Abstract
In a previous study (Lee et al., 1996), we found that the expression of HSP90 in cardiac tissue was substantially reduced in sudden-death pigs with hypertrophic cardiomyopathy (HCM). This was the first evidence correlating a reduced HSP90 level in ventricular tissue with cardiac arrest in HCM pigs. To realize the biochemical characterization, we cloned porcine hsp90 cDNA and determined its heat-inducibility. In addition, we further purified HSP90 to test its phosphorylation ability and using geldanamycin (GA), an inhibitor of HSP90, to study if GA indeed blocks HSP90 function in vitro and in vivo.We have isolated and sequenced cDNA clones encoding a 90-kDa heat shock protein (HSP90) from a porcine brain cDNA library. The sequence of the 2,202- nucleotide coding region showed 88.6% homology with that of the human homologue. Moreover, the deduced amino acid sequence of the porcine hsp90 cDNA was 99.7% identical to that of the human counterpart, with differences of only three amino acids in a total of 733 residues. Expression of the gene was greatly elevated in cultured cells during recovery from heat shock treatment at 45℃ for 60 min. Three major transcripts with 2.2, 3.0, and 4.1 kb in size were detected by Northern blot hybridization. These transcripts were further identified in a whole pig hyperthermia experiment. These three hsp90 transcripts were constitutively expressed in porcine tissues including kidney, liver, brain, and heart, and their levels were markedly enhanced during recovery from 30 min hyperthermia treatment at 43℃. Furthermore, we found that HSP90 was preferentially expressed in pituitary gland, brain, adrenal gland, and testis, in comparison to the other tissues.Therefore, we purified a large quantity of HSP90 from porcine testis by hydroxylapatite (HA-HSP90) and SDS-PAGE/electroelution (eluted-HSP90) to explore the molecular mechanism of HSP90 phosphorylation affecting its metabolism. The purified HSP90 was used as an antigen to raise polyclonal antibodies in rabbits. Immunoblot analysis revealed that most purified HSP90 was HSP90a. Incubation of the purified HSP90 or HSP90 immunoprecipitated from extracts of human A431 cells, Balb/c 3T3 fibroblasts, and porcine testis with [γ-32P]ATP.Mg2+ resulted in phosphorylation of HSP90. However, the eluted-HSP90 lost its phosphorylation ability when incubated with [γ-32P]ATP.Mg2+ alone but could be phosphorylated by various protein kinases. The order of phosphorylation of HSP90 by these kinases is PKA = CKII > AK >> kinase FA/GSK-3α. Geldanamycin (GA) is HSP90-specific inhibitor, which binds the ATP-binding site of HSP90. Therefore, we test if GA could inhibit the phosphorylation of HA-HSP90. The result showed that GA did not inhibit the phosphorylation of HA-HSP90. However, heparin severely inhibited the phosphorylation. This result implied that the phosphorylation of HA-HSP90 was due to the associated CKII. Moreover, high temperatures almost completely inhibited the phosphorylation of HA-HSP90. Accordingly, we suggest that the HA-HSP90 cannot autophosphorylate in our system.The phosphorylated function of HSP90 did not affect by GA in our in vitro system. Therefore, we use COS-7 cells as our in vivo system to test whether GA affect the cellular functions in trichostatin A (TSA)-treated cells. TSA is an inhibitor of histone deacetylase, results in histone H4 hyperacetylation and cell cycle arrest. When using TSA treatment, although caused COS-7 cell death, pretreatment of 0.5 mg/ml GA for 30 min and an addition of 50 ng/ml TSA (GA+TSA) apparently averted cell death. Our results indicated that the cell survival rate was only approximately 20% when prolonged treatment was undertaken with 50 ng/ml TSA (TSA) alone for 24 h. In contrast, the cell survival rate was enhanced by two folds when treating with GA+TSA. Furthermore, DNA fragmentation assay revealed that fragmented DNA was produced 8 h after prolonged treatment with TSA alone. Within 16 h, the apoptotic percentages of TSA-treated cells were between 15-25%. In contrast, the other treatments did not exceed 6%. Furthermore, GA inhibited TSA-induced histone H4 hyperacetylation. Western blotting analysis further demonstrated that the HSP70 levels did not significantly increase in TSA-treated cells. However, the accumulated 70-kDa heat shock protein (HSP70) markedly increased up to 2 to 3 folds at 8 h in GA- and GA+TSA-treated cells, and the maximum amount up to 5 to 7 folds at 20 h. Conversely, HSP90 did not markedly increase in all treatments. Based on the results in this study, we suggest that apoptosis induced by TSA can be prevented by GA-induced increment of heat shock proteins, particularly HSP70.