Abnormal Nuclear Translocation Of Androgen Receptor (AR)Splice Variants

1、The Role of HSP90in nuclear translocation disorder of AR pre-mRNA splice variantsObjective:The aim of present study is to evaluate the role of HSP90in abnormal nuclear translocation of two androgen receptor alternative splice variants (ASVs) upon androgen exposure, one inserted69bp into intron2between exons2and3(ins) whereas the other skipped exon3(del), which were identified in granulose cells (GCs) and exclusively expressed in some of PCOS women.Materials and methods:Nuclear translocation of AR proteins encoded by different variants(wild, ins or del AR) was investigated by transfecting KGN cells with the AR and using immunofluorescence to determine its distribution between nucleus and cytoplasm following androgen exposure. Co-immunoprecipitation and Western Blot were performed to examine the binding affinity of these variants to chaperones. We evaluated the co-localizations of AR variants with HSP90.Results:(1) The co-immunoprecipitation results showed that the unliganded ins and del AR presented increased binding affinity to chaperones compared to wile AR.(2) Stimulated by5a-dihydrotestosterone, ins and del AR showed reduced binding affinity to chaperones, whereas wild AR remained invariable affinity.(3) Subcellular localizations of ins and del AR with HSP90were retained in cytoplasm and significantly reduced upon androgen exposure whereas wild-AR was predominately in nucleus.Conclusion:Our findings indicate that unliganded AR ASVs bind more chaperones to maintain stable status in cytoplasm. The reduced AR ASVs binding affinities for chaperones cause attenuated nuclear translocation, thus resulting in impaired transcriptional activation of AR. This perhaps is the mechanism of ovarian dysfunction of PCOS. 2、The Role of importin a in nuclear translocation disorder of AR pre-mRNA splice variantsObjective:The aim of this study was to evaluate the role of importin ain abnormal nuclear translocation of two androgen receptor alternative splice variants (ASVs) upon androgen exposure, one inserted69bp into intron2between exons2and3(ins) whereas the other skipped exon3(del), which were identified in granulose cells (GCs) and exclusively expressed in some of PCOS women.Materials and methods:Nuclear translocation of AR proteins encoded by different variants(wild, ins or del AR) was investigated by transfecting KGN cells with the AR and using immunofluorescence to determine its distribution between nucleus and cytoplasm following androgen exposure. Co-immunoprecipitation and Western Blot were performed to examine the binding affinity of these variants to importin a. We evaluated the colocalizations of AR variants with importin a.Results:(1) The co-immunoprecipitation results showed that the unliganded ins and del AR presented increased binding affinity to importin a compared to wile AR.(2) The liganded ins and del AR showed reduced binding affinity for importin a whereas wild AR increased.(3) Importin a also presented nuclear translocation disorder in accordance with AR ASVs.(4) Subcellular localizations of ins and del AR with importin a were retained in cytoplasm and significantly reduced upon androgen exposure whereas wild-AR was predominately in nucleus.Conclusion:Our findings indicate that unliganded AR ASVs bind more importin a. The reduced AR ASVs binding affinities for importin a cause attenuated nuclear translocation, thus resulting in impaired transcriptional activation of AR. This perhaps is the mechanism of ovarian dysfunction of PCOS.