Shb (Src homology 2 protein B) is an adapter protein downstream of the vascular endothelial growth factor receptor receptor-2

The Shb adapter protein is a signaling intermediate that operates downstream of vascular endothelial growth factor receptor-2 (VEGFR-2) in endothelial cells. The Shb knockout mouse displays a dysfunctional microvasculature and impaired growth of subcutaneously implanted tumor cells. We decided to investigate tumor growth and angiogenesis in the absence of Shb in an inheritable tumor model, the RIP-Tag2 mouse, which produces insulinomas in a manner highly dependent on de novo angiogenesis. We observed a reduced tumor incidence and burden in both RIP-Tag2 Shb−/− and RIP-Tag2 Shb+/− mice. This correlated with a reduced microvascular density, measured as a percentage of insulinoma area positive for CD31 staining, and altered vascular morphology. However, treatment with a VEGFA blocking antibody was without effect on the Shb mutant tumor volume whereas it significantly inhibited tumor volume in the wild-type mice, suggesting that in mice with reduced Shb expression tumor angiogenesis was primarily sustained by VEGF-A independent pathway(s). This notion was further substantiated by gene expression analysis of angiogenic markers showing reduced VEGF-A expression in Shb-deficient tumors. Considerable heterogeneity with respect to the gene expression profiles of other angiogenic markers and the signal-transduction characteristics was observed between different tumors, suggesting that multiple “rescue” pathways could be operating. The numbers of invasive tumors or metastases were unchanged in the Shb mutant. It is concluded that the Shb mutant background reduces tumor frequency by chronically suppressing VEGF-A dependent angiogenesis. However, VEGF-A independent angiogenesis supports a significant degree of tumor expansion in Shb-deficient mice, indicating heterogeneity in the mechanisms by which tumor expansion is promoted. Interference with Shb signaling may provide novel means for future cancer therapy.

This review will describe the SH2-domain signaling protein Src Homology-2 domain containing protein B (SHB) and its role in various physiological processes relating in particular to glucose homeostasis and b cell function. SHB operates downstream of several tyrosine kinase receptors and assembles signaling complexes in response to receptor activation by interacting with other signaling proteins via its other domains (proline-rich, phosphotyrosine-binding and tyrosine-phosphorylation sites). The subsequent responses are context-dependent. Absence of Shb in mice has been found to exert effects on hematopoiesis, angiogenesis and glucose metabolism. Specifically, first-phase insulin secretion in response to glucose was impaired and this effect was related to altered characteristics of focal adhesion kinase activation modulating signaling through Akt, ERK, b catenin and cAMP. It is believed that SHB plays a role in integrating adaptive responses to various stimuli by simultaneously modulating cellular responses in different cell-types, thus playing a role in maintaining physiological homeostasis.

Hallmarks of the inflammatory process in Type I diabetes are macrophage activation, local release of beta-cell-toxic cytokines and infiltration of cytotoxic T lymphocytes. We have observed recently that mice overexpressing active FRK (fyn-related kinase)/RAK (previously named GTK/Bsk/IYK, where GTK stands for gut tyrosine kinase, Bsk for beta-cell Src-homology kinase and IYK for intestinal tyrosine kinase) in beta-cells exhibit increased susceptibility to beta-cell-toxic events, and therefore, we now attempt to find a more precise role for FRK/RAK in these processes. Phosphopeptide mapping of baculovirus-produced mouse FRK/RAK revealed an autophosphorylation pattern compatible with Tyr-394 being the main site. No evidence for in vitro phosphorylation of the C-terminal regulatory sites Tyr-497 and Tyr-504 was obtained, nor was there any indication of in vitro regulation of FRK/RAK kinase activity. Screening a panel of known tyrosine kinase inhibitors for their ability to inhibit FRK/RAK revealed several compounds that inhibited FRK/RAK, with a potency similar to that reported for their ability to inhibit other tyrosine kinases. Cytokine-induced islet toxicity was reduced in islets isolated from FRK/RAK knockout mice and this occurred without effects on the production of nitric oxide. Addition of the nitric oxide inhibitor nitroarginine to FRK/RAK knockout islets exposed to cytokines decreased cell death to a basal level. In normal islets, cytokine-induced cell death was inhibited by the addition of two FRK/RAK inhibitors, SU4984 and D-65495, or by transfection with short interfering RNA against FRK/RAK. It is concluded that FRK/RAK contributes to cytokine-induced beta-cell death, and inhibition of this kinase could provide means to suppress beta-cell destruction in Type I diabetes.

To cite this version:Björn Åkerblom, Cecilia Annerén, Michael Welsh. A role of frk in regulation of embryonal pancreatic beta cell formation. Molecular and Cellular Endocrinology, Elsevier, 2007, 270 (1-2) This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.A c c e p t e d M a n u s c r i p t A c c e p t e d M a n u s c r i p t 2 SummaryThe Fyn-Related-Kinase is a non-receptor tyrosine kinase expressed in various tissues, among them, the islets of Langerhans. The role of FRK in pancreatic beta cells has been addressed by studies of knockout or FRK-transgenic mice. These experiments have shown that FRK overexpression in beta cells leads to an increased susceptibility to the beta cell toxin streptozotocin and to cytotoxic cytokines, suggesting that FRK may participate in events leading to beta cell destruction.However, these mice also exhibit an increased relative beta cell volume and increased beta cell replication following partial pancreatectomy, suggesting a positive role for FRK in the regulation of beta cell number as well.To further assess the significance of FRK for beta cell replication, we studied the beta cell area and islet cell replication in FRK null mice. We currently observe that the FRK-knockout mouse showed no difference in the insulin positive cell area or in the percentage of Ki67-stained proliferating islet cells at adulthood, when compared to wild-type control. In addition, adult FRK-/-mice performed normally when subjected to an intravenous glucose tolerance test.To elucidate whether FRK affects pancreatic beta cell number during embryogenesis and shortly after birth, pancreata were collected from FRK-/-mice at these stages.Histological analysis of insulin stained pancreatic sections showed that the insulin positive cell area in FRK-/-mice was reduced at embryonal day 15 and at birth to 31% and 70% of that of wild-type mice, respectively. FRK -/-pancreas weight on day 1 neonatally was similar to that of the control, indicating that the obtained resultswere not due to altered pancreatic growth.Taken together these results show that FRK affects beta cell number during embryogenesis and early in life, but is probably redundant for beta cell number and function in adult animals under normal conditions.