Modulation of the Stress‐Induced Synthesis of hsp27 and αB‐Crystallin by Cyclic AMP in C6 Rat Glioma Cells

Abstract

The possible participation of cyclic AMP in the stress‐induced synthesis of two small stress proteins, hsp27 and αB‐crystallin, in C6 rat glioma cells was examined by specific immunoassays, western blot analysis, and northern blot analysis. When C6 cells were exposed to arsenite (50–100 µM for 1 h) or heat (42°C for 30 min), expression of hsp27 and αB‐crystallin was stimulated, with levels of the two proteins reaching a maximum after 10–16 h of culture. Induction of hsp27 was markedly enhanced when cells were exposed to arsenite in the presence of isoproterenol (20 µM) or epinephrine (20 µM) but not in the presence of phenylephrine. The stimulatory effects of isoproterenol and epinephrine were blocked completely by propranolol, an antagonist of β‐adrenergic receptors. Cholera toxin (2 µg/ml), forskolin (20 µM), and dibutyryl cyclic AMP (2.5 mM), all of which are known to increase intracellular levels of cyclic AMP, also stimulated the arsenite‐ or heat‐induced accumulation of hsp27. Treatment of cells with each of these modulators alone did not result in the induction of hsp27. The level of hsp70 in C6 cells, as estimated by western blot analysis, was also enhanced by arsenite or heat stress. However, induction of hsp70 by stress was barely stimulated by isoproterenol. By contrast, induction of αB‐crystallin by heat or arsenite stress was suppressed when isoproterenol, cholera toxin, forskolin, or dibutyryl cyclic AMP was present during the stress period. Northern blot analysis of the expression of mRNAs for hsp70, hsp27, and αB‐crystallin showed that the modulation of the stress‐induced accumulation of the three hsps by the various agents was regulated at the level of the corresponding mRNA. These results indicate that stress responses of hsp70, hsp27, and αB‐crystallin in C6 rat glioma cells are regulated differently and, moreover, that when the level of cyclic AMP increases in cells, the response to stress of hsp27 is stimulated but that of αB‐crystallin is suppressed.

Rho-kinase regulates thrombin-stimulated interleukin-6 synthesis via p38 mitogen-activated protein kinase in osteoblasts

“…A Western blot analysis was performed as previously described (21). The cultured cells were stimulated by thrombin or vehicle in α-MEM containing 0.3% FCS for the indicated periods.…”

AICAR reduces the collagen-stimulated secretion of PDGF-AB and release of soluble CD40 ligand from human platelets: Suppression of HSP27 phosphorylation via p44/p42 MAP kinase

“…For western blot analysis, the pellet was washed twice with phosphate-buffered saline, lysed and immediately boiled in a lysis buffer containing 62.5 mM Tris/Cl (pH 6.8), 2% sodium dodecyl sulfate (SDS), 50 mM dithiothreitol and 10% glycerol. Western blot analysis was performed as previously described (24). Briefly, proteins were separated by SDS-polyacrylamide gel electrophoresis PAGE according to the Laemmli method (25) with 10% or 12% polyacrylamide gel, and were subsequently transferred onto a polyvinylidine fluoride (PVDF) membranes.…”

Rac regulates collagen-induced HSP27 phosphorylation via p44/p42 MAP kinase in human platelets

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