Bone Morphogenetic Protein 3 Controls Insulin Gene Expression and Is Down-regulated in INS-1 Cells Inducibly Expressing a Hepatocyte Nuclear Factor 1A–Maturity-onset Diabetes of the Young Mutation

Caroline Bonner,Angela M Farrelly,Caoimhín G Concannon,Heiko Dussmann,Mathurin Baquié,Isabelle Virard,Hella Wobser,Donat Kögel,Claes B Wollheim,Marjan Rupnik,Maria M Byrne,Hans-Georg König,Jochen H.M Prehn

doi:10.1074/jbc.m110.215525

Abstract

Inactivating mutations in the transcription factor hepatocyte nuclear factor (HNF) 1A cause HNF1A-maturity-onset diabetes of the young (HNF1A-MODY), the most common monogenic form of diabetes. To examine HNF1A-MODY-induced defects in gene expression, we performed a microarray analysis of the transcriptome of rat INS-1 cells inducibly expressing the common hot spot HNF1A frameshift mutation, Pro291fsinsC-HNF1A. Real-time quantitative PCR (qPCR), Western blotting, immunohistochemistry, reporter assays, and chromatin immunoprecipitation (ChIP) were used to validate alterations in gene expression and to explore biological activities of target genes. Twenty-four hours after induction of the mutant HNF1A protein, we identified a prominent down-regulation of the bone morphogenetic protein 3 gene (Bmp-3) mRNA expression. Reporter assays, qPCR, and Western blot analysis validated these results. In contrast, inducible expression of wild-type HNF1A led to a time-dependent increase in Bmp-3 mRNA and protein levels. Moreover, reduced protein levels of BMP-3 and insulin were detected in islets of transgenic HNF1A-MODY mice. Interestingly, treatment of naïve INS-1 cells or murine organotypic islet cultures with recombinant human BMP-3 potently increased their insulin levels and restored the decrease in SMAD2 phosphorylation and insulin gene expression induced by the HNF1A frameshift mutation. Our study suggests a critical link between HNF1A-MODY-induced alterations in Bmp-3 expression and insulin gene levels in INS-1 cells and indicates that the reduced expression of growth factors involved in tissue differentiation may play an important role in the pathophysiology of HNF1A-MODY.

Highlights

Cytokines of the transforming growth factor-␤ (TGF-␤) superfamily, which includes the TGF-␤ isoforms, activins, and the bone morphogenetic proteins (BMPs), regulate gene expression in diverse cell types and are involved in numerous processes including lineage determination, tissue differentiation, cell proliferation, and apoptosis (9 –11)
These include (i) the inducible expression of the common, frameshift mutation, Pro291fsinsC-HNF1A leads to the potent down-regulation of bone morphogenetic protein 3 gene (Bmp-3) at the mRNA and protein level in insulin-secreting
INS-1 cells; (ii) Dual Luciferase Reporter assays reveal that the HNF1A mutant reduces Bmp-3 promoter activity, which results in the decrease in the transcriptional activity of the

Summary

EXPERIMENTAL PROCEDURES

Cell Culture: INS-1 Cells Overexpressing HNF1A in an Inducible System—Rat INS-1 insulinoma cells overexpressing wildtype HNF1A (WT-HNF1A), the HNF1A-MODY-associated. Immunohistochemistry—Paraffin-embedded pancreatic sections from transgenic mice expressing a DN-HNF1A mutant under the control of rat insulin promoter DN-HNF1A and control wild-type C57BL/6JBomTac mice-were deparaffinized [22] and incubated overnight at 4 °C with the rabbit polyclonal antiBMP-3 (Santa Cruz Biotechnology) diluted 1:20). INS-1 cells were induced with doxycycline for 0 – 48 h, and Hnf1a gene expression was determined using absolute real-time qPCR. C and D, Pro291fsinsC-HNF1A INS-1 cells were treated as in A, and L-type Prk gene mRNA expression (C) and Tmem mRNA expression (D) were determined using real-time qPCR. Differences were considered to be significant at p Յ 0.05

RESULTS

Differentially expressed genes classified into distinct functional subgroups

Decreased No change No change No change No change

DISCUSSION