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The Effect Of Shear Stress On Phosphorylation Of PKC ζ In the Membrane Type 1 Matrix Metalloproteinase Signaling Pathway

Introduction and Objectives:Previous studies of our laboratory have shown the increased expression of membrane type 1 matrix metalloproteinase (MT1-MMP) with cyclic strain, but not with shear stress. This phenomenon has been attributed to the increased affinity of Sp1 for MT1-MMP’s promoter site. The atypical protein kinase C (PKC) has been proposed to be responsible for the serine phosphorylation of Sp1,which can increase its affinity. The aim of this study was to determine the effect of PKC ζ on serine phosphorylation and activation of Sp1 in response to shear stress.
Methods:Rat microvascular endothelial cell (MVEC) were cultured and exposed to oscillatory shear stress at 14 dynes/cm2. PKC-ζ were assessed through immunoblotting analysis and by performing activity assays. As PKC-ζ inhibitor, we used chelerytrine chloride and Go6983 . Serine phosphorylation of Sp1, with and without PKC-ζ inhibitor, was determined by immunoprecipitation followed by an anti-phosphoserine-specific antibody (Ab). MT1-MMP protein levels, with and without PKC-ζ inhibitor, were assessed by immunoblotting analysis
Results: Shear stress increased PKC- ζ protein expression and activity (1.73±0.3, 1.4±0.3, and 1.2±0.1 in protein expression, 1.9±0.3, 1.7±0.4, and 1.4±0.2 in activity at 1, 4, and 8h, respectively; n=3, p<0.05). Shear stress stimulation of EC led to an increased Sp1 phosphorylation (1.8-, 3.3-, and 1.8-fold, at 1,4,and 8h n = 2),but when PKC-ζ inhibitor was added, there was no change in the phosphorylation state of Sp1. Shear stress down-regulated MT1-MMP protein in a time-dependent fashion (19%, 45%, and 48% reduction at 1, 4 and 8 h, respectively; n=4,p< 0.05). but MT1-MMP protein level was not affected by shear stress when chelerytrine chloride or Go6983 was added to EC.
Conclusions: These data demonstrate shear stress activation of PKC ζ which increases serine phosphorylation of Sp1. this may be responsible for the decrease in MT1-MMP expression in EC exposed to shear stress.