University of Cincinnati
Date: August 23, 2012
The Role of Sox17 in Normal and Pathological Beta Cell
A dissertation submitted to the Division of Graduate Studies and Research of the
University of Cincinnati
in partial fulfillment of the requirements for the degree of
Doctor of Philosophy
in the Graduate Program in Molecular and Developmental Biology
of the College of Medicine
2012
by
Diva Jonatan
Bachelor of Science, Xavier University, 2006
Committee Chair: James Wells, PhD
Jeffrey Whitsett, MD, PhD
Aaron Zorn, PhD
Jonathan Katz, PhD
Gail Deutsch, MD ABSTRACT
Glucose homeostasis is a complex process involving many regulatory molecules and disruption of this process can result in diabetes. Sox17 is a transcription factor and a key regulator in various developmental and disease contexts. During endoderm development, Sox17 acts, in part, by transcriptionally regulating other important endodermal transcription factors including HNF and
Foxa2, which are also known regulators of postnatal cell function. Our data shows that Sox17 is expressed in all islet cells. Thus, we hypothesize that Sox17 is a key modulator of cell homeostasis.
In the course of this study, we discovered a novel role for Sox17 in regulating insulin trafficking and secretion in normal and pathological cells.
Loss of Sox17 throughout pancreas development in a wildtype background resulted in mice with prediabetes. These mice had higher proinsulin protein content in the ER of the islet cells and dilated secretory organelles in cells. In line with the prediabetes phenotype, these mice went on to develop additional symptoms of diabetes when placed on a high fat diet, including elevated fasting glucose levels and an inability to respond to a glucose challenge. This suggested that Sox17 affects either insulin processing and/or transit through the secretory system. To more directly investigate these possibilities, we used a tetracycline regulated transgenic system to overexpress Sox17 in mature cells in wildtype background. Transgenic Sox17 expression resulted in rapid trafficking and secretion of improperly processed proinsulin. At the transcriptional level, Sox17 altered expression of genes involved in biological processes that regulate
iii hormone transport, secretion, and cellular localization, which led to diabetes after prolonged exposure. This demonstrates that Sox17 affects insulin trafficking throughout the secretory pathway. We therefore wanted to explore the possibility that physiologic levels of Sox17 might be used to positively impact diabetic phenotypes using a genetic model of diabetes (MODY4). We did so by overexpressing Sox17 two-fold in a MODY4 mouse model (Pdx1-tTA hemizygote mice). Increased expression of Sox17 in the -cells of MODY4 animals was sufficient to transiently normalize basal blood glucose and insulin levels as well as restore islet cell organization architecture; however, Sox17 overexpression was not able to rescue the inability of MODY4 animals to properly respond to glucose challenge.
Together, these data demonstrate new and critical role for Sox17 in regulating insulin trafficking and secretion processes in the adult pancreas that are important to ensure proper glucose homeostasis. This study also suggests that modulation of Sox17-regulated pathways can be used therapeutically to improve cell function in the context of diabetes.
iv v Table of Contents
ABSTRACT iii
TABLE OF CONTENTS 1
LIST OF FIGURES AND TABLES 4
CHAPTER 1. Introduction
Glucose homeostasis, prediabetes and the different forms of diabetes 7
MODY and the Islet Transcriptional Factor Network Involved 13
Insulin Biosynthesis, Processing, and Secretion 14
Overview: Functional Roles of Sox Family Proteins 18
Overview of Sox Family of Transcription Factors 18
Sox Proteins in Endocrine Pancreas Development and Function 19
The Roles of Sox17 in Different Biological Contexts 22
References 26
Figure Legends 34
Figures 35
CHAPTER 2. The Role of Sox17 in Insulin Processing and Cell Secretory Pathway
Abstract 39
Introduction 40
Materials and methods 43
Results 47
1 Discussion 59
Acknowledgements 66
Sources of Funding 66
References 67
Figure Legends 73
Figures 78
Supplementary Figure Legends 86
Supplementary Figures 89
CHAPTER 3. The Partial Rescue of MODY4 Phenotypes by Sox17
Summary 115
Introduction 116
Materials and Methods 121
Results 123
Discussion 128
Acknowledgements 130
Sources of Funding 131
References 131
Figure Legends 134
Figures 137
CHAPTER 4. Summary and Discussion
Major Findings 144
2 Sox17 in normal cells 144
Sox17 in pathological cells 146
Experimental limitations and alternative approaches 147
Acknowledgements 149
Sources of funding 150
References 150
Figure Legends 152
Figures 152
3 LIST OF FIGURES AND TABLES
CHAPTER 1.
Figure 1. MODY genes islet transcriptional network 35
Figure 2. Insulin biosynthesis in pancreatic cells 36
Figure 3. Glucose sensing and glucose-stimulated insulin release pathway 37
CHAPTER 2.
Figure 1. Sox17 is not required for cell development 78
Figure 2. Sox17-paLOF results in elevated proinsulin protein in the islets 79
Figure 3. Loss of Sox17 in the pancreas causes accumulation of proinsulin in the ER and structural changes in secretory organelles 80
Figure 4. Sox17-paLOF mice are prediabetic and prone to high fat diet-induced stress of