Fyn mediates stretch- and flow-elicited PECAM-1 phosphorylation in ECs. ECs cultured on an elastic substrate were transiently transfected with Src, Yes, or Fyn siRNA or Lipofectamine 2000 alone. When confluent monolayers were formed, cells were stretched in the presence of Na3VO4 for 10 min and PECAM-1 tyrosine phosphorylation was assayed. Expression of each kinase was checked in whole cell lysates by immunoblotting using actin as a loading control. (A) PECAM-1 phosphorylation was not suppressed in HAECs transfected with Src siRNA. (B) Levels of PECAM-1 phosphorylation in Src siRNA-treated HAECs are quantified and shown relative to unstretched cells (mean ± SEM; n = 4). Student's t test was used to compare stretched to unstretched values. *, P = 0.0315 (LF control) and 0.0264 (Src siRNA). (C) Reduced expression of Yes in BAECs did not suppress PECAM-1 phosphorylation. (D) PECAM-1 phosphorylation was suppressed in BAECs transfected with two different Fyn siRNAs. (E) Levels of PECAM-1 phosphorylation in Yes and Fyn siRNAs treated cells are quantified and shown relative to unstretched cells (mean ± SEM). The sample sizes are n = 4 for Lipofectamine control and Yes siRNA, n = 3 for Fyn siRNA-2, and n = 5 for Fyn siRNA-1. Student's t test was used to compare stretched to unstretched values for each category. *, P = 0.0002 (Lipofectamine control) and 9.8 × 10−7 (Yes siRNA). #, P = 0.1493 (Fyn siRNA-2) and 0.4875 (Fyn siRNA-1). (F) Confluent BAECs transiently transfected with Fyn siRNA or Lipofectamine 2000 alone were subjected to flow (24 dyn/cm2) in the presence of Na3VO4 for 10 min at 37°C, and PECAM-1 phosphorylation was assayed. Flow-induced PECAM-1 phosphorylation was suppressed in Fyn siRNA-treated cells. (G) Quantified results of flow experiments were expressed as the mean ± SEM (n = 4). Student's t test was used to compare shear stressed to static control values. *, P = 0.0311; #, P = 0.0532. (H) Fyn activity was analyzed in BAECs subjected to stretch or flow for 10 min by immunoprecipitation with anti-Fyn and immunoblot with antibodies that recognize the active form of Src family kinases (pY418 SFK). Note that mechanical stress does not increase Fyn activity. Note also that Fyn is active in unstimulated (unstretched and static) cells. One of three independent experimental results is depicted. Black lines indicate that intervening lanes have been spliced out.
