Activated protein C (APC), a plasma serine protease, exerts both anticoagulant and cytoprotective signaling in vitro and in vivo. APC protects cells from apoptosis, stress, and inflammation; it also can reduce or prevent endothelial barrier leakage as well as murine death from lethal levels of radiation. APC induces selective alterations of gene expression, provides neuroprotection following ischemic stroke, and minimizes damage in chronic neurodegenerative disease animal models. When given as bolus doses, APC reduces death in animals from severe sepsis. Receptors that mediate APC-induced beneficial signaling include endothelial cell protein C receptor (EPCR), PAR1, PAR3, integrins and Tie2. We discovered that apolipoprotein E receptor 2 (ApoER2) (aka LRP8), a lipoprotein receptor, which is well known for its effects in the Reelin-signaling pathway for neurons, has remarkable APC-dependent cytoprotective actions for the monocytic U937 cell line. In neurons, ligation of ApoER2 triggers phosphorylation of disabled-1 (Dab1) by Src family kinases that further promulgate signaling via PI3K, Akt and GSK3beta. Thus, here we report studies that test and support the hypothesis that ApoER2 mediates APC-induced cytoprotective signaling in EA.hy926 endothelial cells, which involves the adaptor protein, Dab1, as well as Src family members, Akt, and GSK3beta. We used siRNA and inhibitors to probe requirements for specific receptors for phosphorylation of key signaling pathway components. APC treatment of endothelial cells induced a 2 to 3-fold increase in phosphorylation of Akt at Ser473 and GSK3beta at Ser9 over 30 to 180 min after APC treatment, as well as a substantial rapid increase in ERK1/2 phosphorylation within 5 min of APC treatment. In purified systems, APC binds with good affinity to ApoER2. APC treatment of endothelial cells caused phosphorylation of Dab1 at Tyr232, a phosphorylation known to trigger Dab1-mediated signaling in other types of cells. Knocking down of ApoER2 using siRNA reduced GSK3beta and ERK1/2 phosphorylation by circa 30–40%. Combined knockdown of EPCR and ApoER2 reduced GSK3beta and ERK1/2 phosphorylation by 50–75%. Additional studies implicated involvement of PI3K and Src kinases. When cells were pretreated with PP2, a Src kinase inhibitor, Y232-Dab1 phosphorylation was reduced by > 50%, consistent with the primary paradigm for apoER2-mediated signaling involving phosphorylations of Dab1 and Src-family kinases. Moreover, the PAR1 antagonist SCH79797 markedly reduced APC-induced phosphorylation of Ser473-Akt, Ser9-GSK3beta and ERK1/2, showing that PAR1 was also required for the full extent of these effects. Hence, both PAR1 proteolysis by APC and ligation of ApoER2 by APC appear to contribute significantly and simultaneously to signaling in this endothelial model system. In summary, these data indicate that apoER2 is an endothelial cell receptor that contributes, with other better recognized receptors, to the spectrum of APC-induced signaling that is integrated by the endothelial cell system to achieve one or more of the multiple cytoprotective effects of APC. Thus, there is a remarkable ensemble of potential endothelial receptors for APC that could variably mediate a spectrum of APC's cytoprotective effects.
No relevant conflicts of interest to declare.
Asterisk with author names denotes non-ASH members.