i INVESTIGATING THE ROLE OF CRABP1 IN ADIPOSE BIOLOGY by JOSHUA E. MILLER Submitted in partial fulfillment of the requirements For the degree of Master of Science Thesis Adviser: Dr. Noa Noy Department of Pharmacology CASE WESTERN RESERVE UNIVERSITY May, 2017 ii CASE WESTERN RESERVE UNIVERSITY SCHOOL OF GRADUATE STUDIES We hereby approve the thesis of Joshua E. Miller candidate for the degree of Master of Science*. Committee Chair: Ruth Keri, Ph.D. Committee Member: Hua Lou, Ph.D. Committee Member: Noa Noy, Ph.D. Committee Member: Monica Montano, Ph.D. Committee Member: David Danielpour Date of Defense: January 13th, 2017 *We also certify that written approval has been obtained for any proprietary material contained therein. iii Table of Contents TABLE OF CONTENTS___________________________________________________________iii LIST OF FIGURES____________________________________________________________iv-v ACKNOWLEDGEMENTS_____________________________________________vi-vii ABSTRACT____________________________________________________________1 CHAPTER 1: Background and Statement of Purpose______________________________________________________________2-8 CHAPTER 2: Materials and Methods_____________________________________________________________9-14 CHAPTER 3: Examination of the role of CRABP1 in Adipocytes__________________________________________________________15-30 CHAPTER 4: Conclusions, Discussion and Future Directions___________________________________________________________31-37 Bibliography________________________________________________________38-41 iv LIST OF FIGURES Figure 1.1 Retinoic acid functions through two distinct nuclear hormone receptors_______________________________________________________________3 Figure 3.1. CRABP transcript expression declines upon adipocyte differentiation_________________________________________________________19 Figure 3.2. CRABP1 protein expression is diminished upon adipocyte differentiation_________________________________________________________20 Figure 3.3. CRABP1 transcript expression in WAT greatly exceeds that of D0 3T3- L1 cells__________________________________________________________________21 Figure 3.4. Severe ablation of CRABP1 transcript upon high fat feeding and vitamin A deficient diet__________________________________________________________________22 Figure 3.5. Dietary changes alter the mouse white adipose tissue______________________________________________________________23-24 Figure 3.6. Generation of stable 3T3- L1s__________________________________________________________________25 Figure 3.7. Overexpression of CRABP1 does not significantly alter RAR target genes in WAT_________________________________________________________________26 v Figure 3.8. CRABP1 overexpression does not significantly reduce FABP4 transcript_____________________________________________________________27 Figure 3.9. Retinoic Acid inhibits adipocyte differentiation while CRABP1 overexpression induces less lipid droplet formation_____________________________________________________________28 Figure 3.10. CRABP1 overexpression reduces CEBPα protein expression_____________________________________________________________29 Figure 3.11. CRABP1 overexpression does not affect phosphorylation of HSL at Ser 563___________________________________________________________________30 vi ACKNOWLEDGEMENTS I would like to thank my graduate school mentor, Dr. Noa Noy. Noa contributed greatly to my training as a scientist, both in that she challenged me to convey my ideas concisely and confidently and she trained me to really reflect on the meaning of the data before jumping to conclusions. Her passion and enthusiasm for research drove me to persevere in the face of innumerable failures, and I am a stronger person for it. She provided me with opportunities to interact with leaders in the field and to develop a diverse depth of knowledge. She will be deeply missed. I would like to express my gratitude to the members of the Noy lab, both past and present, who have guided my development as a scientist. I thank you for your patience in teaching me lab techniques, providing me with useful tips and protocols and overseeing the honing of my lab skills. Thank you for your advice when planning experiments and for being generous with lending samples and reagents when I needed them. Finally, thank you for being more than a colleague when I needed a friend. You became like second family to me. To my thesis committee, Drs. Keri, Montano, Lou and Danielpour, I am so grateful for your participation in my development as a scientist. Thank you for your help and guidance throughout my project and your patience when I was following a hypothesis that you may not have believed in. You challenged me to think critically and for that I thank you. I would like to thank my friends and fellow pharmacology graduate students for all of their help and support in the good times and bad. You created a community and I am so grateful that I could be a part of it. To Leslie and Sahil, you have become two of vii my closest friends, and I want you to know that I could not have survived graduate school without you. Finally I would like to thank my family, who have loved and supported me throughout my time in this program. 1 Investigating The Role of CRABP1 In Adipose Biology Abstract by JOSHUA E. MILLER Dysfunctional regulation of adipose tissue is a key risk factor for a number of diseases such as type 2 diabetes(1). Consequently, understanding the role of specific proteins involved in adipose biology is critical to treatment of these serious illnesses. Administration of the vitamin A metabolite, retinoic acid has been shown to improve insulin responses and protect against diet-induced obesity in several mouse studies(2,3). One of its carrier proteins, CRABP2 has been shown to participate in adipose tissue biology by enhancing retinoic acid-induced transcriptional modulation of target genes(4,5). Due to its role in adipose tissue biology, we hypothesized that CRABP1 may also be involved. Our studies show that CRABP1 is down regulated upon induction of adipocyte differentiation. However, high fat diet feeding depletes CRABP1 from mouse white adipose tissue, suggesting that there may be an interesting link between CRABP1 and fat accumulation in the adipose tissue. 2 Chapter 1: Background and Statement of Purpose 1.1 Background Vitamin A Vitamin A (all-trans retinol) is a key nutrient involved in numerous biological processes, ranging from development and vision to maintenance of the immune system(6). Many of its functions are carried out by a key metabolite, known as all- trans retinoic acid (RA), which is synthesized from all-trans retinol by a series of dehydrogenases(7). Retinoic acid is then able to travel to the nucleus, where it binds directly to specific nuclear hormone receptors, including PPARβ/δ and the three isotypes of RAR, known as RARα, RARβ and RARγ(6,8). Binding of RA to these nuclear hormone receptors alters the transcription of specific target genes. Although retinoic acid is largely hydrophobic and is able to traverse the nuclear membrane by itself, its delivery to specific nuclear hormone receptors is facilitated by a group of proteins from the intracellular lipid binding protein family(9). It has been well established that the effect of retinoic acid on a given cell is dependent upon the expression of specific lipid binding proteins and nuclear hormone receptors(10-13). For example, fatty acid binding protein 5 (FABP5) shuttles retinoic acid to peroxisome proliferator activated receptor β/δ, PPARβ/δ, which is a nuclear hormone receptor that regulates genes involved in promoting cell proliferation(8,14- 16). However, when cellular retinoic acid binding protein 2 (CRABP2) is the predominant intracellular lipid binding protein in the cell, it will deliver retinoic acid 3 to the retinoic acid receptor (RAR), which regulates a different set of genes that induce differentiation, growth arrest and cellular apoptosis(17). Figure 1.1 Retinoic acid functions through two distinct nuclear hormone receptors All-trans retinoic acid is capable of binding to either CRABP2 or FABP5 in the cytosol, after which it is shuttled to either RAR or PPARβ/δ respectively. In cells which highly express CRABP2, delivery of retinoic acid to RAR will induce transcription of genes involved in apoptosis and cell cycle arrest. When FABP5 is highly expressed, retinoic acid will be delivered to PPARβ/δ, which will modulate 4 transcription of genes that induce pro-survival pathways. Figure adapted from Vreeland, A. (2015). Cellular Retinoic Acid-Binding Protein 2 Cooperates with HuR to Stabilize RNA and Inhibit Tumor Growth . (Electronic Thesis or Dissertation). Retrieved from https://etd.ohiolink.edu, with contributions from Dr. Liraz Levi and Dr. Noa Noy. CRABPs Among the lipid binding proteins, two homologs, the cellular retinoic acid binding proteins 1 and 2 (CRABPs) share ~75% homology and bind to all-trans retinoic acid with sub-nanomolar affinity(12,18). CRABP2 has been shown to have two distinct functions. In the presence of retinoic acid, CRABP2 enhances delivery of retinoic acid to RAR to modulate transcription of target genes. However, in the absence of retinoic acid, CRABP2 has been shown to directly cooperate with an RNA binding protein known as HuR(19,20). When CRABP2 binds to HuR it enhances the affinity of HuR for target mRNAs that bear an AU-rich region in their 3’UTR(21-24). This interaction with HuR allows CRABP2 to enhance the stability