Cellular Biology The Ras Activator RasGRP3 Mediates Diabetes-Induced Embryonic Defects and Affects Endothelial Cell Migration

Rationale: Fetuses that develop in diabetic mothers have a higher incidence of birth defects that include cardiovascular defects, but the signaling pathways that mediate these developmental effects are poorly understood. It is reasonable to hypothesize that diabetic maternal effects are mediated by 1 or more pathways activated downstream of aberrant glucose metabolism, because poorly controlled maternal glucose levels correlate with the frequency and severity of the defects. Objective: We investigated whether RasGRP3 (Ras guanyl-releasing protein 3), a Ras activator expressed in developing blood vessels, mediates diabetes-induced vascular developmental defects. RasGRP3 is activated by diacylglycerol, and diacylglycerol is overproduced by aberrant glucose metabolism in diabetic individuals. We also investigated the effects of overactivation and loss of function for RasGRP3 in primary endothelial cells and developing vessels. Conclusions: These findings provide the first evidence that RasGRP3 contributes to developmental defects found in embryos that develop in a diabetic environment. The results also elucidate RasGRP3-mediated signaling in endothelial cells and identify endothelin-1 as an upstream input and Ras/MEK/ERK as a downstream effector pathway. RasGRP3 may be a novel therapeutic target for the fetal complications of diabetes. R eceptor-mediated signaling is required for endothelial cell proliferation and migration, processes that are critical to blood vessel formation and function. 1 Signaling downstream of receptor engagement in endothelial cells leads to the activation of several pathways, including phospho-lipase C-␥ activation, to produce diacylglycerol (DAG). DAG in turn activates downstream targets to affect endothelial cell behaviors. Signals that are upregulated in diabetes, such as vascular endothelial growth factor-A (VEGF-A) and endothelin-1 (ET1), generate DAG. DAG is also produced by aberrant glucose metabolism, and DAG levels are elevated in diabetic animals and patients. 2,3 Diabetic individuals have compromised angiogenesis and blood vessel function, and fetuses of diabetic mothers have an increased incidence of birth defects, including vascular defects. 3,4 It is assumed that these vascular defects are mediated, at least in part, by elevated DAG levels, because mouse embryos recovered from diabetic mothers had elevated DAG levels and increased developmental defects 3 ; however, it is not fully understood how elevated DAG leads to vessel dysfunction. The effects of DAG on cell signaling are mimicked by phorbol esters, tumor promoters that also affect endothelial proliferation, cellular morphology, apoptosis, and barrier function. 5–9 DAG and phorbol esters are potent activators of the protein kinase C (PKC) family of proteins. Numerous mamma-lian PKC isoforms fall into several subfamilies, and DAG/ …