Shear stress regulates occludin content and phosphorylation

1 Biomolecular Transport Dynamics Laboratory, Department of Chemical Engineering, The Pennsylvania State University, University Park 16802; and Departments of 2 Cellular and Molecular Physiology and 3 Ophthalmology, College of Medicine, Hershey, Pennsylvania 17033 Previous studies determined that shear stress imposed on bovine aortic endothelial cell (BAEC) monolayers increased the hydraulic conductivity ( L P ); however, the mechanism by which shear stress increases L P remains unknown. This study tested the hypothesis that shear stress regulates paracellular transport by altering the expression and phosphorylation state of the tight junction protein occludin. The effect of shear stress on occludin content was examined by Western blot analysis. Ten dyn/cm 2 significantly reduced occludin content in a time-dependent manner such that after a 3 h exposure to shear, occludin content decreased to 44% of control. Twenty dyn/cm 2 decreased occludin content to 50% of control and increased L P by 4.7-fold after 3 h. Occludin expression and L P depend on tyrosine kinase activity because erbstatin A (10 µM) attenuated both the shear-induced decrease in occludin content and increase in L P . Shear stress increased occludin phosphorylation after 5 min, 15 min, and 3 h exposures. The shear-induced increase in occludin phosphorylation was attenuated with dibutyryl (DB) cAMP (1 mM), a reagent previously shown to reverse the shear-induced increase in L P . We conclude that shear stress rapidly ( 5 min) increases occludin phosphorylation and significantly decreases the expression of occludin over 1-4 h. Alterations in the occludin phosphorylation state and occludin total content are potential mechanisms by which shear stress increases L P . erbstatin A; dibutyryl cAMP; permeability; tight junctions