Fluid Shear Stress Stimulates Mitogen-Activated Protein Kinase in Endothelial Cells

Abstract

Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. Because mitogen-activated protein (MAP) kinases have been shown to be activated by physical forces, we measured the phosphorylation and enzyme activity of MAP kinase to identify the signal events involved in the endothelial cell response to fluid shear stress. Flow at physiological shear stress (3.5 to 117 dynes/cm2) activated 42-kD and 44-kD MAP kinases present in cultured bovine aortic endothelial cells, with maximal effect at 12 dynes/cm2. Activation of a G protein was necessary, as demonstrated by complete inhibition by the nonhydrolyzable GDP analog GDP-beta S. Activation of protein kinase C (PKC) was required, as shown by inhibiting PKC with staurosporine or downregulating PKC with phorbol 12,13-dibutyrate. Both Ca(2+)-dependent and -independent PKC activity, measured by translocation and substrate phosphorylation, increased in response to flow. However, MAP kinase activation was not dependent on Ca2+ mobilization, since Ca2+ chelation had no inhibitory effect. On the basis of these findings, it is proposed that flow activates two signal-transduction pathways in endothelial cells. One pathway is Ca2+ dependent and involves activation of phospholipase C and increases in intracellular Ca2+. A new pathway, described in the present study, is Ca2+ independent and involves a G protein and increases in PKC and MAP kinase activity.

Inhibition of MAP kinase kinase (MEK) blocks endothelial PGI2 release but has no effect on von Willebrand factor secretion or E‐selectin expression

“…In the present study, inhibition of basal and agonist-induced p42 m~pk activation by PD98059 was accompanied by a parallel inhibition of PGI2 release in both 'resting' and stimulated HUVEC. Our ability to detect both p42 m~pk activation and PGI2 release in the absence of agonist is not unexpected; replacement of medium above endothelial cell monolayers subjects the cells to shear stress which is known to promote PGI2 release [23] and to enhance phosphorylation and activation of p42 m~pk [19,20]. Taken together these results demonstrate a central role for p42 m~pk in the control of PGI2 synthesis/release in HUVEC.…”

“…Growing evidence from a number of cell types, including terminally differentiated cells, additionally implicates p42 m~pk in the control of acute responses to receptor stimulation such as exocytosis and arachidonic acid release [14,15]. In endothelial cells p42 mapk can be activated by growth factors [16], GPCA [17,18], shear stress [19,20] or inflammatory cytokines (this study) but the functional relevance of stimulus-induced changes in p42 mapk remains to be determined. In the present study we used a novel inhibitor of MEK to explore the potential involvement of p42 mapk in thrombin-driven PGI2 synthesis and in cytokineinduced expression of E-selectin.…”

Atherosclerosis and calcium signalling in endothelial cells

“…It is well known that the physical forces exerted on EC by fluid flow result in an internal signalling response (Geiger et al, 1992;Tseng et al, 1995). The mechanism whereby the cell detects and responds to the mechanical stimulus is unclear, but there are two (not mutually exclusive) possibilities that have gained some biological evidence.…”

“…The mechanism whereby the cell detects and responds to the mechanical stimulus is unclear, but there are two (not mutually exclusive) possibilities that have gained some biological evidence. One is the existence of Ca 2 + channels in the plasma membrane that are directly gated by shear stress (Kwan et al, 2003); the second is a shear-sensitive mechano-receptor that is linked to the IPa signalling pathway (Tseng et al, 1995). However, very few data exist regarding mechano-transduction via IPa in EC, so we focus on the first of these mechanisms.…”

“…Wall shear stress appears to contribute to Ca 2 + signalling by activating plasma membrane Ca 2 + channels, thus allowing further influx (Kwan et al, 2003). Wall shear stress may also activate a mechano-sensitive surface receptor linked to the IP3 signalling pathway, thus increasing the rate of internal Ca 2 + release (Tseng et al, 1995). Further details of the Ca 2 + signalling response to biochemical and mechanical stimuli may be found in section 3.…”