Activation of Platelet-activating Factor Receptor-coupled Gαq Leads to Stimulation of Src and Focal Adhesion Kinase via Two Separate Pathways in Human Umbilical Vein Endothelial Cells

Abstract

Platelet-activating factor (PAF), a phospholipid second messenger, has diverse physiological functions, including responses in differentiated endothelial cells to external stimuli. We used human umbilical vein endothelial cells (HUVECs) as a model system. We show that PAF activated pertussis toxin-insensitive G␣ q protein upon binding to its seven transmembrane receptor. Elevated cAMP levels were observed via activation of adenylate cyclase, which activated protein kinase A (PKA) and was attenuated by a PAF receptor antagonist, blocking downstream activity. Phosphorylation of Src by PAF required G␣ q protein and adenylate cyclase activation; there was an absolute requirement of PKA for PAFinduced Src phosphorylation. Immediate (1 min) PAFinduced STAT-3 phosphorylation required the activation of G␣ q protein, adenylate cyclase, and PKA, and was independent of these intermediates at delayed (30 min) and prolonged (60 min) PAF exposure. PAF activated PLC␤3 through its G␣ q protein-coupled receptor, whereas activation of phospholipase C␥1 (PLC␥1) by PAF was independent of G proteins but required the involvement of Src at prolonged PAF exposure (60 min). We demonstrate for the first time in vascular endothelial cells: (i) the involvement of signaling intermediates in the PAF-PAF receptor system in the induction of TIMP2 and MT1-MMP expression, resulting in the coordinated proteolytic activation of MMP2, and (ii) a receptor-mediated signal transduction cascade for the tyrosine phosphorylation of FAK by PAF. PAF exposure induced binding of p130 Cas , Src, SHC, and paxillin to FAK. Clearly, PAF-mediated signaling in differentiated endothelial cells is critical to endothelial cell functions, including cell migration and proteolytic activation of MMP2.

Alveolar Macrophages from Septic Mice Promote Polymorphonuclear Leukocyte Transendothelial Migration via an Endothelial Cell Src Kinase/NADPH Oxidase Pathway

“…Previous studies have implicated various individual components of this pathway in endothelial cell oxidant stress and/or monocyte adhesive interactions with endothelium induced by pharmacologic agents (e.g., LPS, PAF, and H 2 O 2 ) (33,46,(57)(58)(59). The significance of our findings is 1) the use of an in vitro construct of the pulmonary vascular-interstitial interface using cells relevant to sepsis-induced ALI/ARDS (e.g., AM), and 2) the sequential positioning of these components into a pathway responsible for endothelial-dependent PMN migration.…”

In spermatozoa, Stat1 is activated during capacitation and the acrosomal reaction

“…Thus, results in capacitation clearly show that for cytosolic Stat1 translocation to occur, all PKA, PKC, and calcium are needed to promote the phosphorylation of Stat1. It is well known that PKA activation increases tyrosine phosphorylation; also the phosphorylated PKA participates in Stat tyrosine phosphorylation (Deo et al 2004, Sakhalkar et al 2005. It is quite possible that PKA, PKC, and calcium are involved (Breitbart 2002).…”

“…In the short term, this would result in the first calcium waves. If that were the case, it would indicate that two different signal pathways converge in a common substrate: a candidate is Stat1 by itself, and another could be a Src-family member (Chang et al 2004, Deo et al 2004, Salonen et al 2006, like c-yes (Leclerc & Goupil 2002). In addition, another non-receptor tyrosine kinase like PYK2 might be involved (Takaoka et al 1999, Chieffi et al 2003.…”

Sequential activation of JAKs, STATs and xanthine dehydrogenase/oxidase by hypoxia in lung microvascular endothelial cells

“…Phosphorylated STATs in nucleus function as transcriptional factors activating transcriptional expression of a number of genes (Levy & Darnell, 2002;Vinkemeier, 2004). STATs are critical mediators of signal transduction and transcription in many cell types, including human umbilical vein endothelial cells, murine aortic endothelial cells and smooth muscle cells (Levy & Darnell, 2002;Vinkemeier, 2004;Deo et al, 2004;Ni et al, 2004). However, whether hypoxia could induce IL release and subsequently activate JAK-STAT pathway and the role of the JAK-STAT signaling pathway in the activation of XDH/XO in lung microvascular endothelial cells (LMVEC), which are critical targets in both hypoxia-and regeneration-mediated lung injury, have not been defined.…”