The behavior of the endogenous heat shock protein 25 (Hsp25) in heat-stressed rat H9c2 myoblasts was studied. After mild or severe heating, this protein became less extractable with Triton X-100 and displayed characteristic immunofluorescence patterns, namely (1) granules in the nucleus, and (2) association with F-actin bundles in the cytoplasm. The intranuclear granulation of Hsp25 and its association with F-actin were sensitive to drugs affecting Hsp25 phosphorylation (cantharidin, sodium orthovanadate, SB203580, SB202190). Isoform analysis of Hsp25 translocated to the nucleus-free cytoskeletal fraction revealed only mono- and biphosphorylated Hsp25 and no unphosphorylated Hsp25. Transfected luciferase with initial localization in the nucleosol became colocalized with the Hsp25-containing granules after a heat shock treatment that denatured the enzyme in the cells. The association of Hsp25 with actin filaments after a mild heat stress conferred protection from subsequent F-actin-damaging treatments with cytochalasins (D and B) or severe heat stress. We hypothesize that (1) the binding of heat-denatured nucleosolic proteins to the Hsp25 contained in specific granular structures may serve for the subsequent chaperoning or degradation of the bound proteins, and (2) the actin cytoskeleton is stabilized by the direct targeting of phosphorylated Hsp25 to microfilament bundles.

These data suggest that CsA preferentially up-regulates the transcription of Ang II receptors, which very likely leads to vasoconstriction in vivo and could be at the origin of CsA-induced hypertension and nephrotoxicity in humans.

To study the cytoprotective capacity of Hsp27 under various cellular stresses, we compared the effects of heating and energy deprivation on its distribution and isoform composition. Cultured endothelial cells from human aorta or umbilical vein were subjected to heat shock (45°C) and ATP-depleting metabolic stress (CCCP or rotenone in a glucose-free medium). Both exposures led to the translocation of Hsp27 into the Triton X-100-insoluble cellular fraction, whereas the immunofluorescent Hsp27 pattern was characteristic for each stress employed. Heating (5-30 min) caused unexpected association of Hsp27 with thick bundles of actin microf'daments (stress fibers). ATP depletion within 30-120 min resulted in the appearance of Hsp27-containing compact granules in the nucleus. The insolubilization and relocallzation of Hsp27 were reversible in both cases. The stress-induced shifts in the Hsp27 isoform spectrum indicate an increase in phosphorylation of Hsp27 in heat-shocked cells and its dephosphorylation in ATP-depleted cells. We suggest that these stresses diversely affect the phosphorylation status of endothelial Hsp27, thus altering its localization, supramolecular organization and functional activity toward actin.

The vascular endothelium response to ischemic depletion of ATP was studied in vitro. Endothelial cells (EC) cultured from human aorta or umbilical vein were incubated in a glucose-free medium containing CCCP or rotenone. Such blockade of energy metabolism caused a drop in ATP, destruction of actin filaments, morphological changes, and eventually disintegration of EC monolayer within 2-2.5 h. While ATP fell and F-actin collapsed, the 27-kDa heat shock protein (Hsp27) lost basal phosphorylation and became Triton X-100-insoluble forming granules inside the cell nuclei. Protein phosphatase (PP) inhibitors (okadaic acid, cantharidin, sodium orthovanadate) did not delay the ATP decrease in energydeprived EC but arrested both the alterations in the Hsp27 status and the changes for the worse in F-actin and cell morphology. Similarly, the Hsp27 dephosphorylation/insolubilization/granulation and the cytoskeletal and morphological disturbances resulting from lack of ATP were suppressed in heat-preconditioned (thermotolerant) cultures, this effect being sensitive to quercetin, a blocker of Hsp induction. The longer preservation of the cytosolic pool of phosphorylated Hsp27 during ATP depletion in the PP inhibitor-treated or thermotolerant EC correlated with the acquired resistance of F-actin and morphology. These data suggest that PP inhibitors as well as heatinducible Hsp(s) can protect ischemia-stressed cells by preventing the ATP loss-provoked protein dephosphorylation and breakdown of the actin cytoskeleton.