Study On The Relation Between Heat Shock Protein 90 And The Difference Of The GC-GR Effect

It's well known to all that glucocorticoid-glucocorticoid receptor(GC-GR) effect would change markedly when bodies suffer with various stress injury including heating and drugs. A special group of chaperones known as heat shock protein 90(HSP90) is a highly conserved and ubiquitous protein in the cell, as well as an important chaperone in GC-GR effect and take part in the nuclear translocation of GR. Under normal conditions HSP90 has been found to be associated with GR and other partner protein including HSP70, HIP, HOP and HSP40 in hormone free heterocomplexes to maintain the activity of GR binding to ligand. After binding of GC and dissociation of the heterocomplexes into individual proteins, the hormone bound ligand translocates into nuclear undergoing various interaction as transcription factor. Thus, as one of key element of the GC-GR signal pathway , the conformational and quantitative change of HSP90 will affect the GC-GR effects directly.It is demonstrated that two strains of mice ,named C57BL/6 and BALB/c,are obviously different in several aspects comprise inflammatory reaction, repairing in trauma, heat endurance,immunity reaction, machinary and radiation injury which are closed to GC-GR effect. Likewise, patients with the same diseases usually display the different therapeutic effect even if administrated the equal dose of glucocorticoid in clinical practice. The difference of glucocorticoid effect may be result from the different genetic factor. Though it is confirmed that there is genetic difference of glucocorticoid receptor in minority of glucocorticoid-resisted genealogy case, majority of clinical case are not found the above-mentioned variation. Heat shock protein 90 is a key molecular in GC-GR pathway ,so the relation between the change of HSP90 and GC-GR effect catches our attention. Our previous experiments found the stress endurance of C57BL/6 mice are stronger than BALB/c. Then 4 missense mutation had been founded by sequencing the full length of HSP84 gene( correspond human HSP90βgene). If the mutation are related to the difference of GC-GR effect? The experiment has been executed to examine the HSP84 expression of two strains mice with the systemic blast injury stress model. At the same time , HSP84 gene of C57BL/6 mice has been transfected in BALB/c fibroblast in order to observe the nuclear translocation of GR under heat stress condition. The data demonstrated HSP84 expression of two strains mice both increased in SBI model and no significant difference between C57BL/6 and BALB/c. But BALB/c fibroblast transfected HSP84 gene of C57BL/6 has stronger ability of nuclear translocation compared to the same cell transfected HSP84 gene of BALB/c and pcDNA3.1 plasmid. So we presume the HSP84 gene mutation have obvious effect on GC-GR effect.It's proved heat shock protein 90 play important role in the ligand binding and nuclear translocation in glucocorticoid- glucocorticoid receptor signal pathway. Now there is only few report about it , so we are interested in the reason why the effect are different.In this study, the fibroblast of two strains of C57BL/6 and BALB/c mice with identical genetic background are considered as study object. Heat stress experiment model are applied to observe the change of glucocoiticoid- glucocoiticoid receptor effect administrated various kinds of drugs and further prove heat shock protein 90 is one of the main factor in the difference effect of stress. At the same time, we have collected a large number of primary nephrotic syndrome case via glucocoiticoid therapy to study the relationship between the glucocoiticoid sensitivity and the mutation frequency of heat shock protein 90. This study adopts the methods of amplification of the dimerization and neuclear translocation domain and gene sequencing to screen the mutation of heat shock protein 90 gene.The main conclusions of the study are as below:1. The stress model induced by heating fibroblast of C57BL/6 and BALB/c mice at 42℃has some advantages in easy producing, low prices, good reproducibility and avoiding infections caused by bacteria.2. The results showed that the survival rate, the proliferation activity of C57BL/6 and BALB/c fibroblast decrease and the release of lactate dehydrogenase and the neuclear translocation of glucocorticoid receptor increase obviously compared with control group. The change extent of C57BL/6 and BALB/c fibroblast are different significantly. The results manifest C57BL/6 fibroblast have stronger adaptability to heat stress.3. Treated with dexamethasone (50nM) the damage of C57BL/6 and BALB/c fibroblast lighten obviously. The neuclear translocation of GR increase significantly. Above results show dexamethasone therapy protect fibroblast of two strains of mice from heat stress by strengthen the neuclear translocation of GR. The protection of C57BL/6 fibroblast are stronger than that of BALB/c.4. Treated with dexamethasone (50nM) and geldenamycin, a specific inhibitor of HSP90, the protection of dexamethasone is abolished in both fibroblast. Low dosage(0.1uM)of geldenamycin increase the damage of BALB/c fibroblast much more than C57BL/6. Significant difference exist in both strains of fibroblast. High dosage(1uM and 10uM) geldenamycin block the neuclear translocation of GR. No significant difference of both fibroblast except the release of lactate dehydrogenase. Which indicated the difference of adaptability to heat stress was disappear after HSP90 was blocked completely. So HSP90 is one of the important factors caused the difference of GC-GR effect.5. By specifically amplifying the dimerization domain and nuclear translocation domain (6923bp-7803bp,reference Genebank sequence J04988) of HSP90βgene and sequencing the product directly, two point mutation had been founded within primary nephrotic syndrome patients treated with glucocorticoid. Two mutations of HSP90βgene are 7489 C>T in intron 10 and 7612 C>T in 3'-untranslated region respectively.6. There are 85 glucocoiticoid-sensitive and 70 glucocoiticoid-resisted patients in 155 primary nephrotic syndrome patients treated with glucocorticoid. The frency of 7489 C>T were 2.4% and 4.3% and 7612 C>T were 2.4% and 2.9% in glucocoiticoid-sensitive and glucocoiticoid-resisted case respectively. No significant difference (P >0.5)of both case indicated the untranslated region mutation of HSP90 gene were not in association with the sensitivity of glucocorticoid. Above results clue the expression quantity of HSP90 has no relation to therapeutic effect because the mutation of untranslated region only affect the expression quantity. It's presumed the constitution differences of HSP90 are main element in glucocoiticoid therapeutic sensitivity.