Exploring The Role Played By HSP90A Isoform In Chaperoning GR Signal Pathway

Glucocorticoids(GCs)are drugs of good effects and have been widely used to treat diseases such as Nephritic Syndrome, bronchial asthma, rheumatic diseases, inflammatory bowel disease,eczema, Multiple Sclerosis, lymphoma and leukemia. But there are some cases that are not sensitive or even resistant to GCs therapy. The problem we research is the way that leads to the status of resisting to GCs.As we know,it is glucocorticoid receptor (GR) that mediates the broad and intricate biological function of GC-GR, which reside in the cytoplasm, form dimmers after binding with GC and then translocation into nucleus where GR regulate the transcription of target genes and thus tune up the cellular function. Heat shock protein 90(HSP90) is the molecular chaperon that play a vital and indispensable role during GC exerting its biological function through GR. Actually HSP90 is involved in each step of the whole process of GC-GR function(figure 1). We presume that the quality (different isoform) and quantity of HSP90 can influence the function of GC-GR directly.HSP90 is one member of the heat shock protein families, accounting for 1%～2% of the whole cytoplasm proteins. There are mainly two isoforms of HSP90 in cytoplasm, HSP90α(inducible form ,corresponding to HSP84 in mice) and HSP90β(constitutive form, corresponding to HSP86 in mice) respectively, while HSPN was recently found. The form of HSP90 is mainly homodimers (αα,ββ), and a few exists as monomers, heterodimers and polymers. In our previous work, we found that C57BL/6 and BALB/c mouse respond to stress differently, and the differences exist within 6 hours. Since there are differences in the HSP84 of the two mice, it shows there is some relation between the HSP84 and the stress. But whether the chang in the HSP84 is the only and sufficient factor? Whether change in other isoform also leads to the difference in the stress endurance? Whether there is no substantial evidence to support the opinion that HSP84 is the only isoform that are involved in the GC-GR signal pathway. The two isoforms locate in different genomes and can be induced in quite different ways (expression of HSP90ɑis enhanced by heat inducement while HSP90βby mitogen).It is evident that the function of different HSP90 isoforms is dissimilarity.There are some phenomena we should be cautious about:1)HSP90ɑis inducible protein while HSP90βis constitutive protein. And as we know, when encountered with stress, HSP90ɑwill be supplied and enhanced quickly while HSP90βwill be enhanced in a much later phase. Does that favor the opinion that HSP90ɑare also involved in the GC-GR signal pathway?2) HSP90ɑand HSP90βlocate in different chromosomes and differ mostly in the C terminal and hinge region which specially serves to bind GR. Can difference in the region shape HSP90βinto the right configuration and thus HSP90βbecoming the only isoform that can help GR to sustain structure and function? We further presume that the different isoform of HSP90 contribute independently to GC-CR effects.To research these questions will benefit to elucidate the influence of quality (isoforms) and quantity of HSP90 on GC-CR effects, and to resolve the problems of GCs resistance, maybe to bring the new hope to these patients.In our research, we firstly construct plasmids to direct HSP90ɑspecific shRNA synthesis in eukaryotic cells. Then the change of GC-GR function were observed in the condition of HSP90ɑbeing knocked down.We mainly got the following results:1 .Three plasmids to be used for HSP90αgene silencing were constructed by inserting chemically synthesized ribonucleotide fragment into linearized pSUPER-EGFP vector and correctness of construction was verified by enzyme digestion and electrophoresis, named as pSuper-HSP90α1, pSuper-HSP90α2 and pSuper-HSP90α3 respectively. The sequences inserted into the recombinant pSuper-HSP90α1 and pSuper-HSP90α2 was proved correct by further sequencing. We have transfected HUVEC,HepG2 and HEK 293 cells with different ratio of plasmids to liposome, and eventually selected HEK 293 as target cells for transfection. By detecting green fluorescence ,a transfection efficiency of 80% was achieved after optimizing transfection conditions in HEK-293 cells.2.It was showed by the means of semi-quantitive RT-PCR that the amount of HSP90αmRNA in HEK-293 cells declined after transfection of the above three plasmids, pSuper-HSP90α1 is obviously decreased。This was observed 24h after transfection and compared with that of negative control groups.3.It was showed by the means of western blot that the amount of HSP90αprotein in HEK-293 cells declined after transfection of the above three plasmids, pSuper-HSP90αis obviously decreased .This was observed 48h after transfection and compared with that of negative control groups.4.The ratio of the GR amount within cellular nucleus versus that in cytoplasm gets higher 2 h after 50nM GC treatments, indicating GR translocation into nucleus to exert regulating function.This ratio change was increased when HEK-293 cells were transfected with pSuper-HSP90α, which observed at 48 h after transfection.It is reasonable to refer HSP90αas the isoform that is not in charge of chaperoning GR signaling.