Date of Award

2009

Document type

Thesis

Degree Name

PhD (Doctor of Philosophy)

First Supervisor

Professor Jochen H.M. Prehn

Keywords

Hepatocyte Nuclear Factor 1-alpha

Abstract

In this project, we were interested in studying HNF1A-induced defects in the expression of genes involved in the differentiation and regeneration of beta-cells in a cellular model of HNFlA-maturity-onset-diabetes-of-the-young(HNF1A-MODY), as well as the signaling pathways leading to such effects. Inactivating mutations in the human gene encoding transcription factor-1 (tcf-l), also known as hepatocyte nuclear factor 1 (hnf) 1a, result in decreased beta-cell mass, defects in glucose-induced insulin secretion, and HNF1A-MODY, the most common monogenic form of diabetes.

We utilised rat INS-1 cells to conditionally overexpress wild-type HNF1A, the frameshift Pro291fsinsC-HNF1A mutant (a common human HNF1A-MODY mutation), and DN-HNFlA all of which were under the control of a reverse tetracycline-dependent transactivator system. Additionally, we utilised paraffin-embedded pancreatic sections on slides from the Rat Insulin Promoter (RIP)-DN-HNF1A transgenic mice and pancreatic sections of the healthy islets of wild-type C57BL/6JBomTac mice as an in-vivo model.

Genes involved in beta-cell differentiation and regeneration were investigated by high density oligonucleotide Affymetrix microarrays the Pro29lfsinsC-HNFlA induced INS-1 cells. These GeneChip expression arrays enabled us to simultaneously monitor genome-wide expression profiles and identify known and novel HNFlA dependent target genes which are involved in a variety of physiological processes. These putative genes were confirmed using qPCR and Western blotting.

We identified a prominent down-regulation of Bone Morphogenic Protein (bmp-3) mRNA expression in induced vs. non-induced mutant INS-1 cells. In contrast, the inducible expression of WT-HNF1A led to a time-dependent increase in bmp-3 mRNA levels. QPCR and Western blotting analysis also identified a compensatory upregulation of bmp-r2 gene/protein and alk4 gene expression in Pro29lfsinsC-HNF1A cells, and coincided with a time-dependent reduction in the expression of the insulin. BMPs represent a large family of growth factors critically involved in tissue differentiation and development, which signal via type I and type I1 BMP receptors. Treatment of INS-1 cells with recombinant BMP-3 restored insulin expression levels which were decreased by the HNF1A frame-shift mutation. This coincided with an increase in the phosphorylation of SMAD 2/3. Furthermore, immunohistochemistry on DN-HNF1A transgenic mice revealed a reduction in double positive staining of BMP-3 / Insulin, in insulin positive cells. Finally, we could demonstrate decreased activation of the bmp-3 promoter using a luciferase construct containing the bmp-3 promoter following expression of the ProfsinsC-HNF1A mutant. These findings suggest a critical link between HNF1A-MODY-induced alterations in gene expression and growth factors that control tissue differentiation and regeneration.

We also identified a significant up-regulation of Pancreatitis Associated Protein III (PAP III) mRNA expression in induced INS-1 cells. We found that of all the reg genes examined (PAP I, PAP II PAP III, and PSP/reg), the expression of Pro29lfsinsCHNF1A mutant led to a rapid and potent induction PSP/reg. Interestingly, PAP III and PSP/reg belong to the "Reg" (Regenerating) family of genes and have been implicated in islet regeneration. We detected a prominent induction of PSP/reg at gene and protein level during DN-HNF1A-induced apoptosis of INS-1 cells, and this induction was inhibited by the caspase inhibitor, zVAD.fmk. Conditioned culture medium from DN-HNF1A-expressing cells, but not from DN-HNF1A-expressing cells treated with zVAD.fmk, was sufficient to induce PSP/reg gene expression in naïve, INS-1 cells. Filtration and flow cytometry experiments suggested that Annexin-V-positive microparticles originating from blebbing membranes of apoptosing INS-1 cells mediated this effect. Treatment with recombinant PSP/reg protein stimulated cell proliferation and reversed the DN-HNF1A-induced decrease in p27 and insulin gene expression. We propose that apoptotic beta-cells secrete microparticles that stimulate PSP/reg induction in neighbouring cells, and facilitate the recovery of beta-cell mass. Collectively, these findings provide key insights into a primary aspect of beta-cell mass regulation, and could open up new avenues for renewal of impaired or decreased beta-cell mass in HNF1A-MODY and Type 2 diabetes.

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Comments

A thesis submitted to National University of Ireland in fulfilment of the requirements for the degree of Doctor of Philosophy to the Royal College of Surgeons in Ireland, 2009

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