Impact of Defined Matrix Interactions on Insulin Production by Cultured Human β-Cells: Effect on Insulin Content, Secretion, and Gene Transcription

Thomas Kaido 1 Mayra Yebra 1 Vincenzo Cirulli 1 Christopher Rhodes 0 Giuseppe Diaferia 1 Anthony M. Montgomery 1 0 -terminal kinase; mAb, monoclonal antibody; pAb, polyclonal antibody. DOI: 10.2337/db06-0120 2006 by the American Diabetes Association. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact 1 Islet Research Laboratory at the Whittier Institute for Diabetes, Department of Pediatrics, University of California at San Diego , La Jolla, California ; and the The impact of extracellular matrix on insulin production needs to be understood both to optimize the derivation of functional -cells for transplantation and to understand mechanisms controlling islet neogenesis and glucose homeostasis. In this study, we present evidence that adhesion to some common matrix constituents has a profound impact on the transcription, secretion, and storage of insulin by human -cells. The integrin-dependent adhesion of fetal -cells to both collagen IV and vitronectin induces significant glucose-independent insulin secretion and a substantial reciprocal decline in insulin content. Collagen IV, but not vitronectin, induces comparable responses in adult -cells. Inhibition of extracellular signal-regulated kinase activation abrogates matrix-induced insulin secretion and effectively preserves the insulin content of adherent -cells. Using real-time PCR, we demonstrate that adhesion of both fetal and adult -cells to collagen IV and vitronectin also results in the marked suppression of insulin gene transcription. Based on these findings, we contend that integrin-dependent adhesion and signaling in response to certain matrices can have a significant negative impact on insulin production by primary human -cells. Such responses were not found to be associated with cell death but may precede -cell dedifferentiation. Diabetes 55: 2723-2729, 2006 - Ibrane adhesion molecules that serve to integrate a ntegrins are a family of heterodimeric transmemcells interior machinery with the extracellular environment. Such integration is achieved through the binding of extracellular matrix components and the subsequent activation of intracellular signaling elements (1). Integrins have been implicated in a plethora of processes required for normal development, including cell survival, proliferation, cytodifferentiation, migration, and spatial segregation (1,2). Complex matrices and individual extracellular matrix components have been shown to strongly affect many aspects of -cell function, including motility (3,4), survival (5,6), proliferation (7,8), and differentiation (9,10). Several recent reports have shown that matrix interactions can also influence insulin secretion (4,11,12). A complex matrix rich in laminin-5 (804G-matrix) has been shown to potentiate insulin secretion in response to glucose (11,12), and we have shown that common constituents of basement membranes, including collagen IV, induce significant insulin secretion by human fetal -cells (4). Although matrix interactions have been shown to be beneficial for insulin secretion in the short term (4,11,12), there is less certainty as to the long-term impact of matrix interactions on insulin content and gene expression. Collagen, matrigel, and fibrin gels have all been reported to induce or maintain insulin content or secretion (1316). However, others have shown that long-term exposure to both purified and complex matrices, particularly in monolayer culture, results in a significant loss of insulin secretion, message, or content (7,1719). Interpreting the role of matrix in these long-term studies is complicated by the addition of different exogenous growth factors or serum, which may themselves influence insulin transcription. The purpose of this study was to define the impact of individual matrix interactions on insulin production by cultured human -cells. To do this, we have exploited assays that allow the rapid and controlled interaction of -cells with individual matrices in the absence of potentially confounding factors such as serum or exogenous growth factors. We show that adhesion of primary human -cells to substrates such as vitronectin and collagen IV results in a dramatic loss of insulin content and insulin production. We show that such losses can be attributed to protracted, extracellular signalregulated kinase (ERK)dependent insulin secretion and simultaneous suppression of insulin gene transcription. The observed loss of insulin production was not associated with cell death but may precede -cell dedifferentiation and anchorage-dependent growth. These observations have important implications for the empirical design and optimization of protocols for the ex vivo expansion of -cells for transplantation. RESEARCH DESIGN AND METHODS Monoclonal antibodies (mAbs) to v 3 (LM609), v 5 (PIF6), and 1 (P4C10) were from Chemicon (Temecula, CA). VNR (vitronectin receptor), anti- v polyclonal antibody (pAb), was generated at the Scripps Research Institute (La Jolla, CA). A guinea pig pAb to insulin was obtained from DakoCytomation (Carpinteria, CA). c-Jun NH2-terminal kinase (JNK) inhibitor SP600125, p38 inhibitor SB203580, ERK-1/2 inhibitors PD8059 and U0126, and the analog control U0124 were all obtained from Calbiochem (San Diego, CA). Vitronectin and fibronectin were from Chemicon, and collagen IV and entactin-free laminin-1 were purchased from BD Biosciences (Bedford, MA). Collagen I was from Upstate Cell Signaling (Lake Placid, NY). Derivation of fetal and adult -cells. Fetal pancreata (20 24 weeks) were obtained from ABR (Alameda, CA). Islet-like cell clusters were generated and cultured in suspension for 2 days as previously described (17,20). Islet-like cell clusters were then transferred and cultured for 4 days on dishes coated with HTB-9 matrix (18) in the presence of 10% fetal bovine serum and hepatocyte growth factor (10 ng/ml) essentially as reported (7,17). Islet-like cell cluster culture on HTB-9 matrix results in some loss of insulin expression (7) but also promotes a gradual transition from spheroidal aggregates to monolayers, which can then be harvested as single cells without compromising -cell viability. Harvesting was performed using a 0.025% trypsin-versene solution (Gibco). Harvested cells typically contained 8590% pancreatic epithelial cells and 12% insulin-positive -cells. Human adult islets (age 4556 years) of 30 40% purity were obtained through the Human Islet Distribution Program (City of Hope National Medical Center, Duarte, CA). Islet clusters were expanded on HTB-9 matrix for 3 4 days and were harvested as described above for fetal cells. Matrix adhesion, ERK inhibition, and insulin release. Fetal or adult cells were added to 96-well high-binding plates (Costar) precoated overnight (4C) with equimolar amounts of vitronectin, collagen IV, (...truncated)