The differentiation of pancreatic exocrine AR42J cells into insulin-expressing endocrine cells has served as an important model for both endogenous in vivo ␤-cell differentiation as well as potential application to ␤-cell engineering of progenitor cells. Exogenous activin, possibly working through intracellular smad 2 and/or smad 3, as well as exogenous exendin-4 (a long-acting glucagon-like peptide-1 agonist) have both been shown to induce insulin-positive/endocrine differentiation in AR42J cells. In this study, we present evidence of significant interplay and interdependence of these two pathways as well as potential synergy between the pathways. In particular, insulin-positive differentiation seems to entail an exendin-4 -induced drop in smad 2 and elevation in smad 3 in RNA levels. The latter appears to be dependent on endogenous transforming growth factor (TGF)-␤ isoform release by the AR42J cells and may serve as a mechanism to promote ␤-cell maturation. The drop in smad 2 may mediate early endocrine commitment. The coapplication of exogenous exendin-4 and, specifically, low-dose exogenous TGF-␤1 led to a dramatic 20-fold increase in insulin mRNA levels, supporting a novel synergistic and codependent relationship between exendin-4 signaling and TGF-␤ isoform signaling. Diabetes 53: 2824 -2835, 2004 M echanisms underlying the differentiation of pancreatic precursor cells into insulin-positive cells are of paramount importance toward our goal of engineering cells to become glucose-responsive ␤-cells for the curative therapy of diabetes. Over the last several years, a well-validated model of such differentiation has been developed using rat AR42J pancreatic epithelial cells (1-3). Early reports used either activin, or activin in combination with hepatocyte growth factor or ␤-cellulin, to generate either endocrine (activin alone) or insulin-positive (activin in combination) cell lines. Activins are members of the transforming growth factor (TGF)-␤ superfamily that form hetero-or homodimers and bind specific type I and type II TGF-␤ superfamily receptors (4). In most cases, activin ligand signaling induces phosphorylation and activation of smad 2 and/or smad 3 transcription factors to initiate downstream signaling (5). Based on the above initial observations of the effects of activin, subsequent AR42J studies showed a potential role for intracellular smad signaling in mediating these activin-induced pathways. Early findings were that smad 2 was necessary for insulin-positive differentiation, but forced overexpression of smad 2 in AR42J cells was not sufficient to induce insulin-positive differentiation (6). More recently, given the incretin (insulin release) and insulinotrophic (insulin cell growth) effects of glucagon-like peptide-1 (GLP-1) (7-15), either GLP-1 protein or a long-acting form of GLP-1 (exendin-4) was used to induce an endocrine phenotype in AR42J cells. Here, multiple islet cell types were seen, with expression of cell-specific markers and the acquisition of an endocrine morphology in culture...

A key goal of cellular engineering is to manipulate progenitor cells to become ␤-cells, allowing cell replacement therapy to cure diabetes mellitus. As a paradigm for cell engineering, we have studied the molecular mechanisms by which AR42J cells become ␤-cells. Bone morphogenetic proteins (BMPs), implicated in a myriad of developmental pathways, have not been well studied in insulin-positive differentiation. We found that the canonical intracellular mediators of BMP signaling, Smad-1 and Smad-8, were significantly elevated in AR42J cells undergoing insulin-positive differentiation in response to exendin-4 treatment, suggesting a role for BMP signaling in ␤-cell formation. Similarly, endogenous BMP-2 ligand and ALK-1 receptor (activin receptor-like kinase-1; known to activate Smads 1 and 8) mRNAs were specifically up-regulated in exendin-4-treated AR42J cells. Surprisingly, Smad-1 and Smad-8 levels were suppressed by the addition of BMP-soluble receptor inhibition of BMP ligand binding to its receptor. Here, insulin-positive differentiation was also ablated. BMP-2 ligand antisense also strongly inhibited Smad-1 and Smad-8 expression, again with the abolition of insulin-positive differentiation. These results demonstrate a previously unrecognized key role for BMP signaling in mediating insulin-positive differentiation through the intracellular Smad signaling pathway. In short, BMP signaling may represent a novel downstream target of exendin-4 (glucagon-like peptide 1) signaling and potentially serve as an upstream regulator of transforming growth factor-␤ isoform signaling to differentiate the acinarlike AR42J cells into insulin-secreting cells.Type 1 diabetes is an insulin deficiency state due to pancreatic destruction of ␤-cells caused by autoimmunity. Several approaches to treat diabetes are being pursued, such as islet cell transplantation, pancreas transplantation, and genetic manipulation. However, a key alternative strategy is cellular engineering to manipulate progenitor cells to become ␤-cells, allowing cellular therapy to cure diabetes. As a paradigm for cell engineering, we have used exendin-4 treatment of AR42J cells, a fairly plastic acinar cell carcinoma-derived cell line, as a model for studying the role of bone morphogenetic protein (BMP) 2 signaling in the induction of insulin-positive differentiation. Exendin-4, a peptide from Helodermatidae venom, is a novel insulinotropic agent and a long acting analogue of glucagon-like peptide-1 (GLP-1). It interacts with endocrine pancreatic islet GLP-1 receptors, inducing a stimulatory effect on insulin secretion. Over the past few decades significant progress has been made in our understanding of the biological function of BMPs, which have been found to regulate a myriad of developmental and differentiation process in the embryo, including epithelial-mesenchymal interactions, cell fate specification, dorsoventral patterning, and apoptosis as well as the secretion of extracellular matrix components (1-5).BMPs are one of the multifunctional cytokines from...

Epithelial-mesenchymal interactions are crucial for the proper development of many organs, including the pancreas. Within the pancreas, the ducts are thought to harbor stem/progenitor cells, and possibly to give rise to pancreatic ductal carcinoma. Little is known about the mechanism of formation of pancreatic ducts in the embryo. Pancreatic mesenchyme contains numerous soluble factors which help to sustain the growth and differentiation of exocrine and endocrine structures. Here, we report that one such morphoregulatory mesenchymal protein, epimorphin, plays an important role during pancreatic ductal proliferation and differentiation. We found that epimorphin is expressed in pancreatic mesenchyme during early stages of development, and at mesenchymal-epithelial interfaces surrounding the ducts at later stages. Strong upregulation of epimorphin expression was seen during in vitro pancreatic duct differentiation. Similarly, in vitro pancreatic duct formation was inhibited by a neutralizing antibody against epimorphin, whereas addition of recombinant epimorphin partially rescued duct formation. Together, our study demonstrates the role of epimorphin in pancreatic ductal morphogenesis.