Mechanism underlying the retarded nuclear translocation of androgen receptor splice variants
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摘要
As shown in our previous study, two alternatively spliced androgen receptor(AR) variants, which are exclusively expressed in the granulosa cells of patients with polycystic ovary syndrome, exhibit retarded nuclear translocation compared with wild-type AR. However, researchers have not yet determined whether these abnormalities correlate with heat shock protein 90(HSP90)and importin α(the former is a generally accepted co-chaperone of AR, and the latter is a component of classical nuclear import complexes). Here, these two variants were mainly retained in cytoplasm with HSP90 and importin α in the presence of dihydrotestosterone(DHT), and their levels in nucleus were significantly reduced, according to the immunofluorescence staining. The binding affinity of two AR variants for importin α was consistently decreased, while it was increased in WT-AR following DHT stimulation, leading to reduced nuclear import, particularly for the insertion-AR(Ins-AR). However, the binding affinities of two AR variants for HSP90 were increased in the absence of DHT compared with WT-AR, which functioned to maintain spatial structural stability, particularly for the deletion-AR(Del-AR). Therefore, the retarded nuclear translocation of two AR variants is associated with HSP90 and importin α, and the abnormal binding affinities for them play critical roles in this process.
As shown in our previous study, two alternatively spliced androgen receptor(AR) variants, which are exclusively expressed in the granulosa cells of patients with polycystic ovary syndrome, exhibit retarded nuclear translocation compared with wild-type AR. However, researchers have not yet determined whether these abnormalities correlate with heat shock protein 90(HSP90)and importin α(the former is a generally accepted co-chaperone of AR, and the latter is a component of classical nuclear import complexes). Here, these two variants were mainly retained in cytoplasm with HSP90 and importin α in the presence of dihydrotestosterone(DHT), and their levels in nucleus were significantly reduced, according to the immunofluorescence staining. The binding affinity of two AR variants for importin α was consistently decreased, while it was increased in WT-AR following DHT stimulation, leading to reduced nuclear import, particularly for the insertion-AR(Ins-AR). However, the binding affinities of two AR variants for HSP90 were increased in the absence of DHT compared with WT-AR, which functioned to maintain spatial structural stability, particularly for the deletion-AR(Del-AR). Therefore, the retarded nuclear translocation of two AR variants is associated with HSP90 and importin α, and the abnormal binding affinities for them play critical roles in this process.
As shown in our previous study, two alternatively spliced androgen receptor(AR) variants, which are exclusively expressed in the granulosa cells of patients with polycystic ovary syndrome, exhibit retarded nuclear translocation compared with wild-type AR. However, researchers have not yet determined whether these abnormalities correlate with heat shock protein 90(HSP90)and importin α(the former is a generally accepted co-chaperone of AR, and the latter is a component of classical nuclear import complexes). Here, these two variants were mainly retained in cytoplasm with HSP90 and importin α in the presence of dihydrotestosterone(DHT), and their levels in nucleus were significantly reduced, according to the immunofluorescence staining. The binding affinity of two AR variants for importin α was consistently decreased, while it was increased in WT-AR following DHT stimulation, leading to reduced nuclear import, particularly for the insertion-AR(Ins-AR). However, the binding affinities of two AR variants for HSP90 were increased in the absence of DHT compared with WT-AR, which functioned to maintain spatial structural stability, particularly for the deletion-AR(Del-AR). Therefore, the retarded nuclear translocation of two AR variants is associated with HSP90 and importin α, and the abnormal binding affinities for them play critical roles in this process.
引文
Alvira,S.,Cuéllar,J.,R?hl,A.,Yamamoto,S.,Itoh,H.,Alfonso,C.,Rivas,G.,Buchner,J.,and Valpuesta,J.M.(2014).Structural characterization of the substrate transfer mechanism in Hsp70/Hsp90 folding machinery mediated by Hop.Nat Commun 5,5484.
Azad,A.A.,Zoubeidi,A.,Gleave,M.E.,and Chi,K.N.(2015).Targeting heat shock proteins in metastatic castration-resistant prostate cancer.Nat Rev Urol 12,26-36.
Azziz,R.,Carmina,E.,Chen,Z.J.,Dunaif,A.,Laven,J.S.E.,Legro,R.S.,Lizneva,D.,Natterson-Horowtiz,B.,Teede,H.J.,and Yildiz,B.O.(2-016).Polycystic ovary syndrome.Nat Rev Dis Primers 2,16057.
Chan,S.C.,Li,Y.,and Dehm,S.M.(2012).Androgen receptor splice variants activate androgen receptor target genes and support aberrant prostate cancer cell growth independent of canonical androgen receptor nuclear localization signal.J Biol Chem 287,19736-19749.
Chen,L.,Li,J.,Farah,E.,Sarkar,S.,Ahmad,N.,Gupta,S.,Larner,J.,and Liu,X.(2016).Cotargeting HSP90 and its client proteins for treatment of prostate cancer.Mol Cancer Therapeut 15,2107-2118.
Cutress,M.L.,Whitaker,H.C.,Mills,I.G.,Stewart,M.,and Neal,D.E.(2008).Structural basis for the nuclear import of the human androgen receptor.J Cell Sci 121,957-968.
Dehm,S.M.,and Tindall,D.J.(2011).Alternatively spliced androgen receptor variants.Endocr Related Cancer 18,R183-R196.
Fang,L.,Ricketson,D.,Getubig,L.,and Darimont,B.(2006).Unliganded and hormone-bound glucocorticoid receptors interact with distinct hydrophobic sites in the Hsp90 C-terminal domain.Proc Natl Acad Sci USA 103,18487-18492.
Forwood,J.K.,Harley,V.,and Jans,D.A.(2001).The C-terminal nuclear localization signal of the sex-determining region Y(SRY)high mobility group domain mediates nuclear import through importinβ1.J Biol Chem 276,46575-46582.
Galigniana,M.D.,Echeverría,P.C.,Erlejman,A.G.,and Piwien-Pilipuk,G.(2010).Role of molecular chaperones and TPR-domain proteins in the cytoplasmic transport of steroid receptors and their passage through the nuclear pore.Nucleus 1,299-308.
Georget,V.,Térouanne,B.,Nicolas,J.C.,and Sultan,C.(2002).Mechanism of antiandrogen action:key role of Hsp90 in conformational change and transcriptional activity of the androgen receptor.Biochemistry41,11824-11831.
Grossmann,C.,Ruhs,S.,Langenbruch,L.,Mildenberger,S.,Str?tz,N.,Schumann,K.,and Gekle,M.(2012).Nuclear shuttling precedes dimerization in mineralocorticoid receptor signaling.Chem Biol 19,742-751.
Hart,R.J.(2016).Physiological aspects of female fertility:role of the environment,modern lifestyle,and genetics.Physiol Rev 96,873-909.
Heemers,H.V.,and Tindall,D.J.(2007).Androgen receptor(AR)coregulators:a diversity of functions converging on and regulating the ARtranscriptional complex.Endocr Rev 28,778-808.
Helsen,C.,Dubois,V.,Verfaillie,A.,Young,J.,Trekels,M.,Vancraenenbroeck,R.,De Maeyer,M.,and Claessens,F.(2012).Evidence for DNA-binding domain-ligand-binding domain communications in the androgen receptor.Mol Cell Biol 32,3033-3043.
Huang,W.,Greene,G.L.,Ravikumar,K.M.,and Yang,S.(2013).Crosstalk between the ligand-and DNA-binding domains of estrogen receptor.Proteins 81,1900-1909.
Hughes,I.A.,Davies,J.D.,Bunch,T.I.,Pasterski,V.,Mastroyannopoulou,K.,and MacDougall,J.(2012).Androgen insensitivity syndrome.Lancet 380,1419-1428.
Ischia,J.,and So,A.I.(2013).The role of heat shock proteins in bladder cancer.Nat Rev Urol 10,386-395.
Jagla,M.,Fève,M.,Kessler,P.,Lapouge,G.,Erdmann,E.,Serra,S.,Bergerat,J.P.,and Céraline,J.(2007).A splicing variant of the androgen receptor detected in a metastatic prostate cancer exhibits exclusively cytoplasmic actions.Endocrinology 148,4334-4343.
Jiménez-Canino,R.,Lorenzo-Díaz,F.,Jaisser,F.,Farman,N.,Giraldez,T.,and Alvarez de la Rosa,D.(2016).Histone deacetylase 6-controlled hsp90 acetylation significantly alters mineralocorticoid receptor subcellular dynamics but not its transcriptional activity.Endocrinology 1-57,2515-2532.
Kaku,N.,Matsuda,K.,Tsujimura,A.,and Kawata,M.(2008).Characterization of nuclear import of the domain-specific androgen receptor in association with the importinα/βand ran-guanosine 5′-triphosphate systems.Endocrinology 149,3960-3969.
Kirschke,E.,Goswami,D.,Southworth,D.,Griffin,P.R.,and Agard,D.A.(2014).Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles.Cell 157,1685-1697.
Liu,L.L.,Xie,N.,Sun,S.,Plymate,S.,Mostaghel,E.,and Dong,X.(2014).Mechanisms of the androgen receptor splicing in prostate cancer cells.Oncogene 33,3140-3150.
Marfori,M.,Mynott,A.,Ellis,J.J.,Mehdi,A.M.,Saunders,N.F.W.,Curmi,P.M.,Forwood,J.K.,Bodén,M.,and Kobe,B.(2011).Molecular basis for specificity of nuclear import and prediction of nuclear localization.Biochim Biophys Acta 1813,1562-1577.
Ni,L.,Llewellyn,R.,Kesler,C.T.,Kelley,J.B.,Spencer,A.,Snow,C.J.,Shank,L.,and Paschal,B.M.(2013).Androgen induces a switch from cytoplasmic retention to nuclear import of the androgen receptor.Mol Cell Biol 33,4766-4778.
Norman,R.J.,Dewailly,D.,Legro,R.S.,and Hickey,T.E.(2007).Polycystic ovary syndrome.Lancet 370,685-697.
Ozanne,D.M.,Brady,M.E.,Cook,S.,Gaughan,L.,Neal,D.E.,and Robson,C.N.(2000).Androgen receptor nuclear translocation is facilitated by the f-actin cross-linking protein filamin.Mol Endocrinol 14,1618-1626.
Pawlowski,J.E.,Ertel,J.R.,Allen,M.P.,Xu,M.,Butler,C.,Wilson,E.M.,and Wierman,M.E.(2002).Liganded androgen receptor interaction withβ-catenin.J Biol Chem 277,20702-20710.
Pemberton,L.F.,and Paschal,B.M.(2005).Mechanisms of receptor-mediated nuclear import and nuclear export.Traffic 6,187-198.
Pratt,W.B.,Gestwicki,J.E.,Osawa,Y.,and Lieberman,A.P.(2015).Targeting Hsp90/Hsp70-based protein quality control for treatment of adult onset neurodegenerative diseases.Annu Rev Pharmacol Toxicol 55,353-371.
Rosenfield,R.L.,and Ehrmann,D.A.(2016).The pathogenesis of polycystic ovary syndrome(PCOS):the hypothesis of PCOS as functional ovarian hyperandrogenism revisited.Endocr Rev 37,467-520.
Savage,N.(2015).Metastasis:resistance fighters.Nature 528,S128-S129.
Shafi,A.A.,Yen,A.E.,and Weigel,N.L.(2013).Androgen receptors in hormone-dependent and castration-resistant prostate cancer.Pharmacol Therapeut 140,223-238.
Shostak,Y.,and Yamamoto,K.R.(2005).Overlapping but separable determinants of DNA binding and nuclear localization map to the C-terminal end of the Caenorhabditis elegans DAF-12 DNA binding domain.J Biol Chem 280,6554-6560.
Sprenger,C.C.T.,and Plymate,S.R.(2014).The link between androgen receptor splice variants and castration-resistant prostate cancer.Horm Cancer 5,207-217.
Sun,M.,Long,J.,Yi,Y.,and Xia,W.(2017).Importin alpha-importin beta complex mediated nuclear translocation of insulin-like growth factor binding protein-5.Endocr J 64,963-975.
Tao,Y.J.,and Zheng,W.(2011).Chaperones and the maturation of steroid hormone receptor complexes.Oncotarget 2,104-106.
Wadosky,K.M.,and Koochekpour,S.(2017).Androgen receptor splice variants and prostate cancer:from bench to bedside.Oncotarget 8,1-8550-18576.
Wang,F.,Pan,J.,Liu,Y.,Meng,Q.,Lv,P.,Qu,F.,Ding,G.L.,Klausen,C.,Leung,P.C.K.,Chan,H.C.,et al.(2015).Alternative splicing of the androgen receptor in polycystic ovary syndrome.Proc Natl Acad Sci USA 112,4743-4748.
Watson,P.A.,Arora,V.K.,and Sawyers,C.L.(2015).Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer.Nat Rev Cancer 15,701-711.
Yang,F.M.,Lin,Y.C.,and Hu,M.C.(2011).Identification of two functional nuclear localization signals mediating nuclear import of liver receptor homologue-1.Cell Mol Life Sci 68,1241-1253.
Zhu,X.,Daffada,A.A.I.,Chan,C.M.W.,and Dowsett,M.(1997).Identification of an exon 3 deletion splice variant androgen receptor m RNA in human breast cancer.Int J Cancer 72,574-580.
Azad,A.A.,Zoubeidi,A.,Gleave,M.E.,and Chi,K.N.(2015).Targeting heat shock proteins in metastatic castration-resistant prostate cancer.Nat Rev Urol 12,26-36.
Azziz,R.,Carmina,E.,Chen,Z.J.,Dunaif,A.,Laven,J.S.E.,Legro,R.S.,Lizneva,D.,Natterson-Horowtiz,B.,Teede,H.J.,and Yildiz,B.O.(2-016).Polycystic ovary syndrome.Nat Rev Dis Primers 2,16057.
Chan,S.C.,Li,Y.,and Dehm,S.M.(2012).Androgen receptor splice variants activate androgen receptor target genes and support aberrant prostate cancer cell growth independent of canonical androgen receptor nuclear localization signal.J Biol Chem 287,19736-19749.
Chen,L.,Li,J.,Farah,E.,Sarkar,S.,Ahmad,N.,Gupta,S.,Larner,J.,and Liu,X.(2016).Cotargeting HSP90 and its client proteins for treatment of prostate cancer.Mol Cancer Therapeut 15,2107-2118.
Cutress,M.L.,Whitaker,H.C.,Mills,I.G.,Stewart,M.,and Neal,D.E.(2008).Structural basis for the nuclear import of the human androgen receptor.J Cell Sci 121,957-968.
Dehm,S.M.,and Tindall,D.J.(2011).Alternatively spliced androgen receptor variants.Endocr Related Cancer 18,R183-R196.
Fang,L.,Ricketson,D.,Getubig,L.,and Darimont,B.(2006).Unliganded and hormone-bound glucocorticoid receptors interact with distinct hydrophobic sites in the Hsp90 C-terminal domain.Proc Natl Acad Sci USA 103,18487-18492.
Forwood,J.K.,Harley,V.,and Jans,D.A.(2001).The C-terminal nuclear localization signal of the sex-determining region Y(SRY)high mobility group domain mediates nuclear import through importinβ1.J Biol Chem 276,46575-46582.
Galigniana,M.D.,Echeverría,P.C.,Erlejman,A.G.,and Piwien-Pilipuk,G.(2010).Role of molecular chaperones and TPR-domain proteins in the cytoplasmic transport of steroid receptors and their passage through the nuclear pore.Nucleus 1,299-308.
Georget,V.,Térouanne,B.,Nicolas,J.C.,and Sultan,C.(2002).Mechanism of antiandrogen action:key role of Hsp90 in conformational change and transcriptional activity of the androgen receptor.Biochemistry41,11824-11831.
Grossmann,C.,Ruhs,S.,Langenbruch,L.,Mildenberger,S.,Str?tz,N.,Schumann,K.,and Gekle,M.(2012).Nuclear shuttling precedes dimerization in mineralocorticoid receptor signaling.Chem Biol 19,742-751.
Hart,R.J.(2016).Physiological aspects of female fertility:role of the environment,modern lifestyle,and genetics.Physiol Rev 96,873-909.
Heemers,H.V.,and Tindall,D.J.(2007).Androgen receptor(AR)coregulators:a diversity of functions converging on and regulating the ARtranscriptional complex.Endocr Rev 28,778-808.
Helsen,C.,Dubois,V.,Verfaillie,A.,Young,J.,Trekels,M.,Vancraenenbroeck,R.,De Maeyer,M.,and Claessens,F.(2012).Evidence for DNA-binding domain-ligand-binding domain communications in the androgen receptor.Mol Cell Biol 32,3033-3043.
Huang,W.,Greene,G.L.,Ravikumar,K.M.,and Yang,S.(2013).Crosstalk between the ligand-and DNA-binding domains of estrogen receptor.Proteins 81,1900-1909.
Hughes,I.A.,Davies,J.D.,Bunch,T.I.,Pasterski,V.,Mastroyannopoulou,K.,and MacDougall,J.(2012).Androgen insensitivity syndrome.Lancet 380,1419-1428.
Ischia,J.,and So,A.I.(2013).The role of heat shock proteins in bladder cancer.Nat Rev Urol 10,386-395.
Jagla,M.,Fève,M.,Kessler,P.,Lapouge,G.,Erdmann,E.,Serra,S.,Bergerat,J.P.,and Céraline,J.(2007).A splicing variant of the androgen receptor detected in a metastatic prostate cancer exhibits exclusively cytoplasmic actions.Endocrinology 148,4334-4343.
Jiménez-Canino,R.,Lorenzo-Díaz,F.,Jaisser,F.,Farman,N.,Giraldez,T.,and Alvarez de la Rosa,D.(2016).Histone deacetylase 6-controlled hsp90 acetylation significantly alters mineralocorticoid receptor subcellular dynamics but not its transcriptional activity.Endocrinology 1-57,2515-2532.
Kaku,N.,Matsuda,K.,Tsujimura,A.,and Kawata,M.(2008).Characterization of nuclear import of the domain-specific androgen receptor in association with the importinα/βand ran-guanosine 5′-triphosphate systems.Endocrinology 149,3960-3969.
Kirschke,E.,Goswami,D.,Southworth,D.,Griffin,P.R.,and Agard,D.A.(2014).Glucocorticoid receptor function regulated by coordinated action of the Hsp90 and Hsp70 chaperone cycles.Cell 157,1685-1697.
Liu,L.L.,Xie,N.,Sun,S.,Plymate,S.,Mostaghel,E.,and Dong,X.(2014).Mechanisms of the androgen receptor splicing in prostate cancer cells.Oncogene 33,3140-3150.
Marfori,M.,Mynott,A.,Ellis,J.J.,Mehdi,A.M.,Saunders,N.F.W.,Curmi,P.M.,Forwood,J.K.,Bodén,M.,and Kobe,B.(2011).Molecular basis for specificity of nuclear import and prediction of nuclear localization.Biochim Biophys Acta 1813,1562-1577.
Ni,L.,Llewellyn,R.,Kesler,C.T.,Kelley,J.B.,Spencer,A.,Snow,C.J.,Shank,L.,and Paschal,B.M.(2013).Androgen induces a switch from cytoplasmic retention to nuclear import of the androgen receptor.Mol Cell Biol 33,4766-4778.
Norman,R.J.,Dewailly,D.,Legro,R.S.,and Hickey,T.E.(2007).Polycystic ovary syndrome.Lancet 370,685-697.
Ozanne,D.M.,Brady,M.E.,Cook,S.,Gaughan,L.,Neal,D.E.,and Robson,C.N.(2000).Androgen receptor nuclear translocation is facilitated by the f-actin cross-linking protein filamin.Mol Endocrinol 14,1618-1626.
Pawlowski,J.E.,Ertel,J.R.,Allen,M.P.,Xu,M.,Butler,C.,Wilson,E.M.,and Wierman,M.E.(2002).Liganded androgen receptor interaction withβ-catenin.J Biol Chem 277,20702-20710.
Pemberton,L.F.,and Paschal,B.M.(2005).Mechanisms of receptor-mediated nuclear import and nuclear export.Traffic 6,187-198.
Pratt,W.B.,Gestwicki,J.E.,Osawa,Y.,and Lieberman,A.P.(2015).Targeting Hsp90/Hsp70-based protein quality control for treatment of adult onset neurodegenerative diseases.Annu Rev Pharmacol Toxicol 55,353-371.
Rosenfield,R.L.,and Ehrmann,D.A.(2016).The pathogenesis of polycystic ovary syndrome(PCOS):the hypothesis of PCOS as functional ovarian hyperandrogenism revisited.Endocr Rev 37,467-520.
Savage,N.(2015).Metastasis:resistance fighters.Nature 528,S128-S129.
Shafi,A.A.,Yen,A.E.,and Weigel,N.L.(2013).Androgen receptors in hormone-dependent and castration-resistant prostate cancer.Pharmacol Therapeut 140,223-238.
Shostak,Y.,and Yamamoto,K.R.(2005).Overlapping but separable determinants of DNA binding and nuclear localization map to the C-terminal end of the Caenorhabditis elegans DAF-12 DNA binding domain.J Biol Chem 280,6554-6560.
Sprenger,C.C.T.,and Plymate,S.R.(2014).The link between androgen receptor splice variants and castration-resistant prostate cancer.Horm Cancer 5,207-217.
Sun,M.,Long,J.,Yi,Y.,and Xia,W.(2017).Importin alpha-importin beta complex mediated nuclear translocation of insulin-like growth factor binding protein-5.Endocr J 64,963-975.
Tao,Y.J.,and Zheng,W.(2011).Chaperones and the maturation of steroid hormone receptor complexes.Oncotarget 2,104-106.
Wadosky,K.M.,and Koochekpour,S.(2017).Androgen receptor splice variants and prostate cancer:from bench to bedside.Oncotarget 8,1-8550-18576.
Wang,F.,Pan,J.,Liu,Y.,Meng,Q.,Lv,P.,Qu,F.,Ding,G.L.,Klausen,C.,Leung,P.C.K.,Chan,H.C.,et al.(2015).Alternative splicing of the androgen receptor in polycystic ovary syndrome.Proc Natl Acad Sci USA 112,4743-4748.
Watson,P.A.,Arora,V.K.,and Sawyers,C.L.(2015).Emerging mechanisms of resistance to androgen receptor inhibitors in prostate cancer.Nat Rev Cancer 15,701-711.
Yang,F.M.,Lin,Y.C.,and Hu,M.C.(2011).Identification of two functional nuclear localization signals mediating nuclear import of liver receptor homologue-1.Cell Mol Life Sci 68,1241-1253.
Zhu,X.,Daffada,A.A.I.,Chan,C.M.W.,and Dowsett,M.(1997).Identification of an exon 3 deletion splice variant androgen receptor m RNA in human breast cancer.Int J Cancer 72,574-580.