Regulation of masculinization: androgen signalling for ...

REVIEWS Regulation of masculinization: androgen signalling for external genitalia development Shoko Matsushita1,5, Kentaro Suzuki1,5, Aki Murashima1,2, Daiki Kajioka1, Alvin Resultay Acebedo1, Shinichi Miyagawa1, Ryuma Haraguchi3, Yukiko Ogino4 and Gen Yamada1*

Abstract | The biology of masculinization is fundamentally important for understanding the embryonic developmental processes that are involved in the development of the male reproductive tract, external genitalia, and also the tumorigenesis of prostate cancer. The molecular mechanisms of masculinization are of interest to many researchers and clinicians involved in varied fields, including molecular developmental biology, cancer research, endocrinology, and urology. Androgen signalling is mediated by the nuclear androgen receptor, which has fundamental roles in masculinization during development. Various modes of androgen signalling, including 5α-dihydrotestosterone-induced regulation of mesenchymal cell proliferation, have been observed in masculinization. Such regulation is essential for regulating urogenital tissue development, including external genitalia development. Androgen-induced genes, such as MAFB, which belongs to the activator protein 1 (AP-1) superfamily of genes, have essential roles in male urethral formation, and disruption of its signalling can interfere with urethral formation, which often results in hypospadias. Another AP-1 superfamily gene, ATF3, could be responsible for some instances of hypospadias in humans. These androgen-dependent signals and downstream events are crucial for not only developmental processes but also processes of diseases such as hypospadias and prostate cancer.

1 Department of Developmental Genetics, Wakayama Medical University, Wakayama City, Wakayama, Japan.

Department of Anatomy, Iwate Medical University, Yahaba, Iwate, Japan. 2

3 Department of Molecular Pathology, Ehime University Graduate School of Medicine, Shitsukawa, Toon City, Ehime, Japan. 4 Faculty of Agriculture, Kyushu University, Higashi-ku, Fukuoka, Japan. 5 These authors contributed equally: Shoko Matsushita, Kentaro Suzuki.

*e-mail: genyama77@ yahoo.co.jp https://doi.org/10.1038/ s41585-018-0008-y

The masculinization of embryos is generally mediated by androgens, and androgen signalling is mediated by the nuclear androgen receptor (AR)1–5. Androgen–AR signalling could have fundamental roles in masculinization. Mechanisms of AR-induced gene regulation have been studied using molecular genetics and developmental biology employing mutant mouse models. Despite the critical functions of androgen signalling in the process of masculinization, regulation of gene expression for masculinization and the downstream molecular mechanisms that occur during organogenesis remain poorly understood. Moreover, the involvement of nongonadal and locally produced masculine factors in the developing organs has not yet been well elucidated. Sexual differentiation depends on the orchestration of the several signalling networks. The biology of masculinization is an important field of study that can affect various research efforts and clinical fields. This Review summarizes current findings that promote understanding of this emerging area of research, with an emphasis on the regulatory genes for external genitalia and the

development of the male reproductive tract (MRT) in comparison with prostate cancer development and the roles of cancer-associated fibroblasts (CAFs).

Androgens in genital formation Androgen–AR signalling is involved in male-type organogenesis and masculinization1–5. During embryogenesis, androgens induce the process of masculinization, initiating formation of the prostate, penis, and tissues of the MRT, such as the epididymis3,6–10. For external genitalia, the genital tubercle (the embryonic anlage) develops as a swelling common to both males and females, eventually diverging to exhibit female or male organogenesis11. AR has a central role in mediating masculinization of the genital tubercle. The development of male external genitalia from the genital tubercle in the embryo is characterized by the formation of a tubular urethra with a well-developed prepuce and the condensation of a bilateral prospective corporal body6,12. In mice, anatomical differences are not prominent until embryonic day 14.5. Sexual

NATURE REVIEWS | UROLOGY © 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

REVIEWS Key points • The molecular mechanisms of masculinization are fundamental topics of many fields of science, including molecular developmental biology, cancer research, endocrinology, and urology. • One of the activator protein 1 (AP-1) superfamily genes, MAFB, has been identified as an androgen target gene and has essential roles in male- type urethral formation. • Mesenchymal cell proliferation can be regulated by testosterone and 5α-dihydrotestosterone via the androgen receptor. • Putatively similar mesenchymal cell characteristics in embryos and prostate-cancer-associated fibroblasts have been described, including the identification of AP-1 superfamily genes. • Genes such as ATF3 that are involved in various signalling pathways are affected by oestrogen receptor- mediated cellular processes.

differentiation in the genital tubercle starts at embryonic day 16.5, when canalization of the urethral plate to form the urethra occurs, which is dependent on androgen signalling13. The male urethra is incorporated into the glans, a process that is mediated by the action of androgens13. In contrast to male development, the female genital tubercle does not form a tubular urethra with an enclosed prepuce at the lower (ventral) midline13. Thus, urethral formation is a useful tool for investigating the mechanisms of masculinization and androgen-dependent signalling cascades. Mice with tissue-specific mutations in Ar are used to analyse the various mechanisms and disruption of masculinization. The testicular feminization mouse model, in which mice lack functional ARs, is useful for analysing the phenotypes of null mutations in Ar, which correspond to human complete androgen-insensitivity syndrome 14. In addition, conditional Ar- knockout mouse models facilitate analysis of the roles of AR in various cell types and organs in male and female reproductive systems15,16. Studies of mice with conditional Ar knockout specifically in Sertoli cells, Leydig cells, or peritubular myoid cells indicate that androgen signalling has essential but different roles in the various cells responsible for spermatogenesis16–20. Mice lacking AR in germ cells have normal spermatogenesis and fertility, suggesting that AR might affect spermatogenesis through indirect paracrine cell–cell communication16. However, androgen–AR signalling has a direct role in spermatogenesis in Sertoli cells, and AR knockout in Leydig cells influences steroidogenic function, resulting in the arrest of spermatogenesis16. Furthermore, mice with conditional AR knockout in Sertoli cells have atrophied testicles, reduced levels of serum testosterone, increased serum luteinizing hormone concentrations, and defective expression of anti-Müllerian hormone17. At the start of sexual differentiation during organogenesis, AR is expressed in the mesenchyme of the embryonic urogenital anlage, including the mesenchyme adjacent to the urethral plate epithelium of the genital tubercle6. The epithelial- specific and mesenchymalspecific functions of AR have been investigated during genital tubercle development21. Conditional knockout of mesenchymal AR in the genital tubercle of male mice causes female-like developmental features, such as reduced overall size of the genital tubercle, defective

urethral fusion, and preputial closure, demonstrating that mesenchymal androgen signalling through AR is essential for genital tubercle masculinization 21. This male- specific genital tubercle patterning occurs at around embryonic day 16.5 in mice and at 12 weeks in human gestation22. Regarding defects in external genitalia formation in humans, hypospadias is characterized by the formation of an abnormal urethral meatus, in which it can locate in several proximal and distal regions of the penis23–31. Phenotypes associated with hypospadias include underdeveloped foreskin, bending of the penis (also known as chordee), and undescended testes32. Timing of AR signalling disruption influences the phenotype of the developmental disorder33–35. For example, genetic deletion of Ar using tamoxifen administered at embryonic day 13.5 caused feminization of the genital tubercle in male mice and subsequent micropenis development at embryonic day 17.5 (REF.33). Disruption of androgen signalling using the antiandrogen flutamide during different developmental windows resulted in different degrees of abnormal genital phenotypes in male rats34. The critical time window of genital development is referred to as the masculinization programme window35 and corresponds to the embryonic stage during which the genital tubercle becomes sensitive to androgen signals. This window is reported to occur between 8 weeks and 12 weeks gestation in humans22. The biology of masculinization and the role of androgen signalling are fundamentally important for understanding embryonic developmental processes of the MRT, external genitalia, and also for tumorigenesis.

Effects of androgens on development Embryonic masculinization can be achieved by dimorphic events such as cell-specific proliferation occurring in the mesenchyme of the MRT36. Augmented cell proliferation has been suggested to occur during the organogenesis of male genitalia37,38. Inhibition of cellular proliferation, including differentiation of mesenchymal cells, is also part of the masculinization process36. Regulation of cell growth by cell cycle regulators is a candidate causative mechanism of the masculinization process. The cyclin- dependent kinase inhibitor p21 (also known as CDKN1A) negatively regulates cell growth39. Ar mutation in non- mitotic cells caused defective development of the sexually dimorphic bulbocavernosus muscle in embryonic mice, probably as a result of reduced numbers of proliferating undifferentiated myofibroblasts37. These cells exhibited upregulation of p21, which was similar to the increase in expression observed in female mouse embryos compared with male mouse tissue37. Expression of p21 in prostate cancer cells can be upregulated by the synthetic androgen R1881 (REF.40). R1881 increased the activity of a promoter in p21, which is suggestive of the presence of an androgen response element (ARE), and further analysis revealed the presence of an ARE at –200 bp proximal to the promoter region. p21 is also expressed in the genital tubercle and is possibly involved in the regulation of mesenchymal cell proliferation. Thus, this gene could be a target of androgen signalling that regulates cell proliferation during genital tubercle development. www.nature.com/nrurol

© 2018 Macmillan Publishers Limited, part of Springer Nature. All rights reserved.

REVIEWS Circulating androgens induce prostatic formation and masculinization of the genital tubercle41,42. Androgens produced locally in the embryonic tissues or in adult tissues include testosterone and 5α-dihydrotestosterone (DHT), which both have essential roles in organogenesis of the genital tubercle36. One study suggested that DHT could negatively regulate cell proliferation in the ventral side of the genital tubercle during urethral formation in a dose-dependent manner36. Notably, type II 5α-reductase is encoded by SRD5A2 (REF.43), which is expressed in the bilateral mesenchyme of the genital tubercle before male-type urethral formation36. This enzyme converts testosterone to DHT in the tissues where SRD5A2 is expressed. DHT production by the conversion of testosterone has also been suggested to be an important factor in genital tubercle development. For example, cellular proliferation is reduced and the levels of testosterone relative to DHT have been shown to alternate in the mesenchyme bilateral to the urethra during male genital tubercle differentiation36. Thus, levels of testosterone and DHT could affect cell proliferation in this tissue. Hence, regulated production of DHT might lead to reduced cell proliferation of the bilateral mesenchyme by regulators (such as transcription factor MAFB, which regulates male-type urethral formation); MAFB is suggested to be a DHT-responsive gene44. Local regulation of DHT production in the genital tubercle requires further investigation to elucidate its role in the masculinization of the genitalia. Furthermore, the concept of local regulation of androgens and the roles of DHT production in the genital tubercle in general require further investigation, as masculinization of the genital tubercle has generally been considered to be mediated by circulating androgens45. In prostate cancer biology, locally produced androgens could regulate the growth of the tumour46,47. High concentrations of androgen have a suppressive effect of LNCaP cell proliferation48. Moreover, the proliferation of AR-transfected PC3 cells is blocked by DHT49. Thus, epithelial AR signalling can also negatively regulate prostate cancer cell proliferation50. Further investigation of the dynamics and local distribution of DHT and their effects on cell proliferation is required. Regulation of cell proliferation by androgens is an important event for masculinization. Different roles for locally converted androgen and DHT in contrast to testosterone are intriguing topics for further investigation.

Growth factor signalling Organogenesis is mediated by epithelial–mesenchymal interactions (EMI) (FIG. 1a) through several growth factors and growth factor signalling. Expression of genes involved in masculinization of the genital tubercle can be identified in the mesenchyme adjacent to the urethra (the bilateral mesenchyme of the genital tubercle) (FIG. 1a) using conditional knockout mouse models. Mouse models of genital masculinization. A feature of the reproductive tract and urogenital organ formation is its prominent dimorphic development from the common anlage of the male–female-type organs. In mammals, sex determination usually occurs first

in the gonads: gonadal hormones such as testosterone subsequently regulate masculinization of the MRT and other organs, including the male- type genital tubercle3,51. The functions of fibroblast growth factor (FGF), Hedgehog, and Wnt signalling have been characterized with regard to their roles in genital tubercle protrusion and outgrowth52–55. These growth factor signals are essential for inducing initial genital tubercle bud formation. For example, disruption of Hedgehog signalling results in genital abnormalities, which affects the interaction between the cloaca and its surrounding mesenchyme, the pericloacal mesenchyme, in mouse models56,57. Generally, such growth factor signals have been considered essential for the initial morphogenesis of the genital tubercle anlage and necessary for its protrusion and further outgrowth before the process of masculinization11. Expression of FGFs and FGF receptors (FGFRs) is often observed in the embryonic mesenchyme and the epithelia of the genital tubercle, respectively, and disruption of FGF signalling can cause genital abnormalities52,58–61. For example, Fgf10-knockout mice have a hypospadiaslike phenotype52,59,61. Moreover, Fgfr2IIIb expression decreases in response to flutamide treatment in an in vitro organ culture of the genital tubercle59. However, further conditional Fgf-mutant mouse models are necessary to increase our understanding of the role of FGF signalling in the masculinization of the genital tubercle. Hedgehog signalling has also been suggested to be involved in genital masculinization and organogenesis62,63. The essential signal mediator of Sonic hedgehog protein (SHH) in the mesenchyme, zinc-finger protein GLI2, has been suggested to be a potential masculinization factor. Shh knockout resulted in complete genital tubercle agenesis64. In Gli2-mutant mice, genital tubercle development was severely defective, with the urethral epithelium exposed to the outer surface64. Hedgehog signalling probably facilitates the masculinization processes by affecting the androgen responsiveness. Gli2+/––Gli3Δ699/+ compound mutant mice have reduced GLI2 activator levels. Constitutive expression of the repressor of transcriptional activator GLI3 causes a spectrum of urogenital malformations. Male embryos and adult male mice displayed shortened anogenital distance, an open ventral urethral groove, and abnormal penile size and structure with incomplete testicular descent65. Treatment with oestradiol benzoate or flutamide reduced Indian hedgehog protein (IHH, an SHH homologue) expression, and Ihh expression was observed in urethral, preputial, and glans epithelial cells in male and female genitalia33. Hedgehog and FGF signalling both have essential roles in genital tubercle protrusion and testis development66,67. Thus, the Hedgehog signalling pathway is considered a possible key factor for initial development and also for sexually dimorphic development of the external genitalia in coordination with androgen signalling. Wnt signals have been observed in the genesis of various organs and many pathological processes68–70. Numerous regulatory factors involved in canonical Wnt signalling have been identified in genital tubercle development52,71,72. Canonical Wnt signals are mediated by factors including β-catenin21,73, and β-catenin activity has been detected during the initiation of


REVIEWS a

MAFB GFP

Male

Female

Male genital tubercle at embryonic day 16.5

Urethral masculinization

Tubular urethra

Bilateral mesenchyme adjacent to urethra

b Testosterone 5αGrowth factors reductase (TGFs, Wnt–β-catenin) DHT

AP-1 superfamily including MAFB

Partner selection

Protein modification

AP-1 superfamily

MAF (MAFB, MAFA, MAF, NRL, MAFK, MAFG, and MAFF) JUN (JUND, JUN, and JUNB) FOS (ATF3, FOSB, FOS, FRA1, and FRA2) ATF (ATF4, ATF5, ATF2, and ATF7) CREB (ATF1, CREB, and CREM)

Fig. 1 | The processes of masculinization of the embryonic external genitalia by Mafb. a | Differential expression of MAFB in male and female embryonic mesenchyme. The masculinization-regulatory gene Mafb (which encodes transcription factor MAFB) belongs to the activator protein 1 (AP-1) superfamily. It shows a differential expression pattern in the male and female embryonic mesenchyme, as visualized by green fluorescent protein (GFP) expression adjacent to the urethra. Mafb is a key regulatory gene for androgen-induced male-type urethral formation in the bilateral mesenchyme (blue region). Regulation of AP-1 superfamily gene expression is mediated by growth factor signalling, including transforming growth factors (TGFs) and Wnt–β-catenin signalling. MAFB could be necessary for the masculinization of the bilateral mesenchyme of the genital tubercle. Male-type urethral formation is achieved by epithelial– mesenchymal interaction (white arrows). The enzyme 5α-reductase converts testosterone in the mesenchymal region into 5α-dihydrotestosterone (DHT). DHT production could regulate DHT target genes in the AP-1 superfamily, including Mafb, in the mesenchyme. The embryonic mesenchyme might possess characteristics similar to those of cancer-associated fibroblasts in prostate cancer. b | AP-1 superfamily components including Mafb. The AP-1 superfamily is a group of dimeric transcription factors that includes activating transcription factors (ATFs), JUN transcription factors, proto-oncogene FOS, and MAF transcription factors. These transcription factors are involved in various signalling cascades, interacting with their partners through the basic leucine-zipper domain and often immediately affecting formation of a signal-induced transcriptional complex upstream of target genes. CREB, cAMP-responsive elementbinding protein; CREM, cAMP-responsive element modulator; FRA, FOS-related antigen; NRL, neural retina-specific leucine-zipper protein.

genital tubercle development at the cloacal membrane71. These canonical Wnt signals are downstream of Hedgehog signals. β- Catenin and Wnt levels in the genital tubercle are associated with SHH levels71. Expression of β-catenin and Wnt in the genital tubercle is downregulated in Shh-null mouse models that show genital tubercle agenesis. However, genital tubercle outgrowth is rescued in Shh-null models with constitutively active β-catenin71. Mice with loss-of-function and gain- of-function mutations in β- catenin displayed abnormal urorectal septum development 72. Wnt–β-catenin signalling has been shown to be essential for regulating genital tubercle outgrowth73. In mice with deficient Wnt signalling, expression of Fg f8 rescues deficient genital tubercle development71,74. Canonical Wnt signalling and β-catenin expression are apparent during masculinization of the mesenchyme adjacent to the urethra. Expression of canonical Wnt signalling antagonists Dkk2 and Sfrp1 is increased in prospective female- type genitalia formation 21. Gain- of-function β- catenin mutations specifically in the mesenchyme of the genital tubercle result in adult male-like external genitalia in female embryos21. How canonical Wnt signalling and AR signalling interact with regards to the masculinization of genitalia has not been extensively studied and requires investigation. Previous investigation has suggested that β- catenin binds to the AR ligand binding domain and increases the transcriptional activity of AR in GnRH neuronal cells75. Signalling for EMI. EMI has an essential role in prostatic and genital tubercle development, prostate cancer cells, and CAFs. Notably, AR expression and function are critical in the mesenchyme in such tissues. Some genes, such as those in the Hedgehog, Wnt, and AP-1 gene families, including MAFB, are also expressed during EMI76,77. CAFs can regulate cell proliferation in adjacent tumours, and CAF–AR signalling could be associated with cancer progression78. Mesenchymal AR inhibits the growth of malignant epithelial cells, possibly through expression of paracrine factors. Such paracrine factors are involved in CAF– epithelial tumour cell interactions. Mesenchymal AR signalling initiated by paracrine factors can induce the expression of genes that have antiproliferative effects79. Studies have shown that the AR-regulated transcriptome in prostate CAFs is distinct from that in prostate cancer. The primary pioneer factor in prostate cancer epithelial cells is typically forkhead box protein A1 (FOXA1), whereas data obtained from CAF-like cell lines indicate that AP-1 has a major role in controlling the AR cistrome77,80. This distinct fibroblast transcriptome is strongly associated with androgen- induced genes that mediate the antiproliferative action of AR77, which is reminiscent of the processes that occur in the genital tubercle mesenchyme (FIG. 1) . Thus, understanding mesenchymal androgen signalling through genes including those in the AP-1 superfamily might contribute to an improved understanding of EMI in the genital tubercle and prostate CAFs. β-Catenin is also involved in the adherens junction complex at the cell membrane and is expressed at the www.nature.com/nrurol


REVIEWS cell membrane in the endoderm, urethral epithelia, and ectoderm of the genital tubercle in mice81. Ectodermal expression of β-catenin has a role in the development of the genital tubercle81. Mutant mice with conditional loss of ectodermal β-catenin do not have proper midventral fusion of the external genitalia. Furthermore, these mice have reduced α-catenin expression and filamentous actin (F-actin) integrity at the cell–cell border of the ectoderm during early preputial uprising81. Growth factor signalling is considered essential for the initial morphogenesis of the genital tubercle and also the process of androgen-dependent masculinization.

MAFB and masculinization Transcription factor MAFB has been identified as an androgen-responsive target gene that regulates maletype external genitalia formation44 (FIG. 1a). Expression of Mafb shows a sexually dimorphic pattern from embryonic day 14.5, before the onset of male- type urethral formation (REF.82) (FIG. 1a). Mafb-knockout mice fail to develop a male-type urethra, which is reminiscent of the phenotype of hypospadias in humans44. Conditional knockout of Ar in the mesenchyme of the genital tubercle resulted in defective male-type urethral formation without Mafb expression. MAFB is a basic leucinezipper (bZIP) transcription factor and is a member of the AP-1 superfamily83,84 (FIG. 1b). The AP-1 superfamily is a group of dimeric transcription factors that includes activating transcription factors such as JUN, protooncogene FOS and transcription factor MAF85–88. These transcription factors are involved in various signalling cascades, including Wnt signalling, and interact with their partners through the bZIP domain, which often immediately affects formation of a signal-induced transcriptional complex upstream of target genes89. The target genes of MAFB during genital tubercle development have not yet been identified. Addition of androgens can induce expression of MAFB. For example, Mafb expression is increased in female mouse embryos when they are exposed to testosterone propionate in utero44. Furthermore, exposure of male mouse and female mouse genital tubercle tissue in organ culture to DHT induced expression of Mafb82. However, the functional importance of the complex formation of MAFB regulation and identification of other partners have been rarely studied. Many genes belonging to the AP-1 superfamily and the possible resultant combinations of partner molecules hamper the identification of the function of such a protein complex. Some candidate partner genes for MAFB have been identified in developmental processes and cellular differentiation. For example, FOS and JUN were identified as MAFB partners involved in the apoptotic regulation of the interdigital regions during chick embryonic limb development90. A role for the MAFB– FOS complex has been shown in monocytes91. Moreover, MAFB overexpression interferes with the DNA-binding ability of FOS, microphthalmia-associated transcription factor, and nuclear factor of activated T cells, cytoplasmic 1 (NFATC1), inhibiting transactivation of NFATC1 and osteoclast-associated immunoglobulin-like receptor during receptor activator of nuclear factor-κB ligand (RANKL; also known as TNFSF11)-induced osteoclast

differentiation92. Expression of MAFB and other members of the MAF family is involved in regulating the embryonic differentiation of α-cells and β-cells during pancreas development93. Studies using mouse models to investigate the functions of members of the AP-1 superfamily during organogenesis are rarely performed owing to the presence of their many partner genes. The roles of MAFB in genital tubercle masculinization have been shown, but the possibility of sequential changes of partner genes and the effects of these changes has not been demonstrated. Protein modification, such as sumoylation, has been reported to occur to various transcriptional regulators, including MAFB 94 . Sumoylation of MAFB can occur at lysines 32 and 297, and repression of MAFB transactivation by transcriptional activator MYB is small ubiquitin- related modifier dependent. Furthermore, MAFB-driven transactivation and macrophage differentiation potential were increased by sumoylation, but cell cycle progression and myeloid progenitor growth were inhibited94. MAFB was originally identified as an oncogene95; however, its involvement in androgendependent prostate cancer is not known. Possibly, certain AP-1 superfamily genes, such as MAFB, could be involved in mesenchymal differentiation of the genital tubercle, and others could be involved in CAF-related androgen signalling. Regulators of masculinization such as MAFB could be involved in the development of other organs in which the dimorphic developmental phenotypes are not obviously observed. Mutations in MAFB are associated with multicentric carpotarsal osteolysis syndrome96. A study published in 2010 reported the proximity of risk variants for cleft lip and cleft palate to MAFB97. Cleft lip and cleft palate have been associated with the presence of hypospadias98. Mafb- mutant mice display cleft palates as well as a hypospadias-like phenotype (K. Suzuki, unpublished observations). Thus, MAFB could be involved in both developmental disorders. Multiple factors are involved in craniofacial and reproductive tissue organogenesis, and associated phenotypes are, therefore, often complex. Thus, further classification and careful consideration of the possible causes of such overlapping phenotypes are necessary. Further analyses are necessary to understand the role of MAFB in disease pathogenesis and genital masculinization.

Potential interactions of AR Canonical Wnt signals might regulate various downstream factors such as lymphoid enhancer-binding factor 1 (LEF1) and Axin 2. The mesenchymal region of the genital tubercle expresses MAFB and canonical Wnt signals21. Thus, locally active canonical Wnt signals could also be involved in the regulation of downstream genetic cascades. Constitutively active β-catenin augments the reporter activity of MAFB in vitro 82. Moreover, the β-catenin conditional knockout mutant genital tubercle shows reduced expression of MAFB, indicating that expression in the genital tubercle is β-catenin dependent (S. Matsushita, unpublished observations). Thus, regulation of MAFB expression mediated by Wnt–β-catenin signalling could be necessary for the


REVIEWS masculinization of the bilateral mesenchyme of the genital tubercle (FIG. 1a). Putative crosstalk between Wnt signalling and androgen signalling has been reported in other contexts, such as androgenetic alopecia99. Thus, AR, Wnt, and MAFB could interact in the genital tubercle during masculinization. Modulations in AR- induced transcription have been frequently attributed to a polyglutamine stretch of variable length in the amino-terminal domain of the receptor. A variable number of CAG triplets in exon 1 of AR, located on the X chromosome, encode this polyglutamine stretch, and pathologically elongated AR CAG repeats have been reported. For example, a pathological expansion of the AR CAG repeat has been observed in Kennedy syndrome, which is an inherited neurodegenerative disease characterized by progressive neuromuscular weakness100. Formation of the AR transcriptional complex requires the functional and structural interaction of AR with its co-regulators, including members of the CREB-binding protein (CBP)–P300 family101. Similarly, β-catenin associates with several nuclear factors, such as transcription factor TCF, forming a nuclear signalling complex in the canonical Wnt signalling pathway. Signalling complexes including such molecules could possibly interact through factors associated with Wnt–β-catenin that mediate downstream cellular processes102. Possible modulation of canonical Wnt activity by AR mutations, such as those found in Kennedy syndrome, should be examined. However, whether nuclear β-catenin–AR interaction has a role in MAFB regulation is unclear (FIG. 1a). Further analyses are necessary for understanding the masculinization processes for the bilateral mesenchyme adjacent to the urethra by MAFB and canonical Wnt signalling. In addition to Wnt signals, other signalling pathways have been identified as potential regulators of MAFB, such as those pathways involving FGF and retinoic acid103,104. Both FGF and retinoic acid signalling are involved in gonadal sexual differentiation105. MAFB is also expressed and functions in the testicular niche, in which it is required for testicular vasculogenesis and testicular cord formation106,107. Steroid receptor chaperones 51 kDa FK506-binding protein (FKBP51; also known as FKBP5) and FKBP52 (also known as FKBP4) also interact with AR 108,109, and FKBP52 is involved in masculinization processes109. FKBP52-deficient mice develop hypospadias and prostate dysgenesis109,110. FKBP52 has been reported to synergize with β-catenin to potentiate AR signalling111. FKBP51 expression is higher in the male genital tubercle than the female genital tubercle112. Overexpression of FKBP51 stimulates AR- mediated transcription in prostate cancer cells113. Androgen treatment of prostate cancer cells induced FKBP51 expression114, but comprehensive understanding of the mechanisms of its regulation in response to androgen signalling is still lacking. Further understanding of the role of FKBPs in response to putative crosstalk between Wnt–β-catenin signalling and androgen signalling during genital tubercle masculinization is required. Analysing the roles of the genetic interactions between androgens and locally expressed growth factor signalling

is an intriguing topic regarding the development of reproductive organs including external genitalia.

Regulation of target genes by AREs Several genes that have roles in masculinization that are regulated by androgens have been identified using differential screening and phenotypic analyses of mouse mutant models during genital tubercle development. These genes include Fgfr2, Ephb2, Fkbp51, Cyp1b1, Mafb, Ctnnb1, and Ihh21,33,59,112,115. The binding of AR to androgen target genes often requires the presence of AREs. Identification of several regulatory elements and ARE characterization suggest that these genes are androgen responsive. Several types of androgeninducible gene regulation have been reported. One type of this regulation depends on the functions of AR–ARE binding. A second type corresponds to indirect binding to DNA by other factors that associate with AR. These two modes of regulation by AR–ARE can be observed in an overlapping manner. Another mode of regulation is mediated by a third-party regulatory signal cascade often indirectly associated with AR for the regulation of various target genes116,117. The role of AR–ARE binding in the regulation of masculinization gene expression has been frequently described82. The presence of AREs has been shown in Mafb. Specifically, two functional AREs in the 3′ untranslated region (UTR) of Mafb have been observed, and the 5′ UTR also showed androgen responsiveness82. ARE sites in the 3′ UTR of Mafb have been shown to be necessary for the androgen–ARinduced expression of the gene in the genital tubercle82. Cooperation of the Mafb 3′ UTR with other regulatory elements, including enhancers in the promoter and distal regions, is necessary for the transcriptional regulation of Mafb (S. Matsushita, unpublished observations). The involvement of androgen signalling in organ development and pathogenesis depends on the type of tissue. A frequently analysed tissue regarding AR-target genes and their enhancers is the epididymis. The epididymis expresses various AR-target genes in the epithelia and mesenchyme (such as Man1a, Ace, Glul, Isyna1, Afar (also known as Akr7a2), and Gpx5)118–121. Furthermore, functional genes that regulate lipid metabolism and sperm maturation have been identified122. For example, one study indicated that TP63 (Trp63 in mice) is a candidate downstream gene of androgen signalling during epididymal development123. Loss of epithelial AR reduced expression of tumour protein p63, which is essential for differentiation of basal cells in its epithelium123. The putative androgendriven enhancer of TP63 contains several AREs (termed the p63 androgen response region)123. p63 could be responsive to androgen–AR signalling, but how p63 expression is regulated in vivo is unknown. The onset of p63 expression is much later than that of AR in the embryonic epididymal epithelia. Thus, androgen–AR signalling might augment or maintain expression of p63 in this tissue. TP63 is also an essential gene in embryonic urethral formation124. Mice with Trp63 knocked out display embryonic urethral defects, including disruption of the urethral-plate-to-urethra formation process without midline fusion. Furthermore, Fgf8 www.nature.com/nrurol


REVIEWS expression, one of the signals regulating development of the genital tubercle, was reduced in the distal urethral epithelia of the genital tubercle of Trp63-knockout mice at embryonic day 11.0 (REF.124). However, whether sexual differences in p63 expression exist during urethral cell differentiation is not known25,60,124. Furthermore, whether androgen signalling can regionally and temporally affect male-type urethral formation via p63 is unclear, as null mutation of Trp63 results in embryonic lethality in mouse models. Mutation of this gene should be studied with regard to urogenital abnormalities and androgen dependency. Several developmental genes for masculinization are regulated by androgens through AREs in regulatory regions during genital tubercle development. Further genome-wide analyses of AR binding are also necessary to understand androgen- mediated masculinization during genital tubercle development.

Disruption of signalling Studies on the disruption of genetic regulation of masculinization and the effects of endocrine disrupting chemicals (EDCs), such as bisphenol A, have been conducted125. Interference of androgen signalling through the modulation of various signal cascades by chemicals could affect MRT tissue development, including that of the external genitalia126,127. The frequency of genital birth defects, such as hypospadias, has been rising128–130, and some reports indicate that EDCs could have a role in the increasing frequency of these defects, with the development of novel technologies and chemicals131,132. However, little is known about the temporal windows of sensitivity to EDCs and the tissue-specific and downstream targets of the AR and oestrogen receptor for external genitalia formation. AP-1 superfamily genes for oestrogenic signalling. Endogenous oestrogen signals influence developmental gene functions; however, both male and female mice with the oestrogen receptor knocked out have no obvious defects during normal organogenesis133. Exposure of female mice to the synthetic oestrogen diethylstilbestrol causes hypospadias134. Oestrogen receptor gene disruption and oestrogen could underlie several congenital penile anomalies33, including hypospadias, chordee, micropenis, and ambiguous genitalia 33,135. Among the genes affected by oestrogens and the oestrogen receptor are cell cycle regulator genes, such as CCNB1 (REF.136). 17β- Oestradiol induces cyclin D expression, which is critical to its mitogenic effects 137. Genes involved in various signalling pathways are also affected by oestrogen receptor-mediated cellular processes, as is the case for ATF genes138. ATF genes belong to the AP-1 superfamily. Among them, ATF3 (a mediator of the stress response) has been suggested to be responsible for some instances of hypospadias in humans139. ATF3 has been shown to be upregulated by the suppression of androgen-induced genital tubercle development with oestrogen treatment in an organotypic genital tubercle culture in vitro138 (FIG. 2). Sequence variants in the 3′ UTR and the coding regions of ATF3 have been observed in patients with hypospadias27 (FIG. 2). Knockout of Atf3

increases prostatic cell proliferation, leading to prostatic hyperplasia in mice140–142. However, the genes and cellular events mediated by activating transcription factor 3 (ATF3) during genital tubercle development have not yet been fully elucidated. Growth factors and growth factor receptors, such as transforming growth factor-β1 (TGFβ1), TGF receptor 3, and frizzled 1, are expressed during external genitalia formation in mouse models143. Notably, TGFβ is involved in ATF3 translocation to the nucleus144. At the protein level, ATF3 might interact with several molecules, such as mothers against decapentaplegic homologue (SMAD), and could undergo nuclear translocation after ligand stimulation145. Nuclear translocation of the ATF3–SMAD3 or the ATF3–SMAD4 heterodimer after oestrogen exposure has been postulated, where the components of this heterodimer could interact with oestrogen receptor and inhibit DNA binding, causing a cellular response that is possibly related to EMI, resulting in hypospadias138. A novel mechanism of ATF3 regulation via upregulation of VAMP7 was reported in 2014 (REF.146). Increased copy numbers of VAMP7 disrupt human male urogenital development by altering oestrogen signalling, and transgenic mice expressing human VAMP7 show cryptorchidism, urethral defects, hypospadias, reduced penile length, and focal spermatogenic anomalies with subfertility 146. VAMP7 colocalized with oestrogen receptor after the treatment with 17β-oestradiol in vitro146. Cellular protein levels of oestrogen receptor were elevated in conditions of VAMP7 overexpression and ligand stimulation, leading to increased transcriptional activity of the receptor and the expression of oestrogen-responsive genes, including ATF3 (REF.146). VAMP7 overexpression underlies the aetiology of a subset of common disorders of male sexual differentiation146. Members of the AP-1 superfamily, including ATF3, dimerize with other AP-1 family members via a characteristic bZIP domain. Dimeric combinations of MAFB, FOS, and JUN are involved in the control of the apoptosis and survival balance in chick limb buds during limb morphogenesis90. However, such dimeric AP-1 superfamily member complexes have not yet been identified during urogenital organ formation. Further investigations using immunoprecipitation and chromatin immunoprecipitation analysis with antibodies against AP-1 super family members should be performed to elucidate whether this process occurs in genital development. Furthermore, studies looking for a potential partner complex in the AP 1 superfamily genes for male-type urethral formation also need to be performed. In fact, members of a gene family such as AP-1 generally form a complex that induces early response in the signalling responsible for initiating downstream signalling cascades, including hormone signalling (FIG. 2). The oestrogen receptor and AP-1 interaction has been reported via FOS, an AP-1 transcription factor component, in breast cancer cells147. This interaction was shown to mediate various transcriptional pathways, and oestrogen receptor might be important for mediating the effects of EDCs or other external signals that influence genitalia formation147. Hypospadias is a multifactorial


REVIEWS Embryonic mouse Genetic factors

Environmental factors

G th ffactors Growth Oestrogen treatment

ER

↑ Atf3

AR

Androgens

" eam signalling " signalling

Hypospadias

Atf3 3′ UTR and coding sequence variants

Fig. 2 | ATF3 gene and upstream signals in hypospadias. The regulatory gene Atf3 (which encodes activating transcription factor 3), belonging to the activator protein 1 (AP-1) superfamily, is responsive to various oestrogenic, androgenic, and growth factor signals. ATF3 could be involved in the development of hypospadias. Sequence variants in the 3′ untranslated region (UTR) and the coding regions of ATF3 have been observed in patients with hypospadias. Hypospadias is a multifactorial disease that is suggested to be caused by multiple factors, including genetic and environmental factors. The involvement of AP-1 superfamily genes in the onset of hypospadias remains unclear. Members of the AP-1 superfamily are involved in many processes in cellular signalling. AR, androgen receptor; ER, oestrogen receptor.

disease, which might be caused by multiple genetic and environmental factors148. The involvement of AP-1 superfamily genes in the onset of hypospadias remains unclear because members of the AP-1 superfamily are involved in many processes in cellular signalling, making the roles of individual members of this family in genital development difficult to delineate. Retinoic acid signalling. Retinoic acid, an active derivative of vitamin A, exerts crucial functions in vertebrate development149. Both excessive and reduced retinoic acid signalling can cause developmental abnormalities, including in the genitalia149. Retinoic acid administration is a good model system for examining teratological effects on external genitalia, as the genital tubercle is susceptible to the toxic effects of external factors150. Exposure of mouse embryos to excessive amounts

of retinoic acid between embryonic day 8.5 and 9.5 induces agenesis of the urethral epithelia on the ventral side of the genital tubercle151. Excessive retinoic acid signalling in mice with mutant cytochrome P450 26B1 (CYP26B1), a retinoic acid-catabolizing enzyme, leads to abnormal cell proliferation and differentiation during genital tubercle development, upregulating the expression of growth factor signalling molecules, such as SHH152. Human genetic studies have also linked genital tubercle agenesis and defects in urethra formation to abnormal retinoic acid levels in patients with NADPH–cytochrome P450 reductase deficiency153. Moreover, retinoic acid receptor-β (RARβ) is suggested to be involved in the regulation of human androgen biosynthesis154. The interactions between testosterone and retinoic acid might regulate the expression of RARs and cell growth in LNCaP cells 155. AR–RAR antagonistic effects could be mechanisms that regulate transcription of genes within the prostate, such as CDCA7L and CDK6 (REF. 156). Thus, investigating the mechanisms underlying the effects of retinoic acid and androgen signalling during genital tubercle masculinization is essential. Interference of hormonal signalling by various chemicals affects MRT development, including the development of external genitalia. Elucidation of the downstream events of such disruptors is necessary for understanding masculinization processes.

Conclusions Androgen–AR signalling is involved in male- type embryogenesis. Sexual differentiation of the male external genitalia is under the influence of androgens. Progress in the analysis of androgens, specifically testosterone and DHT, and their target genes has increased our understanding of the molecular and genetic processes involved in genital masculinization. Comprehension of the function of growth factor signals in EMI during the masculinization of the genital tubercle has also improved. This progress has resulted in the identification of the new AR- related regulatory genes and signalling events, such as MAFB and Wnt–β- catenin signalling, which provokes further questions about the roles of androgen- induced signalling complexes and subsequent phenotypes. Following the identification of regulatory genes belonging to the AP-1 superfamily, including MAFB and ATF3, the mechanisms of partner molecules of AP-1 superfamily genes should be further studied. The regulation of local androgen production and the influence of the DHT production on cellular processes are also intriguing topics. Such studies could reveal a variety of cellular functions related to androgen signalling during genital masculinization. A wide variety of possible regulatory mechanisms in developmental processes and prostate cancer tumorigenesis should be studied, as these mechanisms could provide insight into genital masculinization. Progress in this emerging field will provide increased information on the development of congenital penile anomalies, such as hypospadias. Published online xx xx xxxx

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Acknowledgements The authors thank A. Thomson, L. Baskin, J. Cunha, R. Nishinakamura, S. Takahashi, G. Prins, K.-I. Matsumoto, H. Reutter, and T. DeFalco for their encouragement and

discussion points. The authors also thank T. I. Iba and all laboratory colleagues for their assistance. This work was supported by the Japan Society for the Promotion of Science grants 18K06938, 18K06837, 17K18024, 15H04300, 15K15403, 15K10647, 15K19013, and 15J11033.

Author contributions S.Ma., K.S., and G.Y. discussed the content, wrote the manuscript, and reviewed and edited the manuscript before submission. A.M., D.K., A.R.A., S.Mi., R.H., and Y.O. discussed the content and wrote the manuscript.

Competing interests The authors declare no competing interests.

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