Rennian Wang

β1 integrin/FAK/ERK signalling pathway is essential for human fetal islet cell differentiation and survival

β1 integrin and collagen matrix interactions regulate the survival of cells by associating with focal adhesion kinase (FAK) and initiating MAPK/ERK signalling, but little is known about these signalling pathways during human fetal islet ontogeny. The purpose of this study was to investigate whether β1 integrin/FAK activation of the MAPK/ERK pathway regulates human fetal islet cell expression of endocrine cell markers and survival. Isolated human (18–21 weeks fetal age) islet–epithelial cell clusters, cultured on collagen I, were examined using β1 integrin blocking antibody, β1 integrin siRNA and FAK expression vector. Perturbing β1 integrin function in the human fetal islet–epithelial cell clusters resulted in a marked decrease in cell adhesion, in parallel with a reduction in the number of cells expressing PDX‐1, insulin and glucagon (p < 0.05). β1 integrin blockade disorganized focal adhesion contacts in the PDX‐1+ cells and decreased activation of FAK and ERK1/2 signalling in parallel with an increase in expression of cleaved caspases 9 and 3 (p < 0.01). Similar results were obtained following an siRNA knock‐down of β1 integrin expression. In contrast, over‐expression of FAK not only increased phospho‐ERK and the expression of PDX‐1, insulin and glucagon (p < 0.05) but also abrogated the decreases in phospho‐ERK and PDX‐1 by β1 integrin blockade. This study demonstrates that activation of the FAK/ERK signalling cascade by β1 integrin is involved in the differentiation and survival of human fetal pancreatic islet cells. Copyright

Integrin α3, But Not β1, Regulates Islet Cell Survival and Function via PI3K/Akt Signaling Pathways

β1-integrin is a well-established regulator of β-cell activities; however, the role of its associated α-subunits is relatively unknown. Previously, we have shown that human fetal islet and INS-1 cells highly express α3β1-integrin and that collagens I and IV significantly enhance their survival and function; in addition, blocking β1 function in the fetal islet cells decreased adhesion on collagen I and increased apoptosis. The present study investigates the effect of blocking α3. Using α3 blocking antibody or small interfering RNA, the effects of α3-integrin blockade were examined in isolated human fetal or adult islet cells or INS-1 cells, cultured on collagens I or IV. In parallel, β1 blockade was analyzed in INS-1 cells. Perturbing α3 function in human islet or INS-1 cells resulted in significant decreases in cell function (adhesion, spreading, proliferation and Pdx1 and insulin expression/secretion), primarily on collagen IV. A significant decrease in focal adhesion kinase and ERK1/2 phosphorylation and increased caspase3 cleavage were observed on both collagens. These effects were similar to changes after β1 blockade. Interestingly, only α3 blockade reduced expression of phospho-Akt and members of its downstream signaling cascades (glycogen synthase kinase β and X-linked inhibitor of apoptosis), demonstrating a specific effect of α3 on the phosphatidylinositol 3-kinase/Akt pathway. These results suggest that α3- as well as β1-integrin-extracellular matrix interactions are critical for modulating β-cell survival and function through specialized signaling cascades and enhance our understanding of how to improve islet microenvironments for cell-based treatments of diabetes.

Conditional β1-integrin-deficient mice display impaired pancreatic β cell function.

β1‐Integrin, a critical regulator of β cell survival and function, has been shown to protect against cell death and promote insulin expression and secretion in rat and human islet cells in vitro. The aim of the present study was to examine whether the knockout of β1‐integrin in collagen I‐producing cells would have physiological and functional implications in pancreatic endocrine cells in vivo. Using adult mice with a conditional knockout of β1‐integrin in collagen I‐producing cells, the effects of β1‐integrin deficiency on glucose metabolism and pancreatic endocrine cells were examined. Male β1‐integrin‐deficient mice display impaired glucose tolerance, with a significant reduction in pancreatic insulin content (p < 0.01). Morphometric analysis revealed a significant reduction in β cell mass (p < 0.001) in β1‐integrin‐deficient mice, along with a significant decrease in β cell proliferation, Pdx‐1 and Nkx6.1 expression when compared with controls. Interestingly, these physiological and morphometric alterations in female β1‐integrin‐deficient mice were less significant. Furthermore, β1‐integrin‐deficient mice displayed decreased FAK (p < 0.05) and ERK1/2 (p < 0.001) phosphorylation, reduced cyclin D1 levels (p < 0.001) and increased caspase 3 cleavage (p < 0.01), while no changes in Akt phosphorylation were observed, indicating that the β1‐integrin signals through the FAK–MAPK–ERK pathway in vivo. Our results demonstrate that β1‐integrin is involved in the regulation of glucose metabolism and contributes to the maintenance of β cell survival and function in vivo. Copyright

β1 integrin-extracellular matrix interactions are essential for maintaining exocrine pancreas architecture and function.

Integrin receptors are responsible for integrating extracellular matrix signals inside the cell. The most prominent integrin receptor, β1 integrin, has a role in cell function, survival and differentiation. Recently, we demonstrated a profound in vivo role of β1 integrin expression in the pancreas on glucose homeostasis and islet function. Here, we extend these results by examining the role of β1 integrin in exocrine pancreatic structure and function. Adult C57Bl/6 mice hemizygous for a collagen type Iα2 (Col1a2) promoter-controlled tamoxifen-inducible Cre recombinase gene and homozygous for loxP-β1 integrin were injected with tamoxifen or corn oil to generate mice deleted or not for β1 integrin. Pancreata derived from these male mice were analyzed by quantitative reverse transcriptase-polymerase chain reaction, western blot and immunofluorescence. Our results showed that β1 integrin-deficient mice displayed a significant decrease in pancreas weight with a significant reduction of amylase, regenerating islet-derived protein II and carboxypeptidase-A expression (P<0.05–0.01). Compared with control pancreata, β1 integrin-deficient pancreata showed reduced mRNA expression of extracellular matrix (collagen type Iα2, fibronectin and laminin) genes (P<0.05), detached acini clusters and lost focal adhesion structure. Moreover, β1 integrin-deficient pancreatic acinar cells displayed decreased proliferation (P<0.05) and increased apoptosis (P<0.001). Apoptosis was reduced to that of controls when isolated exocrine clusters were cultured in media supplemented with extracellular matrix proteins. Taken together, these results implicate β1 integrin as an essential component for maintaining exocrine pancreatic structure and function.

c-Kit and stem cell factor regulate PANC-1 cell differentiation into insulin- and glucagon-producing cells

Recent evidence has shown that stem cell factor (SCF) and its receptor, c-Kit, have an important role in pancreatic islet development by promoting islet cell differentiation and proliferation. In this study, we examined the role of c-Kit and SCF in the differentiation and proliferation of insulin- and glucagon-producing cells using a human pancreatic duct cell line (PANC-1). Our study showed that increased expression of endocrine cell markers (such as insulin and glucagon) and transcription factors (such as PDX-1 and PAX-6) coincided with a decrease in CK19+ and c-Kit+ cells (P<0.001) during PANC-1 cell differentiation, determined by immunofluorescence and qRT-PCR. Cells cultured with exogenous SCF showed an increase in insulin+ (26%) and glucagon+ (35%) cell differentiation (P<0.01), an increase in cell proliferation (P<0.05) and a decrease in cell apoptosis (P<0.01). siRNA knockdown of c-Kit resulted in a decrease in endocrine cell differentiation with a reduction in PDX-1 and insulin mRNA, as well as the number of cells immunostaining for PDX-1 and insulin. Taken together, these results show that c-Kit/SCF interactions are involved in mediating islet-like cluster formation and islet-like cell differentiation in a human pancreatic duct cell line.

Reduced Immunogenicity of First-Trimester Human Fetal Pancreas

OBJECTIVE—The use of human fetal pancreatic tissue may provide a potential source of transplantable β-cells as a therapy for type 1 diabetes. Human fetal pancreas has a remarkable capacity to grow and differentiate in vivo and has been shown to reverse diabetes in rodents. However, it is known that human fetal pancreas obtained from the second trimester of gestation is immunogenic and is rejected after transplantation. Tissue obtained from earlier stages might prove to be immune privileged, as has been shown for other tissues. RESEARCH DESIGN AND METHODS—In this study, we determined the immunogenicity of human fetal pancreatic tissue obtained from the first trimester of gestation in a humanized mouse model. A microarray study of immunoregulatory gene expression in first- and second-trimester human fetal pancreas was also undertaken. RESULTS—The analysis of transplanted human fetal pancreata revealed a significantly decreased immunogenicity of the first-trimester tissue. The first-trimester grafts showed only limited cellular infiltration and contained numerous insulin-positive cells, whereas second-trimester tissue was completely infiltrated and rejected. Furthermore an analysis of immunoregulatory genes expressed in first- and second-trimester human fetal pancreas by microarray demonstrated the upregulation of several key immunoregulatory genes in the second-trimester tissue. This might account for the reduced immunogenicity of the younger tissue. CONCLUSIONS—Our results provide the first indication that the use of first-trimester human fetal pancreas for transplantation might increase the survival of the grafts and might decrease the requirement for immunosuppressive drugs.

Expression and function of αβ1 integrins in pancretic beta (INS-1) cells

Integrin-extracellular matrix interactions are important determinants of beta cell behaviours. The β1 integrin is a well-known regulator of beta cell activities; however, little is known of its associated α subunits. In the present study, αβ1 integrin expression was examined in the rat insulinoma cell line (INS-1) to identify their role in beta cell survival and function. Seven α subunits associated with β1 integrin were identified, including α1-6 and αV. Among these heterodimers, α3β1 was most highly expressed. Common ligands for the α3β1 integrin, including fibronectin, laminin, collagen I and collagen IV were tested to identify the most suitable matrix for INS-1 cell proliferation and function. Cells exposed to collagen I and IV demonstrated significant increases in adhesion, spreading, cell viability, proliferation, and FAK phosphorylation when compared to cells cultured on fibronectin, laminin and controls. Integrin-dependent attachment also had a beneficial effect on beta cell function, increasing Pdx-1 and insulin gene and protein expression on collagens I and IV, in parallel with increased basal insulin release and enhanced insulin secretion upon high glucose challenge. Furthermore, functional blockade of α3β1 integrin decreased cell adhesion, spreading and viability on both collagens and reduced Pdx-1 and insulin expression, indicating that its interactions with collagen matrices are important for beta cell survival and function. These results demonstrate that specific αβ1 integrin-ECM interactions are critical regulators of INS-1 beta cell survival and function and will be important in designing optimal conditions for cell-based therapies for diabetes treatment.

Definitive endoderm derived from human embryonic stem cells highly express the integrin receptors αV and β5

Human embryonic stem cells (hESCs) can be directed to differentiate into a number of endoderm cell types, however mature functional cells have yet to be produced in vitro. This suggests that there may be important factors that have yet to be described, which may be essential for the proper derivation of these cells. One such factor is the integrin mediated interactions between a cell and the extracellular matrix (ECM). On this basis, the present study investigated the role of the ECM in the directed differentiation of hESCs to definitive endoderm via analysis of integrin gene expression. The results showed that definitive endoderm can be efficiently and effectively derived from hESCs in a feeder free, single defined ECM of laminin. Analysis of integrin expression also showed that definitive endoderm highly express the integrins αV and β5, which have the ability to bind to vitronectin, whilst expression of the pluripotency related laminin binding integrins α3, α6 and β4 were downregulated. This suggested a potential role of vitronectin binding integrins in the development of definitive endoderm.

SOX9 regulates endocrine cell differentiation during human fetal pancreas development

The transition of pancreatic progenitor cells to mature endocrine cells is regulated by the sequential activation and interaction of several transcription factors. In mice, the transcription factor Sox9 has been shown to support endocrine cell differentiation. However, the functional role of SOX9 during pancreas development in the human has yet to be determined. The present study was to characterize SOX9 expression during human fetal pancreas development and examine its functional role by transfection with SOX9 siRNA or SOX9 expression vectors. Here we report that SOX9 was most frequently expressed in PDX1(+) cells (60-83%) and least in mature endocrine cells (<1-14%). The proliferation of SOX9(+) cells was significantly higher at 8-10 weeks than at 14-21 weeks (p<0.05) or 20-21 weeks (p<0.01). SOX9 frequently co-localized with FOXA2, NGN3 and transcription factors linked to NGN3 (NKX2.2, NKX6.1, PAX6). siRNA knockdown of SOX9 significantly decreased islet-epithelial cell proliferation, NGN3, NKX6.1, PAX6 and INS mRNA levels and the number of NGN3(+) and insulin(+) cells (p<0.05) while increasing GCG mRNA and glucagon(+) cells (p<0.05). Examination of SOX9 associated signaling pathways revealed a decrease in phospho-Akt (p<0.01), phospho-GSK3β (p<0.01) and cyclin D1 (p<0.01) with a decrease in nuclear β-catenin(+) (p<0.05) cells following SOX9 siRNA knockdown. In contrast, over-expression of SOX9 significantly increased the number of islet cells proliferating, NGN3, NKX6.1, PAX6 and INS mRNA levels, the phospho-Akt/GSK3β cascade and the number of insulin(+) cells. Our results demonstrated that SOX9 is important for the expression of NGN3 and molecular markers of endocrine cell differentiation in the human fetal pancreas.

The Emerging Role of SOX Transcription Factors in Pancreatic Endocrine Cell Development and Function

The transition of pancreatic progenitor cells to mature beta cells is regulated by the interaction of several transcription factors, including members of the sex-determining region on Y box (SOX) family of transcription factors. The SOX proteins are widely involved in cell fate determination and the development of several tissues, including bone, heart, gonads, lymphocytes, and glial cells as well as the pancreas. In this review, we will present recent findings that illustrate the critical role of SOX transcription factors in maintaining pancreatic progenitor cell pools and in controlling pancreatic islet morphogenesis and islet function. Interrelationships between the SOX family and other pancreatic transcription factors specific to endocrine lineages will also be discussed in light of islet cell-based therapies for the treatment of diabetes.