Mutations in the Helix 3 Region of the Androgen Receptor Abrogate ARA70 Promotion of 17β-Estradiol-induced Androgen Receptor Transactivation
2002; Elsevier BV; Volume: 277; Issue: 39 Linguagem: Inglês
10.1074/jbc.m202824200
ISSN1083-351X
AutoresTin Htwe Thin, Eungseok Kim, Shuyuan Yeh, Erik R. Sampson, Yei‐Tsung Chen, Loretta L. Collins, Ravi Basavappa, Chawnshang Chang,
Tópico(s)Hormonal and reproductive studies
ResumoThe influence of estrogen on the development of the male reproductive system may be interrupted in a subset of partial androgen insensitivity syndrome (PAIS) patients. PAIS describes a wide range of male undermasculinization resulting from mutations in the androgen receptor (AR) or steroid metabolism enzymes that perturb androgen-AR regulation of male sex organ development. In this study, we are interested in determining if PAIS-derived AR mutants that respond normally to androgen have altered responses to estrogen in the presence of ARA70, a coregulator previously shown to enhance 17β-estradiol E2-induced AR transactivation. The wild-type AR (wtAR) and two PAIS AR mutants, AR(S703G) and AR(E709K), all bind to androgen and E2 and subsequently translocate to the nucleus. Whereas ARA70 functionally interacts with the wtAR and the PAIS AR mutants in response to androgen, E2 only promotes the functional interaction between ARA70 and the wtAR but not the PAIS AR mutants. ARA70 increases E2 competitive binding to the wtAR in the presence of low level androgen and also retards E2 dissociation from the wtAR. ARA70 is present in both the cytoplasm and the nucleus of various mouse testicular cells during early embryogenesis day 16, at postpartum day 0 during estradiol synthesis and in the Leydig cells at postpartum day 49. ARA70 may be unable to modulate the PAIS AR mutants-E2 binding, diminishing the effect of E2 via AR during male reproductive system development in patients with such mutations. Therefore, the presence of ARA70 in the testosterone and E2-producing Leydig cells may enhance the overall activity of AR during critical stages of male sex organ development. The influence of estrogen on the development of the male reproductive system may be interrupted in a subset of partial androgen insensitivity syndrome (PAIS) patients. PAIS describes a wide range of male undermasculinization resulting from mutations in the androgen receptor (AR) or steroid metabolism enzymes that perturb androgen-AR regulation of male sex organ development. In this study, we are interested in determining if PAIS-derived AR mutants that respond normally to androgen have altered responses to estrogen in the presence of ARA70, a coregulator previously shown to enhance 17β-estradiol E2-induced AR transactivation. The wild-type AR (wtAR) and two PAIS AR mutants, AR(S703G) and AR(E709K), all bind to androgen and E2 and subsequently translocate to the nucleus. Whereas ARA70 functionally interacts with the wtAR and the PAIS AR mutants in response to androgen, E2 only promotes the functional interaction between ARA70 and the wtAR but not the PAIS AR mutants. ARA70 increases E2 competitive binding to the wtAR in the presence of low level androgen and also retards E2 dissociation from the wtAR. ARA70 is present in both the cytoplasm and the nucleus of various mouse testicular cells during early embryogenesis day 16, at postpartum day 0 during estradiol synthesis and in the Leydig cells at postpartum day 49. ARA70 may be unable to modulate the PAIS AR mutants-E2 binding, diminishing the effect of E2 via AR during male reproductive system development in patients with such mutations. Therefore, the presence of ARA70 in the testosterone and E2-producing Leydig cells may enhance the overall activity of AR during critical stages of male sex organ development. androgen receptor partial androgen insensitivity 17β-estradiol wild-type AR ligand-binding domain chloramphenicol acetyltransferase phosphate-buffered saline 5α-dihydrotestosterone estrogen receptor glucocorticoid receptor progesterone receptor P0, postpartum day 0 Androgen action via the androgen receptor (AR)1 is critical in regulating male reproductive system development (1Quigley C.A., De Bellis A. Marschke K.B. el-Awady M.K. Wilson E.M. French F.S. Endocr. Rev. 1995; 16: 271-321Crossref PubMed Google Scholar). Insufficient androgen action may cause androgen insensitivity syndrome or male undermasculinized genitalia (2Nitsche E.M. Hiort O. Horm. Res. 2000; 54: 327-333Crossref PubMed Scopus (21) Google Scholar). The androgen insensitivity phenotypes vary from external genitalia that are completely female to degrees of partial masculinization. The possible causes may range from mutations in the AR or 5α-reductase genes to abnormal levels of estrogenversus androgen. Although partial androgen insensitivity syndrome (PAIS)-associated AR mutations usually disrupt the response to androgen, a subset of patients have AR mutations without any apparent defect in androgen signaling (3Radmayr C. Culig Z. Glatzl J. Neuschmid-Kaspar F. Bartsch G. Klocker H. J. Urol. 1997; 158: 1553-1556Crossref PubMed Scopus (23) Google Scholar, 4Gottlieb B. Beitel L.K. Lumbroso R. Pinsky L. Trifiro M. Hum. Mutat. 1999; 14: 103-114Crossref PubMed Scopus (113) Google Scholar).Estrogen, primarily 17β-estradiol (E2), has also been proposed to be involved in both normal and abnormal processes of male reproductive development and associated diseases (5Cunha G.R. Donjacour A.A. Cooke P.S. Mee S. Bigsby R.M. Higgins S.J. Sugimura Y. Endocr. Rev. 1987; 8: 338-362Crossref PubMed Scopus (859) Google Scholar, 6Jarred R.A. Cancilla B. Prins G.S. Thayer K.A. Cunha G.R. Risbridger G.P. Endocrinology. 2000; 141: 3471-3477Crossref PubMed Scopus (75) Google Scholar). Male estrogen is synthesized by aromatization of androgen, (e.g.testosterone), in many tissues including brain, liver, adipose tissue, and prostate (5Cunha G.R. Donjacour A.A. Cooke P.S. Mee S. Bigsby R.M. Higgins S.J. Sugimura Y. Endocr. Rev. 1987; 8: 338-362Crossref PubMed Scopus (859) Google Scholar). The physiological level of circulating E2 in the adult male is ∼0.1 nm (73–184 pmol/liter or 12–34 pg/ml), and in the adult female during pregnancy, the level of E2 is ∼1 nm (367–1285 pmol/liter or 100–350 pg/ml) (7Tanagho E.A. McAninch J.W. Smith's General Urology. 15th Ed. Lange Medical Books McGraw-Hill, New York2000: 813Google Scholar). However, local aromatase activity may cause particular tissue levels of E2 to be higher than the serum level. During pregnancy, maternal exposure to exogenous estrogen may influence the sexual maturation of male offspring, leading to cryptorchidism, epididymal cysts, and retained Mullerian ducts (8Rajfer J. Coffey D.S. Invest. Urol. 1978; 16: 186-190PubMed Google Scholar). Although estrogen action on the male reproductive system is not well understood, there is evidence of an estrogen-imprinting effect that may induce prostatic hyperplasia in male offspring with excess prenatal exposure to estrogen (9Prins G.S. Birch L. Couse J.F. Choi I. Katzenellenbogen B. Korach K.S. Cancer Res. 2001; 61: 6089-6097PubMed Google Scholar).Based on reports using CV-1 (10Kemppainen J.A Lane M.V Sar M. Wilson E.M. J. Biol. Chem. 1992; 267: 968-974Abstract Full Text PDF PubMed Google Scholar, 11Tan J.A. Hall S.H. Petrusz P. French F.S. Endocrinology. 2000; 141: 3440-3450Crossref PubMed Scopus (43) Google Scholar) or PC-3 (12Kokontis J. Ito K. Hiipakka R.A. Liao S. Receptor. 1991; 1: 271-279PubMed Google Scholar) cells, it is possible that estrogen may influence male reproductive system development via the AR. Our previous report (13Yeh S. Miyamoto H. Shima H. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5527-5532Crossref PubMed Scopus (243) Google Scholar) demonstrated that E2-mediated wild-type AR (wtAR) transactivation is enhanced by the addition of the AR coregulator, ARA70, in DU145 cells. In HeLa cells, although E2 could promote the translocation of the wtAR from the cytoplasm to the nucleus, E2 requires selective coregulators, such as ARA70 or SRC-1, to significantly induce the wtAR transactivation (14Agoulnik, I., Stenoien, D., Mancini, M. A., and Weigel, N. (2000) in Keystone Steroid Symposium, Boulder, Colorado, March 28, Abstr. 302, Bristol Myers Squibb Pharmaceutical Research Institute and The Directors Sponsor Fund.Google Scholar). In PC-3 (15Han G. Foster B.A. Mistry S. Buchanan G. Harris J.M. Tilley W.D. Greenberg N.M. J. Biol. Chem. 2001; 276: 11204-11213Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), CV-1 (16Zhou Z.X., He, B. Hall S.H. Wilson E.M. French F.S. Mol. Endocrinol. 2002; 16: 287-300Crossref PubMed Scopus (43) Google Scholar), and TSU-Pr1 (17Truica C.I. Byers S. Gelmann E.P. Cancer Res. 2000; 60: 4709-4713PubMed Google Scholar) cells, ARA70 and β-catenin also dramatically enhance wtAR transactivation in response to E2, although there are conflicting results in CV-1 cells (16Zhou Z.X., He, B. Hall S.H. Wilson E.M. French F.S. Mol. Endocrinol. 2002; 16: 287-300Crossref PubMed Scopus (43) Google Scholar, 18Gao T. Brantley K. Bolu E. McPhaul M.J. Mol. Endocrinol. 1999; 13: 1645-1656Crossref PubMed Scopus (48) Google Scholar). The differing results in CV-1 cells may reflect differences in cell passage number, growth conditions, or expression vectors. Therefore, certain coregulators may enable estrogen to influence AR target gene expression via the AR and thereby potentially contribute to male sexual maturation. Furthermore, there is evidence that the disruption of coactivators may also contribute to hormone resistance during target organ development (19Xu J. Qiu Y. DeMayo F.J. Tsai S.Y. Tsai M.J. O'Malley B.W. Science. 1998; 279: 1922-1925Crossref PubMed Scopus (589) Google Scholar, 20Adachi M. Takayanagi R. Tomura A. Imasaki K. Kato S. Goto K. Yanase T. Ikuyama S. Nawata H. N. Engl. J. Med. 2000; 343: 856-862Crossref PubMed Scopus (146) Google Scholar, 21Hughes I.A. N. Engl. J. Med. 2000; 343: 880-882Crossref Scopus (12) Google Scholar). Thus, certain PAIS-associated AR mutations may disrupt this estrogen-dependent AR transactivation, which is modulated by coregulators, during male reproductive system development.Here we have analyzed the functional interaction of PAIS-derived AR mutants with ARA70 in response to androgen or E2. Using co-immunoprecipitation and mammalian two-hybrid assays, we demonstrate that E2 promotes the interaction of ARA70 with the wtAR but not with the PAIS mutants. By various ligand binding analyses, we also demonstrate that ARA70 increases the E2 competitive binding to the wtAR at the low levels of androgen and retards the dissociation of E2 from the wtAR-LBD but not from the PAIS mutants. ARA70 is present in various fetal mouse testicular cells during early embryogenesis day 16 and postpartum day 0 and is also significantly present in the Leydig cells at postpartum day 49. ARA70 may therefore be unable to stabilize the binding of E2 to the PAIS AR mutants, diminishing the effect of E2 via the AR during male reproductive system development in these patients.DISCUSSIONAndrogen insensitivity syndrome is characterized by a spectrum of deficient male virilization or undermasculinization including testicular feminization (29Felig P. Frohman L.A. Endocrinology and Metabolism. 4th Ed. McGraw-Hill, Inc., New York2001: 779-824Google Scholar). It is known that if androgen action is slightly less than optimal, there will be undervirilization or inefficient spermatogenesis (29Felig P. Frohman L.A. Endocrinology and Metabolism. 4th Ed. McGraw-Hill, Inc., New York2001: 779-824Google Scholar). Although mutation of the AR is often found in PAIS patients, certain mutations associated with PAIS respond normally to androgen (4Gottlieb B. Beitel L.K. Lumbroso R. Pinsky L. Trifiro M. Hum. Mutat. 1999; 14: 103-114Crossref PubMed Scopus (113) Google Scholar). The first mutation analyzed in this study, AR(S703G), was identified in a patient diagnosed with male pseudohermaphroditism, which is associated with PAIS (3Radmayr C. Culig Z. Glatzl J. Neuschmid-Kaspar F. Bartsch G. Klocker H. J. Urol. 1997; 158: 1553-1556Crossref PubMed Scopus (23) Google Scholar). The other mutation we studied, AR(E709K), is associated with Reifenstein syndrome (13Yeh S. Miyamoto H. Shima H. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5527-5532Crossref PubMed Scopus (243) Google Scholar). Reifenstein syndrome is a less severe variant of X-linked PAIS in which affected individuals present as phenotypic males with hypospadias, inadequate pubertal virilization, gynecomastia, and infertility (30Aima J. Griffin J.E. Gazak J.M. Wilson J.D. MacDonald P.C. N. Engl. J. Med. 1979; 300: 223-227Crossref PubMed Scopus (154) Google Scholar).Figs. Figure 1, Figure 2, Figure 3, Figure 4 show that both of these PAIS mutants respond to androgen normally, but have lost the ARA70-dependent response to E2. Although E2 is a nonspecific ligand for the wtAR, certain coregulators, such as ARA70 or SRC-1, can enhance wtAR transactivation in response to E2 (13Yeh S. Miyamoto H. Shima H. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5527-5532Crossref PubMed Scopus (243) Google Scholar, 14Agoulnik, I., Stenoien, D., Mancini, M. A., and Weigel, N. (2000) in Keystone Steroid Symposium, Boulder, Colorado, March 28, Abstr. 302, Bristol Myers Squibb Pharmaceutical Research Institute and The Directors Sponsor Fund.Google Scholar, 15Han G. Foster B.A. Mistry S. Buchanan G. Harris J.M. Tilley W.D. Greenberg N.M. J. Biol. Chem. 2001; 276: 11204-11213Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 16Zhou Z.X., He, B. Hall S.H. Wilson E.M. French F.S. Mol. Endocrinol. 2002; 16: 287-300Crossref PubMed Scopus (43) Google Scholar). E2 and DHT have similar structures except that E2 has a phenolic A-ring. It is therefore possible that although the wtAR may be able to bind to E2, E2 may not be well oriented within the wtAR-LBD pocket leading to weaker binding. Therefore, E2 may easily dissociate resulting in marginal activation of the wtAR. The recruitment of ARA70 or other selective coregulators to the wtAR, however, may change the conformation of the wtAR so that E2 can be trapped within the wtAR-LBD pocket allowing E2 to more effectively activate wtAR transactivation. In terms of mutants, the conformation of E2-bound AR mutant may not be similar to that of E2-bound wtAR. That difference in the conformation may influence on the receptor-interface providing for the coregulator recruitment.Our data in Figs. 5 and 6 show that ARA70 helps E2 competitively bind to the wtAR and subsequently retards E2 dissociation from the wtAR binding pocket. This phenomenon may be important during the prenatal period (7Tanagho E.A. McAninch J.W. Smith's General Urology. 15th Ed. Lange Medical Books McGraw-Hill, New York2000: 813Google Scholar) where high maternal estrogen levels may initiate estrogen signaling via the wtAR in the presence of coregulators, such as ARA70. For example, one report found that plasma concentrations of testosterone and E2 in mothers delivering male babies were 1.2 ± 0.4 and 4.5 ± 0.7 ng/ml, respectively (31Adeyemo O. Jeyakumar H. Afr. J. Med. Med. Sci. 1993; 22: 55-60PubMed Google Scholar), and these sex hormone levels may influence the balance between androgens and estrogens in the male fetus. Studies of 20 fetal rat testis samples collected during genital tract differentiation (gestation) also reported that there is a negligible level of circulating DHT in fetal rat plasma (32Habert R. Picon R. J. Steroid Biochem. 1984; 2: 193-198Crossref Scopus (129) Google Scholar). In addition, male and female rat fetuses had similar plasma levels of DHT and E2, although male fetuses did have higher levels of testosterone (32Habert R. Picon R. J. Steroid Biochem. 1984; 2: 193-198Crossref Scopus (129) Google Scholar). High aromatase activity during the last days of fetal life may also convert testosterone to E2 in male fetuses (32Habert R. Picon R. J. Steroid Biochem. 1984; 2: 193-198Crossref Scopus (129) Google Scholar, 33George F.W. Ojeda S.R. Endocrinology. 1982; 111: 522-529Crossref PubMed Scopus (158) Google Scholar), further altering the balance of these sex hormones. Therefore, although male sexual differentiation and reproductive organ development are critically dependent on androgen-AR signaling, it is possible that estrogen-AR-coregulator signaling may influence androgen action in conditions where DHT levels are negligible and testosterone is readily aromatized to estrogen. The loss of the E2-AR-coregulator pathway may partly disrupt the balance between AR and ER signaling during critical stages in male reproductive system development. Fig. 4 Bdemonstrates that the S703G and E709K PAIS mutants cannot functionally interact with ARA70 in response to E2. Therefore, decreased total receptor activity in AR(S703G) and AR(E709K) PAIS patients may tip the balance in favor of ER signaling, resulting in undermasculinization.Our in vivo data in Fig. 8 show that ARA70 and the AR are both present in the interstitial cells of the mouse E16 testis. Primitive sex cords mature to form testis cords and the rete testis at 13 days postcoitum (34Rugh R. The Mouse: Its Reproduction and Development. Burgess Publishing Company, Minneapolis, MN1968: 282-289Google Scholar). By 15 days, the vas deferens, epididymis, and seminal vesicles arise from the Wolffian duct, and the testis develops prominent sex cords, each of which become a seminiferous tubule (34Rugh R. The Mouse: Its Reproduction and Development. Burgess Publishing Company, Minneapolis, MN1968: 282-289Google Scholar). At this point, the cord contains uniform but undifferentiated cells, which are precursors of spermatogenic or Sertoli cells. As the testis continues to develop, mesenchymal cells between the testis cords become Leydig cells. Leydig cells are responsible for the synthesis and secretion of testosterone, the primary hormone of the testis and a critical regulator of spermatogenesis. Leydig cells also produce 5α-reduced androgens, such as DHT and 3α-diol. E2 can be synthesized through the aromatization of testosterone by aromatase in the Sertoli cells and diffuse back to the Leydig cells (33George F.W. Ojeda S.R. Endocrinology. 1982; 111: 522-529Crossref PubMed Scopus (158) Google Scholar, 34Rugh R. The Mouse: Its Reproduction and Development. Burgess Publishing Company, Minneapolis, MN1968: 282-289Google Scholar). Conversion of testosterone to estrogen also occurs in the Leydig cells. Testicular testosterone and its derivatives then induce masculine extragonadal differentiation. The abundance of ARA70 in testicular interstitial cells, at the time of E2 synthesis, supports our in vitro data showing the modulation of ARA70 on the AR-E2 binding. Moreover, this could imply that ARA70 impacts the development of male structures via not only the androgen-AR-ARA70 pathway but also the E2-AR-ARA70 pathway, to stimulate the development of genital organs, such as the vas deferens, epididymis, and seminal vesicle and the masculinization of the external genitalia.Our data therefore provide one possible molecular basis for a subset of PAIS phenotypes where mutation does not abolish androgen-AR signaling. Recent studies reported that ARA70 is not mutated in an analysis of 27 androgen insensitivity syndrome patients (35Lim H.N. Hawkins J.R. Hughes I.A. Clin. Genet. 2001; 59: 284-286Crossref PubMed Scopus (11) Google Scholar). This finding excludes the possibility of ARA70 mutation in PAIS and supports our assertion that the loss of AR mutant functional interaction with ARA70, due to helix 3 mutations, may affect a subset of PAIS patients where androgen response is normal.In summary, our data provide one of the potential molecular causes to explain the development of PAIS in a subset of patients with AR mutants that respond normally to androgen. Moreover, our results have partially elucidated how ARA70 enhances the activation of the wtAR in response to E2. These findings suggest that certain coregulators may have an important role in the regulation of male reproductive system development specifically in modulation of E2 induction of the AR transactivation. Androgen action via the androgen receptor (AR)1 is critical in regulating male reproductive system development (1Quigley C.A., De Bellis A. Marschke K.B. el-Awady M.K. Wilson E.M. French F.S. Endocr. Rev. 1995; 16: 271-321Crossref PubMed Google Scholar). Insufficient androgen action may cause androgen insensitivity syndrome or male undermasculinized genitalia (2Nitsche E.M. Hiort O. Horm. Res. 2000; 54: 327-333Crossref PubMed Scopus (21) Google Scholar). The androgen insensitivity phenotypes vary from external genitalia that are completely female to degrees of partial masculinization. The possible causes may range from mutations in the AR or 5α-reductase genes to abnormal levels of estrogenversus androgen. Although partial androgen insensitivity syndrome (PAIS)-associated AR mutations usually disrupt the response to androgen, a subset of patients have AR mutations without any apparent defect in androgen signaling (3Radmayr C. Culig Z. Glatzl J. Neuschmid-Kaspar F. Bartsch G. Klocker H. J. Urol. 1997; 158: 1553-1556Crossref PubMed Scopus (23) Google Scholar, 4Gottlieb B. Beitel L.K. Lumbroso R. Pinsky L. Trifiro M. Hum. Mutat. 1999; 14: 103-114Crossref PubMed Scopus (113) Google Scholar). Estrogen, primarily 17β-estradiol (E2), has also been proposed to be involved in both normal and abnormal processes of male reproductive development and associated diseases (5Cunha G.R. Donjacour A.A. Cooke P.S. Mee S. Bigsby R.M. Higgins S.J. Sugimura Y. Endocr. Rev. 1987; 8: 338-362Crossref PubMed Scopus (859) Google Scholar, 6Jarred R.A. Cancilla B. Prins G.S. Thayer K.A. Cunha G.R. Risbridger G.P. Endocrinology. 2000; 141: 3471-3477Crossref PubMed Scopus (75) Google Scholar). Male estrogen is synthesized by aromatization of androgen, (e.g.testosterone), in many tissues including brain, liver, adipose tissue, and prostate (5Cunha G.R. Donjacour A.A. Cooke P.S. Mee S. Bigsby R.M. Higgins S.J. Sugimura Y. Endocr. Rev. 1987; 8: 338-362Crossref PubMed Scopus (859) Google Scholar). The physiological level of circulating E2 in the adult male is ∼0.1 nm (73–184 pmol/liter or 12–34 pg/ml), and in the adult female during pregnancy, the level of E2 is ∼1 nm (367–1285 pmol/liter or 100–350 pg/ml) (7Tanagho E.A. McAninch J.W. Smith's General Urology. 15th Ed. Lange Medical Books McGraw-Hill, New York2000: 813Google Scholar). However, local aromatase activity may cause particular tissue levels of E2 to be higher than the serum level. During pregnancy, maternal exposure to exogenous estrogen may influence the sexual maturation of male offspring, leading to cryptorchidism, epididymal cysts, and retained Mullerian ducts (8Rajfer J. Coffey D.S. Invest. Urol. 1978; 16: 186-190PubMed Google Scholar). Although estrogen action on the male reproductive system is not well understood, there is evidence of an estrogen-imprinting effect that may induce prostatic hyperplasia in male offspring with excess prenatal exposure to estrogen (9Prins G.S. Birch L. Couse J.F. Choi I. Katzenellenbogen B. Korach K.S. Cancer Res. 2001; 61: 6089-6097PubMed Google Scholar). Based on reports using CV-1 (10Kemppainen J.A Lane M.V Sar M. Wilson E.M. J. Biol. Chem. 1992; 267: 968-974Abstract Full Text PDF PubMed Google Scholar, 11Tan J.A. Hall S.H. Petrusz P. French F.S. Endocrinology. 2000; 141: 3440-3450Crossref PubMed Scopus (43) Google Scholar) or PC-3 (12Kokontis J. Ito K. Hiipakka R.A. Liao S. Receptor. 1991; 1: 271-279PubMed Google Scholar) cells, it is possible that estrogen may influence male reproductive system development via the AR. Our previous report (13Yeh S. Miyamoto H. Shima H. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5527-5532Crossref PubMed Scopus (243) Google Scholar) demonstrated that E2-mediated wild-type AR (wtAR) transactivation is enhanced by the addition of the AR coregulator, ARA70, in DU145 cells. In HeLa cells, although E2 could promote the translocation of the wtAR from the cytoplasm to the nucleus, E2 requires selective coregulators, such as ARA70 or SRC-1, to significantly induce the wtAR transactivation (14Agoulnik, I., Stenoien, D., Mancini, M. A., and Weigel, N. (2000) in Keystone Steroid Symposium, Boulder, Colorado, March 28, Abstr. 302, Bristol Myers Squibb Pharmaceutical Research Institute and The Directors Sponsor Fund.Google Scholar). In PC-3 (15Han G. Foster B.A. Mistry S. Buchanan G. Harris J.M. Tilley W.D. Greenberg N.M. J. Biol. Chem. 2001; 276: 11204-11213Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar), CV-1 (16Zhou Z.X., He, B. Hall S.H. Wilson E.M. French F.S. Mol. Endocrinol. 2002; 16: 287-300Crossref PubMed Scopus (43) Google Scholar), and TSU-Pr1 (17Truica C.I. Byers S. Gelmann E.P. Cancer Res. 2000; 60: 4709-4713PubMed Google Scholar) cells, ARA70 and β-catenin also dramatically enhance wtAR transactivation in response to E2, although there are conflicting results in CV-1 cells (16Zhou Z.X., He, B. Hall S.H. Wilson E.M. French F.S. Mol. Endocrinol. 2002; 16: 287-300Crossref PubMed Scopus (43) Google Scholar, 18Gao T. Brantley K. Bolu E. McPhaul M.J. Mol. Endocrinol. 1999; 13: 1645-1656Crossref PubMed Scopus (48) Google Scholar). The differing results in CV-1 cells may reflect differences in cell passage number, growth conditions, or expression vectors. Therefore, certain coregulators may enable estrogen to influence AR target gene expression via the AR and thereby potentially contribute to male sexual maturation. Furthermore, there is evidence that the disruption of coactivators may also contribute to hormone resistance during target organ development (19Xu J. Qiu Y. DeMayo F.J. Tsai S.Y. Tsai M.J. O'Malley B.W. Science. 1998; 279: 1922-1925Crossref PubMed Scopus (589) Google Scholar, 20Adachi M. Takayanagi R. Tomura A. Imasaki K. Kato S. Goto K. Yanase T. Ikuyama S. Nawata H. N. Engl. J. Med. 2000; 343: 856-862Crossref PubMed Scopus (146) Google Scholar, 21Hughes I.A. N. Engl. J. Med. 2000; 343: 880-882Crossref Scopus (12) Google Scholar). Thus, certain PAIS-associated AR mutations may disrupt this estrogen-dependent AR transactivation, which is modulated by coregulators, during male reproductive system development. Here we have analyzed the functional interaction of PAIS-derived AR mutants with ARA70 in response to androgen or E2. Using co-immunoprecipitation and mammalian two-hybrid assays, we demonstrate that E2 promotes the interaction of ARA70 with the wtAR but not with the PAIS mutants. By various ligand binding analyses, we also demonstrate that ARA70 increases the E2 competitive binding to the wtAR at the low levels of androgen and retards the dissociation of E2 from the wtAR-LBD but not from the PAIS mutants. ARA70 is present in various fetal mouse testicular cells during early embryogenesis day 16 and postpartum day 0 and is also significantly present in the Leydig cells at postpartum day 49. ARA70 may therefore be unable to stabilize the binding of E2 to the PAIS AR mutants, diminishing the effect of E2 via the AR during male reproductive system development in these patients. DISCUSSIONAndrogen insensitivity syndrome is characterized by a spectrum of deficient male virilization or undermasculinization including testicular feminization (29Felig P. Frohman L.A. Endocrinology and Metabolism. 4th Ed. McGraw-Hill, Inc., New York2001: 779-824Google Scholar). It is known that if androgen action is slightly less than optimal, there will be undervirilization or inefficient spermatogenesis (29Felig P. Frohman L.A. Endocrinology and Metabolism. 4th Ed. McGraw-Hill, Inc., New York2001: 779-824Google Scholar). Although mutation of the AR is often found in PAIS patients, certain mutations associated with PAIS respond normally to androgen (4Gottlieb B. Beitel L.K. Lumbroso R. Pinsky L. Trifiro M. Hum. Mutat. 1999; 14: 103-114Crossref PubMed Scopus (113) Google Scholar). The first mutation analyzed in this study, AR(S703G), was identified in a patient diagnosed with male pseudohermaphroditism, which is associated with PAIS (3Radmayr C. Culig Z. Glatzl J. Neuschmid-Kaspar F. Bartsch G. Klocker H. J. Urol. 1997; 158: 1553-1556Crossref PubMed Scopus (23) Google Scholar). The other mutation we studied, AR(E709K), is associated with Reifenstein syndrome (13Yeh S. Miyamoto H. Shima H. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5527-5532Crossref PubMed Scopus (243) Google Scholar). Reifenstein syndrome is a less severe variant of X-linked PAIS in which affected individuals present as phenotypic males with hypospadias, inadequate pubertal virilization, gynecomastia, and infertility (30Aima J. Griffin J.E. Gazak J.M. Wilson J.D. MacDonald P.C. N. Engl. J. Med. 1979; 300: 223-227Crossref PubMed Scopus (154) Google Scholar).Figs. Figure 1, Figure 2, Figure 3, Figure 4 show that both of these PAIS mutants respond to androgen normally, but have lost the ARA70-dependent response to E2. Although E2 is a nonspecific ligand for the wtAR, certain coregulators, such as ARA70 or SRC-1, can enhance wtAR transactivation in response to E2 (13Yeh S. Miyamoto H. Shima H. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5527-5532Crossref PubMed Scopus (243) Google Scholar, 14Agoulnik, I., Stenoien, D., Mancini, M. A., and Weigel, N. (2000) in Keystone Steroid Symposium, Boulder, Colorado, March 28, Abstr. 302, Bristol Myers Squibb Pharmaceutical Research Institute and The Directors Sponsor Fund.Google Scholar, 15Han G. Foster B.A. Mistry S. Buchanan G. Harris J.M. Tilley W.D. Greenberg N.M. J. Biol. Chem. 2001; 276: 11204-11213Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 16Zhou Z.X., He, B. Hall S.H. Wilson E.M. French F.S. Mol. Endocrinol. 2002; 16: 287-300Crossref PubMed Scopus (43) Google Scholar). E2 and DHT have similar structures except that E2 has a phenolic A-ring. It is therefore possible that although the wtAR may be able to bind to E2, E2 may not be well oriented within the wtAR-LBD pocket leading to weaker binding. Therefore, E2 may easily dissociate resulting in marginal activation of the wtAR. The recruitment of ARA70 or other selective coregulators to the wtAR, however, may change the conformation of the wtAR so that E2 can be trapped within the wtAR-LBD pocket allowing E2 to more effectively activate wtAR transactivation. In terms of mutants, the conformation of E2-bound AR mutant may not be similar to that of E2-bound wtAR. That difference in the conformation may influence on the receptor-interface providing for the coregulator recruitment.Our data in Figs. 5 and 6 show that ARA70 helps E2 competitively bind to the wtAR and subsequently retards E2 dissociation from the wtAR binding pocket. This phenomenon may be important during the prenatal period (7Tanagho E.A. McAninch J.W. Smith's General Urology. 15th Ed. Lange Medical Books McGraw-Hill, New York2000: 813Google Scholar) where high maternal estrogen levels may initiate estrogen signaling via the wtAR in the presence of coregulators, such as ARA70. For example, one report found that plasma concentrations of testosterone and E2 in mothers delivering male babies were 1.2 ± 0.4 and 4.5 ± 0.7 ng/ml, respectively (31Adeyemo O. Jeyakumar H. Afr. J. Med. Med. Sci. 1993; 22: 55-60PubMed Google Scholar), and these sex hormone levels may influence the balance between androgens and estrogens in the male fetus. Studies of 20 fetal rat testis samples collected during genital tract differentiation (gestation) also reported that there is a negligible level of circulating DHT in fetal rat plasma (32Habert R. Picon R. J. Steroid Biochem. 1984; 2: 193-198Crossref Scopus (129) Google Scholar). In addition, male and female rat fetuses had similar plasma levels of DHT and E2, although male fetuses did have higher levels of testosterone (32Habert R. Picon R. J. Steroid Biochem. 1984; 2: 193-198Crossref Scopus (129) Google Scholar). High aromatase activity during the last days of fetal life may also convert testosterone to E2 in male fetuses (32Habert R. Picon R. J. Steroid Biochem. 1984; 2: 193-198Crossref Scopus (129) Google Scholar, 33George F.W. Ojeda S.R. Endocrinology. 1982; 111: 522-529Crossref PubMed Scopus (158) Google Scholar), further altering the balance of these sex hormones. Therefore, although male sexual differentiation and reproductive organ development are critically dependent on androgen-AR signaling, it is possible that estrogen-AR-coregulator signaling may influence androgen action in conditions where DHT levels are negligible and testosterone is readily aromatized to estrogen. The loss of the E2-AR-coregulator pathway may partly disrupt the balance between AR and ER signaling during critical stages in male reproductive system development. Fig. 4 Bdemonstrates that the S703G and E709K PAIS mutants cannot functionally interact with ARA70 in response to E2. Therefore, decreased total receptor activity in AR(S703G) and AR(E709K) PAIS patients may tip the balance in favor of ER signaling, resulting in undermasculinization.Our in vivo data in Fig. 8 show that ARA70 and the AR are both present in the interstitial cells of the mouse E16 testis. Primitive sex cords mature to form testis cords and the rete testis at 13 days postcoitum (34Rugh R. The Mouse: Its Reproduction and Development. Burgess Publishing Company, Minneapolis, MN1968: 282-289Google Scholar). By 15 days, the vas deferens, epididymis, and seminal vesicles arise from the Wolffian duct, and the testis develops prominent sex cords, each of which become a seminiferous tubule (34Rugh R. The Mouse: Its Reproduction and Development. Burgess Publishing Company, Minneapolis, MN1968: 282-289Google Scholar). At this point, the cord contains uniform but undifferentiated cells, which are precursors of spermatogenic or Sertoli cells. As the testis continues to develop, mesenchymal cells between the testis cords become Leydig cells. Leydig cells are responsible for the synthesis and secretion of testosterone, the primary hormone of the testis and a critical regulator of spermatogenesis. Leydig cells also produce 5α-reduced androgens, such as DHT and 3α-diol. E2 can be synthesized through the aromatization of testosterone by aromatase in the Sertoli cells and diffuse back to the Leydig cells (33George F.W. Ojeda S.R. Endocrinology. 1982; 111: 522-529Crossref PubMed Scopus (158) Google Scholar, 34Rugh R. The Mouse: Its Reproduction and Development. Burgess Publishing Company, Minneapolis, MN1968: 282-289Google Scholar). Conversion of testosterone to estrogen also occurs in the Leydig cells. Testicular testosterone and its derivatives then induce masculine extragonadal differentiation. The abundance of ARA70 in testicular interstitial cells, at the time of E2 synthesis, supports our in vitro data showing the modulation of ARA70 on the AR-E2 binding. Moreover, this could imply that ARA70 impacts the development of male structures via not only the androgen-AR-ARA70 pathway but also the E2-AR-ARA70 pathway, to stimulate the development of genital organs, such as the vas deferens, epididymis, and seminal vesicle and the masculinization of the external genitalia.Our data therefore provide one possible molecular basis for a subset of PAIS phenotypes where mutation does not abolish androgen-AR signaling. Recent studies reported that ARA70 is not mutated in an analysis of 27 androgen insensitivity syndrome patients (35Lim H.N. Hawkins J.R. Hughes I.A. Clin. Genet. 2001; 59: 284-286Crossref PubMed Scopus (11) Google Scholar). This finding excludes the possibility of ARA70 mutation in PAIS and supports our assertion that the loss of AR mutant functional interaction with ARA70, due to helix 3 mutations, may affect a subset of PAIS patients where androgen response is normal.In summary, our data provide one of the potential molecular causes to explain the development of PAIS in a subset of patients with AR mutants that respond normally to androgen. Moreover, our results have partially elucidated how ARA70 enhances the activation of the wtAR in response to E2. These findings suggest that certain coregulators may have an important role in the regulation of male reproductive system development specifically in modulation of E2 induction of the AR transactivation. Androgen insensitivity syndrome is characterized by a spectrum of deficient male virilization or undermasculinization including testicular feminization (29Felig P. Frohman L.A. Endocrinology and Metabolism. 4th Ed. McGraw-Hill, Inc., New York2001: 779-824Google Scholar). It is known that if androgen action is slightly less than optimal, there will be undervirilization or inefficient spermatogenesis (29Felig P. Frohman L.A. Endocrinology and Metabolism. 4th Ed. McGraw-Hill, Inc., New York2001: 779-824Google Scholar). Although mutation of the AR is often found in PAIS patients, certain mutations associated with PAIS respond normally to androgen (4Gottlieb B. Beitel L.K. Lumbroso R. Pinsky L. Trifiro M. Hum. Mutat. 1999; 14: 103-114Crossref PubMed Scopus (113) Google Scholar). The first mutation analyzed in this study, AR(S703G), was identified in a patient diagnosed with male pseudohermaphroditism, which is associated with PAIS (3Radmayr C. Culig Z. Glatzl J. Neuschmid-Kaspar F. Bartsch G. Klocker H. J. Urol. 1997; 158: 1553-1556Crossref PubMed Scopus (23) Google Scholar). The other mutation we studied, AR(E709K), is associated with Reifenstein syndrome (13Yeh S. Miyamoto H. Shima H. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5527-5532Crossref PubMed Scopus (243) Google Scholar). Reifenstein syndrome is a less severe variant of X-linked PAIS in which affected individuals present as phenotypic males with hypospadias, inadequate pubertal virilization, gynecomastia, and infertility (30Aima J. Griffin J.E. Gazak J.M. Wilson J.D. MacDonald P.C. N. Engl. J. Med. 1979; 300: 223-227Crossref PubMed Scopus (154) Google Scholar). Figs. Figure 1, Figure 2, Figure 3, Figure 4 show that both of these PAIS mutants respond to androgen normally, but have lost the ARA70-dependent response to E2. Although E2 is a nonspecific ligand for the wtAR, certain coregulators, such as ARA70 or SRC-1, can enhance wtAR transactivation in response to E2 (13Yeh S. Miyamoto H. Shima H. Chang C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5527-5532Crossref PubMed Scopus (243) Google Scholar, 14Agoulnik, I., Stenoien, D., Mancini, M. A., and Weigel, N. (2000) in Keystone Steroid Symposium, Boulder, Colorado, March 28, Abstr. 302, Bristol Myers Squibb Pharmaceutical Research Institute and The Directors Sponsor Fund.Google Scholar, 15Han G. Foster B.A. Mistry S. Buchanan G. Harris J.M. Tilley W.D. Greenberg N.M. J. Biol. Chem. 2001; 276: 11204-11213Abstract Full Text Full Text PDF PubMed Scopus (105) Google Scholar, 16Zhou Z.X., He, B. Hall S.H. Wilson E.M. French F.S. Mol. Endocrinol. 2002; 16: 287-300Crossref PubMed Scopus (43) Google Scholar). E2 and DHT have similar structures except that E2 has a phenolic A-ring. It is therefore possible that although the wtAR may be able to bind to E2, E2 may not be well oriented within the wtAR-LBD pocket leading to weaker binding. Therefore, E2 may easily dissociate resulting in marginal activation of the wtAR. The recruitment of ARA70 or other selective coregulators to the wtAR, however, may change the conformation of the wtAR so that E2 can be trapped within the wtAR-LBD pocket allowing E2 to more effectively activate wtAR transactivation. In terms of mutants, the conformation of E2-bound AR mutant may not be similar to that of E2-bound wtAR. That difference in the conformation may influence on the receptor-interface providing for the coregulator recruitment. Our data in Figs. 5 and 6 show that ARA70 helps E2 competitively bind to the wtAR and subsequently retards E2 dissociation from the wtAR binding pocket. This phenomenon may be important during the prenatal period (7Tanagho E.A. McAninch J.W. Smith's General Urology. 15th Ed. Lange Medical Books McGraw-Hill, New York2000: 813Google Scholar) where high maternal estrogen levels may initiate estrogen signaling via the wtAR in the presence of coregulators, such as ARA70. For example, one report found that plasma concentrations of testosterone and E2 in mothers delivering male babies were 1.2 ± 0.4 and 4.5 ± 0.7 ng/ml, respectively (31Adeyemo O. Jeyakumar H. Afr. J. Med. Med. Sci. 1993; 22: 55-60PubMed Google Scholar), and these sex hormone levels may influence the balance between androgens and estrogens in the male fetus. Studies of 20 fetal rat testis samples collected during genital tract differentiation (gestation) also reported that there is a negligible level of circulating DHT in fetal rat plasma (32Habert R. Picon R. J. Steroid Biochem. 1984; 2: 193-198Crossref Scopus (129) Google Scholar). In addition, male and female rat fetuses had similar plasma levels of DHT and E2, although male fetuses did have higher levels of testosterone (32Habert R. Picon R. J. Steroid Biochem. 1984; 2: 193-198Crossref Scopus (129) Google Scholar). High aromatase activity during the last days of fetal life may also convert testosterone to E2 in male fetuses (32Habert R. Picon R. J. Steroid Biochem. 1984; 2: 193-198Crossref Scopus (129) Google Scholar, 33George F.W. Ojeda S.R. Endocrinology. 1982; 111: 522-529Crossref PubMed Scopus (158) Google Scholar), further altering the balance of these sex hormones. Therefore, although male sexual differentiation and reproductive organ development are critically dependent on androgen-AR signaling, it is possible that estrogen-AR-coregulator signaling may influence androgen action in conditions where DHT levels are negligible and testosterone is readily aromatized to estrogen. The loss of the E2-AR-coregulator pathway may partly disrupt the balance between AR and ER signaling during critical stages in male reproductive system development. Fig. 4 Bdemonstrates that the S703G and E709K PAIS mutants cannot functionally interact with ARA70 in response to E2. Therefore, decreased total receptor activity in AR(S703G) and AR(E709K) PAIS patients may tip the balance in favor of ER signaling, resulting in undermasculinization. Our in vivo data in Fig. 8 show that ARA70 and the AR are both present in the interstitial cells of the mouse E16 testis. Primitive sex cords mature to form testis cords and the rete testis at 13 days postcoitum (34Rugh R. The Mouse: Its Reproduction and Development. Burgess Publishing Company, Minneapolis, MN1968: 282-289Google Scholar). By 15 days, the vas deferens, epididymis, and seminal vesicles arise from the Wolffian duct, and the testis develops prominent sex cords, each of which become a seminiferous tubule (34Rugh R. The Mouse: Its Reproduction and Development. Burgess Publishing Company, Minneapolis, MN1968: 282-289Google Scholar). At this point, the cord contains uniform but undifferentiated cells, which are precursors of spermatogenic or Sertoli cells. As the testis continues to develop, mesenchymal cells between the testis cords become Leydig cells. Leydig cells are responsible for the synthesis and secretion of testosterone, the primary hormone of the testis and a critical regulator of spermatogenesis. Leydig cells also produce 5α-reduced androgens, such as DHT and 3α-diol. E2 can be synthesized through the aromatization of testosterone by aromatase in the Sertoli cells and diffuse back to the Leydig cells (33George F.W. Ojeda S.R. Endocrinology. 1982; 111: 522-529Crossref PubMed Scopus (158) Google Scholar, 34Rugh R. The Mouse: Its Reproduction and Development. Burgess Publishing Company, Minneapolis, MN1968: 282-289Google Scholar). Conversion of testosterone to estrogen also occurs in the Leydig cells. Testicular testosterone and its derivatives then induce masculine extragonadal differentiation. The abundance of ARA70 in testicular interstitial cells, at the time of E2 synthesis, supports our in vitro data showing the modulation of ARA70 on the AR-E2 binding. Moreover, this could imply that ARA70 impacts the development of male structures via not only the androgen-AR-ARA70 pathway but also the E2-AR-ARA70 pathway, to stimulate the development of genital organs, such as the vas deferens, epididymis, and seminal vesicle and the masculinization of the external genitalia. Our data therefore provide one possible molecular basis for a subset of PAIS phenotypes where mutation does not abolish androgen-AR signaling. Recent studies reported that ARA70 is not mutated in an analysis of 27 androgen insensitivity syndrome patients (35Lim H.N. Hawkins J.R. Hughes I.A. Clin. Genet. 2001; 59: 284-286Crossref PubMed Scopus (11) Google Scholar). This finding excludes the possibility of ARA70 mutation in PAIS and supports our assertion that the loss of AR mutant functional interaction with ARA70, due to helix 3 mutations, may affect a subset of PAIS patients where androgen response is normal. In summary, our data provide one of the potential molecular causes to explain the development of PAIS in a subset of patients with AR mutants that respond normally to androgen. Moreover, our results have partially elucidated how ARA70 enhances the activation of the wtAR in response to E2. These findings suggest that certain coregulators may have an important role in the regulation of male reproductive system development specifically in modulation of E2 induction of the AR transactivation.
Referência(s)