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
Creators: name: Bonner, Caroline
Publisher: name: Royal College of Surgeons in Ireland
Year: 2019
Subjects: Uncategorized

description: 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 <em>hepatocyte nuclear factor</em> 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 <em>in-vivo</em> 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 <em>Bone Morphogenic Protein</em> (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 <em>bmp-3</em> mRNA levels. QPCR and Western blotting analysis also identified a compensatory upregulation of <em>bmp-r2</em> gene/protein and <em>alk4</em> gene expression in Pro29lfsinsC-HNF1A cells, and coincided with a time-dependent reduction in the expression of the <em>insulin</em>. 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 <em>bmp-3 </em>promoter using a luciferase construct containing the <em>bmp-3</em> 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 <em>Pancreatitis Associated Protein</em> <em>III </em><em>(PAP III) </em>mRNA expression in induced INS-1 cells. We found that of all the <em>reg</em> genes examined (<em>PAP I, PAP II PAP III, and PSP/reg</em>), the expression of Pro29lfsinsCHNF1A mutant led to a rapid and potent induction <em>PSP/reg</em>. Interestingly, <em>PAP III </em>and <em>PSP/reg</em> belong to the <em>"Reg"</em> (Regenerating) family of genes and have been implicated in islet regeneration. We detected a prominent induction of <em>PSP/reg </em>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 <em>PSP/reg </em>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 <em>p27</em> and <em>insulin<strong> </strong></em>gene expression. We propose that apoptotic beta-cells secrete microparticles that stimulate <em>PSP/reg </em>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.