Exogenous HSP70 becomes cell associated, but not internalized, by stressed arterial smooth muscle cells.
Cell death within atherosclerotic plaques leads to necrosis and rupture, resulting in vascular occlusion. We have previously demonstrated that addition of exogenous 70 kDa heat shock protein (HSP70) to arterial smooth muscle cells (aSMCs) in vitro can protect against toxins that may initiate necrosis. To determine whether exogenous HSP70 enters aSMCs or acts from outside cells to preserve viability, cultured rabbit aSMCs were stressed by serum deprivation and treated with fluorescently labeled (7-aminomethyl-4-coumarin-3-acetate) or 125I-radiolabeled HSP70. Cell-associated HSP70 was analyzed using Western blotting, fluorescence spectroscopy, and gamma counting/autoradiography. Surface binding of HSP70 to aSMCs was differentiated from uptake by using trypsin treatment to degrade non-internalized HSP70. Specificity of HSP70 binding was tested by inhibiting uptake of 125I-HSP70 with excess unlabeled HSP70 or bovine serum albumin (BSA). The effect of unlabeled exogenous HSP70 on endogenous HSP synthesis was also tested. Exogenous HSP70 increased total cell-associated HSP70 2.9- to 3.6-fold over levels present in unstressed aSMCs. However, < 5% of the exogenous HSP70 was trypsin-insensitive, indicating that bound HSP70 was not internalized. Binding of 125I-HSP70 was inhibited by both unlabeled HSP70 and BSA, implying a non-specific interaction with the plasmalemma. Exogenous HSP70 significantly lowered overall protein synthesis by serum-deprived aSMCs, but it did not specifically inhibit synthesis of endogenous HSPs after heat shock. The results indicate that exogenous HSP70 protects viability of stressed aSMCs through interactions with the cell surface rather than via internalization.
Johnson
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Department of Neurobiology and Anatomy, Bowman Gray School of Medicine of Wake Forest University, Winston-Salem, North Carolina 27157.
Tytell
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D000818:Animals; D001158:Arteries; D001665:Binding Sites; D002417:Cattle; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D006360:Heat-Shock Proteins / Q000378:metabolism* / Q000494:pharmacology; D008297:Male; D009131:Muscle, Smooth, Vascular / Q000166:cytology / Q000378:metabolism*; D011817:Rabbits
D006360:Heat-Shock Proteins