Cross-talk between Bone Morphogenetic Protein and Transforming Growth Factor-β Signaling Is Essential for Exendin-4-induced Insulin-positive Differentiation of AR42J Cells

Abstract

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...

Bone Morphogenetic Protein-2/-4 Upregulation Promoted by Endothelial Cells in Coculture Enhances Mouse Embryoid Body Differentiation

“…Some studies have demonstrated that BMP-4 and its receptor are essential to induce expression of glucose sensor proteins (GLUT2, GKS, SUR1, and Kir6.2) in pancreatic beta cells [31]. Other studies indicate that BMP-2 is essential for the induction of insulin-positive cells in AR 42J cells [32]. Taken together with the present results, the data suggest that both developing factors are essential for beta cell differentiation and function.…”

Mechanisms of action of glucagon-like peptide 1 in the pancreas

“…In a follow-up study the Gittes laboratory found evidence that GLP-1R-induced differentiation of AR42J cells involves activation of the bone morphogenetic protein (BMP) signaling pathway first, followed by the TGFβ isoform signaling mechanism (Fig 4; Yew et al, 2005). BMPs are cytokines that also form part of the TGFβ superfamily (Zhang and Li, 2005).…”

Bone morphogenetic protein‐4 enhances vascular endothelial growth factor secretion by human retinal pigment epithelial cells

“…BMP-4 was found to be neuroprotective of Müller glia in the chicken retina [Fischer et al, 2004], and upregulated in malignant melanoma, promoting cell invasion and migration [Rothhammer et al, 2005]. BMP-2 and TGFb share a signaling pathway of insulin-stimulated differentiation of pancreatic β cells [Yew et al, 2005]. BMP-4 inhibits follicle-stimulating hormone in the pituitary [Faure et al, 2005] and promotes smooth muscle proliferation [Frank et al, 2005].…”