Pancreatic islets of Langerhans contain insulin-secreting beta cells required for maintaining glucose homeostasis. Dysfunction in beta cell activity or survival results in diabetes mellitus, a disease currently affecting millions of Americans with numbers expected to greatly increase. This is creating an enormous economic and health burden. A future strategy for improving diabetic outcomes will include cell-based therapies wherein functional beta cells are generated to replenish those lost during diabetes progression. Success will require better understanding the complex transcriptional programs that specify and differentiate functional beta cells from progenitors. We previously showed that the LIM domain transcription factor, Islet-1, is required for islet endocrine cell development and function. Furthermore, the activity of Islet-1 during late pancreas development requires the interacting coregulator, Ldb1. My published study defined the pancreatic Ldb1 expression pattern and roles in developing endocrine cells. Whereas mice lacking Ldb1 in the developing islets exhibited a phenotype largely overlapping with Islet-1, it appears that Ldb1 also acts independently in early progenitor cells. Furthermore, interacting factors that comprise the beta cell Ldb1-Islet-1 complex remain unknown, although preliminary data suggests that the complexes are very large. In other tissues, Ldb1 binds the SSBP class of coregulators (or Single Stranded DNA-Binding Proteins, including SSBP2-4). These factors impact the transactivation capacity and stability of Ldb1 complexes. However, nothing is known of SSBP expression, interaction or function in islet beta cells. Data generated from my K01 studies utilized immunoprecipitation and mass spectrometry to isolate and identify beta cell line Ldb1 and Islet-1 binding proteins. The SSBP3 proteins appeared on the candidate list, suggesting that they participate in pancreas Ldb1 complexes. My R03 Aims will define roles for Ldb1 complexes during early pancreas progenitor cells as well as in adult beta cells.
Aim 1 will utilize a conditional Ldb1 mutant mouse (Pdx1-Cre;Ldb1F/-) to test for Ldb1 impacts during early pancreas progenitors. These cells are largely devoid of Islet-1, suggesting roles for another Ldb1- interacting LIM transcription factor. The early Ldb1 knockout mice are expected to (at least) phenocopy our published endocrine-specific Ldb1 model, plus have additional impacts on progenitor formation, proliferation, or cell-type allocation.
Aim 2 examines the function of Ldb1-interacting SSBP3 coregulators in beta cells. Approaches will include ChIP, siRNA knockdown, immunofluorescence, reporter gene assays and DNA- binding experiments to examine how SSBP factors impact Ldb1-Islet-1 complexes. With this R03 proposal, I will further test my central hypothesis that Ldb1 complexes are required for pancreatic organogenesis and beta cell function. My extensive experience with LIM factor complexes make me uniquely suited to executing these Aims, which have the promise of generating interesting preliminary data toward R01 funding.
To combat the ever-growing health and economic burdens associated with diabetes, islet biologists are developing cell-based strategies to replenish lost beta-cell mass and improve quality of life for patients. Success in this endeavor will require increased knowledge of the developmental processes and genetic programs that are fundamental for the production of functional pancreatic beta-cells from progenitors. The studies herein will explore the importance of transcriptional complexes mediated by the critical LIM-factor binding coregulator, Ldb1, during early pancreas progenitor formation as well as in adult beta-cell function.
