Abstract
Inhibitor of DNA binding (Id) proteins bind to and inhibit the function of basic helix-loop-helix (bHLH) transcription factors including those that regulate pancreatic development. Moreover, bone morphogenetic proteins (BMPs) regulate the expression of Ids. We hypothesized that BMP4 and Id proteins play a role in the expansion and differentiation of epithelial progenitor cells. We demonstrate that BMP4 induces the expression of Id2 along with the expansion of AR42J pancreatic epithelial cells. Furthermore, neutralization of BMP4 significantly reduced duct epithelial cell expansion in a mouse model of islet regeneration. BMP4 stimulation promotes Id2 binding to the bHLH transcription factor NeuroD, which is required for the differentiation of pancreatic islet cells. Therefore, our results indicate that BMP4 stimulation blocks the differentiation of endocrine progenitor cells and instead promotes their expansion thereby revealing a novel paradigm of signaling explaining the balance between expansion and differentiation of pancreatic duct epithelial progenitors. Understanding the mechanisms of BMP and Id function elucidates a key step during pancreas embryogenesis, which is important knowledge for expanding pancreatic progenitors in vitro.
Highlights
Inhibition of differentiation proteins (Ids), comprised of four members (Id1–Id4), are a family of proteins that are implicated in a number of cellular processes, including control of proliferation and differentiation (5, 6)
We show that BMP4 and Id proteins regulate the expansion of AR42J cells, a pancreatic epithelial cell line with the potential to differentiate into endocrine cells in vitro (24, 25)
BMP4 Regulates Id Expression in AR42J Cells and Induces Cell Proliferation—The AR42J cell line is a pluripotent cell line derived from a rat pancreatic acinar carcinoma, which are acinar-like cells when exposed to dexamethasone (29), and in contrast, convert into insulinproducing cells with betacellulin and activin A treatment (24)
Summary
Western Immunoblotting—AR42J cells were lysed in 2ϫ sample buffer (0.13 mol/liter Tris-base, pH 6.8, 20% glycerol, and 4% SDS). Duct cells were filtered over a 70-m cell strainer and cultured in 10% fetal calf serum Dulbecco’s modified Eagle’s F-12 medium overnight. For BMP4 treatment, duct cells were growth-arrested in 0.5% fetal calf serum Dulbecco’s modified Eagle’s F-12 medium for 24 h before the addition of BMP4 (1–100 ng/ml) for 18 h. BMP4 Neutralizing Antibody Treatment and BrdUrd Count—To ascertain whether BMP4 signaling affects pancreatic duct cell expansion, we injected mice intravenously with a neutralizing antibody to BMP4 (200 g) (R&D Systems, Inc., Minneapolis, MN) three times a week for 2 weeks. A value of p Ͻ 0.05 was considered statistically significant
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