342 (1998).

46. MacDonald, P.N., Bok, D. & Ong, D.E. Localization of cellular retinol-binding protein and retinol-binding protein in cells comprising the blood-brain barrier of rat and human. Proc. Natl. Acad. Sci. U.S.A. 87, 4265-4269 (1990).

47. Shingleton J.L., Skinner M.K. & Ong, D.E Characterization of retinol accumulation from serum retinol-binding protein by cultured Sertoli cells. Biochemistry 28, 9641-9647 (1989).

106 48. Hagen et al. Uptake of vitamin A in macrophages from physiologic transport proteins: role of retinol-binding protein and chylomicron remnants J. Nutr. Biochem. 10, 345-352 (1999).

49. Kawaguchi et al. A Membrane Receptor for Retinol Binding Protein Mediates Cellular Uptake of Vitamin A. Science 315, 820-825 (2007).

50. Giguère, V., Ong, E.S., Segui, P. & Evans, R.M. Identification of a receptor for the morphogen retinoic acid. Nature 330, 624-629 (1987).

51. Petkovich, M., Brand, N.J., Krust, A. & Chambon, P. A human retinoic acid receptor which belongs to the family of nuclear receptor. Nature 330, 444-450 (1987).

52. Siegenthaler, G., Saurat, J.H. & Ponec, M. Retinol and retinal metabolism. Relationship to the state of differentiation of cultured human keratinocytes. Biochemistry Journal 268, 371-378 (1990).

53. Boleda, M.D., Saubi, N., Farrés, J. & Parés, X. Physiological substrate for rat alcohol dehydrogenase classes: aldehydes of lipid peroxidation, hydroxy fatty acids, and retinoids. Archives of Biochemistry and Biophysics 307, 85-90 (1993).

54. Boerman, M.H.E.M. & Napoli, J.L. Effects of sulfhydryl reagents, retinoids and solubilization on the activity of microsomal retinol dehydrogenase. Archives of Biochemistry and Biophysics 321, 434-441 (1995).

55. Napoli, J.L. Physiological insights into all-trans-retinoic acid biosynthesis. Biochimica et Biophysica Acta 1821, 152–167 (2012).

56. Perlmann, T. Retinoid metabolism: a balancing act. Nature Genetics 31, 7-8 (2002).

57. Penzes, P., Wang, X. & Napoli J.L. Enzymatic characteristics of retinal dehydrogenase type I expressed in Escherichia coli. Biochimica et Biophysica Acta 1342, 175–181 (1997).

107 58. Penzes, P., Wang, X. & Sperkova, Z. & Napoli, J.L. Cloning of a rat cDNA encoding retinal dehydrogenase isozyme type I and its expression in E. coli. Gene 191, 167–172 (1997).

59. Bhat, P.V., Labrecque, J., Boutin, J.M., Lacroix, A. & Yoshida A. Cloning of a cDNA encoding rat aldehyde dehydrogenase with high activity for retinal oxidation. Gene 166, 303-306 (1995).

60. Zhai, Y., Sperkova, Z. & Napoli, J.L. Cellular expression of retinal dehydrogenase types 1 and 2: effects of vitamin A status on testis mRNA. J. Cell. Physiol. 186, 220-232 (2001).

61. Lin, M., Zhang, M., Abraham, M., Smith, S.M. & Napoli, J.L. Mouse retinal dehydrogenase 4 (RALDH4), molecular cloning, cellular expression, and activity in 9-cis-retinoic acid biosynthesis in intact cells. Journal of Biological Chemistry 278, 9856-9861 (2003).

62. Boerman, E.M. & Napoli, J.L. Cellular retinol-binding protein-supported retinoic acid synthesis: relative roles of microsomes and cytosol. Journal of Biological Chemistry 271, 5610-5616 (1996).

63. Duester, G. Involvement of alcohol dehydrogenase, short-chain dehydrogenase reductase, aldehyde dehydrogenase and cytochrome P450 in the control of retinoid signaling by activation of retinoid acid synthesis. Biochemistry Journal 35, 12221-12227 (1996).

64. El Akavi, Z. & Napoli, J.L. Rat liver cytosolic retinal deydrogenase: comparison of 13-cis-, 9-cis-, and all-trans-retinal as substrates and effects of cellular retinoid- binding proteins and retinoic acid on activity. Biochemistry Journal 33, 1938- 1943 (1994).

65. Roberts, E.S., Vaz, A.D.N. & Coon, M.J. Role of isozymes of rabbit microsomal cytochrome P450 in the metabolism of retinoic acid, retinol, and retinal. Mol. Pharmacol. 41, 427-433 (1992).

66. White, J.A. et al. Identification of the retinoic acid-inducible all-trans- retinoic acid 4-hydroxylase. Journal of Biological Chemistry 271, 29922-29927 (1996). 108 67. Fujii, H. et al. Metabolic inactivation of retinoic acid by a novel P450 differentially expressed in developing mouse embryos. EMBO Journal 16, 4163- 4173 (1997).

68. Ross, A.C. Cellular metabolism and activation of retinoids: roles of cellular retinoid-binding proteins. FASEB Journal 7, 317-327 (1993).

69. Fiorella, P.D. & Napoli, J.L. Microsomal retinoic acid metabolism: effects of cellular retinoic acid-binding protein (type I) and C18-hydroxylation as an initial step. Journal of Biological Chemistry 269, 10538-10544 (1994).

70. Napoli, J.L. Biochemical pathways of retinoid transport, metabolism, and signal transduction. Clin. Immunol. Immunopathol. 80, S52-S62 (1996).

71. Aström, A. et al. Molecular cloning of two cellular retinoic acid-binding proteins (CRABP). Journal of Biological Chemistry 266, 17662-17666 (1991).

72. Napoli, J.L., Boerman, M.H.E.M., Chai, Y.Z. & Fiorella, P.D. Enzymes and binding proteins affecting retinoic acid concentrations. J. Steroid Biochem. Molec. Biol. 53, 497-502 (1995).

73. Schwartz, S.H., Tan, B.C., Gage, D.A., Zeevaart, J.A. & McCarty, D.R. Specific oxidative cleavage of carotenoids by VP14 of maize. Science 276, 1872-1874 (1997).

74. Bouwmeester, H.J., Roux, C., Lopez-Raez, J.A. & Becard, G. Rhizosphere communication of plants, parasitic plants and AM fungi. Trends Plant Sci. 12, 224-230 (2007).

75. Gomez-Roldan, V. et al. Strigolactone inhibition of shoot branching. Nature 455, 189-194 (2008).

76. Umehara, M. et al. Inhibition of shoot branching by new terpenoid plant hormones. Nature 455, 195-200 (2008).

109 77. Goodman, D.S. & Huang, H.S. Biosynthesis of vitamin A with rat intestinal enzyme. Science 149, 879-880 (1965).

78. Olson, J.A. & Hayaishi, O. The enzymatic cleavage of beta-carotene into vitamin A by soluble enzymes of rat liver and intestine. Proc. Natl. Acad. Sci. U.S.A. 54, 1364-1370 (1965).

79. Zeevart, J.A.D. & Creelman, R.A. Metabolism and physiology of abscisic acid. Annu. Rev. Plant Physiol. Plant Mol. Biol. 39:439-473 (1988).

80. Ruch, S., Beyer, P., Ernst, H. & Al-Babili, S. Retinal biosynthesis in Eubacteria: In vitro characterization of a novel carotenoid oxygenase from Synechocystis sp. PCC 6803. Molecular Microbiology, 55, 1015-1024 (2005).

81. Prado-Cabrero, A., Scherzinger, D., Avalos, J. & Al-Babili, S. (2007). Retinal biosynthesis in fungi: Characterization of the carotenoid oxygenase CarX from fusarium fujikuroi. Eukaryotic Cell 6, 650-657 (2007).

82. Wyss, A. et al. Cloning and expression of beta,beta-carotene 15,15′-dioxygenase. Biochem. Biophys. Res. Commun. 271, 334-336 (2000).

83. Von Lintig, J. & Vogt, K. Filling the gap in vitamin A research. Molecular identification of an enzyme cleaving beta-carotene to retinal. Journal of Biological Chemistry 275, 11915-11920 (2000).

84. Redmond, T.M. et al. Rpe65 is necessary for production of 11-cis-vitamin A in the retinal visual cycle. Nature Genetics, 20, 344-351 (1998).

85. Lindqvist, A. & Andersson, S. Biochemical properties of purified recombinant human β-carotene 15,15′-monooxygenase. Journal of Biological Chemistry 277, 23942-23948 (2002).

86. Kiefer, C. et al. Identification and characterization of a mammalian enzyme catalyzing the asymmetric oxidative cleavage of provitamin A. Journal of Biological Chemistry 276, 14110-14116 (2001).

110 87. Amengual, J. et al. A mitochondrial enzyme degrades carotenoids and protects against oxidative stress. FASEB Journal 25, 948-959 (2011).

88. Mein, J., Dolnikowski, G., Hansgeorg, E., Russel, R. & Wang, X.D. Enzymatic formation of apo-carotenoids from the xanthophyll carotenoids lutein, zeaxanthin and β-cryptoxanthin by ferret carotene-9’,10’-monooxygenase. Archives Of Biochemistry and Biophysics 506, 109-121 (2011).

89. Hu, K.Q., Liu, C., Ernst, H., Krinsky, N.I., Russell, R.M. & Wang, X.D. The biochemical characterization of ferret carotene-9′,10′-monooxygenase catalyzing cleavage of carotenoids in vitro and in vivo. Journal of Biological Chemistry 281, 19327-19338 (2006).

90. Leuenberger, M. G., Engeloch-Jarret, C. & Woggon, W. The reaction mechanism of the enzyme catalyzed central cleavage of β-carotene to retinal. Angew. Chem. 113, 2684-2687 (2001).

91. Schmidt, H., Kurtzer, R., Eisenreich, W. & Schwab, W. The carotenase AtCCD1 from Arabidopsis thaliana is a dioxygenase. Journal of Biological Chemistry 281, 9845-9851 (2006).

92. Redmond, T.M. et al. Identification, expression, and substrate specificity of a mammalian beta-carotene 15,15’-dioxygenase. Journal of Biological Chemistry 276, 6560-6565 (2001).

93. Wyss, A. et al. Expression pattern and localization of beta-beta carotene 15,15’- dioygenase in different tissues. Biochemistry Journal 354, 521-529 (2001).

94. Paik, J. et al. Expression and characterization of a murine enzyme able to cleave β-carotene: The formation of retinoids. Journal of Biological Chemistry 276, 32160-32168 (2001).

95. Yan, W. et al. Cloning and characterization of a human β,β-carotene-15,15′- dioxygenase that is highly expressed in the retinal pigment epithelium. Genomics 72, 193-202 (2001).

111 96. Wang, X.D. et al. Beta-oxidation in rabbit liver in vitro and in the perfused ferret liver contributes to retinoic acid biosynthesis from beta-apocarotenoic acids. Journal of Biological Chemistry 271, 26490-26498 (1996).

97. Hessel, S. et al. CMO1 deficiency abolishes vitamin A production from beta- carotene and alters lipid metabolism in mice. Journal of Biological Chemistry 282, 33553-33561 (2007).

98. Lindqvist, A., He, Y.G. & Andersson, S. Cell type-specific expression of beta- carotene 9′,10'-monooxygenase in human tissues. J. Histochem. Cytochem. 53, 1403-1412 (2005).

99. Van Vliet, T., Van Vlissingen, M.F., Van Schaik, F. & Van Den Berg, H. β- Carotene absorption and cleavage in rats is affected by the vitamin A concentration of the diet. Journal of Nutrition 126, 499-508 (1996).

100. Bachmann, H. et al. Feedback regulation of β,β-carotene 15,15′-monooxygenase by retinoic acid in rats and chickens. Journal of Nutrition 132, 3616-3622 (2002).

101. Choi, M.Y. et al. A dynamic expression survey identifies transcription factors relevant in mouse digestive tract development. Development 133, 4119-4129 (2006).

102. Seino, Y. et al. Isx participates in the maintenance of vitamin A metabolism by regulation of β-carotene 15,15′-monooxygenase (Bcmo1) expression. Journal of Biological Chemistry 283, 4905-4911 (2008).

103. Lobo, G.P. et al. ISX is a retinoic acid-sensitive gatekeeper that controls intestinal β,β-carotene absorption and vitamin A production. FASEB Journal 24, 1656-1666 (2010).

104. Winterstein, A., Studer, A. & Ruegg, R. Neuere ergebnisse der carotinoid for schung. Chem. Ber. 93, 2951-2165 (1960).

105. Ben-Aziz, A., Britton, G. & Goodwin, T.W. Carotene epoxides of Lycopersicon esculentum. Phytochemistry 12, 2759-2764 (1973) 112 106. Kopec, R.E. et al. Identification and quantification of apo-lycopenals in fruits, vegetables, and human plasma. J. Agric. Food Chem. 58, 3290-32906 (2010).

107. Gajic, M., Zaripheh, S., Sun, F. & Erdman, J.W. Jr. Apo-8′-lycopenal and apo- 12'-lycopenal are metabolic products of lycopene in rat liver. Journal of Nutrition 136, 1552-1557 (2006).

108. Lindshield, B.L., Canene-Adams, K. & Erdman, J.W. Jr. Lycopenoids: are lycopene metabolites bioactive? Archives of Biochemistry and Biophysics 458, 136-140 (2007).

109. Erdman, J.W. Jr., Ford, N.A. & Lindshield, B.L. Are the health attributes of lycopene related to its antioxidant function? Archives of Biochemistry and Biophysics 483, 229–235 (2009).

110. Trosko, J.E., Chang, C.C. & Upham, B. & Wilson, M. Epigenetic toxicology as toxicant-induced changes in intracellular signalling leading to altered gap junctional intercellular communication. Toxicol. Lett. 102-103, 71-78 (1998).

111. Stahl, W., Von Laar, J., Martin, H.D., Emmerich, T. & Sies, H. Stimulation of gap junctional communication: comparison of acyclo-retinoic acid and lycopene. Archives of Biochemistry and Biophysics 373, 271-274 (2000).

112. Kotake-Nara, E., Kim, S.J., Kobori, M., Miyashita, K. & Nagao, A. Acyclo- retinoic acid induces apoptosis in human prostate cancer cells. Anticancer Res. 22, 689-695 (2002).

113. Ben-Dor, A. et al. Effects of acyclo-retinoic acid and lycopene on activation of the retinoic acid receptor and proliferation of mammary cancer cells. Archives of Biochemistry and Biophysics 391, 295-302 (2001).

114. Lian, F.Z., Smith, D.E., Ernst, H. Russell, R.M. & Wang, X.D. Apo-10′- lycopenoic acid inhihits lung cancer cell growth in vitro, and suppresses lung tumorigenesis in the A/J mouse model in vivo. Carcinogenesis 28, 1567-1574 (2007).

113 115. Gouranton, E. et al. Apo-10'-lycopenoic acid impacts adipose tissue biology via the retinoic acid receptors. Biochimica et Biophysica Acta 1812, 1105-1114 (2011).

116. Lomb, D.J., Laurent, G. & Haigis, M.C. Sirtuins regulate key aspects of lipid metabolism. Biochimica et Biophysica Acta 1804, 1652-1657 (2010).

117. Jin, Q. et al. C/EBPalpha regulates SIRT1 expression during adipogenesis. Cell Res. 20, 470-479 (2010).

118. Picard, F. et al. (2004) Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-gamma. Nature 429, 771-776 (2004).

119. Chung J. et al. Apo-10'-Lycopenoic Acid, a Lycopene Metabolite, Increases Sirtuin 1 mRNA and Protein Levels and Decreases Hepatic Fat Accumulation in ob/ob Mice. Journal of Nutrition 142, 405-410 (2012).

120. Sharma, R.V., Mathur, S.N. & Ganguly, J. Studies on the relative biopotencies and intestinal absorption of different apo-beta-carotenoids in rats and chickens. J. Biochem 158, 377-383 (1976).

121. Sharma, R.V., Mathur, S.N., Dmitrovskii, A.A., Das, R.C. & Ganguly, J. Studies on the metabolism of beta-carotene and apo-beta-carotenoids in rats and chickens. Biochimica et Biophysica Acta 486, 183-194 (1976).

122. Wang, X.D., Tang, G.W., Fox, J.G., Krinsky, N.I. & Russell R.M. Enzymatic conversion of beta-carotene into beta-apo-carotenals and retinoids by human, monkey, ferret, and rat tissues. Archives of Biochemistry and Biophysics 285, 8- 16. (1991).

123. Barua, A.B. & Olson, J.A. β-Carotene is converted primarily to retinoids in rats in vivo. Journal of Nutrition 130, 1996-2001 (2000).

124. Handelman, G.J., Van Kuijk, F.J., Chatterjee, A. & Krinsky, N.I. Characterization of products formed during the autoxidation of beta-carotene. Free Radic. Biol. Med. 10, 427-437 (1991). 114 125. Ouyang, J.M., Daun, H., Chang, S.S. & Ho, C. T. Formation of carbonyl compounds from β-carotene during palm oil deodorization. J. Food Sci.45, 1214- 1217 (1980).

126. Marty, C. & Berset, C. Factors affecting the thermal degradation of all-trans-β- carotene. J. Agric. Food Chem. 38, 1063-1067 (1990).

127. Rodriguez, E.B. & Rodriguez-Amaya, D.B. Formation of apocarotenals and epoxycarotenoids from β-carotene by chemical reactions and by autoxidation in model systems and processed foods. Food Chem. 101, 563-72. (2007).

128. Fleshman, M.K. et al. Carotene and novel apocarotenoid concentrations in orange-fleshed cucumis melo melons: Determinations of β-carotene bioaccessibility and bioavailability. J. Agric. Food Chem. 59, 4448-4454 (2011).

129. Eroglu, A. et al. Naturally-occurring eccentric cleavage products of provitamin A carotene β-carotene function as antagonists of retinoic acid receptors. Journal of Biological Chemistry 287, 15886-15995 (2012).

130. Ho, C.C., de Moura, F.F., Kim, S.H. & Clifford, A.J. Excentral cleavage of beta- carotene in vivo in a healthy man. Am J Clin Nutr. 85, 770-777 (2007).

131. Shmarakov, I. et al. Hepatic stellate cells are an important cellular site for β- carotene conversion to retinoid. Archives of Biochemistry and Biophysics 504, 3- 10 (2010).

132. Malone, W.F. Studies evaluating antioxidants and β-carotene as chemopreventives. Am J Clin Nutr 53, 305-313 (1991).

133. Krishnan, K., Campbell, S., Abdel-Rahman, F., Whaley, S. & Stone, W.L. Cancer chemoprevention drug targets. Curr Drug Targets 45-54 (2003).

134. Mayne, S.T. Beta-carotene, carotenoids, and disease prevention in humans. FASEB Journal 10, 690-70 (1996).

115 135. Krinsky, N.I. Anticarcinogenic activities of carotenoids in animals and cellular systems. In Emerit, I. Chance, B. eds. Free Radicals and Aging Birkhäuser Basel 1992, 227-234 (1992).

136. Alpha-Tocopherol, Beta Carotene Cancer Prevention Study Group. The effect of vitamin E and beta carotene on the incidence of lung cancer and other cancers in male smokers. N. Engl. J. Med. 330, 1029-1035 (1994).

137. Omenn, G.S. et al. Effects of a combination of beta carotene and vitamin A on lung cancer and cardiovascular disease. N. Engl. J. Med. 334, 1150 -1155. (1996).

138. Prakash, P. et al. Beta-carotene and beta-apo-14'-carotenoic acid prevent the reduction of retinoic acid receptor beta in benzo[a]pyrene-treated normal human bronchial epithelial cells. Journal of Nutrition 134, 667-673 (2004).

139. Benkoussa, M., Brand, C., Delmotte, M.H., Formstecher, P. & Lefebvre, P. Retinoic acid receptors inhibit AP1 activation by regulating extracellular signal- regulated kinase and CBP recruitment to an AP1-responsive promoter. Mol. Cell. Biol. 22, 4522-4534 (2002).

140. Tibaduiza, E.C., Fleet, J.C., Russell, R.M. & Krinsky, N.I. Excentric cleavage products of β-carotene inhibit estrogen receptor positive and negative breast tumor cell growth in vitro and inhibit activator protein-1-mediated transcriptional activation. Journal of Nutrition 132, 1368-1375 (2002).

141. Zile, M. Induction of differentiation of human promyelocytic leukemia cell line HL-60 by retinoyl glucuronide, a biologically active metabolite of vitamin A. Proc. Natl. Acad. Sci. U.S.A 84, 2208-2212 (1987).

142. Collins, S.J., Gallo, R.C. & Gallagher, R.E. Continuous growth an differentiation of human myeloid leukemic cells in suspension culture. Nature 270, 347-349 (1977).

143. Breitman, T.R., Selonick, S.E. & Collins, S.J. Induction of differentiation of the human promyelocytic leukemia cell line (HL-60) by retinoic acid. Proc. Natl. Acad. Sci. U.S.A 77, 2936-2940 (1980).

116 144. Breitman, T.R., Collins, S.J. & Keene, B.R. Terminal differentiation of human promyelocytic leukemic cells in primary culture in response to retinoic acid. Blood 57, 1000-1004 (1981).

145. Gross, M., Bishop, T., Belcher, J. & Jacobs, D.R. Jr. Induction of HL-60 differentiation by carotenoids. Nutr. Cancer 27, 169-173 (1997).

146. McDevitt, T.M., Tchao, R., Harrison, E.H. & Morel, D.W. Carotenoids normally present in serum inhibit proliferation and induce differentiation of a human monocyte/macrophage cell line (U937). Journal of Nutrition 135, 160-164 (2005).

147. Winum, J.Y. et al. Synthesis and biological activities of higher homologues of retinoic acid. Farmaco 52, 39-42 (1997).

148. Suzuki, T., Matsui, M. & Murayama, A. Biological activity of (all-E)-beta-apo- 12'-carotenoic acid and the geometrical isomers on human acute promyelocytic leukemia cell line HL-60. J. Nutr. Sci. Vitaminol. 41, 575-585 (1995).

149. Rühl, R. Induction of PXR-mediated metabolism by β-carotene. Biochimica et Biophysica Acta 1740, 162-169 (2005).

150. Kliewer, S.A. The Nuclear Pregnane X Receptor Regulates Xenobiotic Detoxification. Journal of Nutrition 133, 2444S-2447S (2003).

151. Rühl, R. et al. Carotenoids and their metabolites are naturally occurring activators of gene expression via the pregnane X receptor. Eur. J. Nutr. 3, 692-703 (2004).

152. Ziouzenkova, O. et al. Asymmetric Cleavage of β-Carotene Yields a Transcriptional Repressor of Retinoid X Receptor and Peroxisome Proliferator- Activated Receptor Responses. Mol. Endocrinol. 21, 77-88 (2007).

153. Kawada, T. et al. Carotenoids and retinoids as suppressors on adipocyte differentiation via nuclear receptors. Biofactors 13, 103-109 (2000).

117 154. Shulman, A.I. & Mangelsdorf, D.J. Retinoid X receptor heterodimers in the metabolic syndrome. N. Engl. J. Med. 353, 604–615 (2005).

155. Schlicht, M. et al. D’orenone blocks polarized tip growth of root hairs by interfering with the PIN2-mediated auxin transport network in the root apex. Plant J. 55, 709-717 (2008).

156. Booker, J. et al. MAX3/CCD7 is a carotenoid cleavage dioxygenase required for the synthesis of a novel plant signaling molecule. Curr Biol 14, 1232-1238 (2004).

157. Winterhalter, P. & Rouseff, R.L. Carotenoid-Derived Aroma Compounds. American Chemical Society (2002).

158. Simkin, A.J., Schwartz, S.H., Auldridge, M. & Taylor, M.G. The tomato carotenoid cleavage dioxygenase 1 genes contribute to the formation of the flavor volatiles-ionone, pseudoionone, and geranylacetone. The Plant Journal 40, 882- 892 (2004).

159. Germain, P. et al International Union of Pharmacology. LX. Retinoic acid receptors. Pharmacol. Rev. 58, 712-725 (2006).

160. Germain, P., Staels, B., Dacquet, C., Spedding, M. & Laudet, V. Overview of nomenclature of nuclear receptors. Pharmacol. Rev. 58, 685-704 (2006).

161. Bour, G., Taneja, R. & Rochette-Egly, C. Mouse embryocarcinoma F9 cells and retinoic acid: A model to study the molecular mechanisms of endodermal differentiation. Advances in Developmental Biology 16, 211-253 (2006).

162. Benbrook, D., Lernhardt, E. & Pfahl, M. A new retinoic acid receptor identified from a hepatocellular carcinoma. Nature (London) 333, 669-672 (1988).

163. Krust, A., Kastner, P., Petkovich, M., Zelent, A. & Chambon, P. A third human retinoic acid receptor, hRAR-γ. Proc. Natl. Acad. Sci. U.S.A 86, 5310-5314 (1989).

118 164. Germain, P. et al. International Union of Pharmacology. LXIII. Retinoid X Receptors. Pharmacol. Rev. 58, 760-772 (2006).

165. Chambon, P. (1996) A decade of molecular biology of retinoic acid receptors. FASEB J. 10, 940-954 (1996).

166. Mark, M., Ghyselinck, N.B. & Chambon, P. Function of retinoic acid receptors during embryonic development. Nuclear Receptor Signal 7, e002 (2009).

167. Dawson, M.I. & Xia, Z. The Retinoid X receptors and their ligands. Biochimica et Biophysica Acta 1821, 21-56 (2012).

168. Rastinejad, F. Retinoid X receptor and its partners in the nuclear receptor family. Curr. Opin. Struct. Biol. 11, 33-38 (2001).

169. Naar, A.M., et al. The orientation and spacing of core DNA-binding motifs dictate selective transcriptional responses to three nuclear receptors. Cell 65, 1267-79 (1991).

170. Mangelsdorf, D. J. et al. A direct repeat in the cellular retinol-binding protein type II gene confers differential regulation by RXR and RAR. Cell 66, 555-61 (1991).

171. Durand, B., Saunders, M., Leroy, P., Leid, M. & Chambon, P. All-trans and 9-cis retinoic acid induction of CRABPII transcription is mediated by RAR-RXR heterodimers bound to DR1 and DR2 repeated motifs. Cell 71, 73-85 (1992).

172. Smith, W.C., Nakshatri, H., Leroy, P., Rees, J. & Chambon, P. A retinoic acid response element is present in the mouse cellular retinol binding protein I (mCRBPI) promoter. EMBO J. 10, 2223-30 (1991).

173. de The, H. et al. Identification of a retinoic acid responsive element in the retinoic acid receptor β gene. Nature 343, 177-180 (1990).

119 174. Loudig, O. et al. Cytochrome P450RAI (CYP26) promoter: a distinct composite retinoic acid response element underlies the complex regulation of retinoic acid metabolism. Mol. Endocrinol. 14, 1483-1497. (2000).

175. Dupe, V. et al. In vivo functional analysis of the Hoxa-1 3' retinoic acid response element (3'RARE). Development 124, 399-410 (1997).

176. Qian, A., Cai, Y., Magee, T.R. & Wan, Y.J. Identification of retinoic acid- responsive elements on the HNF1alpha and HNF4alpha genes. Biochem. Biophys. Res. Commun. 276, 837-842 (2000).

177. Mangelsdorf, D.J. & Evans, R.M. The RXR heterodimers and orphan receptors. Cell 83, 841-850 (1995).

178. Gehin, M. et al. Structural basis for engineering of retinoic acid receptor isotype- selective agonists and antagonists. Chem. Biol. 6, 519-29 (1999).

179. Klaholz, B.P., Mitschler, A. & Moras, D. Structural basis for isotype selectivity of the human retinoic acid nuclear receptor. J. Mol. Biol. 302, 155-170 (2000).

180. Klein, E.S. et al. Identification and functional separation of retinoic acid receptor neutral antagonists and inverse agonists. Journal of Biological Chemistry 271, 22692-22696 (1996).

181. le Maire, A. et al. A unique secondary-structure switch controls constitutive gene repression by retinoic acid receptor. Nature Structural Molecular Biology 17, 801–807 (2010).

182. Perez, E., Bourguet, W., Gronemeyer H. & de Lera A.R. Modulation of RXR function through ligand design. Biochimica et Biophysica Acta 1821, 57-69 (2012).

183. Brzozowski, A.M. et al. Molecular basis of agonism and antagonism in the oestrogen receptor. Nature 389, 753-58 (1997).

120 184. Shiau, A.K. et al. The structural basis of estrogen receptor/coactivator recognition and the antagonism of this interaction by tamoxifen. Cell 95, 927-37 (1998).

185. Darimont, B.D. et al. Structure and specificity of nuclear receptor-coactivator interactions. Genes Dev. 12, 3343-56 (1998).

186. Mak, H.Y., Hoare, S., Henttu, P.M. & Parker, M.G. Molecular determinants of the estrogen receptor-coactivator interface. Mol. Cell Biol. 19, 3895-903 (1999).

187. Klaholz, B.P. et al. Conformational adaptation of agonists to the human nuclear receptor RARγ. Nature Structural Biology 5, 199–202 (1998).

188. Glass, C.K. & Rosenfeld, M.G. The coregulator exchange in transcriptional functions of nuclear receptors. Genes Dev. 14, 121-141 (2000).

189. Lefebvre, P. et al. Transcriptional activities of retinoic acid receptors. Vitam. Horm. 70, 199-264 (2005).

190. Perissi, V. et al. Molecular determinants of nuclear receptor-corepressor interaction. Genes Dev. 13, 3198-208 (1999).

191. Bannister, A.J. & Kouzarides, T. The CBP-coactivator is a histone acetyltransferase. Nature 384, 641-643 (1996).

192. Ogryzko, V.V., Schiltz, R.L., Russanova, V., Howard, B.H. & Nakatani, Y. The transcriptional coactivators p300 and CBP are histone acetyltransferases. Cell 87, 953-959 (1996).

193. Chen, H. & Evans, R.A. transcriptional corepressor that interacts with nuclear hormone receptors. Nature 377, 454-457 (1995).

194. Horlein, A.J. et al. Ligand-independent repression by the thyroid hormone receptor mediated by a nuclear receptor corepressor. Nature 377, 397-404 (1995).

121 195. Dressel, U. et al. Alien, a highly conserved protein with characteristics of a corepressor for members of the nuclear hormone receptor superfamily. Mol. Cell Biol. 19, 3383-3394 (1999).

196. Perissi, V. et al. Molecular determinants of nuclear receptor-corepressor interaction. Genes Dev. 13, 3198-3208 (1999).

197. Ordentlich, P., Downes, M. & Evans, R.M. Corepressors and nuclear hormone receptor function. Curr. Top Microbiol. Immunol. 254, 101-116 (2001).

198. Horwitz, K.B. et al. Nuclear receptor coactivators and corepressors, Mo.l Endocrinol. 10, 1167-1177 (1996).

199. Nagy, L. et al. Mechanism of corepressor binding and release from nuclear hormone receptors. Genes Dev. 13, 3209-3216 (1999).

200. Heinzel, T. et al. A complex containing N-CoR, mSin3 and histone deacetylase mediates transcriptional repression. Nature 387, 43-48 (1997).

201. Alland, L. et al. Role for NCoR and histone deacetylase in Sin3-mediatedd transcriptional repression. Nature 387, 49-55 (1997).

202. Jenuwin, T. & Allis, C.D. Translating the histone code. Science 293, 1074-1080 (2001).

203. Delva, J.N. et al. Physical and functional interactions between cellular retinoic acid binding protein II and the retinoic acid-dependent nuclear complex. Mol. Cell. Biol. 19, 7158–7167 (1999).

204. Ito, M. & Roeder, R.G. The TRAP/SMCC/mediator complex and thyroid hormone receptor function. Trends Endocrinol. Metab. 12, 127-134 (2001).

205. Rachez, C. & Freedman, L.P. Mediator complexes and transcription. Curr. Opin. Cell Biol. 13, 274-80 (2001).

122 206. Muscat, G.E., Burke, L.J. & Downes, M. The corepressor N-CoR and its variants RIP13a and RIP13Delta1 directly interact with the basal transcription factors TFIIB, TAFII32 and TAFII70. Nucleic Acids Res. 26, 2899-907 (1998).

207. Wong, C.W. & Privalsky, M.L. Transcriptional repression by the SMRT-mSin3 corepressor: multiple interactions, multiple mechanisms, and a potential role for TFIIB. Mol. Cell Biol. 18, 5500-10 (1998).

208. Römer, S. & Fraser, P.D. Recent advances in carotenoid biosynthesis, regulation, and manipulation. Planta 221, 1533-1535 (2005).

209. Spurgeon, S.L. & Porter, J.W. Biosynthesis of carotenoids, Biosynthesis of lsoprenoid Compounds. Wiley & Sons, New York 2, 1-122 (1983).

210. Olson, J.A. & Krinsky, N.I. Introduction: the colorful, fascinating world of the carotenoids: important physiologic modulators, FASEB Journal 9, 1547-1550 (1995).

211. Harrison, E.H. Mechanism of Digestion and Absorption of Dietary Vitamin A. Ann. Rev. Nutr. 35, 87-103 (2005).

212. Allegretto, E.Z. et al. Transactivation properties of retinoic acid and retinoid X receptors in mammalian cells and yeast. Journal of Biological Chemistry 26, 625- 633 (1993).

213. Keidel, S., LeMotte, P. & Apfel, C. Different agonist- and antagonist-induced conformational changes in retinoic acid receptors analyzed by proteases mapping. Mol. Cell Biol. 14, 287-298 (1994).

214. Ostrowski, J., Hammer, L., Roalsvig, T., Pokornowski, K. & Reczek, P.R. The N- terminal portion of domain E of retinoic acid receptors and is essential for the recognition of retinoic acid and various analogs. Proc. Natl. Acad. Sci. 92, 1812- 1816 (1995).

215. Mangelsdorf, D.J., Ong, E.S., Dyck, J.A. & Evans, R.M. Nuclear receptor that identifies a novel retinoic acid response pathway. Nature 345, 224-229 (1990). 123 216. Rowe, A., Eager, N.S. & Brickell, P.M. A member of the RXR nuclear receptor family is expressed in neural-crest-derived cells of the developing chick peripheral nervous system. Development 111, 771-778 (1991).

217. Yu, V.C. et al. RXRβ: a coregulator that enhances binding of retinoic acid, thyroid hormone, and vitamin D receptors to their cognate response elements, Cell 67, 1251-1266 (1991).

218. Heyman, R.A. et al. 9-cis Retinoic acid is a high affinity ligand for the retinoid X receptor. Cell 68, 397-406 (1992).

219. Leid, M et al. Purification, cloning, and RXR identity of the HeLa cell factor with which RAR or TR heterodimerizes to bind target sequences efficiently. Cell 68, 377-395 (1992).

220. Levin, A.A. et al. 9-cis Retinoic acid stereoisomer binds and activates the nuclear receptor RXRα. Nature 355, 359-361 (1992).

221. Mangelsdorf, D.J. et al. Characterization of three RXR genes that mediate the action of 9-cis retinoic acid. Genes Dev. 6, 329-344 (1992).

222. Kliewer, S.A., Umesono, K., Mangelsdorf, D.J. & Evans, R.M. Retinoid X receptor interacts with nuclear receptors in retinoic acid, thyroid hormone and vitamin D3 signaling. Nature 355, 446-449 (1992).

223. Kliewer, S.A., Umesono, K., Noonan, D.J., Heyman, R.A. & Evans, R.M. Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors. Nature 358, 771-774 (1992).

224. Forman, B.M., Umesono, K., Chen, J. & Evans, R.M. Unique response pathways are established by allosteric interactions among nuclear hormone receptors. Cell 81, 541-550 (1995).

225. Mangelsdorf, D.J. et al. A direct repeat in the cellular retinol-binding protein type II gene confers differential regulation by RXR and RAR. Cell 66, 555-561 (1991). 124 226. Mader, S. et al. The patterns of binding of RAR, RXR and TR homo- and heterodimers to direct repeats are dictated by the binding specificities of the DNA binding domains. EMBO Journal 12, 5029-5041 (1993).

227. Wan, Y.J. et al. Hepatocyte-specific mutation establishes retinoid X receptor ∀ as a heterodimeric integrator of multiple physiological processes in the liver. Mol. Cell. Biol. 20, 4436-4444 (2000).

228. Boehm, M.F. et al. Synthesis and structure-activity relationships of novel retinoid X receptor-selective retinoids. J. Med. Chem. 37, 2930-2941 (1994).

229. Boehm, M.F. et al. Design and synthesis of potent retinoid X receptor selective ligands that induce apoptosis in leukemia cells. J. Med. Chem. 38, 3146-3155 (1995).

230. Heald, P. The treatment of cutaneous T-cell lymphoma with a novel retinoid. Clin. Lymphoma S1, S45-S49 (2000).

231. Hurst, R.E. Bexarotene ligand pharmaceuticals. Curr. Opin. Investig. Drugs 4, 514- 523 (2000).

232. Kempf, W., Kettelhack, N., Duvic, M. & Burg, G. Topical and systemic retinoid therapy for cutaneous T-cell lymphoma. Hematol. Oncol. Clin. North Am. 17, 1405-1419 (2003).

233. Zhang, C. & Duvic, M. Retinoids: therapeutic applications and mechanisms of action in cutaneous T-cell lymphoma. Dermatol. Ther. 16, 322-330 (2003).

234. Corey, E.J., Gilman, N.W. & Ganem, B.E. New method for the oxidation of aldehydes to carboxylic acids and esters. J. Am. Chem. Soc. 90, 5616-5617 (1968).

235. Kithsiri, W.E.M. et al. Synthesis and preliminary biological evaluations of β- carotene and retinoic acid oxidation products. Bioorg. Med. Chem.14, 7875-7879 (2006).

125 236. Promega Corporation, Madison, WI, USA, pRL-TK Vector, Technical Bulletin No. TB240 (2001).

237. Gampe, R.T. et al. Structural basis for autorepression of retinoid X receptor by tetramer formation and the AF-2 helix. Genes Dev. 14, 2229-2241 (2000).

238. Egea, P.F. et al. Crystal structure of the human RXRα ligand-binding domain bound to its natural ligand: 9-cis retinoic acid. EMBO Journal 19, 2592-2601 (2000).

239. Jain, A.J. Surflex-Dock 2.1: Robust performance from ligand energetic modeling, ring flexibility, and knowledge-based search. J. Comput. Aided Mol. Des. 21, 281- 306 (2007).

240. Tang, G., Wang, X.D., Russell, R.M. & Krinsky, N.I. Characterization of β-apo- 13-carotenone and β-apo-14'-carotenal as enzymatic products of the excentric cleavage of β-carotene. Biochemistry 30, 9829-9834 (1991).

241. Boehm, M.F. et al. Synthesis of high specific activity [3H]-9-cis-retinoic acid and its application for identifying retinoids with unusual binding properties. J. Med. Chem. 37, 408-414 (1994).

242. Goldstein, A., Aronow, L. & Kalman, S.M. Principles of Drug Action: The Basis of Pharmacology 2nd edition. Wiley, New York (1987).

243. Kenakin, T.P. Pharmacologic Analysis of Drug-Receptor Interaction. Raven Press, New York (1974).

244. Schachtschabel, D., Schimek, C., Wöstemeyer, J. & Boland, W. Biological activity of trisporoids and trisporoid analogues in Mucor mucedo (-). Phytochemistry 66, 1358- 136 (2005).

245. Goodwin, T.W. The Biosynthesis of Vitamins and Related Compounds. Chapter 14, 270-319, Academic Press, London (1963).

126 246. Repa, J.J., Hanson, K.K. & Clagett-Dame, M. All-trans retinol is a ligand for the retinoic acid receptors. Proc. Natl. Acad. Sci. USA 90, 7293-7297 (1993).

247. Rarey, M., Kramer, B. & Lengauer, T. Docking of hydrophobic ligands with interaction-based matching algorithms. Bioinformatics 15, 243-250 (1999).

248. Marsh, R.S. et al. β-Apocarotenoids do not significantly activate retinoic acid receptors α or β. Experimental Biology and Medicine 235, 342-348 (2010).

249. Tay, S., Dickmann, L., Dixit, V. & Isoherranen, N. A comparison of the roles of peroxisome proliferator-activated receptor and retinoic acid receptor on CYP26 regulation. Mol. Pharmacol. 77, 218-227 (2010).

250. de Lera, A.R., Bourguet, W., Altucci, L. & Gronemeyer, H. Design of selective nuclear receptor modulators: RAR and RXR as a case study. Nat. Rev. Drug Discovery 6, 811-820 (2007).

251. Eroglu, A., Hruszkewycz, D.P., Curley, R.W. & Harrison, E.H. The eccentric cleavage product of β-carotene, β-apo-13-carotenone, functions as an antagonist of RXRα. Archives of Biochemistry and Biophysics 504, 11-16 (2010).

252. Wang, X.D. et al. Retinoid signaling and activator protein-1 expression in ferrets given β-carotene supplements and exposed to tobacco smoke. J. Natl. Cancer Inst. 91, 60-66 (1999).

253. Russell, R.M. The enigma of β-carotene in carcinogenesis: What can be learned from animal studies. J. Nutr. 134, 262S-268S (2004).

254. Omenn, G.S. et al. Risk factors for lung cancer and for intervention effects in CARET, the beta-carotene and retinol efficacy trial. J. Natl. Cancer Inst. 88, 1550-1559 (1996).

255. Liu, C. et al. Effects of physiological versus pharmacological β-carotene supplementation on cell proliferation and histopathological changes in the lungs of cigarette smoke-exposed ferrets Carcinogenesis 21, 2245-2253 (2000).

127 256. Canene-Adams, K., Campbell. J.K., Zaripheh, S., Jeffery, E.H. & Erdman, J.W. Jr. The tomato as a functional food. Journal of Nutrition 135, 1226-1230 (2005).

257. Voutilainen, S., Nurmi, T., Mursu, J. & Rissanen, T.H. Carotenoids and cardiovascular health. Am J Clin Nutr. 83, 1265-1271 (2006).

258. Levy, J. et al. Lycopene is a more potent inhibitor of human cancer cell proliferation than either alpha-carotene or beta-carotene. Nutr Cancer 24, 257-266 (1995).

259. Prakash, P., Jackson, C.L. & Gerber, L.E. Subcellular accumulation of beta- carotene and retinoids in growth-inhibited NCI-H69 small cell lung cancer cells. Nutr Cancer 34, 76-82 (1999).

260. Pastori, M., Pfander, H., Boscoboinik, D. & Azzi, A. Lycopene in association with alpha-tocopherol inhibits at physiological concentrations proliferation of prostate carcinoma cells. Biochem Biophys Res Commun. 250, 582-585 (1998).

261. Tang, L., Jin, T., Zeng, X. & Wang, J.S. Lycopene inhibits the growth of human androgen-independent prostate cancer cells in vitro and in BALB/c nude mice. J Nutr. 135, 287-290 (2005).

262. Amir, H. et al. Lycopene and 1,25-dihydroxyvitamin D3 cooperate in the inhibition of cell cycle progression and induction of differentiation in HL-60 leukemic cells. Nutr Cancer 33, 105-112 (1999).

263. Tonucci, L.H. et al. Carotenoid content of thermally processed tomato-based food products. J. Agric. Food Chem. 43, 579-586 (1995).

264. Giovannucci, E. A review of epidemiologic studies of tomatoes, lycopene, and prostate cancer. Exp. Biol. & Med. 227, 852-859 (2002).

265. Shi, J. Lycopene in tomatoes: chemical and physical properties affected by food processing. Critical Reviews in Food Science and Nutrition 40, 1-42 (2000).

128 266. Willis, M.S. & Wians, F.H. The role of nutrition in preventing prostate cancer: a review of the proposed mechanisms of action of various dietary substances. Clinica Chimica Acta 330, 57-83 (2003).

267. Erdman, J.R. Jr., Bierer, T.L. & Gugger, E.T. Absorption and transport of carotenoids. In: Canfield, L.M., Krinsky, N.I. & Olson, J.A. (eds). Carotenoids in Human Health New York Academy of Sciences: New York, 691, 76-85 (1993).

268. Boileau, A.C. et al. Cis-lycopene is more bioavailable than trans-lycopene in vitro and in vivo in lymph-cannulated ferrets. Journal of Nutrition 129, 1176–1181 (1999).

269. Gartner, C., Stahl, W. & Sies, H. Lycopene is more bioavailable from tomato paste than from fresh tomatoes. American Journal of Clinical Nutrition 66, 116- 122 (1997).

270. Stahl, W., Schwarz, W., Sundquist, A.R. & Sies, H. cis-trans Isomers of lycopene and beta-carotene in human serum and tissues. Arch. Biochem. Biophys. 294, 173- 177 (1992).

271. Rao, A.V., Ray, M.R. & Rao L.G. Lycopene. Adv. Food Nutr. Res. 51, 99-164 (2006).

129