The role of Shb in ES cell differentiation, angiogenesis and tumor growth

Abstract: Shb is a ubiquitously expressed adaptor protein with the ability to bind several tyrosine kinase receptors and intracellular signaling proteins. Previous studies have implied a wide spectrum of Shb-mediated cellular responses, which motivated me to further investigate the role of Shb in differentiation and angiogenesis. Embryonic stem (ES) cells differentiate into endoderm and mesoderm from a bipotent mesendodermal cell population. Interregulatory signals between these germlayers are required for further specification. ES cells overexpressing Shb with an inactive SH2 domain (R522K-Shb) altered the expression of endodermal genes as a consequence of upregulated FGF expression. This response was enhanced by addition of activin A, suggesting a synergistic mechanism operative between FGF and activin A signaling in endoderm specification. To investigate a role for Shb in mesodermal specification, Shb knockout ES cells were established. These cells showed a reduced ability to form blood vessels after VEGF stimulation and delayed downregulation of genes associated with mesendoderm, indicating a reduced capacity for these cells to enter later stages.To assess a role for Shb in tumor cell apoptosis, Shb expression was silenced in angiosarcoma endothelial cells. FAK-phosphorylation was reduced in Shb knockdown cells and this made them more susceptible to apoptotic stimuli both in vitro and in vivo.Shb knockout microvasculature in mouse kidney, liver, and heart showed irregular endothelial linings with cytoplasmic projections toward the lumen, a feature that was also related to increased vascular permeability. VEGF treatment failed to stimulate vascular permeability in Shb knockout mice.In order to elucidate whether these features relate to reduced angiogenesis, tumor growth was examined. Tumors grown in knockout mice showed reduced growth capacity and lower vessel density. In conclusion, Shb is a multifunctional adaptor protein that may be involved in several cellular responses both during embryonic development and adult life.