Mayra Yebra

Suppression of p53 activity and p21WAF1/CIP1 expression by vascular cell integrin alphaVbeta3 during angiogenesis.

Induction of p53 activity in cells undergoing DNA synthesis represents a molecular conflict that can lead to apoptosis. During angiogenesis, proliferative endothelial cells become apoptotic in response to antagonists of integrin alphavbeta3 and this leads to the regression of angiogenic blood vessels, thereby blocking the growth of various human tumors. Evidence is presented that administration of alphavbeta3 antagonists during angiogenesis in vivo selectively caused activation of endothelial cell p53 and increased expression of the p53-inducible cell cycle inhibitor p21WAF1/CIP1. In vitro studies revealed that the ligation state of human endothelial cell alphavbeta3 directly influenced p53 activity and the bax cell death pathway. Specifically, agonists of endothelial cell alphavbeta3, but not other integrins, suppressed p53 activity, blocked p21WAF1/CIP1 expression, and increased the bcl-2/bax ratio, thereby promoting cell survival. Thus, ligation of vascular cell integrin alphavbeta3 promotes a critical and specific adhesion-dependent cell survival signal during angiogenesis leading to inhibition of p53 activity, decreased expression of p21WAF1/CIP1, and suppression of the bax cell death pathway.

Requirement of Receptor-bound Urokinase-type Plasminogen Activator for Integrin αvβ5-directed Cell Migration

The urokinase plasminogen activator (uPA) interacts with its cell surface receptor (uPAR), providing an inducible, localized cell surface proteolytic activity, thereby promoting cellular invasion. Evidence is provided for a novel function of cell surface-associated uPA·uPAR. Specifically, induction of cell surface expression of uPA·uPAR by growth factors or phorbol ester was necessary for vitronectin-dependent carcinoma cell migration, an event mediated by integrin αvβ5. Cell migration on vitronectin was blocked with either a soluble form of uPAR, an antibody that disrupts uPA binding to uPAR, or a monoclonal antibody to αvβ5. Moreover, plasminogen activator inhibitor type 2 blocked this migration event but did not affect adhesion, suggesting a direct role for uPA enzyme activity in this process and that migration but not adhesion of these cells is regulated by uPA·uPAR. Growth factor-mediated induction of uPA·uPAR on the carcinoma cell surface promotes a specific motility event mediated by integrin αvβ5, since cells transfected with the β3 integrin subunit expressed αvβ3 and migrated on vitronectin independently of growth factors or uPA·uPAR expression. This relationship between αvβ5 and the uPA·uPAR system has significant implications for regulation of motility events associated with development, angiogenesis, and tumor metastasis.

Recognition of the Neural Chemoattractant Netrin-1 by Integrins α6β4 and α3β1 Regulates Epithelial Cell Adhesion and Migration

Netrins, axon guidance cues in the CNS, have also been detected in epithelial tissues. In this study, using the embryonic pancreas as a model system, we show that Netrin-1 is expressed in a discrete population of epithelial cells, localizes to basal membranes, and specifically associates with elements of the extracellular matrix. We demonstrate that alpha6beta4 integrin mediates pancreatic epithelial cell adhesion to Netrin-1, whereas recruitment of alpha6beta4 and alpha3beta1 regulate the migration of CK19+/PDX1+ putative pancreatic progenitors on Netrin-1. These results provide evidence for the activation of epithelial cell adhesion and migration by a neural chemoattractant, and identify Netrin-1/integrin interactions as adhesive/guidance cues for epithelial cells.

An integrin β 3 –KRAS–RalB complex drives tumour stemness and resistance to EGFR inhibition

Tumour cells, with stem-like properties, are highly aggressive and often show drug resistance. Here, we reveal that integrin αvβ3 serves as a marker of breast, lung and pancreatic carcinomas with stem-like properties that are highly resistant to receptor tyrosine kinase inhibitors such as erlotinib. This was observed in vitro and in mice bearing patient-derived tumour xenografts or in clinical specimens from lung cancer patients who had progressed on erlotinib. Mechanistically, αvβ3, in the unliganded state, recruits KRAS and RalB to the tumour cell plasma membrane, leading to the activation of TBK1 and NF-κB. In fact, αvβ3 expression and the resulting KRAS–RalB–NF-κB pathway were both necessary and sufficient for tumour initiation, anchorage independence, self-renewal and erlotinib resistance. Pharmacological targeting of this pathway with bortezomib reversed both tumour stemness and erlotinib resistance. These findings not only identify αvβ3 as a marker/driver of carcinoma stemness but also reveal a therapeutic strategy to sensitize such tumours to RTK inhibition.

Regulation of Human β-Cell Adhesion, Motility, and Insulin Secretion by Collagen IV and Its Receptor α1β1

Collagens have been shown to influence the survival and function of cultured β-cells; however, the utilization and function of individual collagen receptors in β-cells is largely unknown. The integrin superfamily contains up to five collagen receptors, but we have determined that α1β1 is the primary receptor utilized by both fetal and adult β-cells. Cultured β-cells adhered to and migrated on collagen type IV (Col-IV), and these responses were mediated almost exclusively by α1β1. The migration of cultured β-cells to Col-IV significantly exceeded that to other matrix components suggesting that this substrate is of unique importance for β-cell motility. The interaction of α1β1 with Col-IV also resulted in significant insulin secretion at basal glucose concentrations. A subset of β-cells in developing islets was confirmed to express α1β1, and this expression co-localized with Col-IV in the basal membranes of juxtaposed endothelial cells. Our findings indicate that α1β1 and Col-IV contribute to β-cell functions known to be important for islet morphogenesis and glucose homeostasis.

A Novel Approach for the Derivation of Putative Primordial Germ Cells and Sertoli Cells from Human Embryonic Stem Cells

Using human embryonic stem cells (hESCs), we describe a novel method for the rapid derivation and enrichment of cells that are comparable to primordial germ cells (PGCs) and Sertoli cells. The methodology described is based on modest changes to the growth conditions commonly used to expand hESCs and does not require genetic manipulation or complex three‐dimensional culture. Remarkably, we have determined that simply reducing the size of cultured ESC colonies and manipulating the number of feeding cycles, results in the rapid emergence of cells that are comparable to migratory PGCs. Importantly, these cells can be monitored and purified on the basis of the expression of the chemokine receptor CXCR4. Under more stringent differentiating conditions these cells mature and upregulate the expression of specific germ cell markers. Importantly, this process is accompanied by the development of Sertoli‐like support cells. Such cells normally provide trophic support and immunoprotection to developing germ cells and may have significant clinical utility in the prevention of graft rejection. The putative Sertoli‐germ cell cocultures generated in this study may ultimately be developed to study and manipulate interactions and processes involved in human gametogenesis. STEM CELLS 2009;27:68–77

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

The impact of extracellular matrix on insulin production needs to be understood both to optimize the derivation of functional β-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 β-cells. The integrin-dependent adhesion of fetal β-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 β-cells. Inhibition of extracellular signal–regulated kinase activation abrogates matrix-induced insulin secretion and effectively preserves the insulin content of adherent β-cells. Using real-time PCR, we demonstrate that adhesion of both fetal and adult β-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 β-cells. Such responses were not found to be associated with cell death but may precede β-cell dedifferentiation.

Endothelium-derived Netrin-4 supports pancreatic epithelial cell adhesion and differentiation through integrins α2β1 and α3β1.

Background Netrins have been extensively studied in the developing central nervous system as pathfinding guidance cues, and more recently in non-neural tissues where they mediate cell adhesion, migration and differentiation. Netrin-4, a distant relative of Netrins 1–3, has been proposed to affect cell fate determination in developing epithelia, though receptors mediating these functions have yet to be identified. Methodology/Principal Findings Using human embryonic pancreatic cells as a model of developing epithelium, here we report that Netrin-4 is abundantly expressed in vascular endothelial cells and pancreatic ductal cells, and supports epithelial cell adhesion through integrins α2β1and α3β1. Interestingly, we find that Netrin-4 recognition by embryonic pancreatic cells through integrins α2β1 and α3β1 promotes insulin and glucagon gene expression. In addition, full genome microarray analysis revealed that fetal pancreatic cell adhesion to Netrin-4 causes a prominent down-regulation of cyclins and up-regulation of negative regulators of the cell cycle. Consistent with these results, a number of other genes whose activities have been linked to developmental decisions and/or cellular differentiation are up-regulated. Conclusions/Significance Given the recognized function of blood vessels in epithelial tissue morphogenesis, our results provide a mechanism by which endothelial-derived Netrin-4 may function as a pro-differentiation cue for adjacent developing pancreatic cell populations expressing adhesion receptors α2β1 and α3β1 integrins.

Inhibitors of Src and Focal Adhesion Kinase Promote Endocrine Specification IMPACT ON THE DERIVATION OF β-CELLS FROM HUMAN PLURIPOTENT STEM CELLS

Background: Signaling mechanisms regulating the endocrine specification of stem cell derivatives. Results: Inhibiting Src family kinase (SFK) and focal adhesion kinase (FAK) activity promotes endocrine commitment. Conclusion: Select inhibitors of SFK/FAK signaling increase the derivation of β-cells. Significance: Enhanced derivation of insulin-producing β-cells for the cell-based therapy of diabetes. Stepwise approaches for the derivation of β-cells from human embryonic stem cells have been described. However, low levels of endocrine specification limit the final yield of insulin-producing β-cells. In this study, we show that the pyrrolo-pyrimidine Src family kinase (SFK) inhibitor PP2 effectively promotes the endocrine specification of human embryonic stem cell derivatives based on its capacity to induce the expression of proendocrine transcription factors (NGN3, NEUROD1, NKX2.2, and PAX4) and to significantly increase the final yield of insulin-positive cells. We further demonstrate that PP2 inhibits the activation of focal adhesion kinase (FAK), and selective inhibition of this kinase is also sufficient to induce early endocrine commitment based on increased expression of NGN3, NEUROD1, and NKX2.2. Additional studies using dominant negative constructs and isolated human fetal pancreata suggest that c-Src is at least partially responsible for inhibiting early endocrine specification. Mechanistically, we propose that inhibition of SFK/FAK signaling can promote endocrine specification by limiting activation of the TGFβR/Smad2/3 pathway. Moreover, we show that inhibition of SFK/FAK signaling suppresses cell growth, increases the expression of the β-cell-associated cyclin-dependent kinase inhibitor p57kip2, and simultaneously suppresses the expression of Id1 and Id2. This study has important implications for the derivation of β-cells for the cell-based therapy of diabetes and sheds new light on the signaling events that regulate early endocrine specification.

Impact of integrin–matrix interaction and signaling on insulin gene expression and the mesenchymal transition of human β-cells†

A critical shortage of donor pancreata currently prevents the development of a universal cell‐based therapy for type I diabetes. The ex vivo expansion of insulin‐producing β‐cells offers a potential solution but is problematic due to the inherent tendency of these cells to transition into mesenchymal‐like cells that are devoid of function. Here, we demonstrate for the first time that exposure to elements of the extracellular matrix (ECM) directly potentiates the mesenchymal transition of cultured fetal β‐cells and causes associated declines in insulin gene expression. Individual ECM constituents varied in their ability to induce such responses, with collagen‐IV (C‐IV) and fibronectin inducing strong responses, whereas laminin‐1 had no significant effect. Mesenchymal transition and concomitant losses in insulin gene expression observed on C‐IV were found to be dependent on β1‐integrin ligation and were augmented in the presence of hepatocyte growth factor. Importantly, selective inhibition of c‐Src, c‐Jun N‐terminal kinase (JNK), and extracellular signal‐regulated kinase (ERK) prior to exposure to C‐IV prevented mesenchymal transition and effectively preserved insulin expression. Fetal β‐cells undergoing mesenchymal transition were found to acquire α1β1 expression, and ligation of this integrin then promotes declines in insulin gene expression and a marked increase in β‐cell motility. Inhibition of Src‐, ERK‐, or JNK‐dependent signaling combined with the selective regulation of matrix exposure may ultimately facilitate the development of more effective β‐cell expansion protocols. J. Cell. Physiol. 224:101–111, 2010