Regulation of Translation and the Unfolded Protein Response in Pituitary Gonadotropes
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UC San Diego UC San Diego Electronic Theses and Dissertations Title Stress, mixed messages, and hormone signaling : regulation of translation and the unfolded protein response in pituitary gonadotropes Permalink https://escholarship.org/uc/item/00q985gk Author Do, Minh-Ha T. Publication Date 2008 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California UNIVERSITY OF CALIFORNIA, SAN DIEGO Stress, Mixed Messages, and Hormone Signaling: Regulation of Translation and the Unfolded Protein Response in Pituitary Gonadotropes A dissertation submitted in partial satisfaction of the Requirements for the degree Doctor of Philosophy in Biomedical Sciences by Minh-Ha T. Do Committee in charge: Professor Mark Lawson, Chair Professor Don Cleveland Professor Pamela Mellon Professor Daniel O’Connor Professor Nick Webster 2008 Copyright Minh-Ha T. Do, 2008 All rights reserved. The Dissertation of Minh-Ha T. Do is approved, and it is acceptable in quality and form for publication on microfilm: _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ _____________________________________________________________________ Chair University of California, San Diego 2008 iii TABLE OF CONTENTS Signature Page …………………………………………………………………………...................... iii Table of Contents …………………………………………………………………………………........ iv List of Figures …………….……………………………………………………………....................... v List of Tables ................……………………………………………………………………………...... vii Acknowledgements …………….................................................................................................. viii Vita ……...................................................................................................................................... ix Abstract …................................................................................................................................... xi Chapter 1 Introduction …...…………………………………………………………………..…. 1 Chapter 2 Activation of the unfolded protein response by GnRH …………………………. 8 Chapter 3 Specificity of the regulation of translation by GnRH ......................................... 41 Chapter 4 Conclusions .................................…………………….………………………..…. 82 Chapter 5 Appendix …………………………………………………………………………….. 95 References ……………………………………………………………………………………………... 166 iv LIST OF FIGURES Figure 2-1. Expression of ER stress sensors and activation of PERK by GnRH in LβT2 gonadotropes …………………………..…………………………….……………… 29 Figure 2-2. GnRH causes splicing of the UPR transcription factor Xbp1 mRNA in LβT2 and mouse primary gonadotropes …………………………….………………….. 30 Figure 2-3. GnRH exposure causes an accumulation of RNP complexes in LβT2 cells … 31 Figure 2-4. Accumulation of RNP complexes is mimicked by disruption of intracellular calcium ……………………………………………………………………………….. 32 Figure 2-5. Lhb, Cga and Gapdh mRNAs redistribute to RNP complexes in response to GnRH …………………………………………………………………………………. 33 Figure 2-6. Accumulation of RNP complexes by GnRH does not involve transcriptional events ………………………………………………………………………………… 34 Figure 2-7. GnRH attenuates translation by redistributing ribosomes to RNP complexes .. 35 Figure 2-8. Translation of Lhb, Cga and Gapdh mRNAs is attenuated …………………….. 36 Figure 2-9. Attenuation of Lhb, Cga and Gapdh translation by GnRH is transient ………… 37 Figure 2-10. A 10-minute pulse of GnRH is sufficient to cause accumulation of RNP complexes and phosphorylation of eIF2α ………………………………………… 38 Figure 2-11. A 10-minute pulse of GnRH is sufficient to cause and sustain phosphorylation of PERK ...……………………………………………………………………………. 39 Figure 2-12. Proposed model for transient attenuation of translation induced by GnRH ….. 40 Figure 3-1. Gnrhr, Egr1 and Cmyc mRNAs differentially redistribute in ribosome complexes in response to GnRH ……………………………………………………………….. 65 Figure 3-2. Gnrhr, Egr1 and Cmyc are differentially sensitive to translational and transcriptional regulation by GnRH ……………………………………………….. 66 Figure 3-3. mRNA redistribution in ribosomal complexes is reproducible and not global … 67 Figure 3-4. GnRH causes bidirectional redistribution of a specific subset of mRNAs …….. 68 Figure 3-5. The top regulated genes show distinct patterns of mRNA redistribution ……… 69 Figure 3-6. Summary of KEGG pathways identified by the top 5% of bioweight genes ….. 70 Figure 3-7. Inhibition of ERK activation by GnRH exacerbates RNP accumulation ………. 71 Figure 3-8. ERK activation by GnRH contributes to association of Dusp1 with polysomes …………………………………………………………………………………………. 72 v Figure 3-9. Role of ERK activation on Cmyc and Egr1 recruitment to polysomes is not clear …………………………………………………………………………………... 73 Figure 3-10. PKC activation promotes polysomal recruitment but not RNP accumulation of an mRNA ………………………………………………………………………….. 74 Figure 3-11. Activation of the UPR does not induce polysomal recruitment of Dusp1 mRNA by GnRH …………………………………………………………………….. 75 Figure 3-12. The 5’UTR of mouse Lhb is not sufficient to confer sensitivity to translational regulation by GnRH ………………………………………………………………… 76 Figure 3-13. The signal peptide of LHβ is not sufficient to confer sensitivity of Lhb mRNA to translational regulation by GnRH ………………………………………………. 77 Figure 3-14. GnRH does not stimulate Lhb mRNA recruitment to polysomes ……………… 78 Figure 3-15. Contribution of Lhb and Cga mRNA transcription on intracellular LH protein levels …………………………………………………………………………………. 79 Figure 4-1. Summary of specificity of acute translational regulation by GnRH ………….... 94 Figure 5-1. ATF6 Western blotting attempts ………………………………………………….. 96 Figure 5-2. Gnrhr and Gapdh mRNA levels do not change in response to GnRH treatment …………………………………………………………………………….. 97 Figure 5-3. Effect of various starvation conditions on ribosomal remodeling ……………… 98 Figure 5-4. Effect of various starvation conditions on GnRH-induced ribosomal redistribution of specific mRNAs ………………………………………………….. 99 Figure 5-5. An acute increase in protein synthesis is not responsible for activation of ER stress by GnRH …………………………………………………………………….. 100 Figure 5-6. One hour of tonic 10 nM GnRH treatment does not cause apoptosis-induced DNA fragmentation ………………………………………………………………… 101 vi LIST OF TABLES Table 3-1. Top 100 bioweight (BW) genes ………………………………………………….. 80 Table 5-1. Top 800 bioweight (BW) genes ………………………………………………….. 102 Table 5-2. Genes identified by SAM …………………………………………………………. 123 Table 5-3. Genes in common between bioweight (BW) and SAM ……………………….. 144 Table 5-4. KEGG pathways identified by bioweight ………………………………………... 156 vii ACKNOWLEDGEMENTS I would like to thank: My advisor, Professor Mark A. Lawson, for his never-ending encouragement, for ensuring I had and took opportunities to advance my training, and for providing a lab environment that was easy to come in to everyday for the past 5 years. Professor Pamela Mellon, for providing a fresh mind in critiquing my work and keeping on top of my progress. I would also like to thank the members of the Mellon lab for bearing with me during lab meetings and always being available for advice. Thank you in particular to Dr. Djurdjica Coss, for answering all my questions, large and small. The rest of the members of my dissertation committee, Professor Nick Webster, Professor Don Cleveland, and Professor Daniel O’Connor, for asking me the difficult questions and thus providing me with the ideas and guidance I needed to complete this body of work. Dr. Cleveland, thank you for trying to ensure that I was sufficiently toughened up. The Biomedical Sciences program has provided an invaluable opportunity to learn and train through the expertise of the faculty, support of the staff, and a body of classmates and friends who have supported me in so many ways. I would especially like to thank Theresa Operaña for being so generous with her time, support, thoughts and care. My colleagues, Sharon Santos, Dr. Amy Navratil, Dr. Kathryn Nguyen, and Janine Low. I am so grateful to have been able to pick their minds, laugh with them, and have their voices of reason to hear. My friends back home, I cannot express enough how all the potlucks, adventures, and emails have supported me. The drive to LA is worth it every time. And finally: Thank you to Jonathan Tang, who patiently waited for me all these years, drove down to San Diego all this time, gave up so many weekends, and who listened, understood, and supported me in every way he could and knew how. At last! To my sisters, who have turned into women I am so proud of and have motivated me by setting such a high standard of achievement. And last, but furthest from least, thank you to my parents. Only recently have I begun to truly realize and appreciate what all their hard work and sacrifice has provided me and our family. I hope each day fulfills another part of the dream they had in mind and heart when choosing to come to this country. Chapter 2, in part, has been submitted for publication of the material as it may appear in Molecular Endocrinology, 2008, Minh-Ha T. Do, Sharon J. Santos and Mark A. Lawson. The dissertation author was the primary investigator and author of this paper. Chapter 4, in part, has been submitted for publication of the material as it may appear in Molecular Endocrinology, 2008, Minh-Ha T. Do, Sharon J. Santos and Mark A. Lawson. The dissertation