Gene expression profiling of molecular target genes which are regulated by hepatocyte nuclear factor 1 Alpha (HNF1A) in rat insulin-secreting cells: role in HNF1A-MODY
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.
First SupervisorProfessor Jochen H.M. Prehn
CommentsA 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
Published CitationBonner, C. Gene expression profiling of molecular target genes which are regulated by hepatocyte nuclear factor 1 Alpha (HNF1A) in rat insulin-secreting cells : role in HNF1A-MODY [PhD Thesis]. Dublin: Royal College of Surgeons in Ireland; 2009.
- Doctor of Philosophy (PhD)