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Winter 2020
THE ROLE OF GLI3 IN INFLAMMATION
Stephan Josef Matissek University of New Hampshire, Durham
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Recommended Citation Matissek, Stephan Josef, "THE ROLE OF GLI3 IN INFLAMMATION" (2020). Doctoral Dissertations. 2552. https://scholars.unh.edu/dissertation/2552
This Dissertation is brought to you for free and open access by the Student Scholarship at University of New Hampshire Scholars' Repository. It has been accepted for inclusion in Doctoral Dissertations by an authorized administrator of University of New Hampshire Scholars' Repository. For more information, please contact [email protected]. THE ROLE OF GLI3 IN INFLAMMATION
BY
STEPHAN JOSEF MATISSEK
B.S. in Pharmaceutical Biotechnology, Biberach University of Applied Sciences,
Germany, 2014
DISSERTATION
Submitted to the University of New Hampshire
in Partial Fulfillment of
the Requirements for
the Degree of
Doctor of Philosophy
In
Biochemistry
December 2020
This dissertation was examined and approved in partial fulfillment of the requirement for the degree of Doctor of Philosophy in Biochemistry by:
Dissertation Director, Sherine F. Elsawa, Associate Professor
Linda S. Yasui, Associate Professor, Northern Illinois University
Paul Tsang, Professor
Xuanmao Chen, Assistant Professor
Don Wojchowski, Professor
On October 14th, 2020
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ACKNOWLEDGEMENTS
First, I want to express my absolute gratitude to my advisor Dr. Sherine
Elsawa. Without her help, incredible scientific knowledge and amazing guidance I would not have been able to achieve what I did. It was her encouragement and believe in me that made me overcome any scientific struggles and strengthened my self-esteem as a human being and as a scientist. There was never a time where her door was not open, and she was always reachable even at late hours or holidays. Her heartful manner and positive presence was one of the main reasons that kept me motivated in my
Ph.D. studies and her scientific skillset and plethora of knowledge was always a standard I tried to achieve. Dr. Elsawa taught me that no goal is too big to be reached as long as I keep working consistently ,always try to improve and always believe in myself and it is without a doubt that without her I would not be sitting here finishing my dissertation. I consider my Ph.D. as one of the biggest achievements in my life which I would have never reached without the guiding hand of Dr. Elsawa.
Besides Dr. Elsawa I also would like to deeply express my gratitude to my committee members, Dr. Linda Yasui, Dr. Don Wojchowski, Dr. Paul Tsang and Dr. Xuanmao Chen for their professional insights, kind help and amazing scientific suggestions.
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This research was supported by grant P20GM113131 from the National
Institute of General Medical Sciences of the NIH. I also would like to thank the
NIU Office of Vice President for Research and Innovation Partnerships
(OVPRIP) for financial support for this project. Additionally, I would like to thank
UNH for proving travel funds which allowed me to attend several conferences.
These conferences were very valuable experiences from which I gained many important connections. Finally, I want to thank UNH for providing graduate student teaching assistantships and linked financial support.
I absolutely want to thank my family and friends for their never-ending emotional (but also financial) support in my studies. Doing a Ph.D. can be very exhausting and without them supporting me and believing in me I would have never finished my studies. In particular, I want to thank following people: My mother, for always believing in me especially when I had trouble believing in myself and for being the most supportive person in my life. There was never a struggle that I faced without having my mother’s encouraging words in my ear.
She is my role model for how to deal with problems since without her teaching me to be patient with myself and solving issues by putting one foot in front of the other I would not be where I am today. My sister Karina for being an extremely accomplished scientist who never fails to achieve any goal she sets.
Her love to science motivated me to start my Ph.D. My sister Astrid whose discipline seeks its equal and motivated me to push through any late-night shifts in lab. My father, for being such a tough person and him seeing how he
iv pushed through problems gave me, besides other things, the strength to push through mine.
I also want to express my gratitude to past and present members of the Elsawa
Lab. I want to thank for all the professional help provided, for the fun times we shared and contributions to my project.
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TABLE OF CONTENT
ACKNOWLEDGEMENTS ...... iii TABLE OF CONTENT ...... vi LIST OF FIGURES...... ix ABSTRACT ...... xi
CHAPTER 1 ...... 1 Inflammation, a double-edged sword ...... 1 Acute inflammation; the good response ...... 2 General principles of an acute inflammatory response ...... 2 Principles of inflammation: para-inflammation ...... 3 Principles of inflammation: acute inflammation ...... 4 Exogenous stimuli ...... 5 Infection ...... 5 Toll-like receptors (TLRs) and their signaling pathways ...... 6 Extracellular TLRs ...... 7 TLR2 only complexes with TLR1 or TLR6...... 7 TLR4 ...... 9 MYD88 pathway ...... 10 TRIF pathway ...... 11 TLR5 ...... 12 TLR10 ...... 13 Intracellular TLRs ...... 13 TLR3 ...... 13 TLR7/8 ...... 14 TLR9 ...... 15 Endogenous stimuli ...... 17 Role of adaptive immune system in inflammation ...... 18 Chronic inflammation, the bad one ...... 19
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Infections related to chronic inflammation ...... 20 Cellular stress related to chronic inflammation (DAMPs) ...... 21 Molecular characteristics involved in diseases caused by chronic inflammation ...... 22 Autoimmunity ...... 22 Cancer ...... 25 Molecular pathways linking chronic inflammation to cancer ...... 26 NF-κB, STAT and SHH...... 26 GLI3: A mediator of genetic diseases, development and cancer ...... 28 Regulation and Structure ...... 29 Hedgehog ligands and their function ...... 29 Classic (canonical) Hh signaling ...... 30 In mammals ...... 31 Non-classical (non-canonical) HH signaling ...... 34 Regulation of GLI3...... 38 Gene locus and domains of mouse and human Gli3/GLI3 ...... 38 Processing of GLI3-FL to GLI3-R ...... 40 GLI3-FL, stabilization and degradation ...... 41 Role in Development, Immune system and Cancer ...... 42 Development ...... 42 GCPS, PHS and Tibial Hemimelia ...... 43 Brain development ...... 44 Lung development ...... 46 Role of GLI3 in the immune system ...... 46 In Cancer ...... 48 MicroRNA (miRNA) and GLI3 ...... 59 In cancer, liver fibrosis and spermatogenesis ...... 59 Conclusions ...... 61 CHAPTER 2 ...... 70 A novel mechanism of regulation of the transcription factor GLI3 by toll-like receptor signaling ...... 70
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Introduction ...... 70 Experimental Procedures ...... 72 Cell lines and primary cells ...... 72 Reagents ...... 73 RNA isolation and Quantitative RT-PCR (qPCR) ...... 73 Chromatin immunoprecipitation (ChIP) assay ...... 74 Plasmid constructs and cell transfections ...... 75 Immunoblotting ...... 75 Statistical analysis ...... 76 Results ...... 76 LPS stimulation induces GLI3 expression ...... 76 LPS/TLR4-induced GLI3 occurs through TRIF signaling ...... 77 TLR4-TRIF regulates GLI3 through the transcription factor IRF3 ...... 78 IRF3 directly binds the GLI3 promoter ...... 79 IRF3 is required for TRIF-induced GLI3 ...... 80 Discussion...... 80 CHAPTER 3 ...... 96 BIOLOGICAL ROLE OF TLR-INDUCED GLI3 ...... 96 Introduction ...... 96 Experimental Procedures ...... 98 ELISA ...... 98 Mice ...... 98 RNA seq ...... 98 Bioinformatic analysis of RNA-seq data ...... 99 Cell culture and Reagents ...... 99 Statistical analysis ...... 100 Plasmid constructs ...... 100 RNA isolation and qPCR ...... 100 DNA extraction method ...... 101 Results ...... 101 Knockdown of GLI3 reduces cytokines expression in vitro ...... 101 Gli3 is required for TLR-TRIF-induced cytokine secretion ...... 102
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RNA-seq analysis reveals Gli3 modulates inflammation ...... 103 Discussion...... 103 SUMMARY/CONCLUSION ...... 117 LIST OF REFERENCES ...... 120 APPENDIX ...... 160
LIST OF FIGURES
Figure 1-1: Biochemical domains of GLI family members ...... 63
Figure 1-2: Biochemical domains of human GLI3 with posttranslational
modifications ...... 65
Figure 1-3: Regulation of GLI3-FL and GLI3-R...... 67
Figure 1-4: Alignment of human and mouse GLI3/Gli3 protein sequences...... 69
Figure 2-1: LPS induces GLI3 expression in a HH independent manner...... 85
Figure 2-2: Functional TLR4 is required for GLI3 expression...... 87
Figure 2-3: LPS-induced GLI3 is regulated by TRIF downstream of TLR4...... 89
Figure 2-4: GLI3 is regulated by IRF3 downstream of TRIF...... 91
Figure 2-5: IRF3 regulates GLI3 by directly binding to IRF3 binding sites...... 93
Figure 2-6: Model for TLR-TRIF-induced GLI3...... 95
Figure 3-1: GLI3 reduces cytokine expression in monocytes ...... 108
Figure 3-2: Breeding schematic to generate mice which lack Gli3 in
macrophages and confirmation of lack of Gli3 by PCR and Western Blot...... 110
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Figure 3-3: RNA-seq and pathway analysis reveal Gli3 being a potential major
regulator of inflammation...... 112
Figure 3-4: GLI3 deficient macrophages secrete significantly less
proinflammatory cytokines than macrophages that have functional GLI3...... 114
Figure 3-5: RNA-seq analysis strongly suggests Gli3’s involvement in
macrophage polarization...... 116
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ABSTRACT
THE ROLE OF GLI3 IN INFLAMMATION
By
Stephan Matissek
University of New Hampshire
Inflammation is an important and healthy mechanism that our body uses for wound-healing and fighting off infections by utilizing molecular pathways of immune and non-immune cells. However, when inflammation is persistent or chronic it can have severe consequences that can lead to auto-immune diseases or cancer. Pathogen-associated molecular patterns (PAMPs), such as Lipopolysaccharide (LPS), present on gramnegative bacteria, or double- stranded RNA (dsRNA), present in RNA-viruses, were shown to be potent inducers of inflammation. These PAMPs are known ligands of Toll like receptor
4 (TLR4), activated by LPS and Toll-like receptor 3 (TLR3), activated by dsRNA which are carried on the outside and inside of immune cells. Interaction of
PAMPs with their receptors lead to production of cytokines such as Interleukin
6 (IL6), Tumor necrosis factor α (TNFα) or C-C Motif Chemokine Ligand 2
(CCL2), which bind to other receptors on immune and non-immune cells which leading to an amplification of the inflammatory signal inside the host. Besides
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TLR signaling, studies have found other important signaling events involved in inflammation and chronic inflammation. One of those pathways is the
Hedgehog (HH) signaling pathway. At the center of HH signaling are Patched1
(PTCH1) and Smoothened (SMO) receptor system but also GLI transcription factor family members GLI1, GLI2 and GLI3. Whilethe role of GLI1 and GLI2 was described very frequently in inflammation and inflammation related diseases such as cancer, the role of GLI3 was barely studied. This dissertation focuses on the role of GLI3 in inflammation.
Upon activation of TLR4 signaling by LPS, either Myeloid Differentiation
Primary Response Protein (MYD88) or TIR-domain-containing adapter- inducing interferon-β (TRIF) dependent signaling can occur. Downstream of
MYD88, Nuclear Factor-κB (NFκB) and Mitogen-Activated Protein Kinase
(MAPK) pathways are activated while downstream of TRIF, Interferon
Regulatory Factor 3 (IRF3) pathway in addition to NFκB and MAPK signaling are initiated. These transcription factors bind to promoter regions of cytokine related genes and initiate cytokine expression and secretion. The TRIF cascade also applies to TLR3 signaling, which is the only adaptor molecule involved in this signaling and which can be initiated by Poly (I:C), a dsRNA homolog. We found that upon stimulation of monocytes with LPS, GLI3 expression is upregulated and that this upregulation is highly dependent on the
TLR-TRIF-cascade. Additionally, we found that IRF3 is the transcription factor which regulates GLI3 expression levels downstream of TRIF by directly binding to its promoter region. The role of IRF3 regulation of GLI3 expression was xii further confirmed by treatment of monocytes with Poly(I:C) which robustly induced GLI3 levels and significantly increased binding of IRF3 to the GLI3 promoter. This strongly supports a role for GLI3 in inflammation. To further investigate the role of GLI3 in inflammation we utilized the CreloxP system to generate conditional GLI3 knock-out mice, lacking GLI3 expression in macrophages termed as M-GLI3-/- . We isolated peritoneal macrophages from wild-type (WT) mice and M-GLI3-/- mice and treated those with LPS and found a significant difference in expression of inflammation related genes by RNA- seq analysis. Furthermore, we identified GLI3 asa new candidate in M2 macrophage polarization, an anti-inflammatory macrophage subset highly involved in promoting tumor growth. Finally, we identified GLI3 as a regulator of inflammatory response since isolated macrophages from M-GLI3-/- mice secreted significantly less proinflammatory cytokines IL6, CCL2 and TNFα than macrophages from WT mice.
Therefore, our studies identified TLR signaling as novel molecular mechanism in regulating GLI3 expression and GLI3 as regulator of inflammation.
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CHAPTER 1
INTRODUCTION
Inflammation, a double-edged sword
Research on inflammation is as old as humankind itself. Hippocrates and the roman physician Galen described several symptoms such as redness, pain, heat, swelling and disturbance of function, as hallmarks of inflammation [1]. In addition, Menkinand
Warner(1937) identified hypoxia and the resulting shift to an acidic cellular environment as another hallmark [2]. These hallmarks were indentified by John Hunter (1994) as conditions that must be met so that a wound heals; meaning inflammation is an important response of our body to re-establish homeostasis. In the context of healthy or acute inflammation, molecular counterparts (anti-inflammatory pathways) turn inflammation off after the wound is healed. However, this process is not flawless. Due to genetic predispositions or external stress factors, homeostasis can be disrupted which can lead to a prolonged form of inflammation known as chronic/disease associated inflammation.
In the following section, we will discuss molecular pathways and principles involved in acute inflammation which are disrupted therefore leading to chronic inflammation – a form of inflammation that promotes infectious, autoimmune or malignant diseases.
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Acute inflammation; the good response
Acute inflammation is a necessary mechanism used by the body to fight an infection or heal a wound. The literature differentiates between basal homeostatic conditions, para-inflammation and true/acute inflammation [3] [4] [5]. Basal homeostatic condition is referred to as a non-inflammatory state, while para-inflammation is a state of inflammation between true inflammation/acute inflammation and homeostatic conditions.
Para-inflammation is oftentimes related to aging and is induced by tissue stress or malfunction. On the other hand, true/acute inflammation is induced by tissue damage or by pathogenic infections [6]. Another difference between para-inflammation and true inflammation is the recruitment of different immune cell types to the site of disruption and the level of cytokines (mediators of inflammation) produced which will be discussed later in more detail.
General principles of an acute inflammatory response
Based on current data, certain components were proposed to be the center of a generic inflammatory pathway including: inducers, sensors, mediators and effectors. An inducer is considered anything which disrupts the homeostatic state of the body; meaning infectious agents such as bacteria or viruses, or non-infectious stimuli such as toxins or damaged tissue. Sensors are molecules triggered molecules by the inducer and activate the production of mediators. Sensors can be surface receptors found on immune cells
2 such as the pattern recognition receptor (PRR) toll-like receptor 4 (TLR4) found on most myeloid cells including macrophages or dendritic cells (DCs). PRRs are activated by
Pathogen-associated molecular patterns (PAMPs; ligands found on the surface of microbes) which are inducers. Mediators are molecules that activate pain sensors in addition to activating effectors. Effectors can be cells or tissues. Mediators can be cytokines which are ligands that bind specific receptors expressed on immune cells and other cells such as fibroblasts or epithelial cells. The severity of an inflammatory response depends on the degree of damage or infection and how fast homeostasis can be restored.
Principles of inflammation: para-inflammation
The idea of para-inflammation was first suggested by Medzhitov (2008). It can be considered an inflammatory state between homeostasis and acute inflammation due to tissue damage or infection. It occurs when stress stimuli compromises tissue (inducer) which activates tissue-resident macrophages (sensor) to secrete cytokines and chemokines, mediators of inflammation, and to attract inflammatory monocytes (effector) from blood vessels [7]. These monocytes help to re-establish homeostasis by possibly differentiating into macrophages themselves and at some point, by producing anti- inflammatory components to reduce inflammation. Interestingly neutrophils, which are essential inflammatory cells, are not involved in the parainflammatory process. Since the idea of para-inflammation is relatively new, to date, there is no research to characterize the mechanisms/ molecular steps from stress induction to stress relief. Para-inflammation is mostly reported to be important in dealing with agerelated stress on tissue. It has also
3 been linked to obesity, atherosclerosis, Alzheimer, Parkinson’s and Age Related Macular
Degeneration (AMD) when it becomes pathological [8][9][10][11][12][13]. In addition, multiple studies suggest an effect of para-inflammation in positively regulating cancer development and progression [14][15][16]. Chronic inflammation is now recognized as a hallmark of cancer and pathological parainflammation might be a subset of inflammation that leads to cancer initiation and growth[17].
Principles of inflammation: acute inflammation
True or acute inflammation is established following signs that occur together ,such as redness, pain, heat, swelling and disturbance of function. In one study, the term true inflammation was adapted from the publication by Chen et al (2015) to differentiate it from para-inflammation. However, acute inflammation and true inflammation are interchangeable terms. In comparison to para-inflammation, a tissue needs to be damaged or an infection needs to occur in order for this inflammatory mechanism to be activated. Exogenous and endogenous stress signals induce the activation and recruitment of immune cells which are the main effectors of acute inflammation. It is considered a healthy response to restore homeostasis and multiple complex systems are involved in resolving acute inflammation which will be described and discussed in this section.
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Exogenous stimuli
Infection
Once an infection is established by pathogens such as bacteria or viruses, PAMPs are recognized by immune cells via PRRs. The immune cells that initially recognize these pathogens are part of the innate immune system. This innate immune system which then forwards inflammation-related signaling to the adaptive immune cells. Resident macrophages and mast cells are myeloid innate immune cells, which start secreting chemokines, cytokines, vasoactive amines andeicosanoids in response to recognizing pathogenic microbes by Toll-like receptors (TLRs) or nucleotide-binding oligomerizationdomain protein (NOD)-like receptors (NLRs) [18]. This leads to activation of the endothelium which leads to selective release of plasma proteins and neutrophils to the extravascular tissue at the site of infection [19]. Neutrophils start producing and secreting reactive oxygen species (ROS) in addition to proteinase 3, elastase, cathepsin
G and reactive nitrogen species whichterminate the pathogen [20]. After clearance of the infectious site, mostly tissue resident macrophages initiatethe repair phase, meaning neutrophils undergo apoptosis and macrophages phagocytose those apoptotic cells [21].
In addition to the phagocytic clearance of neutrophils, their permeabilization is inhibited by the production of lipoxins, proctins, resolvins and TGF- leading to the initiation of the mechanisms involved in tissue repair [22]. Inflammation can also be triggered without the involvement of dedicated receptors by metabolites produced by pathogens. For example, exotoxins which are secreted by gram-positive bacteria, lead to pore formation and efflux of K+ ions. This triggers NACHT, leucine-rich repeat- and pyrin-domain-
5 containing protein (NALP3) inflammasome which is a multimeric protein complex that leads to release of cytokines such as IL-1β and IL18 to fight infections by causing cell death [23][24].
Toll-like receptors (TLRs) and their signaling pathways
As previously mentioned, resident macrophages and neutrophils form a major response to bacterial infections which leads to inflammation. In addition to macrophages and neutrophils, dendritic cells are also involved in inducing inflammation in response to infection. All innate immune cells carry PRRs that recognize PAMPs, which are unique ligands solely presented by pathogens. One group of PRRs are TLRs. There are 10 subtypes of TLRs (TLR1-10) in humans and 12 in mice (TLR1–TLR9, TLR11–TLR13)
[25]. TLR1-10 can be classified in two groups, extracellular TLRs and intracellular TLRs.
Extracellular TLRs are TLR1, TLR2, TLR4, TLR5, TLR6 , which are known to recognize ligands presented by microbes, while TLR3, TLR7, TLR8 and TLR9 are intracellular receptors that react to microbial-specific nucleic acids [26]. The following section will describe the role of TLRs, including their specificity and their respective molecular pathways involved in response to infections.
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Extracellular TLRs
TLR2 only complexes with TLR1 or TLR6
To date, there is no evidence that TLR2, TLR1 or TLR6 can act as receptors that mediate signaling on their own. However, they can form complexes that were found to be expressed on neutrophils, natural killer (NK) cells, B cells and monocytes [27].
TLR2TLR1/TLR2-TLR6 complexes form M-shaped structures, where TLR1 or TLR6 is the decisive component for binding di- or tri-acylated lipopeptides. This difference arises from the fact that TLR6 has no hydrophobic channel that could bind to a lipid chain which is present in TLR1. TLR2 binds to two out of three lipid chains from tri-acylated lipopeptides, thus the unbound lipid chain can interact with TLR1 and not with TLR6. Thus TLR2-TLR6 complexes can only recognize diacylated lipopeptides while TLR2-TLR1 complex can bind to tri-acylated lipopeptides [28]. Additionally, cluster of differentiation 36 (CD36) interacts with TLR2-TLR6 complex and dectin-1 can interact with TLR2 which induces internalization of microbes [29][30]. Due to TLR2’s interaction partners, it allows it to recognize both gram- negative and gram-positive bacteria, as well as fungi (through zymosan) and certain viruses, such as measles virus through its hemagglutinin protein
[31][32]. In addition to its involvement in fighting off Tuberculosis (Mycobacterium tuberculosis), TLR2 is also involved in fighting off infections of non-bacterial origin caused by parasites such as malaria (P. falciparum) , leishmaniasis (L. major), trypanosomiasis
(Trypanosoma cruzi) and filariasis [33].
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TLR2-TLR1/TLR2-TLR6 interactions solelysignal through Myeloid
Differentiation Primary Response Protein (MYD88) - Toll/Interleukin-1 Receptor
Domaincontaining Adapter Protein (TIRAP) Adaptor Protein complex. Once this pathway is activated, Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) is recruited which activates Interleukin-1 Receptor-Associated Kinase 1 (IRAK1) and Interleukin-1
Receptor-Associated Kinase 2 (IRAK2). Subsequently, IRAK4 and Tumor Necrosis
Factor Receptor-Associated Factor 6 (TRAF6) interact, which leads to polyubiquitination at Lys63 of IRAK4 and TRAF6. This effect mediated by TRAF6 with help of
UbiquitinConjugating Enzyme E2 13 (Ubc13) and Ubiquitin-Conjugating Enzyme E2
Variant 1A (Uev1A). This degree of ubiquitination at position 63 of Lys is crucial for further signaling events. Acquired ubiquitination ascertains binding of TGF-β-Activated Kinase 1 and MAP3K7-Binding Protein 2 (TAB2) and TGF-β-Activated Kinase 1 and MAP3K7-
Binding Protein 3 (TAB3) followed by activation of TGF-β-Activated Kinase 1 (TAK1).
Additionally, the activation of Interferon Regulatory Factor 5 (IRF5) is enabled. This post translational modification leads to interaction with Nuclear Factor-κB Essential Modulator
(NEMO), crucial regulatory element of the IκB Kinase (IκK) complex (IκKα-IκKβ).
Ultimately, this leads to phosphorylation of Inhibitor of κ B (IκB) and its subsequent ubiquitination and degradation which releases Nuclear Factor-κB (NF-κB) and induces transcriptional activity [34]. TAK1 also activates the Mitogen-Activated Protein Kinase
(MAPK) pathway leading to Extracellular Signal-Regulated Protein Kinases 1 and 2
(Erk1/2), p38 and Jun
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N-Terminal Kinase (Jnk) activation, finally activating the transcription factor Activating
Protein 1 (AP-1). The activation of NF- B and AP-1 as a result of TLR2/TLR1 or
TLR2/TLR6 complexes induce expression and secretion of proinflammatory cytokines such as Interleukin 6 (IL-6) and Interleukin 12 (IL-12) p40; however, they do not regulate the expression of type 1 interferons which are essential for antiviral responses.
TLR4
TLR4 is the only member of the TLR family which binds to lipopolysaccharide
(LPS), a ligand expressed on the outer membrane of gram-negative bacteria. TLR4 is expressed on both innate (monocytes, macrophages, dendritic cells, neutrophils, natural killer cells, mast cells, basophils) and adaptive (B-and T-cells) immune cells as well as on non-immune cells such as endothelial cells [35][36][37][38][39][40][41][42][43]. In addition to LPS, TLR4 also recognizes the presence of mycobacteria (e.g. Mycobacterium tuberculosis), fungi (e.g. Saccharomyces cerevisiae), protozoa (e.g. Leishmania major) and viruses (e.g. Respiratory syncytial virus (RSV) [44][45][46]. Most information about
TLR4-dependent immunological response was generated from research focusing on
LPS-TLR4 signaling. Therefore, it is not clear if the molecular steps involved in TLR4 recognition of gram-negative bacteria are the same for other microbes as well. The following section describes the molecular pathways activated upon interaction between
TLR4 and LPS.
LPS can be structurally divided into an inner leaflet with phospholipids and an outer leaflet containing lipid A, which is the endotoxin recognized by the innate immune system
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[47][48]. However, it’s not only TLR4 alone which is involved in recognizing lipid A. Several crucial components involved in LPS recognition were identified including: LPS binding protein (LBP), CD14 and MD-2. The first-in-line to recognize lipid A is LBP, which is a plasma protein that aggregates after LPS binding and moves it towards CD14 [49]. CD14 is present as a homodimer and transfers LPS to MD-2 which is present as a complex with
TLR4 [50][51]. MD-2 is the major binding component for LPS [52]. Upon LPS recognition, the TLR4-MD-2 complex forms a dimer with another TLR4-MD-2 complex and initiates transmembrane signaling [53]. Another protein TLR4 interacts with is 4-1BBL, which was shown to regulate cytokines downstream of TLR4 signaling and thus regulates the TLR4- dependent inflammatory response [54].
TLR4 is the only surface-TLR which can signal through both MYD88 and TRIF.
Both pathways are activated; however, MYD88 pathway activation is considered to occur prior to TRIF-dependent pathway activation [55]. TRIF-dependent pathway activation leads to TLR4 endocytosis which cancels out the MYD88 dependent pathway [56].
MYD88 binds to the adaptor protein TIRAP which regulates the expression of proinflammatory cytokines while TRIF interacts with the adaptor protein TRAM which leads to expression of type 1 interferons, in addition to proinflammatory cytokines [57].
MYD88-dependent TLR4 signaling does not regulate type 1 interferon expression [58].
MYD88 pathway
Upon ligand binding with TLR4-MD2 complex, TIRAP is recruited and binds
MYD88 leading to initiation of signaling [59]. Downstream of this event, IRAK4, IRAK1
10 and IRAK2 are recruited which subsequently leads to activation of TRAF6. This activates either IRF5, MAPK or NF-κB signaling which ultimately leads to the regulation of expression of proinflammatory cytokines. While there is difference in ligand specificity between TLR2 and TLR4, the MYD88-dependent signaling cascade is the same in those two TLR pathways.
TRIF pathway
In contrast to TLR2, TLR4 can also signal in a MYD88-independent manner by recruiting TRIF-TRAM adaptor proteins. As mentioned earlier, this signaling event occurs after the activation of MYD88 and leads to suppression of MYD88-dependent signaling since TLR4 is endocytosed at the beginning of the TRIF-signaling pathway [55]. After endocytosis, TRIF associates with TRAF6, TNFR1-Associated Death Domain Protein
(TRADD), the signal-responsive E3 ubiquitin ligase Pellino 1 (PELI1) and Receptor
Interacting Protein-1 (RIP-1) [60][61]. This interaction is important for transforming growth factor-activated kinase-1 (TAK-1) activation which is crucial for the regulation of NF-κB and MAPK signaling [62]. Additionally, TRIF is also able to interact with tumour necrosis factor receptor-associated factor 3 (TRAF3) [63]. Activation of TRAF3 leads to phosphorylation and activation of interferon regulatory factor 3 (IRF3) by recruitment of
TANK Binding Kinase (TBK-1) and IKKε [64]. Since TRIF activates both NF-κB and MAPK signaling in addition to activating IRF3 signaling, it leads to expression of both proinflammatory cytokines and type 1 interferons.
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TLR5
TLR5 is the only surface TLR which binds to the PAMP flagellin (a crucial component of bacterial motility). Flagella are oftentimes referred to as having a whip-like structure and enable bacterial movement by performing propeller-like rotation. These structures are anchored to the bacterial cell wall [65]. They are attached to the hookbasal- body (HBB) which genes are expressed first followed by the expression of the gene flagellin [66]. More than 30,000 flagellin structures can be assembled at the end of the hook. TLR5 recognizes flagella based on their conserved D1domain responsible for filament assembly[67] [68].
There are no reports on TLR5 interacting with other TLRs for signal transduction.
Additionally, no other co-receptors are known to be important for the TLR5-flagellin interaction. TLR5 is expressed on a wide variety of cells which comprises immune cells
(T-cells, monocytes, macrophages, dendritic cells, neutrophils, natural killer cells, basophiles and mast cells) and non-immune cells such as epithelial cells of the lung and intestines as well as on hepatocytes and adipocytes [69][70][71][72][73][74][75][76].
Important pathogen recognition associated with the presence of TLR5 include recognition of Pseudomonas aeruginosa, Eschericia coli, Salmonella typhimurium [77].
Once TLR5 binds to flagellin, it induces signal transfer through the MYD88 adaptor protein. Interestingly, the adaptor protein TIRAP is not required in TLR5 signal induction
[78]. After MYD88 activation, the signaling cascade follows the same signaling pattern as
TLR4 and TLR2, therefore ultimately activates the transcription factors IRF7, NF-κB and
AP-1 leading to the regulation of proinflammatory cytokines.
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TLR10
Very little research has been performed to define the role of TLR10 in an infectioninduced inflammatory reaction. Neither specific ligands nor definitive signaling pathways have been described and the regulation of inflammatory mediators such as proinflammatory cytokines or type 1 interferons has not been well described. To date,
TLR10 is described to be able to form homodimers with itself or heterodimers with either
TLR1 or TLR2 [79][80][81]. Additionally, TLR10 is mainly expressed in tissues such as spleen, thymus, tonsils and lymph nodes and cells that express TLR10 can be immune cells (e.g. monocytes, dendritic cells, neutrophils, eosinophils, B cells and T cells) or nonimmune cells such as trophoblasts [82][83][84][85]. TLR10’s function is not well known, however certain studies suggest an anti-inflammatory rather than a pro- inflammatory role by inducing the expression of IL-1R through the PI3K-AKT pathway and by negatively regulating MYD88-dependent and independent pathways [86][87]. In addition, TLR10 is involved in infections by Heliobacter Pylori, M. tuberculosis, HIV,
Influenza and Listeria Monocytogenes [88][89][90][91][92].
Intracellular TLRs
TLR3
TLR3 signaling occurs at the endosome and is mostly known for its requirement in fighting viral infections. TLR3 recognizes double-stranded RNA (dsRNA) which culminates in the production of type 1 interferons and proinflammatory cytokines. Its
13 ectodomain has a shape of a horseshoe and is responsible for binding viral dsRNA where residues His539 and Asn541 were described to be especially important [93]. After binding of dsRNA to TLR3, TLR3 forms a homodimer which leads to activation of the adaptor protein TRIF. MYD88 is not involved in inducing TLR3 signaling however it was described to negatively regulate TLR3-dependent downstream signaling events [94]. TLR3 is expressed in B cells, T cells, monocytes, macrophages, dendritic cells, natural killer cells, mast cells, eosinophils [95][96][97][98][99][100][101][102]. In contrast to previously mentioned TLRs, TLR3 is only able to recognize viruses (e.g. West Nile virus (WNV),
Poliovirus (PV), Herpes simplex virus 1 (HSV-1)) and is not involved in bacterial-induced inflammation.
Once TLR3 is activated it associates with c-Src tyrosine kinase and PI3K in addition to TRIF [103][104]. In addition, it follows the same signaling pattern as described for TLR4-TRIF mediated signaling which results in the activation of the transcription factors NF-κB, AP-1 and IRF3 and leads to expression of proinflammatory cytokines and type 1 interferons. In addition to MYD88, SAM and ARM-containing Protein (SARM) and deubuquitination enzyme A (DUBA) were also described to negatively regulate TLR3 signaling.
TLR7/8
In addition to being genetically very similar, both TLR7 and TLR8 are involved in the recognition of single stranded RNA (ssRNA) and dsRNA. They are another member of the TLR family which is facilitates the fight against viral infections with TLR7 being more
14 important than TLR8 [105]. In addition, both TLR 7/8 also recognize bacterial RNA [106].
In addition to being involved in viral defense such as against HIV and influenza virus
[107][108], TLR7 and TLR8 regulate the immune response to bacterial infections such as
E. coli and Mycobacterium bovis [109][110]. It is reported that TLR7 is highly expressed in plasmacytoid dendritic cells (pDCs) and B cells with inducible expression in macrophages, myeloid dendritic cells, hepatocytes and keratinocytes [111][112][113].
TLR8 is most abundantly expressed in monocytes, macrophages and myeloid dendritic cells [83]. It also has been shown that TLR8 acts synergistically with TLR3, TLR4 and
TLR2 and that R848 (TLR7/8 ligand) upregulates CD14 expression, an important coreceptor in TLR4 signaling [114][115][116]. On the contrary, TLR8 was reported to inhibit activation of TLR7 and TLR9 signaling [117].
Upon activation of TLR8 or TLR7, the adaptor protein MYD88 is recruited which activates IRAK4 and TRAF6 similar to TLR2, TLR4 and TLR5 signaling [106][118].
Downstream of this event, IRF7 is phosphorylated and activated which regulates the expression of type 1 interferons. In addition to IRF7, IRF5 is also activated which leads to expression of pro inflammatory cytokines such as IL-6 and IL12p40 [119].
TLR9
TLR9 is the only TLR which recognizes CpG-DNA, which is bacterial DNA with a much higher frequency of unmethylated CG dinucleotides in comparison to vertebrate
DNA [120]. Interestingly not all CpG patterns are equal in their potency to activate the
TLR9 signaling pathway. CpG-A-type has a central palindromic CG pattern which is
15 located between a polyG and a phosphodiester backbone. It is reported to have weak induction of B cell activity but it is very potent in activating the production of IFNα/β in dendritic cells [121]. There also exist CpG-B-type which has a phosphorothioate backbone with strong activation of B cells and less induction of IFNα/β production and
CpG-C-type known to initiate production and secretion of IL-6 and IgM by B cells and strong secretion of IFNα by dendritic cells [122]. In addition to bacteria, certain viruses are also known to contain CpG motifs [123]. Cytomegalovirus (CMV), as well as herpes simplex virus (HSV1/HSV2) were reported to activate TLR9 and stimulate type 1 IFN production in dendritic cells [124][125].
TLR9 was reported to be expressed in intestinal epithelial cells, B cells, basophils, mast cells, natural killer cells, neutrophils, macrophages, dendritic cells
[126][127][128][129][130][131]. Therefore, TLR9 is expressed in both innate and adaptive immune cells as well as in non-immune cells. In addition to regulation of TLR9 by TLR8, which was mentioned in the previous paragraph, CD14 has been shown to be a coreceptor of TLR9 [132].
Once TLR9 is activated by CpG it dimerizes which recruits MYD88 [133]. This leads to activation of IRAK1, IRAK4 and TRAF6 in non-pDCs [134]. This leads to activation of TAK1 and IκB phosphorylation which activate NF-κB and AP-1 transcription factors [135]. Interestingly in pDCs the signaling cascade culminates in the activation of
IRF7 which is a known transcription factor for IFNα/β expression [136].
16
Endogenous stimuli
Endogenous stimuli are referred to as initiators of inflammation independent of the presence of a pathogen. Endogenous stimuli occur when damaged cells from the body secrete proteins into their environment which makes non-immune and immune cells respond to this signal with inflammation. The theory of endogenous stimuli being the cause of inflammation was first proposed by Polly Matzinger in 1994 in the “danger theory” and later those stimuli were termed damage-associated molecular patterns (DAMPs) by
Walter Land in 2003 [137][138]. Although the origin of inflammation is different,
DAMPrelated inflammation still follows the same principles as infection-related inflammation by activating non-immune cells such as epithelial, endothelial and fibroblasts in addition to innate immune cells (neutrophils, macrophages, dendritic cells). This leads to activation of the adaptive immune system which initiates additional molecular cascades that culminate in the resolution of inflammation.
Several DAMPs were reported to initiate activation of a variety of immune receptors, including TLRs [139]. In terms of TLRs, released intracellular proteins or cleaved matrix components can be recognized by TLR2 and TLR4, while intracellular
TLR3, TLR7 and TLR9 recognize DNA and RNA released from necrotic cells [140].
Highmobility group box 1 protein (HMGB1) can act as a DAMP by activating both TLR2 and TLR4 signaling while several S100 proteins and HSPs solely activate TLR4 and biglycan and endoplasmin activate TLR2 [139][141][142][143][144][145][146]. In addition, small interfering RNA (siRNA), RNA and DNA have been reported as DAMPs for TLR7,8 and TLR9 [147][148][149].
17
Role of adaptive immune system in inflammation
For a healthy immune response, the adaptive immune system, which consists of
B- and T-cells, is as important as the innate immune system. Although the innate immune system is the first to act against an infection, the adaptive immune system acts more specifically by utilizing antigen-specific soluble proteins, called antibodies. Upon infection, resident dendritic cells (DCs) phagocytose the pathogen which they biochemically take apart and present on surface receptors called major histocompatibility complex class II
(MHC II) receptor. Subsequently, activated DCs and other antigen presenting cells
(APCs) travel through the lymphatic system until they reach lymphoid organs such as the lymph nodes or spleen where they encounter naïve T cells. Naïve T cells are activated by cytokines secreted by dendritic cells, which make naïve T cells differentiate into either
T-helper subtype Th1, Th2, Th17 after binding of CD28 to CD80, which is expressed on
DCs. While IL-12 is known to generate Th1 cells, secretion of IL-10 with IL-4 or IL-13 induces Th2 cell differentiation. Th17 differentiation is initiated after secretion of IL-6 or
IL-23. All T-helper cells are known to activate B-cells by direct interaction and secretion of cytokines. This activation leads to heavy proliferation of B cells and the generation of plasma cells that produce antibodies, which are soluble proteins that very specifically target pathogens.
In addition to T-helper cell lineages, which express the coreceptor CD4, naïve T cells can also express the coreceptor CD8, which differentiate into cytotoxic T cells. These types of T cells interact with MHC I antigen presenting receptor, which in comparison to
MHC II, is expressed on all nucleated cells in the body. Based on MHC recognition, 18 cytotoxic T cells can target and kill cells that are infected with virus or even cancerous cells. Therefore, it is very important that both the innate and adaptive immune systems are intact and are communicating appropriately. Dysregulation of one or both components can have severe effects on how inflammation is regulated, which can lead to chronic inflammation which is associated with autoimmune disorders and cancer. The following section includes a discussion of chronic inflammation more in detail and the molecular pathways involved.
Chronic inflammation, the bad one
Acute inflammation is an important aspect of restoring homeostasis and fighting off infections. Both immune cells and non-immune cells (both secrete proinflammatory cytokines) play an important role in a healthy inflammatory response. However, sometimes inflammation-related signaling persists which can have a disastrous outcome for the body. Initiators of chronic inflammation are the same for acute inflammation such as PAMPs and DAMPs. However due to certain infectious characteristics of a pathogen
(PAMPs) or continuous cellular stress (DAMPs) certain molecular pathways are not turned off any more which can lead to severe disease manifestations such as autoimmune disorders or cancer. In this section we will apply the principles of inflammation on chronic inflammation, including related molecular pathways and diseases related to chronic inflammatory response.
19
Infections related to chronic inflammation
It comes with no surprise that over time, reports have accumulated that suggest a strong relationship between infections with certain microbes and chronic inflammation.
Pathogens can be persistent in maintaining an infection although inflammatory pathways of the body are active and can even induce autoimmunity. One example is an infection with Streptococcus pyogenes (S. pygoenes) in which the similarity of the M protein to the myosin protein of the let the immune system cross-react which results, when untreated, in rheumatic heart disease (RHD) [150]. Another example for infection-related chronic inflammation is the persistent infection of Helicobacter Pylori (H. pylori) in the human stomach [151]. By changing the pH of its environment with its metabolites, H. pylori is able to persist in the stomach where it induces a strong immune reaction and inflammation
[152]. This continuous gastric inflammation can lead to stomach ulcers or even stomach cancer and there are even reports that suggests an autoimmune response of the body due to molecular mimicry [153]. Another example of pathogens that can induce chronic inflammation are certain types of viruses. The human papillomavirus (HPV) was reported to be the cause of invasive cervical cancer by utilizing viral E6 and E7 proteins which induce genetic instability and favor oncogenic behavior of epithelial cells. In addition to the oncogenic aspect of its infection, HPV was also shown to influence the immune system and activation of inflammation. Kemp et al (2010) has shown that proinflammatory cytokines are upregulated in patients with persistent infection in comparison to healthy individuals [154]. This might arise from the fact that persistent HPV inside the epithelial cells renders them resistant to the cytokine Tumor necrosis factor alpha (TNFα) which is released by innate immune cells such as macrophages to target HPV [155][156]. HPV
20 infection has been linked to chronic inflammation and this resistance towards TNFα might be the inducer that leads to constant immune cell infiltration and inflammation [157].
Another example of a persistent viral infection that causes a chronic inflammatory response is Hepatitis C virus (HCV) infection. HCV is a known inducer of liver cancer which is also related to chronic inflammation [158]. Liver tumors have been shown to have an upregulation of Th1 related cytokines such as IL-6 and TNFα and chronically infected tumors showed an increase in immune cell infiltration and IL-4 and IL-13 secretion [159]
[160] [161] [162]. This induction of cytokines in the presence of a persistent HCV infection was shown to tightly correlate with the occurrence of liver cancer which is consistent with the theory of chronic inflammation being a hallmark of cancer [158][17].
Cellular stress related to chronic inflammation (DAMPs)
Necrotic cells are known to release intracellular components into their microenvironment which recruits immune cells to the site of cellular stress. It is not surprising that this concept is involved in chronic inflammation as well. HMGB1 is a known
DAMP and inducer of inflammation and cytokine production and tightly linked to chronic inflammation and related disease manifestations [163]. It is a known ligand of TLR4 but also for Receptor for Advanced Glycation End products (RAGE). HMGB1 binding to
RAGE was shown to induce activation of NF-κB and MAPK pathways which are known to induce expression and secretion of proinflammatory cytokines and also important in promoting tumor growth [164][165]. RAGE has been shown to be overly expressed in immune cells and surrounding tissues in chronic inflammation; thus its involvement in
21 inflammation- related diseases can be extrapolated [166]. Indeed, RAGE expression is increased in multiple types of tumors, as well as in autoimmune diseases such as arthritis or macrophage activation syndrome [167][168][169]. Another DAMP that has been linked to cancer growth and autoimmunity was extracellular Adenosine Tri-phosphate (ATP).
ATP binds to purinergic P2 receptors which are commonly expressed on a wide variety of cell types and regulate innate and adaptive inflammatory responses [170]. P2Y2R in particular, has the potential to lead to uncontrolled inflammatory signaling events and indeed it was shown to regulate the secretion of cytokines such as IL-33 and IL-8 during allergic airway disease and DCs in asthma [171][172]. In addition to ATP’s role as a DAMP in autoimmunity, there are also several lines of evidence that show its role in tumor growth.
P2Y2R was also shown to induce growth of prostate cancer cells by inducing reactive oxygen species and lymphocytic leukemia cells showed less proliferation after blockage of P2X7, another ATP receptor [173][174].
Molecular characteristics involved in diseases caused by chronic inflammation
Autoimmunity
One common autoimmune disorder is atopic dermatitis (AD) [175][176]. It is a common inflammatory skin disease which leads to a severe skin rash [177]. Immune cellssuch as Th2 and Th17 cells are known to cluster at the site of inflammation in acute dermatitis while Th1 are mostly present at chronic dermatitis [178][179]. Additionally, the presence of IL-4, IL-5, IL-16 and Granulocyte-Macrophage Colony-stimulation factor (GM-
CSF) in acute dermatitis and the presence of IFNγ and IL-12 in addition to IL-4, IL5, IL-16 and GM-CSF in chronic dermatitis was reported [180]. One molecular pathway that might
22 be involved in AD is the JAK-STAT pathway. It is reported to be involved in the regulation of several proinflammatory cytokines such as IL-13 and is activated by IL-4, a main inducer of atopic dermatitis in mice [181][182]. In addition, it is involved in regulating Th2 cell growth and suppresses immunosuppressive regulatory T cells (Tregs) [183]. Indeed,
JAK inhibitors such as PF-04965842 or BARICITINIB have been shown to be clinically applicable for the treatment of atopic dermatitis [184]. Another pathway that might be involved in regulating atopic dermatitis is the MAPK pathway. In a study by Sredni-
Kenigsbuch et al (2008) p38 was suggested as a regulator of AD after treatment of peripheral blood mononuclear cells (PBMCs) with AS101. AS101 inhibited IL-10 and IL-
4, which inhibits Th2 responses, thus p38 might regulate this effect [185]. The
Hedgehog (HH) signaling pathway has also been shown to play a significant role in AD.
In a study by Papaioannou et al (2019), HH signaling was shown to reduce the severity of AD by activating Tregs and that the protective effect of HH signaling occurs through
GLI2, a transcriptional activator of HH signaling pathway [186].
Rheumatoid arthritis (RA) is another autoimmune disease that is associated with chronic inflammation. It is caused by leukocyte migration into the synovial compartment with Th1, Th17 and macrophages at its center, which keep secreting proinflammatory cytokines into their environment leading to the generation of synovial inflammatory tissues
[187][188]. STAT signaling has been linked to chronic inflammation in rheumatoid arthritis. STAT1 has been found to be upregulated in RA patients and STAT3 regulates growth and survival of synovial cells in RA [189][190]. Blockage of cytokines such as IL6,
IL-1 and TNFα were shown to reduce joint inflammation [191]. These are known targets
23 of p38 which makes p38 a potential target for RA therapy. Indeed, inhibition of p38 decreased IL-6, IL-1 and TNFα expression which decreased RA in mice [192].
Additionally, HH signaling has been shown to be involved in RA. In a study by Qin et al
(2016), important components in this pathway were upregulated in rats with RA and when
HH signaling was targeted, proliferation of synovial fibroblasts (SF) was reduced and apoptosis was increased [193].
Type 1 diabetes occurs rarely (5-10% of all diabetes cases) but has a disastrous outcome for patients suffering from this autoimmune disorder. Due to an abnormal activation of T cells in islets of Langerhans and antibody production against β-cells by B cells, patients suffer from critically low insulin levels or even insulin deficiency [194][195].
In addition to its severe effect on insulin production, type 1 diabetes also impairs coronary vascular function due to increased arginase 1 expression [196]. Arginase 1 was found to be downstream of p38, which when inhibited, reversed diabetes 1-induced upregulation of arginase 1 in rats [197]. Another pathway involved in type 1 diabetes is NF-κB pathway.
Interestingly, a difference in TLR7/8 and TLR4 signaling was shown in patients with type
1 diabetes versus healthy individuals [198]. This difference leads to an upregulation of proinflammatory cytokine secretion in monocytes and DCs which was due to upregulation of NF-κB levels inside those cells [198]. Shh signaling was reported to be of importance for blood flow and neovascularization [199]. Since type 1 diabetes leads to cardiovascular issues, HH signaling could be used as a therapeutic target in type 1 diabetes [200].
Indeed, in vivo activation of HH signaling by the Shh ligand SAG increased the number of endothelial progenitor cells in affected limbs in mice and therefore reduced type 1 diabetes related ischemic effect. 24
Cancer
Cell growth is well controlled in our bodies; when growth goes out of control itcan cause severe harm. In addition to oncogenes that are either highly active due to viral promoters or mutations or inactive tumor suppressors, the tumor microenvironment (TME) was reported to be of significance for cancer cell growth and survival. This microenvironment is known to create beneficial and crucial factors for the cancer, which includes chronic inflammation. In addition to cancer associated fibroblasts, endothelial cells, adipocytes, pericytes and mesenchymal stem cells, a high percentage of immune cells from both the innate and adaptive immune system are present which highly regulate inflammation on site [201]. T cell subtypes were shown to inhibit tumor progression or induce tumor growth. While CD8+ cytotoxic T cells and Th1 cells were reported to have anti-tumor function by attacking tumor cells, Th2, Th17 and regulatory/immunosuppressive T cells (Tregs) were mostly shown to be tumor promoting
[202][203][204][205]. The B cell population in the TME also shows characteristics in which they can either be tumor suppressive, such as in ovarian or breast cancer, or tumor promoting, such as in skin cancer [206][207]. In general, it seems to be that most immune cells play the role of a double-edged sword in the TME. Macrophages are present in abundance in proximity of the tumor in which they favor tumor growth and act as tumor associated macrophages (TAM). TAMs are known to directly interact with cells crucial for tumor growth, such as cancer stem cells (CSC), or indirectly promote tumor progression by secreting proinflammatory and proangiogenic factors or anti-inflammatory cytokines into the TME depending on the stage of cancer [208]. In general, macrophages can be divided into two classes, the M1 phenotype which is proinflammatory and the M2
25 phenotype which is anti-inflammatory, and which is generally considered as TAM [209].
However, there are several lines of evidence that suggest that both M1 and M2 phenotype play an important role in cancer growth [210][211][212]. It seems that the TAM phenotype depends on the stage of the cancer. The M1 phenotype, which is known to secrete a great abundance of proinflammatory cytokines such as IL-1β, TNFα and IL-6 was suggested to be involved in tumor initiation and chronic inflammation while M2 macrophages are mostly present in later stages of the tumor, where they highly secrete IL-10 and TGF-β and inhibit anti-cancer immune response and induce angiogenesis and metastasis [208]. Cytokines act as mediators of cancer supporting a role for immune cells.
However, they are also activating tumor promoting pathways within the cancer cells in a paracrine fashion [213]. Tumor-promoting and cytokine-related pathways of immune cells and other cell types which activate pre-cancerous molecular cascades within tumor cells will be discussed below.
Molecular pathways linking chronic inflammation to cancer
NF-κB, STAT and SHH
TNFα, IL-6 and IL-1 are cytokines which are known to be involved in chronic inflammation and cancer growth. NF-κB is a known transcription factor in immune cells where it regulates the secretion of those cytokines, therefore regulating their effect.
Binding of those cytokines to respective receptors on cancer cells induces NF-κB signaling as well [214][215][216]. NF-κB is involved in anti-apoptotic signaling by targeting
FLICE-like inhibitory protein (FLIP) and some members of the BCL-2 family but also in
26 proliferation and invasiveness by targeting cyclin D1 and c-myc [217][218][219][220].
Therefore, NF- B regulates tumor growth by regulating both inflammation and proliferation.
STAT3 signaling has been shown to be involved in regulating inflammation and the resulting cancer growth as well [208]. STAT3 signaling is activated upon stimulants known to increase the risk to develop cancer such as UV light, cigarette smoke and H. pylori infection [221][222][223]. STAT3 was shown to regulate expression and secretion of the proinflammatory cytokines IL-6 and IL-1β, which activates STAT3-axis in cancer cells close by. This activation leads to increased survival and proliferation in addition to secretion of IL-6 by cancer cells [224][225]. Based on this positive feedback loop, the cancer phenotype progresses, and it reflects one of the reasons why STAT3 signaling is cancer promoting.
Another pathway which has been shown to play an important role in regulating cancer growth through the TME is the HH signaling pathway [226]. To date most studies, suggest an importance of HH signaling in regulating malignant cells in a paracrine fashion by it regulating cytokine secretion of the stroma [227]. Central components of HH signaling are the transcription factors GLI1, GLI2 and GLI3. The chemokine CCL5 was reported to induce IL-6 secretion by bone marrow stroma cells mediated by GLI2 and this IL-6 secretion mediated IgM secretion by malignant B cells from Waldenstrom macroglobulinemia (WM), a lymphoma known to induce complications in patients by secreting abundant amounts of IgM [228]. Another study showed the importance of GLI2 in regulating CD40L expression and secretion by bone marrow stromal cells [229], and
27 the regulation of TGF-β by GLI2 in T cells was also reported [230]. These studies reflect the importance of HH signaling in inflammation. In addition to GLI2, GLI3 showed potential in inducing malignant cell behavior as well; however, its role in inflammation has not been reported [231].
This dissertation will focus on the role of GLI3 in inflammation, another transcription factor important for HH signaling. The following sections will describe GLI3 signaling in the context of HH signaling and other signaling cascades and its relevance in development, immune system and cancer.
GLI3: A mediator of genetic diseases, development and cancer
The GLI gene was first identified in humans as a highly expressed gene in human glioma [232]. Using cDNA probes for the zinc finger region of the GLI gene, Ruppert et al
(1988), identified two additional GLI family members, GLI2 and GLI3 [233]. Further characterization of human GLI3 revealed it to be a 190 kDA protein located on chromosome 7p13 and binds to consensus sequences similar to those of GLI1 [234]. The most updated data on the National Center for Biotechnology Information (NCBI) and new publications, mapped human GLI3 to chromosome 7p14.1 (Gene ID:2737)[235]. GLI3 was identified as a gene in which mutations in GLI3 cause GCPS, a disease leading to craniofacial and limb maldevelopment. In a study by Vortkamp et al (1991), 2 translocations in GLI3 were identified, which interrupt GLI3 expression and cause GCPS
[236]. Point mutations in the human GLI3 locus in GCPS patients were identified as a main cause of GCPS disease manifestation [237]. In 1996, GLI3 was described as a protein that is regulated in response to the sonic hedgehog (SHH) signaling pathway
28 where it was described to compete in binding with GLI1 [238]. In the same study, GLI3 was characterized as a negative regulator of SHH signaling [238]. In the following year,
GLI3 was recognized as the cause of PHS, a disease characterized by developmental malformations including polydactyly (extra digits) [239]. Follow-up studies described Gli3 as both an activator and repressor, similar to the Gli2 family member, in response to Shh signaling [240]. Since then, research on mouse and human Gli3/GLI3 mostly focused on its role in brain and limb development with certain exceptions of Gli3/GLI3’s role in angiogenesis, colorectal and liver cancer, TRAIL-dependent apoptosis and its role in regulating the IL-6/JAK2 pathway [241][242][243][244][245].
Regulation and Structure
Hedgehog ligands and their function
The Hh signaling pathway plays a role in embryonic development and homeostasis of stem cells in normal tissues [246]. Dysregulations of Hh signaling cause genetic defects such as holoprosencephaly and polydactyly and are tightly linked to cancer development and progression [247][248]. In addition, a role for Hh signaling in hematopoiesis and in the immune system has been described [249][250][251]. The Hh signaling pathway is activated by 3 ligands: Sonic Hedgehog (Shh/SHH), Indian Hedgehog (Ihh/IHH) or Desert
Hedgehog (Dhh/DHH) (mouse/human respectively) [252]. These ligands are roughly 45 kDA with a N-terminal biologically active domain and an autocatalytic C-terminus, which is cleaved to generate the final Hh ligand form [253][254]. After cleavage, a cholesterol moiety is added to the C-terminus and palmitate is linked to the N-terminus [255]. This
29 allows exogenous Hh ligands to travel far distances to activate Hh signaling in various cells/tissues in the body [256]. Shh is the ligand with the highest expression and therefore is the major inducer of most Hh-related functions such as brain, limb and spinal cord development [257][258]. Ihh was linked to chondrogenesis and negatively regulates chondrocyte differentiation [259]. Dhh null male mice are infertile while there was no visible effect in female mice suggesting a role for Dhh in spermatogenesis [260].
Additionally, Dhh was shown to play a role in peripheral nerve ensheathment [261]. Shh is mostly expressed in epithelia while Dhh is expressed in Schwann and Sertolli precursors and Ihh is expressed in the cartilage and in the gut [262]. All Hh ligands bind to the same receptor Ptch1 and initiate Hh-related signaling. However, Shh has been shown to be the most potent inducer of this pathway [263].
Classic (canonical) Hh signaling
Many components known to be involved in vertebrate Hh signaling were initially identified in Drosophila. The main components of Hh signaling in Drosophila are 1)
Patched (Ptc) a 12-transmembrane protein which binds Hh ligand; 2) Smoothened (Smo), a receptor that is repressed by Ptc and released to activate the pathway once Hh ligand binds Ptc; and 3) Cubitus interruptus (Ci) which is the Drosophila analog of Gli proteins in vertebrates [264]. In the absence of Hh signaling Ci, Costal-2 (cos-2) and Fused (Fu) form the Hedgehog Signaling complex (HSC). This leads to proteasomal degradation of
Ci from a 155 to 75 kDA form through phosphorylation of Protein Kinase A (Pka/PKA),
Glycogen synthase kinase-3 beta (Gsk3β/GSK3β) and Casein Kinase 1 (Ck-1/CK-1)
[265]. The degraded form of Ci subsequently acts as a repressor of Hh signaling. Once
Hh signaling is activated by ligand binding to Ptc, and inhibits its repressor function, Smo 30 is activated which leads to stabilization of Ci and expression of Hh related genes. The signaling cascade is well conserved between invertebrates and vertebrates, therefore the information gained from Drosophila is useful in predicting the signaling events in mice and humans.
In mammals
In mammals, Hh signaling occurs at the primary cilia of cells [266]. The intraflagellar transport (IFT) proteins: Intraflagellar transport protein 172 homolog (Ift172),
Kinesin-like protein KIF3A (Kif3a), dynein cytoplasmic heavy chain 2 (Dnchc2) and
Polaris, are extremely important for the formation and function of cilia, and are essential for the regulation of Hh activation or inactivation [267][268][269]. In addition, the kinesinlike protein Kif7, which is involved in cilia tip assembly, was shown to be essential for proper activation of this pathway and is therefore involved in positively and negatively regulating Hh-dependent functions [270][271]. Several co-receptors for Ptch1 have been identified and were shown to be indispensable for proper Hh function. These include growth-arrest-specific 1 (Gas1), CAM-related/downregulated by oncogenes (Cdo), and brother of Cdo (Boc) which positively regulate Hh-related signaling events [272]. In addition, the Hh interaction protein (Hhip) binds and sequesters all three Hh ligands and therefore acts as a negative regulator of the Hh pathway [273].
In the absence of binding of one of the Hh ligands (Shh, Dhh or Ihh) to Ptch1, Smo is localized intracellularly [274]. The mechanism by which Ptch1 inhibits Smo is not completely understood. It was hypothesized to occur by either binding of a Smo activator
31 or delivery of a Smo small molecule inhibitor [275]. Another hypothesis suggested that in the absence of Hh ligand, Ptch1 translocates to the base of the primary cilia and inhibits localization of Smo to the cilia [276][277]. However, further experiments are needed to elucidate the mechanism by which Ptch1 inhibits Smo. Without SMO being activated, suppressor of fused (Sufu/SUFU) sequesters GLI transcription factors in the cytoplasm
[278]. These GLI proteins are essential effectors of the HH pathway. There are 3 members of the GLI family of transcription factors: GLI1, GLI2 and GLI3. In the absence of Hh activation by ligand binding to Ptch1, Sufu is phosphorylated and recruits G-protein coupled receptor 161 (Gpr161/GPR161), PKA, GSK3β, and beta-tranducin repeat containing E3 ubiquitin protein ligase (βTrCP) complex to proteolytically cleave Gli2 and
Gli3 into smaller proteins with a repressor function [279][280]. Ck-1 negatively regulates
Hh signaling in Drosophila [281]. Since many Hh signaling molecules in Drosophila are evolutionary conserved in mammals, it is probable that Ck-1 negatively regulates mammalian HH signaling as well. In fact, mutations of conserved Ck-1 sequences from
Drosophila showed less phosphorylation of mammalian GLI3 and less GLI3 processing into its repressor form [282]. However, based on our current understanding, it remain unclear what role CK-1 plays in vertebrate HH signaling since there are also reports of positive regulation of HH signaling by CK-1α and CK-1ε [283][284][285]. Interestingly, in
Drosophila, inhibition of Ck-1α or Ck-1ε alone did not significantly affect Ci processing
[286]. However, when both Ck-1 subtypes were inhibited, a significant decrease of Ci repressor was observed. Therefore, it is probable that CK-1α and CK-1ε alone positively regulate HH signaling, while when forming an alpha-epsilon complex, they negatively regulate this pathway by phosphorylating GLI3. This however, is highly speculative, and
32 more research is needed to validate the role of CK-1 in HH signaling. GLI1 is not cleaved in this process but completely degraded [287]. While Gli2 is not very stable in its repressor form and completely degrades after cleavage, the Gli3 repressor form is more stable and translocates to the nucleus where it inhibits Hh-related responses [288]. As such, in the context of Hh/HH signaling, GLI3 is known to be the most potent inhibitor of HH signaling within the GLI family.
Upon ligand binding to Ptch1, Smo translocates to the primary cilia, a process mediated by β-arrestin [289]. This leads to the association of Smo with Ellis-van Creveld syndrome protein/Ellis-van Creveld syndrome protein 2 (Evc/Evc2) complex (a complex that regulates chondrocyte proliferation and differentiation of osteoblasts) in which
SufuGli2/3 complex moves to the tip of the cilia where Sufu dissociates from Gli2/3
[290][291][288]. Sufu is then degraded and Gli2/3 full-length accumulate at the ciliary tip and can translocate into the nucleus to activate a Hh-related gene expression program
[292][280]. Gli1, Gli2-FL, and Gli3-FL have been shown to induce Hh related gene functions. Because GLI2-FL and GLI3-FL induce GLI1 expression, GLI1 therefore acts as an amplifier of the HH signal [293]. However, Gli2 is considered the most potent activator of Hh signaling [240][294]. All members of the GLI protein family (GLI1, GLI2 and GLI3) contain five zinc finger domains which are very similar to those of Ci [295]. Although all
GLI members share biochemical domains, GLI1 does not contain a Nterminal repressor domain which is present in both GLI2 and GLI3 (Figure 1-1). Therefore, biochemically,
GLI1 can only act as transcriptional activator while GLI2 and GLI3 can potentially act as transcriptional activators or repressors.
33
In humans, different isoforms of GLI1 and GLI2 have been reported (Figure 1-1).
To date, there are no reports of different GLI3 isoforms in humans. GLI1 has three isoforms: isoform 1 is 1106 amino acids (AA) long and is considered the full length GLI1 protein (NP_005260.1). GLI1 isoform 2 sequence lacks exons 2 and 3, which leads to a truncated GLI1ΔN form (978 AA) (NP_001153517.1). This isoform was reported in healthy and malignant tissue and showed no binding to SUFU [296]. The third isoform tGLI1 is a cancer-associated form of GLI1 [297], and is not detected in normal cells but is found in glioblastoma multiforme (GBM) and other cancers. It lacks exon 3 and part of exon 4
(1065 AA), but retains most of its function; however, it is associated with increased motility and invasiveness [297]. To date, four GLI2 isoforms have been described, in addition to the full length GLI2 (1586 AA) [298][299]. These isoforms include GLI2α (1258
AA), GLI2β (1241 AA), GLI2γ (829 AA) and GLI2δ (812 AA). GLI2α is also known as
ΔNGLI2 and shows significantly higher activity in vitro than GLI2 full length [300]. GLI2β mRNA was identified to be highly expressed in basal cell carcinoma and both GLI2α and
GLI2β were shown to regulate the expression of the human T cell lymphoma virus 1 gene
[301][298]. To date, no functions for GLI2γ and GLI2δ have been reported.
Non-classical (non-canonical) HH signaling
PTCH1 and SMO are major regulators of the canonical HH signaling pathway and therefore of GLI transcription factors. However, several studies report that GLIs
(especially GLI1 and GLI2) can be regulated by the cross talk with other signaling pathways in various types of cancers including melanoma, gastric cancer, colon cancer,
34 multiple myeloma, medulloblastoma, pancreatic cancer, glioblastoma, and osteosarcoma
[302][303][304][305][306][307][308][309].
The EGFR-RAS-RAF-MEK1 pathway has been frequently investigated in regulating GLI1 and GLI2 expression levels. Constitutively active MEK1 and oncogenic
KRASV12 induce GLI1 protein expression and activity [310][302]. In addition to upregulating positive regulators of HH signaling, GLI2 was shown to be stabilized upon activation of EGF-ERK1/2 signaling [311]. Utilization of the A3 adenosine receptor agonist, resulted in a decrease in breast cancer stem cell survival which correlated with a decrease in activated ERK1/2 and GLI1 [312][313]. Therefore, EGFR signaling through
MAPK can regulate GLI1 and GLI2 at the level of expression and activity.
The PI3K pathway has been closely linked to HH-related functions. Several studies suggest a role of PI3K-AKT axis in the regulation of GLI1/2 transcriptional activators
[314][315][316]. PI3K-AKT activity induced an increase in the expression of GLI1 and
GLI2 in renal cell carcinoma and chronic lymphocytic leukemia (CLL), leading to increased cancer cell growth and progression [314][315]. The regulation of GLI by
PI3KAKT axis was shown to be induced by CCL5-CCR3 signaling in the tumor microenvironment (TME) as well [316]. In addition, the interaction between PI3K and HH has been reported to be important for tamoxifen resistant breast cancer and combined targeting of both GLI1/2 (using the GLI antagonist GANT61) and PI3K/mTOR (using
PI103) synergistically increased apoptosis and reduced tumor growth [317][318].
In addition to ERK1/2 and PI3K, TGF-β has been shown to be involved in regulating
GLI1/2 expression without the involvement of SMO signaling
35
[319][320][321][322][323]. In the last few years several studies have reported a role for the TGF-β-GLI1/2 axis, partially Smad3 dependent, in fibroblast activation, melanoma, stroke (ischemia/reperfusion injury), hepatocellular carcinoma, and gastric cancer
[319][320][321][322][323]. In these studies, both GLI1 and GLI2 regulate tumor growth, survival and epithelial-to-mesenchymal transition (EMT) upon stimulation by TGF-β1/2 in a SMO independent manner. Interestingly, TGF-β2, an inducer of nephrons (a unit required for functional kidneys) lies downstream of GLI3-R [324]. This suggests that a negative feedback loop exists between TGF-β-dependent HH activation in which GLI1 and GLI2 act as positive regulators while GLI3 acts as a negative regulator. However, more experimental evidence is needed to validate this hypothesis. Additionally, in a pan cancer analysis, a positive correlation between TGF-β-related gene expression and GLI1 and GLI2 expression was observed [325]. Notably, GLI1/2 expression was more of a prognostic factor for TGF-β and EMT-related genes than for HH-related genes, which might suggest that GLI1/2 regulate tumor formation by regulating TGF-β-related gene expression rather than HH-induced gene expression.
Protein kinase C (PKC), a known stimulator of cell proliferation, was also reported to regulate GLI expression and function [326][327]. Although PKC was shown to phosphorylate and activate SMO at the beginning of the HH signaling cascade, it was also reported to be able to compensate for SMO inhibition and induce GLI activation
[328][329]. Interestingly, PKC-mediated activation of GLI proteins occurs independently of primary cilia and MEK-1 in NIH3T3 cells [330]. PKC also positively regulates
GLI1HDAC1 association [331] and targeting HDAC1 with the HDAC inhibitor Vorinostat, only resulted in a decrease in cell proliferation at high inhibitor doses. In combination with 36
Vorionstat, PKC inhibition enhanced the therapeutic effect and increased the therapeutic window in basal cell carcinoma [332].
The intracellular membrane trafficking protein Rab23, the serine/threonine kinase involved in cell proliferation and survival CK2α, the inhibitor of transcription SLTM, the proliferative proteins Dual Specificity Tyrosine Phosphorylation Regulation Kinases 1
(DYRK1) and DYRK2, the metabolic proteins MAP3K10 and AMPK all regulate GLI1 expression and function as well [333][334][335][336][337]. These regulators are, in some way, involved in cancer growth and progression, which shows the importance of studying the crosstalk between HH signaling and other pathways, particularly in GLI-directed therapeutic approaches.
Most studies investigating non-canonical HH signaling primarily focus on GLI1 and
GLI2 and oftentimes did not consider GLI3 in their experimental approach. This is understandable since GLI1 and GLI2 are positive regulators of HH ligand-induced gene expression and can therefore be considered major drivers of HH-related genes [338].
However, since various studies describe GLI activation by other signaling pathways which are activated by different ligands (other than SHH, IHH or DHH), the regulation of GLIs might be different in comparison to their regulation in the canonical HH pathway. For example, PKC has been shown to activate GLIs in a cilia-independent manner and additionally, pan cancer analysis revealed that GLIs account for TGF-β-induced gene expression rather than genes induced by classical HH pathway [330][325]. In addition, there are multiple examples that are discussed in this review of the role of GLI3 in development, in the immune system and in cancer where GLI3 often acts as a positive
37 regulator of those pathways, although GLI3 did not show potent HH-gene induction in the canonical HH pathway. It might be that activation, regulation and function of GLIs varies depending on which pathway triggers them, which raises the question of whether or not we can apply the principles of knowledge of the canonical pathway to the non-canonical pathways. Could it be that GLI3-FL acts as a more potent gene activator than GLI2-FL depending on which non-canonical pathway is active? Are post translational changes involved in increasing GLI3-FL function or are GLI3 mRNA levels induced? Is GLI3-FL processing into its repressor form always dependent on PKA, GSK3β or βTrCP or could these factors be substituted, differentially regulated or inhibited in different pathways of non-canonical HH signaling? This report aims to give insights into different cellular functions of GLI3 which are oftentimes in crosstalk with other important signaling pathways.
Regulation of GLI3
Gene locus and domains of mouse and human Gli3/GLI3
Several publications have reported that human GLI3 is located on chromosome
7p13 [236][254][239]. However as mentioned earlier, recent updates on NCBI have mapped human GLI3 to chromosome 7p14.1 (Gene ID: 2737) [339][340] [235]. The human GLI3 gene is 276261 bp in length (NC_000007.14) and the mRNA sequence is composed of 15 exons that are 8405 bp in length (16 Dec, 2019) (NM_000168). However, the consensus coding sequence (CCDS) is 4743 nucleotides (nt) (nt 282-5024) with a transcription start site located in exon 2 (CCDS5465.1). Due to an error in sequencing, older literature characterized GLI3-FL to be 1596 AA in size which was later corrected to
38
1580 AA [341]. This is also listed in the protein database entry NP_000159.3 which shows
GLI3 protein to be 1580 AA with a molecular weight of 190 kDa (GLI3-FL)(NP_000159.3).
Human GLI3 contains a N-terminal repressor domain which is mapped to AA 106-235
(exons 3-6, mRNA nt 649-986) [342]. GLI3 also has a 5 zinc finger DNA-binding domain
(AA 480-632 that span exons 10-13; nt 1719-2177), and a C-terminal transcriptional activation domain that is dependent on CREB-binding protein (CBP) binding (AA 8271132 in exon 15; nt 2760-3677)(Figure 1-2) in addition to transactivation domain 2
(TA2)(AA 1044-1322 in exon 15; mRNA nt 3411-4247) and transactivation domain 1
(TA1)(AA 1376-1580 in exon 15; mRNA nt 4407-5024) [293][343]. The proteasomal cleavage site spans exons 13 and 14 (AA 650-750; nt 2229-2531) which leads to the truncated GLI3-R (677 AA with a molecular weight of 83 kDa) [344][345][346]. In addition to other references listed, mapping of DNA binding domain, cleavage site and activator domain was performed with the help of the publication by Krauß et al (2009) [347].
Although it can be extrapolated that the stated AA range corresponding to each domain is accurate, studies other than that describing the repressor domain [342], that precisely define GLI3’s biochemical domains, are lacking.
In mice, Gli3 is located on chromosome 13 with 266304 bp in length (NC_000079) (last update 19-OCT-2010). The mRNA sequence is 8427 nt long and comprises 15 exons
(NM_008130) (last update: 30-Dec-2019). Between exons 2 and 15 (nt 431-5182) lies the consensus coding sequence which is 4752 nt long and encodes a 1583 AA protein
(mouse GLI3-FL/mGLI3-FL) (CCDS36603.1)(NP_032156.2). Using Align Sequences
Protein BLAST, we aligned amino acid sequences of mouse Gli3 and human GLI3
(Figure 1-4). This revealed the biochemical domains of mouse Gli3-FL are 86% 39 conserved in human Gli3-FL. All domains show high similarity with the exception of the transactivation domain (Similarity of human to mouse: GLI3-R domain: 98%, DNA binding domain: 100%, Cleavage site: 95%, CBP binding domain: 79%, TA2 domain: 68%, TA1 domain: 85%, Total transactivation site: 76%). Interestingly all phosphorylation sites from
PKA, GSK3β and βTrCP are 100% conserved between mouse and human GLI3-FL
(Figure 1-2). This is not surprising since multiple studies show similar function of mouse
Gli3-R in comparison to human GLI3-R regarding inhibition of HH signaling
[348][344][349]. Amino acid sequences that are important for biochemical domains are usually conserved between species. Since the human transactivation domain shows relatively low similarity with the mouse transactivation domain of Gli3-FL, this raises the question whether or not this domain is sufficiently defined in humans yet. Hopefully, future studies will further characterize transactivation site in human GLI3 and possibly identify novel interaction partners at this protein locus.
Processing of GLI3-FL to GLI3-R
Gli2 and Gli3 show 95% amino acid similarity and both can potentially have HH activator and repressor functions [350]. However, Gli2 proteasomal cleavage is not very efficient and the cleavage product that is produced rapidly degrades while Gli3-R is very stable [351]. Therefore, GLI2 is considered an activator in response to HH signaling while
GLI3 is considered a repressor in that context. In the absence of Hedgehog signaling,
GLI3-FL is sequestered in the cytoplasm by Sufu (Figure 1-3) [352]. Sufu recruits Gpr161 which activates Pka and allows it to phosphorylate Gli3 [353]. PKA phosphorylates human
GLI3 at serine residues 849, 865, 877, 907, 980 and 1006 (PKA sites: RRXS) (Figure 1-
40
3) [353]. This leads to additional phosphorylation of GLI3 by GSK3β (at AA 861, 873 and
903)(GSK3β: SXXXpS) [354]. Subsequently, GLI3 binds βTrCP at AA 855, 856, 864 and
894, which recruits Skp1-Cul1-F-box protein-βTrCP complex (SCFTrCP) [355] (Figure 13).
This results in ubiquitination of GLI3 at lysine residues 773, 778, 784 and 800 by
SCFTrCP resulting in GLI3 processing into its repressor form (GLI3-R) (Figure 1-3) [355]. Binding of βTrCP was also reported to occur at the N-terminus and at the C-terminus, however this binding is PKA-independent (binding sequence: AA 1-395 and 1100-1595)
[355]. GLI3-R translocates to the nucleus where it suppresses HH related functions [356].
GLI3-FL, stabilization and degradation
It is generally accepted that GLI3-FL is the transcriptional activator form of GLI3
[357][358][293]. However, GLI3-FL has been reported to directly interact with the androgen receptor and stimulation of pancreatic cancer cells with the synthetic androgen
“R1881” (methyltrienolone) leads to the nuclear translocation of GLI3-FL and subsequent promoter activation of specific GLI consensus sequences [359]. In this study, the androgen receptor was also shown to stabilize GLI3-FL. Despite the role of Sufu in sequestering Gli3 in the cytoplasm to suppress its transcriptional activity, the presence of
Sufu has also been shown to stabilize Gli3 protein [360]. In addition, the Speckle-type
POZ protein (SPOP) induces proteasomal degradation of GLI3-FL but not of GLI3-R
[360], Protein phosphatase 2A (PP2A) directly binds GLI3-FL, and the degradation of
PP2A leads to increased nuclear localization of GLI3-FL [358]. GLI3-FL has also been reported to interact with CBP in a phosphorylation-independent manner at AA 827 and
1132 in the C-terminal end of GLI3. This interaction is important for activation of the GLI1
41 promoter by GLI3-FL and was only detectable in the presence of SHH [293]. In addition,
Mediator of RNA polymerase II transcription subunit 12 (MED12) physically interacts with
GLI3 at AA 1090-1596 and facilitates gene transcription upon HH stimulation [361].
Finally, SET domain containing 7 (SET7) increases the stability of GLI3-FL and its ability to bind the GLI1 promoter region upon SHH treatment [362]. In summary, multiple studies have investigated the regulation of GLI3-FL in response to canonical HH signaling.
Although this information is very valuable, it does not address the possibility that GLI3-FL is regulated in a non-canonical pathway similar to other GLI family members. Therefore, further studies are needed to determine if GLI3-FL regulation is similar in canonical (HH) and non-canonical signaling pathways.
Role in Development, Immune system and Cancer
Development
The role of GLI3 in regulating development is frequently described. In addition to its importance in brain and lung development, GLI3 is a key player in the manifestation of
GCPS, PHS and Tibial Hemimelia, [363][364][365]. These genetic diseases are known for the formation of an extra digit (postaxial polydactyly) consistent with the Gli3Δ699 mouse model (mice with mutated Gli3 that have similar phenotypes compared to PHS in humans)
[366]. Several mutations of GLI3 which compromise GLI3 function, are tightly linked to these developmental disorders, emphasizing that GLI3 is essential for proper human development. In the following section, we provide a more detailed discussion to further elaborate on the role of GLI3 in development and genetic diseases.
42
GCPS, PHS and Tibial Hemimelia
Individuals suffering from GCPS develop postaxial polysyndactyly (limb duplication) of the hands, preaxial polysyndactyly of the feet, and macroencephaly (enlarged head). This genetic disorder is caused by a deletion of a region located on chromosome 7p14.
Vortkamp et al (1992) identified GLI3 as the protein responsible for this disorder and the related phenotype [367]. That study reports GLI3 to be present on chromosome 7p13, however, as stated earlier, recent updates correctly identify the location of GLI3 on chromosome 7p14 [339][340][235]. SHH is responsible for limb development in the anteroposterior axis [368]. The cause for the polydactyly phenotype is a mutation within the GLI3 protein which disrupts the equilibrium between GLI3-R and GLI3 activator
(GLI3FL) forms, resulting in the lack of negative regulation of SHH signaling. In GCPS, mutations in GLI3 can occur throughout the whole protein and can vary from missense to splicing mutations, although deletions, insertions and translocations have been documented [369]. N-terminally truncated versions of GLI3 protein, which completely or partially lack the zinc finger DNA binding domain, are common in GCPS. There are also reports where families have a missense mutation in His601Arg which is located near the
C-terminal end of the 5 zinc finger DNA binding domain [370]. Another reported mutation in GLI3 protein is located at AA 903-934 which are within the transactivation domain [371].
Thus, GCPS-related mutations in GLI3 occur throughout the whole protein and cannot be reduced to one mutation. In addition, the involvement of other proteins in GCPS cannot be ruled out.
PHS is clinically identified by mesoaxial (central) polydactyly and hypothalamic hamartoma (benign tumor of the hypothalamus) [372]. Mutations related to PHS are 43 usually frameshift or nonsense mutations. These mutations lead to a truncated version of
GLI3 which is 691 AA long [364]. Transgenic mice expressing truncated Gli3 (699 AA long) show PHS characteristics and this truncated Gli3 exhibits inhibitory functions similar to Gli3-R in the context of Shh signaling [366]. It can therefore be inferred that mutations in PHS lead to expression of a truncated version of Gli3 similar to Gli3-R, mimicking its function and leading to PHS (probably also due to the lack of Gli3-FL). Kang et al (1997) suggested that PHS was caused by a deletion of a guanosine at nt 2023 in exon 12 of
GLI3 [373]. This exon spans the cleavage site for GLI3-FL to be processed into the GLI3R form. Several additional mutations have been identified and can be found between AA
717-1297 [372]. This spans roughly the beginning and the end of the transactivation domain and includes ubiquitination and phosphorylation sites of GLI3 (Figure 1-2).
Another clinical condition involving GLI3 is tibial hemimelia, a genetic disorder which leads to hypoplastic or absent tibia [365]. Deimling et al (2016) reported a deletion of exon 7 of GLI3 upstream of the DNA binding domain and the transactivation domain
[374]. This deletion leads to a 30 kDA truncated version of GLI3 that is no longer capable of negative regulation of PTCH1 or GLI1 in response to HH signaling. These studies highlight the important role of GLI3 in limb development.
Brain development
In the late 90’s Gli3 was shown to be involved in development of the Dentate gyrus and hippocampus, 2 regions which are responsible for emotional development and memory [375]. In this study XtJ mice show a loss of Homeobox protein Emx1/2 (a
44 transcription factor complex that regulates brain development) expression and show differences in fibroblast growth factor 8 (Fgf8) and bone morphogenetic protein 4 (Bmp4) expression compared to WT mice [375]. Gli3 also plays a role in the development of dorsal telencephalon. In a study that used the extra toes (XtJ) mouse model, an impairment in the development of dorsal di-telencephalic junction was reported [376]. This also led to a decrease in the size of the neocortex and missing parts of the hippocampus.
Gli3 also controls growth and expansion of the cerebral cortex. A study using Kinesin-like protein Kif3a (Kif3a) mutant mice (where loss of Kif3a leads to the degradation of primary cilia) found a dysregulation of the expansion of cerebral cortex. The loss of primary cilia disrupts Gli3 processing which changes expression of the Shh target genes cyclin D1 and fibroblast growth factor 15 (fgf15) [377]. Petrova et al (2013) reported that the Gli3-R mediates proliferation in the dorsal and ventral subventricular zone [378]. Gli3-R mediates
Shh signaling in astrocytes in the forebrain. Gli3 was also shown to be involved in regulating corpus callosum formation by interacting with Slit homolog 2 protein (Slit2) (a protein important for neural development) and therefore regulating Fgf and Wnt/β-catenin expression [379]. In mice, Gli3-R regulates the differentiation of the upper layer of cortical neurons [380]. Furthermore, using Shh-/- Gli3-/- mice embryos, Gli3 was found to play a role in the development of mature oligodendrocytes [381]. Finally, Gli3-R was shown to suppress V0 and V1 interneurons since restoration of motor neurons and V2 interneurons was detected in Shh-/- Gli3-/- embryos in comparison with single Shh-/- knockout embryos
[382]. These studies define an important role for Gli3 in brain development and furthers our understanding of the developmental role of this transcription factor.
45
Lung development
There is evidence for a role of Gli3 in the embryonic stages of mouse lung development [383]. High levels of Gli3 expression were reported in E11.5 mouse embryos in the tip of the accessory lobe. In addition, using Gli3XtJ mice, Gli3 deficiency at E13.5 led to a reduction in size and changes in the shape of three of the five lung lobes [383]. The role of Gli3 in lung development was confirmed by another group who described the impact of Gli2 deficiency in mice lung development to be substantially increased when
GLI3 was knocked out as well. They showed that Gli2-/- Gli3-/- mice embryos lacked lungs, tracheae and esophagi [384]. Therefore, in addition to its role in limb and brain development, Gli3 also regulates lung development.
Role of GLI3 in the immune system
A role for GLI3 in the immune system has been suggested in several studies
[385][386][387][388][389]. Based on current knowledge, the regulatory role of GLI3 in the immune system spans both innate and adaptive immunity. GLI3 has been shown to regulate immune cell development and may play a role in inflammation induced by bacterial infections. A role for GLI3 in the immune system was first described in 2005, in which Gli3 regulates the immune response by modulating T cell development [385]. In this report, Gli3 regulated fetal double negative 1 (DN1) to DN2 transition and the progression of developing thymocytes from double negative (DN) to double positive (DP) state was impaired in the absence of Gli3. This regulatory effect of Gli3 occurs after preTCR signaling. These studies suggest that Gli3 is important for T cell development.
46
However, whether this results in a biological effect of Gli3 loss on T cell responses to an immunological challenge remains unknown. The Cluster of Differentiation 155 (CD155; also known as poliovirus receptor) is a member of the immunoglobulin superfamily and binds to the leukocyte adhesion molecule DNAM1. Both of these molecules are expressed on the surface of NK cells [387]. Treatment of cells with the Shh ligand induced
CD155 expression [388][389]. Further analysis indicated the core promoter region of
CD155 contains candidate GLI binding consensus sequences which, when mutated, results in reduced promoter activation by both Gli1 and Gli3 [389]. This suggests a role for Gli3 in regulating NK cell activation and function in the immune system via regulation of CD155 expression [389]. A role for Gli3 in fetal B cell commitment was suggested in a study where a dose-dependent reduction in CD19+, B220+ and CD19+ B220+ B cell numbers in Gli3 WT, Gli3+/- and Gli3-/- embryos was observed [390]. In addition, the total number of CD93+ B cells and the number of ckit+ CD127+, CD43+ CD19+ HSA+ BP-1+ and
CD19+ HSA+ μH+ B cell subsets was significantly reduced in Gli3-/- E18.5 mouse embryos
[390]. Taken together, these studies suggest Gli3 as a player in lymphocyte development.
However, the biological significance of this reduced lymphocyte populations has yet to be elucidated.
In a study using RAW264.5 mouse macrophages that were challenged with lipopolysaccharide (LPS) to identify novel inducible genes, a muscle specific transcription factor Myoblast determination protein 1 (MyoD) and a major regulator of cardiac development Homeobox protein Nkx-2.5 (Nkx2.5) were reported to be upregulated. More interestingly, the expression of Gli3, but not Gli1 or Gli2, was upregulated in response to
47
LPS suggesting a novel mechanism of regulation of Gli3 by the TLR4 signaling pathway
[391]. Although this is the only report to suggest a regulatory role for Gli3 in Tlr4 signaling, it is probable that Gli3 might regulate innate inflammatory effects based on aforementioned studies. Additionally ,in a shRNA library screening during the development of myeloid cells Gli3 was suggested to play a role in cell proliferation [386].
In Cancer
GLI3 regulates various biological processes that are important for cancer cell growth and progression. Several studies found that GLI3 regulates anchorage independent growth, proliferation and migration of cancer cell lines [392][393][394].
Studies also reported increased GLI3 expression in patient samples compared with healthy donors [395][394][396]. A role for GLI3 as a tumor suppressor was reported in medulloblastoma and AML [397][398]. Although these studies are infrequent, this raises the question of how GLI3 can act as both a tumor initiator and tumor suppressor. The following section summarizes data in which GLI3 was identified as either a pro- or anticancerous protein and discusses what is known and what needs to be further investigated to delineate the role of GLI3 in cancer.
In glioblastoma, GLI3 was downregulated upon anti-cancer drug treatment [399].
In this study the focal adhesion kinase (FAK) autophosphorylation inhibitor ‘Y15’ effectively decreased tumor growth of the glioblastoma cell lines DBTRG and U87, especially in combination with temozolomide (a chemotherapeutic agent that induces
G2/M arrest and apoptosis). In a microarray gene profiling analysis where glioblastoma cell lines DBTRG and U87 were treated with ‘Y15’ in combination with temozolomide, the 48 expression of GLI3 was downregulated [400]. This downregulation was not visible in the presence of either drug alone suggesting that downregulation of GLI3 is a result of synergistic inhibitory effect of this drug combination [400]. It can be inferred that GLI3 downregulation is involved in the synergistic anti-tumor effect of both treatments, however the molecular mechanism resulting in this downregulation remains unclear.
In the earlier described PHS, mutations in GLI3 cause formation of polydactyly of the limbs [401]. However patients suffering from PHS also have a high recurrence of hypothalamic hamartoma, suggesting a role of GLI3 in regulating benign brain tumor formation [401][402][403]. In a study by Saitsu et al (2008), two somatic mutations in
GLI3 in hypothalamic hamartoma patients were identified and showed reduced GLI promoter activity from the floor plate enhancer HNF3b in C3H10T1/2 cells [404]. This suppressor activity of mutated GLI3 might be related to hypothalamic hamartoma formation, however further studies are necessary to confirm the role of GLI3-R and HH signaling in this tumor development.
In addition to the involvement of GLI3 in hamartoma, GLI3 plays a role in Oral squamous cell carcinoma (OSCC) [392]. PCR array results comparing CD44high vs
CD44low OSCC cell populations using SCC4 and SCC9 cells revealed a greater than 6fold increase in GLI3 expression in the CD44high cancer stem cell subtype [392]. This upregulation of GLI3 expression was also confirmed in vivo in OSCC patient samples
[392][405]. In addition, a significant reduction in proliferation and invasion was observed in vitro and a positive correlation between GLI3 expression and tumor size was confirmed in vivo [392]. In this publication GLI3 knockdown had no effect on apoptotic cell death and
49 there was no link between GLI3 expression and metastasis or blood lymphatic or perineural invasion. Although patients with high GLI3 expression seem to survive less, data presented in the Kaplan–Meier blot was not statistically significant. These reports show that GLI3 regulates cancer stem cells in OSCC by regulating relevant markers for
EMT. Additionally, GLI3 regulates cell survival and invasion and correlates with tumor size in vivo. However, whether GLI3-FL or GLI3-R are regulating this effect, is not clear and the involvement of classical HH signaling has not been investigated. Since HH signaling is involved in regulating cancer stem cell subtypes in several cancers, it is plausible that
SHH regulates this subtype partially via GLI3 in a paracrine manner [246]. However, additional studies are needed in order to reach this conclusion. Since cancer stem cells are resistant to most chemotherapy due to irregular cell division, GLI3 might be a potential target to sensitize those cells to chemotherapeutic agents. For example, since GLI3 negatively regulates S100A9 in OSCC cancer stem cells and this has been shown to induce cell death in cancer cell lines [406], targeted depletion of GLI3 in OSCC might be an effective strategy in the treatment of OSCC.
In addition to OSCC cancer stem cells, gastric cancer stem cells, that were sorted based on CD44+ CD24+ surface expression, showed 80-fold higher GLI3 mRNA expression than the CD44lo CD24lo phenotype which positively correlated with mRNA expression of SHH and PTCH1 [395]. Since multiple studies report the involvement of
SHH in gastric cancer [407][408], it is possible that GLI3 is mediating SHH-induced effects. However additional studies are needed to confirm the specific regulatory role of
GLI3 in gastric cancer development and progression. Additional evidence supporting a role for GLI3 as an oncogene was provided by Li et al (2018) who used bladder cancer 50 tissue samples to show higher GLI3 expression in tumor samples vs benign tissue by performing microarray gene expression analysis [394]. Interestingly, GLI3 was identified as a prognostic factor for poor survival in patients suffering from bladder cancer and, consistent with aforementioned studies, GLI3 was shown to regulate cell proliferation, invasion, migration and proteins involved in EMT such as E-cadherin and N-cadherin
[392][393][394]. miR-7-5p was identified as a negative regulator of GLI3 in bladder cancer
[394]. However, no experiments were performed to identify possible downstream targets of GLI3. Additionally, given the role of GLI3 in regulating cancer stem cells in OSCC and gastric cancer, it might be useful to determine the role of GLI3 in regulating bladder cancer stem cell characteristics and survival as well.
Post translational modifications of GLI3-FL (but not GLI3-R) by SET7 has been reported to increase the stability of GLI3-FL and its ability to bind the promoter region of
GLI1 upon SHH treatment [393]. SET7 lysine 436 (K436) and -K595 dependent methylation of GLI3-FL regulates cell viability and colony formation of the non-small cell lung cancer (NSCLC) cell line A549 as well. Additionally, A549 xenografts in mice, expressing GLI3-FL with mutations in SET7 methylation sites, showed less migration, invasiveness and decreased tumor volume in comparison to mice injected with A549 xenografts expressing WT GLI3. This supports the oncogenic role of GLI3 and provides information on post-translational modification-dependent tumorigenicity of GLI3 in
NSCLC. Furthermore, targeting SET7 may be a viable therapeutic strategy to decrease
GLI3’s oncogenic effect in NSCLC as a strategy to antagonize SHH-dependent signaling.
51
In colon cancer, GLI3 transcripts were significantly higher than in healthy tissue
[409]. Treatment of colon carcinoma cell lines RKO and LOVO with GLI3 siRNA led to a significant decrease in cell proliferation. Furthermore, GLI3 levels inversely correlated with p53 levels within those cell lines. Additional evidence from in vitro experiments showed that siGLI3 treatment decreased p53 and MDM2 interaction and reduced ubiquitination of p53 [409]. However, it is unclear if this effect is due to GLI3-FL or GLI3R and warrants further investigation [409]. In another study, GLI3-FL (but not GLI3-R) induced more anchor-independent growth in the human colorectal cancer cell lines
HCT116, HT29, SW480 and DLD-1 which was also visible upon SHH stimulation suggesting GLI3 regulation of colony formation occurs in a paracrine manner as well
[410]. In this study, the oncogenic role of GLI3 in solid tumors was further validated when
GLI3-FL-overexpressing DLD-1 and HT29 mouse xenografts showed significantly increased tumor formation compared to control cells. Interestingly, GLI3 did not seem to be affected by any known cancer-related signaling molecules such as p53, WNT or MAPK signaling [410].
In pancreatic cancer cells such as PANC-1, targeting GLI3 with siRNA reduced cell viability. Additionally, siRNA mediated GLI3 knockdown appeared to sensitize PANC-
1 cells to cyclopamine treatment in vitro [411]. Since cyclopmine’s reduction of cell viability is due to the initiation of pro-apoptotic cell machinery, the question arises as to what degree GLI3 is involved in regulating the apoptotic pathway in pancreatic cancer cells.
52
In a more recent study, GLI3-FL was suggested to be involved in the induction of castration-resistant prostate cancer (GRPC) through a mutated form of MED12 which is common in prostate cancer [412]. MED12 negatively regulates GLI3-FL activation role by directly interacting with GLI3 at AA 1090-1596 [361]. Due to a mutation in MED12, its constraining activity toward GLI3 is inactive, which leads to activation of SHH-GLI3 related gene expression in the absence of androgen [412]. Since a common therapy for prostate cancer is androgen depletion, and in the presence of mutated MED12 SHH induced GLI3 gene expression is activated when androgen is absent, new or additional therapeutic approaches might be necessary to reduce the risk of prostate cancer relapse.
Furthermore, the question remains whether there is a direct interaction between GLI3 and
MED12 in prostate cancer. Although this interaction between MED12 and GLI3 is probable, there are no reports of MED12 interacting with GLI3 to regulate HH related gene expression. In addition, it also remains unclear if MED12 is required for GLI3 phosphorylation. Further research is needed to elucidate the mechanism of MED12-GLI3 regulation.
It can cautiously be suggested that GLI3 may play an oncogenic role in germ cell tumors. In a study by Kuleszo et al (2017), eight germ cell tumors from children were used to perform genomic profiling and GLI3 was identified to have additional copies of its gene in the germ cell tumor group compared with healthy individuals [413]. Although no experiments were performed that directly identify GLI3 as an oncogenic protein in germ cell tumors, the higher GLI3 copy numbers in germ cell tumors is indicative of more HH activation and SHH related gene expression which is known to drive tumor growth and
53 progression. The presence of higher copies of SMO and SHH, in addition to lower copy numbers of PTCH1 (which inhibits SMO activity) in germ cell tumors are another supporting fact of the involvement of HH signaling in germ cell cancer formation.
In addition to regulating proliferation and invasiveness in cancer, GLI3 was also reported to modulate vacuole membrane protein (VMP1), a known regulator of autophagy
[414]. Lo Ré et al (2012) reported that oncogenic KRAS (KRASG12D) leads to activation of
VMP1 by signaling through PI3K-AKT1-GLI3. In this pathway, GLI3 physically interacts with p300 which facilitates binding of GLI3 to the promoter region of VMP1. GLI3 mRNA expression was upregulated upon transfection with oncogenic KRASG12D and coexpression of GLI3 with constitutively active forms of PI3K and AKT1 showed higher
VMP1 promoter activity. These studies show the regulation and requirement for GLI3 in autophagy; however, it is unclear which transcription factor is involved in regulating GLI3 expression downstream of AKT1. In addition, it would be interesting to determine if post translational modifications of GLI3 are required for binding and activating VMP1 promoter activity. This could be accomplished by targeting known regulators of GLI3 such as PKA or GSK3β, in addition to activating this pathway. Additionally, it is unclear if GLI3-FL or
GLI3-R is responsible for the regulation of VMP1 and the biochemical domains mediating the interaction between GLI3 and p300. Depending on the binding site for the interaction between GLI3 and p300, this may determine if GLI3-FL or GLI3-R regulates the
KRASG12D-PI3K-AKT1-VMP1 pathway.
In addition to the oncogenic role of GLI3 in solid tumors, GLI3 was shown to play a similar role in hematologic malignancies. Elevated GLI3 expression was reported in
54
Hodgkin lymphoma cell lines [415]. This was validated using immunohistochemistry in primary patient biopsies where GLI3 was found in Hodgkin/Reed Sternberg cells. GLI3 was also suggested to pay a role in Diffuse large B cell lymphoma (DLBCL). This is an aggressive lymphoma that is divided into 2 subtypes based on gene expression profiling studies: activated B-cell (ABC) and germinal center B-cell (GCB) subtypes. GLI3 expression was examined in a cohort of DLBCL cell lines and was found to be elevated in cell lines belonging to the GCB DLBCL subgroup [416]. Using gene expression datasets using DLBCL patient samples, the authors also report increased GLI3 expression in the GCB subtype. Knockdown of GLI3 reduced cell proliferation suggesting that GLI3 promotes cell growth in GCB DLBCL. Further characterization of the biological role of GLI3 in these cancers will enhance our understanding of the biological significance of GLI3.
Although rare, anti-cancerous activity of GLI3 has been reported as well. GLI3 was found to be present in 94% of neuronal differentiation and glial and neuronal differentiation medulloblastoma, while it could not be detected in any of the differentiationfree medulloblastoma [397]. Additionally, patients with differentiation free medulloblastoma showed significantly less survival. This suggests a role for GLI3 as a prognostically favorable factor in medulloblastoma. The role of SHH in medulloblastoma has been reported and therapeutic success has been shown after inhibiting HH signaling
[417][418]. In theory, as a result of inhibition of HH signaling, GLI3-R should accumulate within the cell and negatively regulate HH-related gene expression and therefore cancer growth. However, whether the presence of GLI3-R form specifically correlates with a higher overall and event free survival has yet to be determined. Additionally a number of
55 studies have reported that GLI transcription factors can be regulated in a HH independent manner [419][326][311][336][420][229][316][421]. Therefore, this raises the possibility that GLI3 may regulate gene expression in a HH-independent manner to mediate its anticancerous functions.
Another study reported GLI3 as a tumor suppressor protein using bone marrow from AML patients. Both GLI3-FL and GLI3-R were shown to be expressed at significantly lower levels in AML patient samples [398]. Based on their data, the absence of GLI3 in
AML is due to hypermethylation of its promoter region. Global demethylation showed an increase in protein expression of both GLI3-FL and GLI3-R in vitro and ex vivo which correlated with decreased proliferation of the AML cell lines (K562 and KG1a) and primary
AML blasts and with mouse survival after K562 xenograft transplantation. This increase in GLI3-FL and GLI3-R was also visible when AML cell lines were treated with the HH inhibitor PF-04449913 (a SMO inhibitor). Therefore, it might be worth investigating whether PF-04449913 might regulate demethylation of GLI3 promoter in its reported synergistic effect with decitabine in AML [398]. A cross-talk between GLI3-R, AKT1 and
ERK1/2 protein has also been reported. Overexpression of GLI3-R negatively correlated with AKT1 but positively correlated with ERK1/2 [422]. AKT1 was reported to be important for GLI3-R effect on negatively influencing cell proliferation [422]. However, it was not determined if GLI3-R regulates proliferation through ERK1/2 as well. ERK1/2 is known to activate MAPK pathway however to what degree GLI-R is involved in this pathway has not been investigated. It might also be of interest to determine the role of GLI3-AKT1 axis in response to HH inhibitor treatment such as PF-04449913 or cyclopamine and if demethylation increased the activity of this pathway. 56
Most studies that have investigated GLI3 in cancer described the cancer supporting role of GLI3 to occur by positively regulating proliferation, survival and invasiveness. Reports suggesting GLI3’s tumor suppressive role have been rare but raise the question where the contradictory attributes of GLI3 in cancer come from. It is possible the role of GLI3 in cancer is tissue specific. In addition, the majority of studies discussed in this part of the review, describe a role for total GLI3 levels in regulating cancer cell growth and progression. However, it is unknown if it is GLI3-FL or GLI3-R that regulates these effects. This is possibly hindered by the lack of antibodies that reliably identify
GLI3FL or GLI3-R (Matissek et al, unpublished observation). Studies that focused on these two forms of GLI3 have suggested that GLI3-FL induces cancerous behavior while
GLI3R reduces cancer associated attributes [393][410][412][398]. However, to date, there is only one study that solely compares the effect of GLI3-FL vs GLI3-R on cancerous characteristics [423]. In this study, GSK3 beta activity was increased which induced higher concentrations of GLI3-R and an anti-cancerous effect.
Based on previous mentioned work by Chaudhry et al (2015), hypermethylation of
GLI3 promoter region leads to a loss of GLI3 and tumor suppressor function in AML [398].
In addition to epigenetic modifications that suppress GLI3 tumor function, it might also be that a disruption of GLI3-FL/GLI3-R equilibrium within the cell leads to a cancerous phenotype and tumor growth. Different expression and activation levels of GLI3-FL regulators (SPOP, androgen receptor, PP2A, CBP, SET7, MED12) or regulators of GLI3-
R (PKA, GSK3β, βTrCP) in cancer cells in comparison to healthy cells might also promote cancer cell growth and survival.
57
GLI3 levels also positively correlated with higher expression of cancer stem cell markers and shRNA-mediated knockdown of GLI3 led to a decrease in the CD44high population (cancer stem cells) while the CD44low population was unaffected in OSCC
[392]. The small cancer stem cell population within a tumor has been shown to be very potent in inducing tumor growth in OSCC [424]. Therefore, GLI3’s tumorigenic role may be due to its regulation of cancer stem cell survival. However, whether GLI3-FL or GLI3R regulates this effect is also not fully explored. There are multiple HH inhibitors
(cyclopamine, PF-04449913, GDC0449, Saridegib, Vismodegib) that showed promising results in fighting HH-driven cancers. These inhibitors target SMO to inhibit HH-related gene expression. However, since HH-independent regulation of GLI transcription factors has been reported, this might rescue the effects induced by SMO inhibition [425].
Therefore, therapies such as GANT61, which inhibit binding of GLI1 and GLI2 transcription factors to DNA, are promising since they act directly on GLI but not on SMO
[426]. GLI3 expression was not affected by GANT61, therefore, targeting modulators of
GLI3-R by making them more active, might decrease PTCH1-SMO-independent HH gene expression and increase the therapeutic effect of GANT61 treatment [329]. Furthermore, a thorough understanding of the signaling pathways that regulate GLI3 independent of
HH will allow therapeutic targeting of GLI3 by targeting other molecular regulators of this protein.
58
MicroRNA (miRNA) and GLI3
As described earlier, GLI3 can interact with several different proteins, an interaction that is required for either stabilization or degradation of GLI3 protein. However, proteins are not the only molecules involved in regulating GLI3. Several lines of evidence have been reported in which GLI3 is regulated at the RNA level by microRNAs which are described below. MicroRNAs were reported to bind complementary sequences of GLI3 RNA and negatively regulate its gene expression.
In cancer, liver fibrosis and spermatogenesis
GLI3 was shown to be regulated by miR-7-5p in bladder cancer tissue. A significant upregulation of GLI3 in bladder cancer was reported by microarray gene expression profiling. Using the online software (TargetScan, PITA, miRanda) the authors found that miR7-5p was the most potent candidate for GLI3 regulation and later confirmed its regulatory role using in vitro experiments. In those studies, a physical interaction between miR-7-5p and GLI3 was identified (GLI3 interaction site: GUCUUCCA), which leads to a negative regulation of GLI3 and to a less cancerous phenotype of bladder cancer cells
[394].
In other studies, miR494 and 506 were shown to regulate tGLI3 oncogenic function in pancreatic and cervical cancers [396]. In pancreatic cancer, GLI3 was regulated by miR-494 [396]. GLI3 mRNA expression was significantly upregulated in tissue from bladder cancer patients and there was an inverse correlation between GLI3 and miR-494.
Furthermore, miR-494 negative regulation of GLI3 was confirmed in vitro in which inhibition of GLI3 by miR-494 led to a decrease in cell viability and cancer cell migration 59
[396]. A similar negative regulation of GLI3 was also detected by miR-506 in cervical cancer [427]. This was reported in both primary patient biopsy samples and in cervical cancer cell lines using western blotting. GLI3 protein was increased upon miR-506 inhibition which led to reduced apoptosis and increased proliferation. In addition, mice xenografts with Casci cell stably expressing sh-miR-506 showed significantly higher tumor weights in comparison to control mice.
In addition to their role in tumorigenesis, micro RNAs appear to regulate GLI3 in liver fibrosis. Both miR-152 and miR-378a-3p have been shown to negatively regulate
GLI3 in liver fibrosis in vitro and in vivo [428][429]. These studies used the LX-2 hepatic stellate cells where they overexpressed miR-152 or miR-378a-3p and found a reduction in GLI3 mRNA and protein expression. Using rodent models of liver fibrosis where fibrosis was chemically induced with CCI4 in mice, GLI3 expression was increased (miR378a-3p downregulated) in tissue isolated from animals suffering from liver fibrosis suggesting
GLI3 positively regulates liver fibrosis.
The role of GLI3 in spermatogenesis in mice was established in 1997 [430].
However, in 2016, a role for miR-133b in regulating Sertoli cells by targeting GLI3 was discovered [431]. miR-133b was significantly upregulated in Sertoli cells from patients suffering from Sertoli-cell-only syndrome (SCOS) and using TargetScan, GLI3 was identified as a potential target for miR-133b. This was confirmed by in vitro experiments showing the regulatory role of miR-133b in which an increase in miR-133b led to reduction of GLI3 mRNA and protein expression [431].
60
Conclusions
The importance of HH signaling in many biological pathways such as those related to development, cancer and inflammation, has been frequently reported. The involvement of HH signaling in positive regulation of these biological functions has focused on GLI1 and GLI2 which are known activators of HH signaling. Their role as a positive regulator in non-canonical HH pathways has been frequently described as well. However, based on this review, GLI3 also shows high potential in regulating non-canonical pathways positively. In addition to early reports of the role of GLI3 in the development of the brain, lungs, sperm and in genetic diseases, the biological significance of GLI3 was also suggested in diseases such as liver fibrosis, cancer, and in the immune system. In cancer,
GLI3’s behavior seems to be bipolar since it was linked to cancer promoting and inhibitory effect in cells which can be explained by GLI3-FL activator and GLI3-R inhibitory function in gene expression. In addition, recent research shows that negative regulation of GLI3 through microRNA reverses cancerous behavior which emphasizes GLI3 as a potential oncogenic target in cancer therapy. Future studies focusing on determining the role of
GLI3 in the immune response and in cancer will clarify its biological significance and lay the foundation to target this molecule to reprogram immune and cancer cells.
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Figure 1-1: Biochemical domains of GLI family members.
62
Figure 1-1: Biochemical domains of GLI family members Schematic diagram of the biochemical domains of human GLI1, GLI2 and GLI3 showing the relative repressor (RD), DNA binding and transactivation (TD) domains. HH-related transcriptional activator GLI1 is the smallest protein of the GLI family with 1106 AA (GLI2:
1586 AA; GLI3: 1580 AA).
In comparison to GLI2 and GLI3, GLI1 lacks a transcriptional repressor domain. GLI1 and
GLI2 isoforms have been described. There is no direct evidence to describe the exact position of the transactivation domain and zinc finger region of human GLI2
63
Figure 1-2:Biochemical domains of human GLI3 with posttranslational modifications
64
Figure 1-2: Biochemical domains of human GLI3 with posttranslational modifications GLI3 can be phosphorylated by PKA and GSK3β which leads to binding by βTrCP and ubiquitination by SCFTrCP. Indicated phosphorylation and ubiquitination sites are shown and are crucial for GLI3 processing into its repressor form (red box). βTrCP binds GLI3 at the C- and N-terminus and at its core domain (activation domain; green). Only binding to GLI3 core domain is important for GLI3 processing into GLI3-R. Processing site to generate Gli3-R and zinc finger domain are completely conserved between mouse and human. Differences in amino acid sequences between mice and human are represented by asterisk and oval structure
65
Figure 1-3:Regulation of GLI3-FL and GLI3-R.
66
Figure 1-3: Regulation of GLI3-FL and GLI3-R. GLI3-FL (but not GLI3-R) is proteasomally degraded upon overexpression of SPOP, while binding by CBP, Androgen receptor, MED12, SET7 and release of PP2A stabilizes
GLI3-FL and facilitates its translocation to the nucleus. GLI3-R generation is facilitated by
SUFU recruitment of Gpr161, which activates PKA. PKA phosphorylates GLI3 at AA residues 849, 865, 877,
907, 980 and 1006. This phosphorylation leads to hyperphosphorylation by GSK3β (AA
841-880) and binding of βTrCP to GLI3. βTrCP then recruits SCFTrCP which ubiquitinates
GLI3 leading to proteasomal cleavage and generation of GLI3-R which can translocate to the nucleus to regulate gene expression
67
Figure 1-4: Alignment of human and mouse GLI3/Gli3 protein sequences.
68
Figure 1-4: Alignment of human and mouse GLI3/Gli3 protein sequences. Alignment of human (NP_000159.3) and mouse (NP_032156.2) GLI3/Gli3 shows 86 % similarity in amino acid sequence.
69
CHAPTER 2
A novel mechanism of regulation of the transcription factor GLI3 by toll-like receptor signaling
Introduction
Hedgehog (HH) signaling is well known for its role in embryonic development, cancer and inflammation[247][432][433][434]. At the center of HH signaling are the 2 receptors patched (PTCH1) and smoothened (SMO) along with GLI transcription factors
[227]. In the absence of HH ligand, PTCH1 inhibits SMO. Upon ligand binding to PTCH1, the inhibition of SMO is removed, and SMO transduces signal ultimately leading to the activation of GLI proteins [227]. Multiple studies demonstrated that SMO-independent
(non-canonical) pathways can regulate GLI proteins[302][305][303][229][435] leading to inflammation and cancer cell growth. Therefore, there is a need to understand the molecular pathways that regulate GLI proteins independent of SMO signaling.
There are 3 members of the GLI family of transcription factors (TFs): GLI1, GLI2 and
GLI3. While GLI1 and GLI2 act primarily as transcriptional activators of SMO-dependent
HH signaling, GLI3 is known for its transcriptional inhibitory effect where it negatively regulates HH signaling [436][240][288]. Despite the identification of several
SMOindependent pathways that regulate GLI1 and GLI2 [435][314], little is known about
SMOindependent pathways that regulate GLI3. GLI3 is known to play a role in developmentassociated diseases such as Greig cephalopolysyndactyly (GCPS) and
70
Pallister-Hall Syndrome (PHS)[437][384][237][239] . More recent studies suggest a role for GLI3 in the immune system[231].
Immune cells, such as monocytes recognize pathogens through their pattern recognition receptors (PRR). Toll-like receptors (TLR) are PRR known to be strong inducers of inflammation and immune clearance [438][439]. Of the TLRs, TLR4, is known to bind
Lipopolysaccharide (LPS) which is expressed on the outer membrane of gram-negative bacteria. Upon LPS binding to TLR4, downstream signaling occurs through either MyD88 or TRIF adapter molecules [440]. Signaling through MyD88 activates NF-κB and MAPK, while signaling through TRIF activates IRF3 in addition to MAPK and NF-κB
[441][442][443]. All other TLRs signal through MyD88 except TLR3 which signals through
TRIF [26]. Using either adapter molecule, activation of these transcription factors leads to their nuclear translocation where they regulate the expression of inflammatory genes
[444][445].
Here, we identify GLI3 as a downstream target of TLR signaling in monocytes. Stimulation of TLR4 with LPS induces an increase in GLI3 mRNA and protein expression independent of signaling through SMO. Further analysis identified TRIF as the adapter molecule used by TLR4 to modulate GLI3 expression. Using pharmacological inhibitors to target signaling molecules downstream of TRIF, we identify IRF3 as the transcription factor mediating TLR-TRIF-induced GLI3. This was further validated using the TLR4 ligand monophosphoryl lipid A (MPLA) and the TLR3 ligand polyinosinic:polycytidylic acid 71
(Poly(I:C)) which exclusively activate TRIF signaling[446]. Both MPLA and Poly(I:C) increased GLI3 expression and overexpression of IRF3 increased GLI3 expression. We found that IRF3 directly binds the GLI3 promoter region upstream of exon 1 and this binding was increased upon activation of TRIF-IRF3 signaling. Finally, in mouse embryonic fibroblasts (MEFs) lacking IRF3 expression (IRF3-/- MEFs), activation of
TRIFIRF3 signaling was no longer able to increase GLI3. Taken together, this study identifies TLR-TRIF-IRF3 signaling as a novel pathway that regulates GLI3 expression.
Experimental Procedures
Cell lines and primary cells
The cell line Mono-Mac-6 (MM6) was purchased from DSMZ and cultured in RPMI
1640 supplemented with 10% FBS, 1% 2mM L-glutamine, non-essential amino acids,
1mM Sodium Pyruvate, and 10μg/ml human insulin. THP-1 and U937 cell lines were purchased from ATCC. THP-1 cells were cultured in RPMI 1640 with 10 % FBS supplemented with 0.05 mM β-Mercaptoethanol and U937 cells were grown in RPMI 1640 with 10% FBS. An antibiotic-antimycotic was added to all cell culture media. Wild-type
(WT) and IRF3-/- MEFs were a generous gift from Benjamin R. tenOever, and were generated as previously described (30). MEFs were maintained in Dulbecco’s Modified
Eagle’s Medium (DMEM) (Corning, NY) supplemented with 10% v/v fetal bovine serum
(FBS) (HyClone) and 1% v/v penicillin-streptomycin (Corning). Poly(I:C) HMW stimulations (10 μg/mL) were performed with Lipofectamine 2000 according to the manufactures recommendations using a ratio of 1 μg:0.2 μL (Poly(I:C):Lipofectamine
2000).
72
Primary human monocytes were purified from peripheral blood mononuclear cells
(PBMCs) obtained from buffy coats purchased from the blood bank of New York (NYC,
NY) or the Oklahoma Blood Institute (Oklahoma City, OK). Experiments using cells from different donors are numbered D1-D6. Monocytes were isolated by negative selection using the monocyte enrichment kit from STEMCELL Technologies (Cambridge, MA). Cell purity was confirmed by flow cytometry using CD14+ staining and was found to be >90%.
Primary cells (CD14+) were plated and treated immediately after purification as described.
Reagents
Lipopolysaccharide (LPS) (E.coli, Serotype 0111;B4) and Poly(I:C) were purchased from
MilliporeSigma (St. Louis, MO). CpG was purchased from Integrated DNA Technologies
(Coralville, IO). The TLR4 signaling inhibitor CLI-095) and MPLA were purchased from
Invivogen (San Diego, CA). The MAPK inhibitor (PD98059), NF-κB inhibitor (QNZ), TBK1 inhibitor (BX-795) and SMO inhibitor (Cyclopamine) were purchased from SelleckChem
(Houston, TX).
RNA isolation and Quantitative RT-PCR (qPCR)
For total RNA isolation, TRIsure reagent (Bioline, obtained through Thomas Scientific,
Swedensboro, NJ) was used following the manufacturer’s recommendations as previously published [420][229]. cDNA synthesis was performed using Moloney Murine
Leukemia Virus Reverse Transcriptase (MML-V)(Promega Corporation, Madison, WI).
ViiA-7 instrument (Life Technologies, Grand Island, NY) was used to perform qPCR analysis. Amplification results from GLI1, GLI2 and GLI3 were normalized to human 73
GAPDH. The following qPCR primers were used: GAPDH,
5’CTCGACTTCAACAGCGACA-3’ (forward) and 5’-GTAGCCAAATTCGTTGTCATACC-
3’
(reverse). GLI1, 5’-TCACGCCTCGAAAACCTGAA-3’ (forward) and 5’-
AGCTTACATACATACGGCTTCTCAT-3’ (reverse) ; GLI2, 5’-
GGAGAAGCCATATGTGTGTGAG-3’ (forward) and 5’-
CAGATGTAGGGTTTCTCGTTGG-3’ (reverse); GLI3, 5’-
GGGACCAAATGGATGGAGCA-3’ (forward) and 5’-TGGACTGTGTGCCATTTCCT-3’
(reverse).
Chromatin immunoprecipitation (ChIP) assay
Briefly, 10x106 cells were cross-linked using 0.37% formaldehyde for 10 min at room temperature then lysed and sonicated to shear DNA to ~ 500 bp (QSonica, Q800R3).
Samples were immunoprecipitated using anti-IRF3 antibody or isotype control (Biolegend,
San Diego, CA) using magnetic Protein A/G beads (ThermoFisher Scientific) at 4oC overnight. Samples were washed and reverse crosslinked overnight in 5M NaCL at 65°C.
After RNase A and Proteinase K digestion, DNA was purified using GeneJET PCR
Purification Kit (ThermoFisher Scientific) and used for qPCR analysis using the following primers: Binding site 1 (BS1), 5’-GGCTAAGAATGGAGTGTTTGGA-3’ (forward) and 5’-
CACCACTGGTTTAGCTACATACAT-3’ (reverse); Binding site 2 & 3 (BS2/3),
5’CTGGGCAACAGAGTGAGAC-3’ (forward) and 5’-CACCCGGAACCTCAATGTTA-3’
(reverse); Binding site 4 (BS4), 5’-CTGCCCTTGGGACTCAC-3’ (forward) and
5’CAAAGGGCACTGACAAAGTATT-3’ (reverse). To determine the effect of TLR-TRIF
74 signaling on IRF3-mediated regulation of GLI3, 10x106 cells were cultured at a density of
5x106 cells/ml and treated with 10μg/ml Poly(I:C) for 1h or left alone followed by fixation and ChIP analysis as described earlier.
Plasmid constructs and cell transfections
Short hairpin RNA targeting TRIF and MYD88 (shTRIF, shMYD88) were purchased from
OriGene Technologies (Rockville, MD). The plasmid expressing dominant negative TRIF
(dnTRIF) was purchased from Invivogen (San Diego, CA). Transfections were performed by electroporation using BTX ECM 630 (Holliston, MA) using the following parameters:
MM6: 240v/25ms and U937: 300v/10ms. Five million cells were electroporated with 10μg shTRIF, shMYD88 or scrambled control (shScr) or 5μg dnTRIF or empty vector in
OPTIMEM followed by culture for 48h.
Immunoblotting
Immunoblotting was performed as previously described [420][229]. Cell lines and primary monocytes (5x106 cells/ml) were treated as indicated and harvested in 100 μl RIPA buffer
(ThermoFisher Scientific). Total protein concentration was determined using a BCA assay
(ThermoFisher Scientific). For GLI3, 5% self-prepared SDS gels were used while for all other proteins 10% gels were used. Transfer to nitrocellulose membranes was performed using a turbo transblot system (Biorad, Hercules, CA). GLI3 antibody was purchased from abcam (cat# ab123495), β-actin antibody was purchased from Milipore Sigma (cat#
75
A3854), IRF3, phospho-IRF3, ERK, phospho-ERK, IκBα, TRIF and MYD88 antibody were purchased from cell signaling (cat#4302, cat# 4947, cat# 9102, cat# 9101, cat# 9242, cat#4596, cat#4283 respectively).
Statistical analysis
Statistical analysis was performed using GraphPad Prism software (San Diego, CA). A two-tailed t test was used to determine statistical significance between two variables and a two-way ANOVA was used to compare more than 2 variables. Statistical significance is indicated on each figure as follows: * (p<0.05), ** (p<0.01), *** (p<0.001), and ****
(p<0.0001).
Results
LPS stimulation induces GLI3 expression
We stimulated human monocyte cell lines MM6, THP-1 and U937 with LPS and investigated the effects on GLI expression. We found that LPS stimulation induces an increase in GLI3 mRNA expression in all 3 cells lines (Fig. 2-1A). GLI1 expression was not altered, while GLI2 expression was only induced in THP-1 cells. To determine the role of HH signaling in LPS-induced GLI3, we pretreated cells with the SMO inhibitor
Cyclopamine, followed by stimulation with LPS and evaluated the expression of GLI genes. As expected, there was a reduction in GLI1 mRNA expression following treatment
76 with Cyclopamine (Fig. 2-1B). However, when we examined GLI2 and GLI3 expression in the presence of Cyclopamine, GLI3 expression was still significantly induced upon LPS stimulation (Fig. 2-1B), suggesting LPS-induced GLI3 occurred in a HH-independent manner. Using CD14+ cells purified from peripheral blood mononuclear cells (PBMCs), we confirm that LPS stimulation induces GLI3 expression in primary human monocytes
(Fig. 2-1C). The increase in GLI3 expression upon LPS stimulation occurred in a time and dose-dependent manner (Fig. 2-1D). This increase in GLI3 expression also resulted in an increase in GLI3 protein in response to stimulation with LPS (Fig. 2-1E). Taken together, these results propose the TLR4 signaling as a novel non-canonical HH pathway that can regulate GLI3 expression.
LPS/TLR4-induced GLI3 occurs through TRIF signaling
To elucidate the signaling mechanism downstream of TLR4 that results in increased GLI3 expression, we used the TLR4 signaling inhibitor (CLI-095), which inhibits TLR4 signaling intracellularly and found that monocyte cell lines and CD14+ cells from PBMCs treated with the TLR4 signaling inhibitor had reduced GLI3 expression (Fig. 2-2A). Furthermore, in the presence of the TLR4 signaling inhibitor, LPS was not able to induce GLI3 expression (Fig 2-2B) suggesting that an active TLR4 signaling pathway was required for
LPS-induced GLI3.
LPS-TLR4 signaling can utilize one of 2 adapter proteins, MyD88 or TRIF [28]. To determine the signaling mechanism downstream of TLR4 that regulates GLI3, we used 77
RNAi targeting either MyD88 or TRIF to determine the effect on GLI3 expression. When cells were transfected with RNAi targeting TRIF (but not MyD88), there was a significant reduction in GLI3 expression (Fig. 2-3A). To validate this, we utilized a dominant negative form of TRIF (dnTRIF) that lacks N- and C-terminus and harbors a proline to histidine mutation at amino acid 434 which is crucial for TLR-dependent signaling. We found that in cells expressing dnTRIF, there was a reduction in GLI3 protein expression (Fig. 2-3B).
Additionally, LPS stimulation was no longer able to induce GLI3 protein expression in cells transfected with shTRIF (Fig.2-3 C). Taken together, these results suggest that LPSTLR4- induced GLI3 occurs via signaling through TRIF.
TLR4-TRIF regulates GLI3 through the transcription factor IRF3
Following activation of TRIF, three different downstream signaling pathways can be initiated: NF-κB pathway, MAPK pathway and IRF3 pathway [28]. We used pharmacological inhibitors to target each of these pathways to identify the contribution of each in TLR4-TRIF-mediated regulation of GLI3. Western blot analysis shows that only inhibition of IRF3 signaling using the TBK1 inhibitor BX795 reduced basal levels of GLI3
(Fig. 2-4A). Pretreatment of monocytes with BX795 also resulted in a loss of LPS-induced
GLI3 (Fig. 2-4B). To further validate the role of IRF3 as a regulator of GLI3, we overexpressed IRF3 in U-937 cells and found an increase in GLI3 expression (Fig. 2-4C).
Furthermore, we stimulated monocytes with monophosphoryl lipid A (MPLA), a TLR4 ligand that signals exclusively through TRIF and found an induction in GLI3 expression
78
(Fig. 2-4D). Finally, we compared GLI3 expression in response to LPS (TLR4 agonist which signals though MyD88 and TRIF), CpG (TLR9 agonist which signals through
MyD88) and Poly(I:C) (TLR3 agonist which signals through TRIF) and found a robust increase in GLI3 expression in response to Poly(I:C), further supporting a role for
TRIFIRF3 signaling in the regulation of GLI3 (Fig. 2-4E). Taken together, these data suggest that GLI3 is a novel target of TRIF-IRF3 signaling and can be regulated by TLR3 and TLR4.
IRF3 directly binds the GLI3 promoter
Although several studies have characterized the promoter region of GLI1 and GLI2
[447][448], less is known about the GLI3 promoter or it’s regulation. The GLI3 gene is composed of 15 exons with transcription of the gene beginning in exon 2 [231]. Therefore, we examined approximately 3000 bp upstream of ATG of the GLI3 gene for candidate
IRF3 binding sequences and identified 4 consensus sequences (AANNGAAA) that IRF3 may bind to regulate GLI3 expression (Fig. 2-5A). Using MM6 cells (which have the highest copy number of GLI3 [data not shown]), we performed chromatin immunoprecipitation (ChIP) assay to determine IRF3 binding to the GLI3 promoter region and found IRF3 binding to all candidate sites (Fig. 2-5B). Activation of TRIF signaling in
MM6 cells using Poly(I:C) resulted in a significant increase in IRF3 binding to all candidate
IRF3 binding sites (Fig.2-5C). Furthermore, using CD14+ cells purified from PBMCs that were treated with Poly(I:C), there was a significant increase in IRF3 binding to the GLI3 promoter in response to Poly(I:C) stimulation (Fig. 2-5D); and stimulation of CD14+ cells
79 from the same donor with Poly(I:C) increases GLI3 expression (Fig. 2-6E). Taken together, these results suggest that TLR-TRIF-mediated regulation of GLI3 occurs via direct binding of IRF3 to the GLI3 promoter and identifies IRF3 as a novel transcription factor that regulates GLI3 independent of HH signaling.
IRF3 is required for TRIF-induced GLI3
To confirm the role of IRF3 in TLR-TRIF-induced GLI3, we used mouse embryonic fibroblasts (MEFs) that lack IRF3 (IRF3-/-) or wild-type (WT) MEFs stimulated with
Poly(I:C). Consistent with previous results in monocytes, in WT MEFs treated with
Poly(I:C), there was a time-dependent increase in Gli3 expression (Fig. 2-6A). This
Poly(I:C)-induced Gli3 was lost in IRF3-/- MEFs. This finding was consistent with an increase in interferon beta (IFN-β) expression in the WT MEFs, but not in the IRF3-/-
MEFs (Fig. 2-6A). Taken together, these results support the requirement for IRF3 in
TLRTRIF-induced GLI3.
Discussion
The regulation of GLI transcription factors by canonical HH signaling is well established
[449]. Several studies have shown that the GLI2 family member is regulated in a
HHindependent manner [229][435][421]. Additional research shows GLI3’s role in the p53 and AKT pathway [409][450] suggesting GLI3’s potential to regulate pathways outside of
HH signaling as well. Here, we show that GLI3 is a downstream target of TLR signaling where the TRIF-IRF3 axis directly regulates GLI3 expression (Fig. 2-6B). TLRs are important immune receptors which are crucial for the innate immune response to pathogens [445][27]. TLRs are expressed on a variety of immune and non-immune cells
80 including monocytes. We found a significant induction in GLI3 expression upon challenge of monocytes with LPS (Figure 2-1). This was found to be due to activation of TRIF and subsequently IRF3 (Figure 2-3 - 2-5). The role of TRIF in regulating GLI3 was validated using the TLR3 ligand Poly(I:C) (which exclusively signals through TRIF) and the TLR4 ligand MPLA (which exclusively activates TRIF but not MyD88) [446]. Therefore, we have identified GLI3 as a novel target of TLR-TRIF-IRF3 signaling in monocytes.
Several studies have suggested a role for GLI3 in the immune system, spanning both innate and adaptive immunity [385][386]. GLI3 was found to regulate B- and T-cell development as well as the expression of CD155 on NK cells [385][451][389]. More interestingly, in a cDNA microarray analysis, GLI3 was one of the upregulated genes when bone marrow derived macrophages and RAW264 cells were challenged with LPS
[452]. Therefore, our findings are consistent with other studies and define the regulatory steps involved in the regulation of GLI3 by TLR signaling.
Upon stimulation of TLR4, signaling can occur through either MYD88 or TRIF adapter proteins [445][453]. We identified TRIF as the adaptor protein mediating TLR4 signaling to regulate GLI3 expression in both our cell line models and in primary CD14+ cells from
PBMCs. Additionally, our data suggests a negative regulation of GLI3 by MYD88, since knockdown of MYD88 using RNAi results in an increase in GLI3 protein expression (Fig.
2-3A). Downstream of MYD88, NF-κB and MAPK signaling can be activated [26]. While targeting MAPK with pharmacological inhibitors did not change GLI3 levels, inhibition of
NF-κB resulted in an increase in GLI3 protein expression (Figure 2-4). This suggests a potential role for MYD88 as a negative regulator of GLI3 by activating NF-κB to decrease
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GLI3 levels. MYD88 is known to be a protein regulating inflammatory cytokine production while TRIF-IRF3 signaling is known to regulate the production of interferons in addition to proinflammatory cytokines [454]. Siednienko et al (2011) suggest a negative regulatory effect of MYD88 on interferon production since the absence of MYD88 increased
TLR3dependent phosphorylation of IRF3 [455]. Therefore, it can be extrapolated that
MYD88 negatively regulates GLI3 by compromising IRF3 phosphorylation. However, it remains unclear what role NF-κB plays in this inhibitory effect and future studies focused on analyzing the role of NF-κB in MYD88-dependent inhibition of GLI3 would increase our understanding of this potential negative feedback signaling.
Several transcription factors were shown to regulate the promoter of GLI1 and/or GLI2
[229][435][421][456][457]. However, our understanding of the regulation of the GLI3 promoter is significantly lacking. The human GLI3 gene is composed of 15 exons with transcription start signal (ATG) located in exon 2. Here we examined approximately 3000 bp sequences upstream of exon 1 and exon 2 and identify candidate IRF3 consensus sequences upstream of exon 2 (which contains ATG signal) (Figure 2-5). In our cell lines,
IRF3 binds to the GLI3 promoter region and this binding is enhanced upon TLR-TRIF stimulation (Fig. 2-5A & 2-5B). However, in resting CD14+ cells from PBMCs, IRF3 does not bind the GLI3 promoter unless TLR-TRIF signaling is activated (Fig. 2-5C). Therefore, these studies identify a novel TF (IRF3) that regulates GLI3 by binding to the GLI3 promoter region.
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In summary, we identified TLR signaling as novel HH-independent signaling pathway that regulates GLI3 and showed this regulation is dependent on TRIF-IRF3 signaling axis.
Additionally, to our knowledge, this is the first report of the regulatory components of the
TLR-GLI3 axis where IRF3 was identified as a novel transcription factor that can modulate the promoter region of GLI3. These results increase our understanding of the signaling molecules that can regulate GLI3 in a HH-independent manner. Since TLR signaling is crucial for an inflammatory response, future studies to characterize the role of
TLRinduced GLI3 in inflammation would enhance our understanding of the biological role of GLI3 in response to TLR-TRIF-IRF3 activation.
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Figure 2-1: LPS induces GLI3 expression in a HH independent manner.
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Figure 2-1: LPS induces GLI3 expression in a HH independent manner. 5 (A) Human monocyte cell lines (MM6, THP-1, U937) (2 x 106 cells/ml) were treated with/without 100 ng/ml LPS (0111; B4 E.coli) for 1 h followed by determination of GLI expression by qPCR. (B) THP-1 cells were pretreated with 1 μM cyclopamine or DMSO control for 1 h and subsequently treated with 100 ng/ml LPS for or left untreated. After 1 h, GLI expression was determined by qPCR. (C) CD14+ cells from PBMCs (2 x 106 cells/ml) were stimulated with 100 ng/ml LPS for 1 h followed by qPCR to determine
GLI3 expression. (D) MM6 cells (2 x 106 cells/ml) were stimulated with 100 ng/ml LPS for the indicated times, or with the indicated doses of LPS for 1 h followed by determination of GLI3 expression by qPCR. (E) Human monocytes (5 x 106 cells/ml) were stimulated with LPS for 12 h followed by determination of GLI3 protein expression by immunoblotting. All experiments were repeated at least three times. Data are presented as average of at least three independent experiments ± SEM.
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Figure 2-2: Functional TLR4 is required for GLI3 expression
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Figure 2-2: Functional TLR4 is required for GLI3 expression. 6 (A) Monocytes (5x106 cells) were treated with 1 μg/ml TLR4 signaling inhibitor (CLI095) for 12h followed by determination of GLI3 expression by western blot. (B) Monocytes
(5x106 cells) were pretreated with CLI095 for 12 h followed by stimulation with 100 ng/ml
LPS for an additional 12 h. GLI3 expression was determined by western blot. These experiments were repeated 3 times.
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Figure 2-3:LPS-induced GLI3 is regulated by TRIF downstream of TLR4.
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Figure 2-3: LPS-induced GLI3 is regulated by TRIF downstream of TLR4. 7 (A) U937 cells (10x106 cells) were transfected with 10 g of either shMYD88, shTRIF or scrambled controls (shScr). After 2 days, cells were lysed and lysates were used to determine protein expression by western blot. (B) Monocytes (10x106 cells) were transfected with a dominant negative form of TRIF (dnTRIF) or empty vector (Ctrl) for 2 days followed by determination of protein expression by western blot. (C) MM6 cells
(10x106 cells) were transfected with shTRIF or shScr and cultured for 30 h, followed by treatment with 100ng/ml LPS. After an additional 12 h, cells were lysed and lysates were used for western blot to determine protein expression.
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Figure 2-4: GLI3 is regulated by IRF3 downstream of TRIF. 8 (A) THP-1 cells (5x106 cells) were treated with 50μM Erk inhibitor (PD98059) or DMSO control for 30 min, 500 nM NF-kB inhibitor (QNZ) or DMSO for 2 h, or 1 μM TBK1 inhibitor (BX795) or DMSO for 1 h. Cells were harvested, lysed and lysates were used to determine protein expression by western blot. (B) THP-1 cells (5x106 cells) were pretreated with 1 μM BX795 for 1 h, followed by stimulation with 100 ng/ml LPS for 12h.
Cells were lysed and lysates were used to determine protein expression by western blot. (C) U937 cells (10x106 cells) were transfected with an IRF3 expression construct or empty vector (EV) for 2 days followed by lysis and western blot. (D) Monocytes (5x106 cells) were treated with 1 μg/ml TLR4 agonist MPLA for 12 h followed by western blot to determine GLI3 expression. (E)
Monocytes (5x106 cells) were treated with 10 μg/ml CpG, 10 μg/ml Poly(I:C), 100 ng/ml
LPS or left untreated (Ctrl) for 12h followed by western blot to determine GLI3 expression.
Data shown are representative of three independent experiments.
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Figure 2-5: IRF3 regulates GLI3 by directly binding to IRF3 binding sites.
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Figure 2-5: IRF3 regulates GLI3 by directly binding to IRF3 binding sites. 9 (A) Schematic diagram of ~ 3000 bp upstream of ATG located in exon 2 of GLI3. Several candidate IRF3 binding sites (BS) were identified. (B) Untreated MM6 cells (10x106) were lysed and a chromatin immunoprecipitation (ChIP) assay was performed followed by qPCR using three primer sets for the areas containing IRF3 binding sites upstream of
ATG of GLI3.(C) MM6 cells (10x106) were treated with 10μg/ml Poly(I:C) for 1h prior to
ChIP and qPCR.(D) Primary human monocytes (D3) were treated with 10μg/ml Poly(I:C) for 1h prior to ChIP and qPCR.(E) Primary human monocytes (D3) were challenged with
10μg/ml Poly(I:C) for 12h prior to western blot analysis.
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Figure 2-6: Model for TLR-TRIF-induced GLI3.
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Figure 2-6: Model for TLR-TRIF-induced GLI3. 10 (A) WT and IRF3-/- MEFs (1.5x105) were treated with 10 μg/mL Poly(I:C) for the indicated time followed by qPCR to determine GLI3 expression. (B) Model of TLR-TRIF-IRF3- dependent regulation of GLI3.
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CHAPTER 3
BIOLOGICAL ROLE OF TLR-INDUCED GLI3
Introduction
Chronic inflammation has been shown to be tightly connected to many diseases including autoimmune diseases and cancer [458][459]. Chronic inflammatory conditions can be activated through several mechanisms which are oftentimes driven by TLR signaling [460]. TLR signaling can be activated through either pathogenic infections or through DAMPs [461]. H. pylori, a gram-negative bacteria, was shown to induce its pathogenic inflammatory effects through TLR4 while S .pygoenes, a gram-positive bacteria, was reported to signal through TLR3 [462][463]. In addition to TLR signaling, other pathways were reported to be important for sustaining inflammation. NF-κB signaling was shown to regulate cytokine expression in immune cells and resulting cancer growth in adjacent tissue. STAT3 signaling is also involved in inflammation-related cancer growth as well [464]. Finally, HH signaling cascades have been reported to be tightly linked to inflammation [226] [227] [228] [229] [230] [231].
In addition to immune cells, stromal cells are another important cell type that secretes cytokines and regulate inflammation. HH signaling was reported to regulate cytokine secretion such as IL-6 and CD40L in bone marrow stromal cells [435][229][420].
One major component of the HH signaling cascade is the activation of GLI transcription factors GLI1, 2 and 3, and GLI2 was shown to regulate the CCL5-induced IL-6 levels
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[435]. GLI2 also regulates CD40L expression and secretion in bone marrow cells as well
[229]. Han et al (2017) reported that the CCR3-PI3K-AKT axis regulates GLI2 in bone marrow stromal cells and that GLI2 directly interacts with the CD40L cytokine promoter to regulate its expression [229]. In addition to IL-6 and CD40L, TGFβ is another cytokine that was linked to GLI2 as well as GLI1 [230][421].
In addition to GLI1 and GLI2, GLI3 is another important transcription factor that is involved in HH signaling. It is mostly known for its importance in genetic diseases, as well as brain and lung development [231]. However, there are also studies that strongly suggest a role of GLI3 in both the adaptive and innate immune system. For example, it was shown that the lack of Gli3 impaired T and B cell development in mice and in a microarray study, Gli3 was an inducible gene by LPS RAW264.7 mouse macrophages
[385][390][391]. In the context of HH signaling, GLI3 is known for its transcriptional repression of HH-related gene expression [465]. However, it was shown that GLI3 can activate HH related gene expression by being present as GLI3-FL. Indeed, GLI3 has been shown to positively regulate gene expression in several lines of cancer and driving cancerous phenotype [231].
In chapter 2 we showed GLI3 is a direct downstream target of TRIF-IRF3 signaling, which is initiated by signaling through TLR4 and TLR3. IRF3 is known for its involvement in cytokine expression and inflammation. We hypothesized that IRF3-mediated inflammation is regulated by GLI3. In this chapter, we are investigating the role of GLI3 in inflammation and its regulatory role in cytokine secretion and related diseases.
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Experimental Procedures
ELISA
Mouse IL-6 ELISA (R and D; DY406), mouse CCL2 ELISA (R and D; DY479) and mouse
TNFα ELISA (R and D; DY410) were used to quantify cytokine secretion in cultured peritoneal macrophages. Cells (1x106) were plated in 1 ml per well in a 24-well plate and were allowed to adhere for 1h. Cells were then gently washed with DPBS, and 1 ml of fresh medium
(IMDM+10%FBS+1%A/A) was added with either DPBS or 100 ng/ml LPS for 24h.
Supernatants were harvested and diluted 1:40 for use inELISA.
Mice
Gli3fl/fl mice and LysM-cre mice were obtained from Jackson Labs (Bar Harbor, ME). Mice were maintained and bred at the UNH animal resource office (ARO). Mice were crossed to generate mice with conditional deletion of Gli3 in myeloid cells (M-Gli3-/-). Progeny were genotyped at p6-p9 using toe clipping and were separated into M-GLI3-/- or WT mice. All experiments were approved by the UNH IACUC following guidelines of the National
Institutes of Health. Mice were used for experiments when they reached 8-14 weeks of age.
RNA seq
Peritoneal macrophages were harvested via peritoneal lavage 3 days following intraperitoneal injection of 500 ul incomplete Freund’s adjuvant (IFA; Sigma, St. Louis,
MO). Cells (1x106 per well in 1 ml of medium (IMDM+10% FBS+1% A/A) were allowed
98 to adhere for 1h. After 1h, cells were washed with DPBS and treated with 100ng/ml LPS or
DPBS control for 1h. Cells were then harvested and RNA was isolated using TRIsure reagent (Invitrogen,CA) . RNA was quantified and sent to the Hubbard Center for Genome
Studies (HCGS) at the University of New Hampshire for library preparation and
RNAsequencing.
Bioinformatic analysis of RNA-seq data
RNA-seq data analysis was performed using the New Tuxedo package
(http://ccb.jhu.edu/people/salzberg/docs/Pertea_et_al-2016-Nature_Protocols.pdf). Using a
generalized linear model in edgeR, we identified 495 genes with significant interaction effects
between genotype and LPS treatment. Ingenuity Pathway Analysis of the interaction genes
revealed “Inflammatory Response” and “Immune Cell Trafficking” pathways as most significantly
enriched in interaction genes. We are currently working with Dr. Katja Koeppen at Dartmouth for
further data analysis.
Cell culture and Reagents
The cell line Mono-Mac-6 (MM6) was purchased from DSMZ and cultured in RPMI 1640 supplemented with 10% FBS, 1% 2mM L-glutamine, non-essential amino acids, 1mM
Sodium Pyruvate, and 10μg/ml human insulin. THP-1 cells were purchased from ATCC and cultured in RPMI 1640 with 10 % FBS supplemented with 0.05 mM
βMercaptoethanol.
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Statistical analysis
Statistical analysis was performed using GraphPad Prism software (San Diego, CA). A two-tailed t test was used to determine statistical significance between two variables and a two-way ANOVA was used to compare more than 2 variables. Statistical significance is indicated on each figure as follows: * (p<0.05), ** (p<0.01), *** (p<0.001), and ****
(p<0.0001).
Plasmid constructs
RNAi targeting GLI3 (shGLI3) (TG312754) and scrambled RNAi (shScr) (in pGFP-V-RS vector) (TR30007) were purchased from Origene Technolgoies (MD). Cells (4x106) cells were transfected with 10 µg of shGLI3 or shScr by electroporation and incubated for 48h prior to RNA isolation and qPCR.
RNA isolation and qPCR
TRIsure Reagent (Bioline, London, UK) was used to isolate RNA following manufacturer’s recommendation. To perform reverse transcription reaction, the reverse transcriptase from the Moloney murine leukemia virus (MMLV) was used (Promega, Madison, WI). For qPCR, the ViiA 7 real-time PCR system (Life Technologies, Grand Island, NY) was used.
For the qPCR following primer pairs were utilized: GAPDH,
5’CTCGACTTCAACAGCGACA-3’ (forward) and 5’-GTAGCCAAATTCGTTGTCATACC-
3’
(reverse). GLI3, 5’-GGGACCAAATGGATGGAGCA-3’ (forward) and 5’-
TGGACTGTGTGCCATTTCCT-3’ (reverse). Amplification results were quantified relative to GAPDH expression. CCL2, 5’-GCCACCTTCATTCCCCAAGG-3’ (forward) and
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5’GCTTCTTTGGGACACTTGCTGC-3’ (reverse). IL6, 5’-TCCAAAGATGTAGCCGCCC-
3’ (forward) and 5’-CAGTGCCTCTTTGCTGCTTTC-3’ (reverse). TNFα,
5’CCAGGGACCTCTCTCTAATCA-3’ (forward) and 5’-TCAGCTTGAGGGTTTGCTAC-3’
(reverse).
DNA extraction method
For DNA extraction from mice toes for genotyping the Phire Animal Tissue Dircet PCR Kit
(F-140WH) was used. For genotyping-PCR the following primer pairs were utilized:
Cre, 5’- GCGGTCTGGCAGTAAAAACTATC-3’ (forward) and 5’-
GTGAAACAGCATTGCTGTCACTT-3’ (reverse). Recombined Gli3 (rGLI3), 5’-
CTGGATGAACCAAGCTTTCCATC-3’ (forward) and 5’- CAGTAGTAGCCTGGTTACAG-
3’ (reverse). Floxed Gli3 (Gli3fl), 5’- GATGAATGTGATCCAGGGC-3’ (forward) and 5’-
GTCATATTGTGCCCAGTAGTAGC-3’ (reverse).
Results
Knockdown of GLI3 reduces cytokines expression in vitro
We knocked down GLI3 in human cell lines MM6 and THP-1 by utilizing a short hairpin
RNA targeting GLI3 (shGLI3) or scrambled control (shScr). This knock down of GLI3 resulted in a significant reduction of the proinflammatory cytokines CCL2, IL-6 and TNFα
(Figure 3-1). These cytokines were investigated because they are known to be induced by TLR signaling.
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Gli3 is required for TLR-TRIF-induced cytokine secretion
To determine the requirement for GLI3 in TLR-TRIF-induced inflammation, we generated conditional Gli3 knockout mice which lack Gli3 expression in myeloid cells including (M-Gli3-/-) by utilizing the Cre-lox system. Gli3 KO was confirmed by genotyping using the presence of cre and the recombined Gli3 in the DNA from M-GLI3-/- or WT toe clip samples by end-point PCR analysis (Figure 3-2 A). Additionally, lack of Gli3 protein expression was confirmed by performing western blot analysis on peritoneal macrophages from both M-GLI3-/- and WT mice (Figure 3-2 B).
Using IFA-elicited peritoneal macrophages from M-Gli3-/- or WT, we treated cells with
100ng/ml LPS (E.coli; Serotype 0111;B4) or DPBS control for 24h and harvested supernatants for further analysis by ELISA. As expected, LPS challenge led to a strong significant increase of CCL2, IL-6 and TNFα (Figure 3-3). However, this increase in cytokine secretion was significantly decreased in macrophages from M-Gli3-/- mice compared with macrophages from WT mice (Figure 3-3). This strongly suggests that Gli3 is required for TLR-TRIF-induced cytokine secretion. Interestingly, only basal levels of
CCL2 were significantly reduced in the absence of Gli3 suggesting Gli3 regulates both basal and TLR-TRIF-induced levels of CCL2. In contrast, basal TNFα levels were unaffected by Gli3 loss and IL-6 levels were significantly elevated in the absence of Gli3 suggested Gli3 negatively regulates IL-6 levels and positively regulates TLR-TRIFinduced
IL-6 levels.
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RNA-seq analysis reveals Gli3 modulates inflammation
To further elucidate the role of Gli3 in inflammation, IFA-elicited macrophages from MGli3-
/- or WT mice were isolated and treated with 100 ng/ml LPS or DPBS control for 1h. RNA was isolated and used for RNA-seq experiments. Analysis of RNA-seq data revealed 495 differentially regulated genes in the absence of Gli3 upon LPS/TLR-TRIF stimulation.
Intriguingly, further analysis strongly suggested inflammatory genes being affected with
25 genes (Adipoq, Acacb, Nqo2, Tnfsf13, Lrrc8a, Tlr8, Rnf122, Cgas,
Hmgn5, Rapgef3, Cd226, IL12b, CCl25, Mertk, Tnfrsf18, Tnfsf13b, Slc6a4,
Cx3cl1,Ccl1,Fry, Sort1, Gstk1, Timd4, Ccne1, Trem2) being mostly influenced by the absence of Gli3 (Figure 3-4). Furthermore, ingenuity pathway analysis revealed that inflammatory response is among the top significant pathways affected by Gli3 (Figure 34) and Gli3 was suggested to regulate M2 macrophage polarization (Figure 3-5).
Discussion
Inflammation is an important response of our body towards fighting infection and wound healing. However, when chronic inflammation occurs, it can be harmful rather than beneficial to our body as it creates an environment that allows the establishment of diseases including autoimmune diseases and cancer. Therefore, a thorough understanding of the molecular pathways that regulate inflammation is needed. This has the potential to allow the development of novel treatments for inflammation-related diseases.
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In chapter 2, we identified GLI3 as a direct downstream target of TLR-TRIF-IRF3 signaling axis. IRF3 is a transcription factor known for its important function in regulating the expression and secretion of inflammatory cytokines . We hypothesized that GLI3 is required for TLR-TRIF-IRF3-dependent inflammatory responses. IRF3 can regulate the expression of the proinflammatory cytokines CCL2, IL-6 and TNFα [466][467][443]. IRF3 is also known to regulate the antiviral cytokines IFN and IFN [468]. To test if Gli3 is involved in the expression of proinflammatory cytokines, we knocked down GLI3 in our cell lines and investigated gene expression. Our results show a significant decrease of human CCL2, IL-6 and TNFα mRNA expression in cells with GLI3 knockdown. This suggested a role for GLI3 in regulating these inflammatory cytokines. To further examine the role of GLI3 in inflammation we generated conditional knockout mice lacking Gli3 expression in macrophages (M-GLI3-/-). Using macrophages from these mice or their WT littermates, we found that in the absence of Gli3, TLR-TRIF-induced cytokine secretion was significantly impaired (Figure 3-4). This data supports a role for Gli3 in regulation inflammation downstream of TLR-TRIF signaling.
In an effort to further characterize the role of Gli3 in regulating inflammation and identify the entire set of TLR-TRIF-induced genes that require Gli3, we performed
RNAseq on RNA isolated from macrophages from M-Gli3-/- or WT mice stimulated with
LPS to activate TLR-TRIF signaling. RNA-seq analysis revealed Gli3 to be involved in the regulation of 25 inflammatory genes and further ingenuity pathway analysis showed that
Gli3 is involved in mostly inflammation-related pathways. This further confirms supports a role for Gli3 in inflammation and the inflammatory response system of our body.
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A role GLI3 in M2 macrophage polarization was suggested by RNA-seq analysis, suggesting a role of GLI3 as anti-inflammatory component in addition to be an inflammatory component. This is mainly due to a reduction in the anti-inflammatory cytokine IL-10. However, a role for Gli3 in macrophage polarization will need to be validated experimentally before a conclusion is reached as to whether Gli3 promotes an inflammatory M1 or anti-inflammatory M2 response.
Several cytokines were identified downstream of Gli3 and suggested to modulate inflammation. cGas was shown not to be inducible by LPS in the absence of Gli3. cGas is involved in sensing cytosolic DNA which leads to activation of STING, which subsequently activates IRF3 and NF-κB and leads to cytokine and interferon production
[469]. cGAS-STING pathway has been reported to involved in Ataxia-Telangiectasia, in which it regulates autoinflammation and elevated type 1 interferon levels [470]. cGAS-
STING pathway has also been closely related to cancer. Cancer can be attacked by CD8+
T cells after activation by DCs and the cGAS-STING pathway was shown to regulate this activation [471]. Indeed, STING deficient mice failed to reject tumor growth of injected cancer cells and treatment with exogenous cGAMP (STING agonist) led to tumor regression [472][473]. Based on our RNA-seq analysis Gli3 potentially acts as an activator of the cGAS-STING pathway by regulating cGAS expression levels. Based on our results from CHAPTER 2 this would suggest a positive feedback loop between STING activating
IRF3 and IRF3 regulating Gli3 expression levels. Based on our results from CHAPTER 3 this might be a pathway in which Gli3 regulates CCL2, IL-6 and TNFα expression and secretion.
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Interleukin 12 is a cytokine which is secreted by macrophages and dendritic cells and leads to proliferation of Th1 cells [474][475]. Therefore, IL-12 plays an essential role in general immune responses. IL-12 consists of two subunits which are expressed by the gene IL-12a and IL-12b [474]. Our RNA-seq data suggest that Gli3 positively regulates
IL-12b expression which makes Gli3 important for IL-12 related functions. Gli3’s role in T cell development was previously described [231]. Our RNA-seq data strongly suggests an additional indirect role for GLI3 in T cell functions and subsequent immune response.
CCL25 is a ligand that selectively binds to CCR9, a receptor expressed on intestinal CD4+ T cells [476]. Vercirnon, a small molecule drug developed by
ChemoCentryx, is a CCR9 antagonist and currently in phase 3 clinical trials for intestinal inflammatory diseases such as Crohn’s disease (CD) or celiac disease [476]. Since our
RNA-seq data suggests a regulation of CCL25 by Gli3, Gli3 might be involved in regulating inflammatory intestinal diseases, which makes Gli3 a potential target for CD or other related diseases.
Additionally, ingenuity pathway analysis suggested a role of Gli3 in M2 macrophage development. M2 macrophages are anti-inflammatory immune cells, that are present in proximity of many advanced tumors and are therefore termed TAMs. Gli3 directly regulates tumor growth and apoptosis in several lines of cancer [231]. Based on our data Gli3 might even be involved indirectly in tumor progression, by regulating macrophage polarization into the M2 phenotype or TAMs. This makes Gli3 an attractive target for cancer therapy, since several publications already showed that tumor cells are not able to grow without the presence of TAMs [477][478][479].
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In summary, Gli3 might be regulating inflammation-related diseases such as autoimmune diseases or cancer which makes Gli3 a potential future target for autoimmune or cancer therapy.
Figure 3-1: GLI3 KD reduces cytokine expression in monocytes
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Figure 3-1: GLI3 reduces cytokine expression in monocytes 11 MM6 and THP-1 cells (4*106) were transfected with 10µg shRNA targeting GLI3 (shGLI3) or scrambled control (shScr) and incubated for 48h. After 48h, RNA extraction, cDNA synthesis and qPCR were performed as earlier described. Three sets of experiments were performed, each representing one biological replicate.
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Figure 3-2: Breeding schematic to generate mice which lack Gli3 in macrophages and confirmation of lack of Gli3 by PCR and Western Blot.
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Figure 3-2: Breeding schematic to generate mice which lack Gli3 in macrophages and confirmation of lack of Gli3 by PCR and Western Blot. 112 (A) Mice were toe clipped after 6-8 days of birth and DNA was isolated using earlier described DNA extraction method and PCR was performed. (B) IFA elicited macrophages were cultured as earlier described and used for Western Blot analysis.
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Figure 3-3: RNA-seq and pathway analysis reveal Gli3 being a potential major regulator of inflammation.
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Figure 3-3: RNA-seq and pathway analysis reveal Gli3 being a potential major regulator of inflammation. 13 IFA elicited macrophages (1x106 cells/ml) were treated with DPBS or 100ng/ml LPS
(E.coli, Serotype 0111;B4) for 1h and then harvested for RNA isolation. RNA-seq analysis shows that there are 495 differentially regulated genes in the absence of Gli3, which mostly are involved in inflammation.
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Figure 3-4: Gli3 deficient macrophages (M-GLI3-/-) secrete significantly less proinflammatory cytokines than macrophages that have functional Gli3 (WT).
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Figure 3-4: GLI3 deficient macrophages secrete significantly less proinflammatory cytokines than macrophages that have functional GLI3. 14 IFA elicited macrophages (1x106 cells/ml) were treated with DPBS or 100ng/ml LPS (E.coli,
Serotype 0111;B4) for 24h and then supernatant was removed for further ELISA analysis.
Each dot represents a biological replicate.
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Figure 3-5: RNA-seq analysis strongly suggests Gli3’s involvement in macrophage polarization.
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Figure 3-5: RNA-seq analysis strongly suggests Gli3’s involvement in macrophage polarization. 15 Further bioinformatical analysis of RNA-seq datasets strongly suggest an involvement of
Gli3 in M2 macrophage polarization.
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SUMMARY/CONCLUSION
Inflammation is a double-edged sword that is of importance for maintaining our health; however it can also have a negative effect on our body. In addition to exogenous antigen such as PAMPS (e.g. bacteria or viruses), we also discussed the impact of endogenous factors, defined as DAMPs (e.g. intracellular proteins or DNA) on inflammation and on human health. These two factors can trigger proinflammatory molecular pathways which can promote inflammation-related diseases such as autoimmune diseases (e.g. atopic dermatitis, rheumatoid arthritis and type 1 diabetes) or cancer, when constantly turned on or chronic.
In addition to the role of STAT and NFκ-B signaling in chronic inflammation, the
HH signaling pathway plays a role in inflammation and cancer. The importance of HH signaling in regulating cancer growth directly or indirectly through inflammation is frequently referenced in scientific literature. For example, mutations in the negative regulator of HH signaling, PTCH1, have been shown to correlate with the occurrence of basal cell carcinoma (BCC) [1]. Additionally, GLI1 and GLI2, known positive regulators of canonical HH signaling, were linked to supporting tumorigenesis. GLI1 was reported to drive glioma growth and prostate cancer, while GLI2 was shown to support pancreatic cancer growth as well in addition to breast cancer, colorectal and prostate cancer
[2][3][4][5] [6] [7]. In addition to direct involvement of HH signaling in cancer progression, its involvement in the tumor microenvironment and inflammation has been established.
Zheng et al showed that GLI1 regulates TGF-β expression, a known cytokine involved in
117 epithelial to mesenchymal transition (EMT) and the driver of metastasis [8]. Additionally,
GLI2 was shown to regulate IL-6 and CD40L secretion in bone marrow stromal cells, an importance cell type in the bone marrow microenvironment and therefore important in cancers such as WM, Multiple Myeloma and Chronic Lymphocytic Leukemia [9][10]. GLI3 was reported to be frequently directly involved in cancer progression as well as in the regulation of innate and adaptive immune cells. These immune cells are another important cell in the tumor microenvironment and play a role in inflammation. For example, methylated GLI3 was reported to drive NSCLC cancer while in colon carcinoma,
GLI3 seems to induce tumor growth since knockdown of GLI3 reduced cancer cell proliferation [11] [12]. Additionally, targeting GLI3 with microRNA reduced cancerous phenotype [13] [14] [14] [15]. In the immune system GLI3 was shown to regulate B- and
T-cell development and was induced in response to LPS treatment in macrophages [16]
[17] [18].
In addition to the published data that shows the direct involvement of GLI3 in cancer, our data suggest a role of GLI3 in inflammation, one of the hallmarks of cancer.
We have shown that GLI3 is induced by LPS in monocytes and that this induction is highly dependent on TLR4-TRIF axis. Downstream of TRIF, three transcription factors can be activated, which are AP-1, NFκB and IRF3 and we have shown that IRF3 regulates GLI3 downstream of TRIF by directly binding to the GLI3 promoter region. IRF3 is known to regulate proinflammatory cytokines; proteins that mediate the inflammatory response. We hypothesized that GLI3 is regulating cytokine expression and therefore inflammation. To validate our hypothesis, we generated M-Gli3-/- mice, which lack Gli3 expression in macrophages. We challenged peritoneal macrophages from these mice with LPS and 118 performed RNA-seq. Our data strongly suggested a role of Gli3 in inflammation since pathway analysis revealed inflammatory pathway as pathway most significantly affected by Gli3 knockout in macrophages. The role of Gli3 in inflammation was further confirmed by ex vivo experiments in which peritoneal macrophages from mice were challenged with
LPS and cytokine levels were quantified by ELISA. In these experiments, CCL2, IL-6 and
TNFα secretion by macrophages was significantly reduced in the absence of Gli3, which warrants further investigation of the role of Gli3 in other aspects of inflammation. In addition to its inflammatory role, Gli3 may play a role in anti-inflammatory immune response, since our RNA-seq data suggested a role of Gli3 in M2 macrophage polarization. Additionally, M2 macrophages are known to drive tumor growth as TAMs, which suggest Gli3 may be an indirect driver of advanced tumor growth by regulating
TAMs.
Chronic inflammation is a known driver of cancer. Most immune cells play a double-edged sword in cancer growth, besides macrophages which were found to only induce tumor growth [19]. While most literature suggest a role of M2 macrophages in supporting cancer growth, it was also mentioned that M1 macrophages, which are proinflammatory immune cells, were found to induce cancer [20]. There are several lines of evidence suggesting GLI3 plays a direct role in promoting tumor growth [21].
Additionally, our data suggest GLI3 plays a role in inflammation as well as in M2 macrophage polarization. This makes Gli3 an attractive target for immunotherapy of M1 or M2 macrophage subset and therefore an attractive target for inflammation-induced diseases such as cancer.
119
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APPENDIX
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University of New Hampshire
Research Integrity Services, Service Building 51 College Road, Durham, NH 03824-3585 Fax: 603-862-3564 08-Jul-2020
Elsawa, Sherine F
Molecular, Cellular, & Biomedical Sciences
Rudman Hall Rm 291
Durham, NH 03824-2618
IACUC #: 200507 Project: Understanding the Role of Gli2 and Gli3 in Hematopoiesis
Approval Date: 08-Jul-2020
The Institutional Animal Care and Use Committee (IACUC) reviewed and approved the
protocol submitted for this study under Category E in Section V of the Application for
Review of Vertebrate Animal Use in Research or Instruction - Animal use activities that
involve accompanying pain or distress to the animals for which appropriate anesthetic, analgesic,
tranquilizing drugs or other methods for relieving pain or distress are not used.
Approval is granted for a period of three years from the approval date above. Continued
approval throughout the three year period is contingent upon completion of annual reports
on the use of animals. At the end of the three year approval period you may submit a
new application and request for extension to continue this project. Requests for extension
must be filed prior to the expiration of the original approval. 161
Please Note: 1. All cage, pen, or other animal identification records must include your IACUC # listed above. 2. Use of animals in research and instruction is approved contingent upon participation in the UNH Occupational Health Program for persons handling animals. Participation is mandatory for all principal investigators and their affiliated personnel, employees of the University and students alike. Information about the program, including forms, is available at http://unh.edu/research/occupationalhealth-program-animal-handlers.
If you have any questions, please contact either Dean Elder at 862-4629 or Susan Jalbert
at 862-3536.
For the IACUC,
Julie Simpson, Ph.D. Director
cc: File
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