PLASMA-MEMBRANE CHOLESTEROL IS A KEY MOLECULE IN SHEAR STRESS-DEPENDENT ACTIVATION OF EXTRACELLULAR SIGNAL-REGULATED KINASE

Shear stress, the dragging force generated by fluid now, differentiall y activates extracellular signal-regulated kinase (ERK) and c-Jun NH2- terminal kinase (JNK) in bovine aortic endothelial cells (BAEC) (Jo, H ., Sipos, IL, Go, Y. M, Law, R, Rong, J., and McDonald, J.M. (1997) J. Biol. Chem. 272, 1395-1401). Here, we examine whether cholesterol-enn iched compartments in the plasma membrane are responsible for such dif ferential regulation. Pretreatment of BAEC with a cholesterol-binding antibiotic, filipin, did not inhibit shear-dependent activation of JNK In contrast, filipin and other membrane-permeable cholesterol-binding agents (digitonin and nystatin), but not the lipid-binding agent xyla zine, inhibited shear-dependent activation of ERK The effect of choles terol-binding drugs did not appear to be due to membrane permeabilizat ion, since treatment of BAEC with a detergent, Triton X-100 which also permeabilizes membranes, did not inhibit shear-dependent activation o f ERK. Furthermore, shear-dependent activation of ERK but not JNK, was inhibited by cyclodextrin, a membrane-impermeable cholesterol-binding agent, which removes cell-surface cholesterol Moreover, the effects o f cyclodextrin were prevented by adding cholesterol during the incubat ion. These results indicate that cholesterol or cholesterol-sensitive compartments in the plasma membrane play a selective and essential rol e in activation of ERK but not JNK by shear stress. Although exposure to shear stress (1 h) increased the number of caveolae by 3-fold, trea tment with filipin had no effect in either control or shear-exposed ce lls suggesting that caveolae density per se is not a crucial determina nt in shear-dependent ERK activation In summary, the current study sug gests that cholesterol-sensitive microdomains in the plasma membrane, such as caveolae-like domains,play a critical role in differential act ivation of ERK and JNK by shear stress.