Steroid Receptor Coactivator-1 Is Not Required for ... - Semantic Scholar

Reproductive Neuroendocrinology Neuroendocrinology 2003;78:45–51 DOI: 10.1159/000071705

Received: March 10, 2003 Accepted after revision: May 5, 2003

Steroid Receptor Coactivator-1 Is Not Required for Androgen-Mediated Sexual Differentiation of Spinal Motoneurons Douglas A. Monks a Jianming Xu b Bert W. O’Malley b Cynthia L. Jordan a a Neuroscience b Department

Program and Department of Psychology, Michigan State University, East Lansing, Mich., and of Molcular and Cellular Biology, Baylor College of Medicine, Houston, Tex., USA

Key Words Steroid receptor coactivators W Spinal nucleus of the bulbocavernosus W Levator ani W Sexual dimorphism W Neuromuscular system W Gonadal steroids W Gonadal steroid receptors W Transgenes W Mice

Abstract Steroid receptor coactivator-1 (SRC-1) amplifies genomic steroid hormone signal transduction and has been implicated in steroid-mediated sexual differentiation of the mammalian nervous system. We investigated the possible effect of an SRC-1 null mutation on 2 morphological endpoints of androgenic signaling: the number and size of motoneurons within the spinal nucleus of the bulbocavernosus (SNB). In wild-type C57/BL6 mice, SRC-1 immunoreactive nuclei were observed within the SNB and one of its target muscles, the levator ani. However, SRC-1 null mice were indistinguishable from sexmatched wild-type littermates in both SNB number and cross-sectional area of SNB motoneurons. Similarly, we found no difference between SRC-1 null and wildtype littermates in the number or size of motoneurons in the retrodorsolateral nucleus, a motor pool that is not typically sexually differentiated in either number or size. These results demonstrate that SRC-1 is not essential for the development and maintenance of a sexually dimorphic neuromuscular system.

Introduction

Steroid hormone receptors are major effectors of the sexual differentiation of the mammalian brain and behavior [1]. Canonical steroid hormone signal transduction includes accessory molecules, referred to as nuclear receptor coregulators [2, 3]. Coactivators are a class of coregulators that serve to enhance the transactivation of nuclear hormone receptors. Steroid receptor coactivator-1 (SRC1) was the first to be identified [4] and remains the most extensively characterized coactivator of steroid hormone receptors. SRC-1 is a phosphoprotein [5] that is thought to increase the transcriptional efficiency of steroid receptor transcription factors and may do so by remodeling chromatin [6, 7] and by recruiting other transcription coactivators, such as p300 [8]. Recent in vivo evidence implicates SRC-1 in steroid hormone-driven sexual differentiation and adult function of the hypothalamus [9, 10]. Sexual dimorphism of the mammalian central nervous system is not limited to the hypothalamus, but rather is present in many structures of the brain and spinal cord. Notably, the sexually dimorphic spinal nucleus of the bulbocavernosus (SNB) [11] has been studied extensively as a model for steroid-mediated sexual differentiation of neural systems. SNB motoneurons and their target muscles, bulbocavernosus (BC) and levator ani (LA), mediate male copulatory reflexes [12, 13]. SNB motoneurons are more numerous and larger in adult male rats [14] and mice [15,

Copyright © 2003 S. Karger AG, Basel

ABC Fax + 41 61 306 12 34 E-Mail [email protected] www.karger.com

© 2003 S. Karger AG, Basel

Accessible online at: www.karger.com/nen

Cynthia L. Jordan Neuroscience Program and Department of Psychology Michigan State University East Lansing, MI 48824 (USA) Tel. +1 517 432 1764, Fax +1 517 353 1652, E-Mail [email protected]

16], and the target muscles are present in adult males but absent or vestigial in adult females. This sexual dimorphism comes about through the action of perinatal androgens on androgen receptors (ARs) present in the BC/LA muscle, where the system is rescued from apoptosis [17]. The idea that cofactors may play a key role in AR signaling in the SNB system was prompted by the observation that differences in AR expression alone cannot account for androgen sensitivity of SNB somata size [18]. The SNB is comprised of 2 distinct groups of motoneurons: the group that responds morphologically to androgen projects to the sexually dimorphic BC/LA muscles, whereas the group that does not respond morphologically to androgens projects to the sexually monomorphic external anal sphincter [19]. Although it is clear that androgens act via ARs in SNB motoneurons to increase the size of their somata [20], both androgen-responsive and androgen-unresponsive subpopulations of SNB motoneurons express AR [18]. Taken together, these results indicate that AR expression within motoneurons is necessary but not sufficient for androgen to increase the size of SNB motoneurons. These studies prompted a search for accessory molecules that participate in AR-mediated morphological plasticity of the SNB system. Recent evidence suggests that SRC-1 might participate in androgenic signaling in this system. For example, exogenous SRC-1 amplifies androgenic signaling in a variety of cell lines [21–24]. Additionally, SRC-1 is expressed in SNB motoneurons, and this expression decreases during aging, in concert with a decreased androgen sensitivity of the system [25]. As an initial step in characterizing the role of steroid receptor cofactors in SNB-BC/LA development and function, we asked whether SRC-1 is necessary for androgen-mediated sexual differentiation of the SNB using SRC-1 null mutant mice (SRC-1 KO) [26]. Here we report that the number and somata size of SNB motoneurons in mice is unaffected by deletion of the SRC-1 gene, indicating that SRC-1 is dispensable for androgen-driven sexual differentiation of the SNB system.

Methods Animals SRC-1 knockout mice were initially generated from embryonic stem cells with a targeted deletion of F9 kb of SRC-1 genomic sequence encoding 446 amino acids from Met-381 to Thr-826. Transcription of the genomic DNA downstream the deletion region was disrupted and terminated by the insertion of a transcriptional termination and poly A addition signaling sequences following the neo expression cassette. In the SRC-1 knockout mice, all functional domains of SRC-1 protein for transcriptional activation, histone ace-

46

Neuroendocrinology 2003;78:45–51

tyltransferase activity, and interactions with nuclear receptors and other coactivators, such as CBP/p300 and pCAF, were absent. Therefore, no SRC-1 proteins and mRNA could be detected in SRC-1 knockout mice by Western blot, in situ hybridization or real-time RT-PCR in all examined tissues including liver, kidney, brain and pituitary although these tissues express SRC-1 in wild-type mice [26– 28]. Since the SRC-1 knockout mouse line harbors the disrupted SRC-1 alleles, no functional SRC-1 protein can be produced in any tissues. In this study, all mice were genotyped by the PCR method as previously described [26]. Twenty-six adult offspring of SRC-1+/– mice were used in these studies. The genotype of these mice was determined by PCR to be as follows: wild-type SRC-1+/+ (WT) males (n = 6), knock-out SRC1–/– (KO) males (n = 6), WT females (n = 7) or KO females (n = 7). Animals were perfused intracardially with 0.9% NaCl, followed by 10% phosphate-buffered formalin. Lumbosacral spinal cords of these mice were then dissected and postfixed for at least 30 days in 10% formalin. Spinal cords were sectioned coronally at 30 Ìm and stained with thionine according to standard histological procedures. BC/LA muscles and seminal vesicles were dissected and weighed from male carcasses and postfixed in 10% phosphate-buffered formalin for 66 months. Three additional WT C57/BL6 males were used to characterize SRC-1 immunoreactive (SRC-1-IR) nuclei in the SNB and LA muscles (described below). Morphological Measures The number of motoneurons in the SNB and retrodorsolateral nucleus (RDLN) was counted bilaterally in adjacent spinal cord sections throughout the rostral-caudal extent of the SNB at 100! magnification on a compound microscope. Cells were defined as motoneurons only if they were large, densely Nissl-stained, and had a clearly visible nucleus. Motoneurons defined by these criteria that were localized medially in the ventral horn of the lumbar spinal cord were defined as SNB motoneurons (fig. 1). Motoneurons in the retrodorsolateral aspect of the ventral horn were defined as RDLN motoneurons (fig. 1). The Konigsmark correction for split nuclei [29] was applied to raw SNB and RDLN counts. The size of the soma and nucleus of each motoneuron in every third section that exhibited a distinct cell and nuclear membrane was measured until 20 or more motoneurons were sampled. Size measurements were made by tracing the external (somal) and internal (nuclear) Nissl-stained boundaries of identified SNB or RDLN motoneurons using a camera lucida. Tracings were then scanned and imported into NIH image where cross-sectional area of somata and nuclei of individual motoneurons were measured. All tracing sessions included a tracing of a scale bar to insure that the camera lucida magnification was consistent across samples. SRC-1 Immunohistochemistry Following perfusion with 0.9% phosphate-buffered saline and 0.1 M phosphate-buffered 4% paraformaldehyde (pH 7.4), the lumbosacral spinal cords and LA muscles of mice were removed and postfixed in the same fixative for an additional 30 min. After cryoprotection overnight at 4 ° C in phosphate-buffered 20% sucrose, 30Ìm coronal sections of spinal cord and 60-Ìm longitudinal sections of LA muscle were cut on a freezing microtome. Free-floating sections were then immunostained as described below. One sixth of all sections were processed for SRC-1, and another sixth was processed as a no primary control using the avidin-biotin horseradish peroxidase immunohistochemical method. The rabbit

Monks/Xu/O’Malley/Jordan

Fig. 1. Relative position of the SNB and RDLN in the mouse. This

photomicrograph depicts the relative positions of the SNB and RDLN in the mouse. The SNB is distributed along the medial aspect of the ventral horn of the lumbar spinal cord, whereas the RDLN is a cohesive nucleus in the retrodorsolateral ventral horn. Coronal sections (30 Ìm) were cut from the 5th and 6th lumbar segments and stained with thionine. Fig. 2. SRC-1 immunoreactivity in the SNB and LA. SRC-1 immunoreactive nuclei (arrows) were observed in sections taken throughout the lower spinal cord (A, B) and LA muscle (C, D), but only in sections exposed to primary antibody (A, C). Scale bars = 200 Ìm. * Position of the central canal (A, B). A SRC-1 immunoreactivity in the lumbar spinal cord, including an SRC-1 immunoreactive SNB motoneuron (arrow). B No primary antibody control of an alternate section from lumbar spinal cord containing unstained SNB motoneurons. C SRC-1 immunoreactivity in a longitudinal section taken from the LA muscle. D No primary antibody control of a longitudinal section taken from the LA muscle.

1

2

SRC-1 primary antibody PA1-840 (Affinity Bioreagents, Golden, Colo., USA) is directed against the common N terminal region of both SRC-1a and SRC-1e and was used at a 1/1,000 dilution. SRC-1 was visualized using a peroxidase Elite ABC kit (Vector Laboratories, Burlingame, Calif., USA) and nickel-enhanced 3,3)-diaminobenzidine as the chromagen. Omission of the primary antibody eliminated all nuclear staining (fig. 2).

Statistics All morphological measures were analyzed using a 2 ! 2 ANOVA design (sex by genotype) followed by planned comparisons for testing differences between means. All statistical calculations were performed using SPSS (version 9) and · was set at p ^ 0.05 for all comparisons.

SNB in SRC-1 KO Mice


47

Results

SRC-1 Is Expressed in the SNB-BC/LA of Mice SRC-1-IR nuclei were observed throughout the lumbar spinal cord, including the SNB and in its peripheral target, the LA (fig. 2). SRC-1 immunoreactivity was restricted to cell nuclei in both spinal cord and muscle. SRC-1-IR nuclei were observed in virtually all SNB motoneurons. Consistent with reportedly low SRC-1 mRNA expression in femoral skeletal muscle [27], SRC-1-IR nuclei in the LA were considerably less abundant than in spinal cord, although uniformly distributed throughout the muscle. SRC-1-IR nuclei in the LA appear to be either muscle fiber or fibroblast nuclei. The presence of SRC-1 IR nuclei within the SNB and LA is consistent with a role for SRC-1 in the androgen-mediated maintenance and growth of the SNB system and prompted an analysis of the SNB in SRC-1 KO mice. WT and SRC-1 KO Male Mice Have Similar Indices of Peripheral Masculinization Despite elevated plasma testosterone concentrations in SRC-1 KO males, presumably due to impaired negative feedback of pituitary gonadotropin secretion, SRC-1 KO males are fertile and without obvious demasculinization of peripheral structures [26]. In agreement with previous reports, we found that seminal vesicle weight, a commonly used urogenital bioassay of androgenic action, as well as BC/LA weight, were equivalent between WT and KO male mice (table 1; independent samples t test, p = 0.28 and 0.69, respectively), suggesting that androgenic signaling was sufficiently effective in KO mice to result in fully masculine peripheral end points.

Fig. 3. The number and size of SNB and RDLN motoneurons in WT and SRC-1 KO male and female mice. As expected, there was a significant main effect of sex on the number (A) and size (B, C) of SNB but not RDLN motoneurons, with males having more and larger SNB motoneurons, regardless of SRC-1 status. Significantly, there was no main effect of the SRC-1 KO on any of these parameters, nor was there an interaction between sex and genotype. Graphs represent the mean B SEM, based on n = 6–7/group. * Significant main effect of sex: p ^ 0.05.

48



Table 1. Somatic measures of SRC-1 KO

mice (mean B SEM)

Weights Body, g Brain, mg BC/LA, mg Seminal vesicles, mg

L5/L6 Motoneuron Number and Soma Size Is Unaffected by SRC-1 Null Mutation Possible involvement of SRC-1 in sexual differentiation of the SNB was examined by determining the number and size of these motoneurons. As a control, we also counted and measured the size of motoneurons in the RDLN that is located at the same lumbar level as the SNB and is neither sexually dimorphic nor androgen-responsive in rats [30, 31]. As expected, the SNB was sexually differentiated in number, soma size and in the size of nuclei (main effect for sex F(1, 22) = 247.54 (number), 14.66 (soma size), 9.92 (size of nuclei); all p ^ 0.01), with males having more numerous SNB motoneurons with larger somata and nuclei than do females (planned comparisons; all p ^ 0.005). These differences were independent of genotype (all p ^ 0.05) and no interaction between sex and genotype was observed for SNB number, soma size or nuclear size (all p 6 0.05). No effects of sex or genotype on RDLN number, somata size or nuclear size were apparent (all p 1 0.05; fig. 3 for a summary of all results).

WT male 21.58B0.92 468.5B6.99 118.83B4.92 229.0B26.0

KO male 21.74B1.83 452.5B6.68 115.67B6.09 215.3B26.03

WT female

KO female

23.46B1.11 486.7B7.80 NA NA

21.56B0.63 462.9B3.56 NA NA

The present results provide strong evidence that, unlike estrogen-mediated sexual differentiation of the rat sexual dimorphic nucleus of the preoptic area (SDNPOA) [9], SRC-1 is dispensable for the normal sexual differentiation of the SNB system. This suggests that SRC-1 is either not normally involved in androgenic signaling in this system, or that other cofactors redundant with SRC-1 compensate for its absence in the null mutants. There are two major differences between this study and previous research that may explain why sexual differentiation of the SNB does not require SRC-1 but sexual differentiation of the SDN-POA does. Firstly, sexual differentiation of the SNB system depends on AR-mediated signaling, whereas sexual differentiation of the SDN-POA is estrogen receptor-mediated. Whereas SRC-1 can en-

hance the transcriptional activity of the AR in vitro and in cell culture, it remains possible that SRC-1 does not normally influence transactivation of the AR in the spinal cord or skeletal muscle in vivo. Consistent with this view, SRC-1 knockdown via injection of antisense oligonucleotides into the developing rat hypothalamus interferes with defeminization, which has been ascribed primarily to estrogenic action, but not masculinization, which has been ascribed primarily to androgenic action [9, 32]. Therefore, SRC-1 may be essential for estrogen receptormediated, but not AR-mediated steroid hormone action in the CNS. Secondly, the previous methodology employed an acute, local disruption of SRC-1, whereas in the present study, the disruption of SRC-1 is chronic and global. Transient, localized disruption of SRC-1 may avoid compensatory recruitment and/or expression of related cofactors, such as transcription intermediary factor-2, as is the case with SRC-1 null mutants [26, 27]. In support of this view, female KO mice show a typical feminine response of receptive sexual behavior to priming with estradiol and progesterone, and this facilitation of sexual behavior can be abolished by knocking-down SRC2 expression in the hypothalamus using antisense oligonucleotides [32]. The apparent dispensability of SRC-1 for androgenmediated trophic action in the SNB system raises the question of what accessory molecules participate in this signaling pathway. Studies in our laboratories and in others have identified several steroid receptor cofactors that are expressed in the SNB system. Specifically, SRC-1, p300, CBP and c-Jun [33] are all abundantly expressed in SNB motoneurons [25, 35], and SRC-2 and SRC-3 mRNA is relatively abundant in femoral skeletal muscle [27] raising the possibility that these cofactors participate in AR-mediated trophic action in this system. In addition to these cofactors, future experiments will also determine whether other candidate AR coactivators (e.g. ARA 70) form stable associations with the AR and can modulate AR expression in the SNB and BC/LA, as they do in vitro [2].



Discussion

49

In summary, the data presented herein indicate that AR signaling can occur in vivo in the absence of SRC-1, but do not exclude the possibility that SRC-1 normally participates in the steroid-mediated sexual differentiation of the SNB system. Taken in conjunction with evidence that the SRC family members are promiscuous and may compensate for SRC-1, these data are consistent with a model of steroid receptor cofactor action in the SNB system that is multifactorial and includes redundancy.

Acknowledgments These studies were funded by National Institutes of Health (NIH) R01 operating grants NICHD/U54, NS045795, DK58242 and NS28421 as well as a postdoctoral fellowship (D.A.M.) from the Canadian Institutes of Health Research (CIHR).

References 1 Cooke B, Hegstrom CD, Villeneuve LS, Breedlove SM: Sexual differentiation of the vertebrate brain: principles and mechanisms. Front Neuroendocrinol 1998;19:323–362. 2 Heinlein CA, Chang C: Androgen receptor (AR) coregulators: An overview. Endocr Rev 2002;23:175–200. 3 McKenna NJ, Xu J, Nawaz Z, Tsai SY, Tsai MJ, O’Malley BW: Nuclear receptor coactivators: Multiple enzymes, multiple complexes, multiple functions. J Steroid Biochem Mol Biol 1999;69:3–12. 4 Onate SA, Tsai SY, Tsai MJ, O’Malley BW: Sequence and characterization of a coactivator for the steroid hormone receptor superfamily. Science 1995;270:1354–1357. 5 Rowan BG, Weigel NL, O’Malley BW: Phosphorylation of steroid receptor coactivator-1. Identification of the phosphorylation sites and phosphorylation through the mitogen-activated protein kinase pathway. J Biol Chem 2000;275:4475–4483. 6 Muller WG, Walker D, Hager GL, McNally JG: Large-scale chromatin decondensation and recondensation regulated by transcription from a natural promoter. J Cell Biol 2001;154:33– 48. 7 Spencer TE, Jenster G, Burcin MM, Allis CD, Zhou J, Mizzen CA, McKenna NJ, Onate SA, Tsai SY, Tsai MJ, O’Malley BW: Steroid receptor coactivator-1 is a histone acetyltransferase. Nature 1997;389:194–198. 8 Liu YH, Wong J, Tsai SY, Tsai MJ, O’Malley BW: Sequential recruitment of steroid receptor coactivator-1 (SRC-1) and p300 enhances progesterone receptor-dependent initiation and reinitiation of transcription from chromatin. Proc Natl Acad Sci USA 2001;98:12426– 12431. 9 Auger AP, Tetel MJ, McCarthy MM: Steroid receptor coactivator-1 (SRC-1) mediates the development of sex-specific brain morphology and behavior. Proc Natl Acad Sci USA 2000; 97:7551–7555. 10 Molenda HA, Griffin AL, Auger AP, McCarthy MM, Tetel MJ: Nuclear receptor coactivators modulate hormone-dependent gene expression in brain and female reproductive behavior in rats. Endocrinology 2002;143:436–444.

50

11 Breedlove SM, Arnold AP: Hormone accumulation in a sexually dimorphic motor nucleus of the rat spinal cord. Science 1980;210:564– 566. 12 Sachs BD: Role of striated penile muscles in penile reflexes, copulation, and induction of pregnancy in the rat. J Reprod Fertil 1982;66: 433–443. 13 Wallace SJR, Hart BL: The role of the striated penile muscles of the male rat in seminal plug dislodgement and deposition. Physiol Behav 1983;31:815–821. 14 Breedlove SM, Arnold AP: Sexually dimorphic motor nucleus in the rat lumbar spinal cord: Response to adult hormone manipulation, absence in androgen-insensitive rats. Brain Res 1981;225:297–307. 15 Forger NG, Howell ML, Bengston L, MacKenzie L, DeChiara TM, Yancopoulos GD: Sexual dimorphism in the spinal cord is absent in mice lacking the ciliary neurotrophic factor receptor. J Neurosci 1997;17:9605–9612. 16 Wee BE, Clemens LG: Characteristics of the spinal nucleus of the bulbocavernosus are influenced by genotype in the house mouse. Brain Res 1987;424:305–310. 17 Christensen SE, Breedlove SM, Jordan CL: Sexual differentiation of a neuromuscular system; in Matsumoto A (ed): Sexual Differentiation of the Brain, ed 1. New York, CRC Press, 2000, pp 150–173. 18 Jordan C: Androgen receptor (AR) immunoreactivity in rat pudendal motoneurons: implications for accessory proteins. Horm Behav 1997; 32:1–10. 19 Collins WF 3rd, Seymour AW, Klugewicz SW: Differential effect of castration on the somal size of pudendal motoneurons in the adult male rat. Brain Res 1992;577:326–330. 20 Watson NV, Freeman LM, Breedlove SM: Neuronal size in the spinal nucleus of the bulbocavernosus: Direct modulation by androgen in rats with mosaic androgen insensitivity. J Neurosci 2001;21:1062–1066. 21 Bevan CL, Hoare S, Claessens F, Heery DM, Parker MG: The AF1 and AF2 domains of the androgen receptor interact with distinct regions of SRC1. Mol Cell Biol 1999;19:8383–8392.


22 Masiello D, Cheng S, Bubley GJ, Lu ML, Balk SP: Bicalutamide functions as an androgen receptor antagonist by assembly of a transcriptionally inactive receptor. J Biol Chem 2002; 277:26321–26326. 23 Yeh S, Kang HY, Miyamoto H, Nishimura K, Chang HC, Ting HJ, Rahman M, Lin HK, Fujimoto N, Hu YC, Mizokami A, Huang KE, Chang C: Differential induction of androgen receptor transactivation by different androgen receptor coactivators in human prostate cancer DU145 cells. Endocrine 1999;11:195–202. 24 Ikonen T, Palvimo JJ, Janne OA: Interaction between the amino- and carboxyl-terminal regions of the rat androgen receptor modulates transcriptional activity and is influenced by nuclear receptor coactivators. J Biol Chem 1997;272:29821–29828. 25 Matsumoto A: Age-related changes in nuclear receptor coactivator immunoreactivity in motoneurons of the spinal nucleus of the bulbocavernosus of male rats. Brain Res 2002;943: 202–205. 26 Xu J, Qiu Y, DeMayo FJ, Tsai SY, Tsai MJ, O’Malley BW: Partial hormone resistance in mice with disruption of the steroid receptor coactivator-1 (SRC-1) gene. Science 1998;279: 1922–1925. 27 Nishihara E, Yoshida-Komiya H, Chan CS, Liao L, Davis RL, O’Malley BW, Xu J: SRC-1 null mice exhibit moderate motor dysfunction and delayed development of cerebellar Purkinje cells. J Neurosci 2003;23:213–222. 28 Sadow PM, Koo E, Chassande O, Gauthier K, Samarut J, Xu J, O’Malley BW, Seo H, Murata Y, Weiss RE: Thyroid hormone receptor-specific interactions with steroid receptor coactivator-1 in the pituitary. Mol Endocrinol 2003; 17:882–894. 29 Konigsmark BW: Methods for the counting of neurons; in Nauta WJH, Ebbeson SOE (ed): Contemporary Research Methods in Neuroanatomy. New York, Springer-Verlag, 1970, pp 315–340. 30 Jordan CL, Breedlove SM, Arnold AP: Sexual dimorphism and the influence of neonatal androgen in the dorsolateral motor nucleus of the rat lumbar spinal cord. Brain Res 1982;249: 309–314.


31 Jordan CL, Christensen SE, Handa RJ, Anderson JL, Pouliot WA, Breedlove SM: Evidence that androgen acts through NMDA receptors to affect motoneurons in the rat spinal nucleus of the bulbocavernosus. J Neurosci 2002;22: 9567–9572. 32 Apostolakis EM, Ramamurphy M, Zhou D, Onate S, O’Malley BW: Acute disruption of select steroid receptor coactivators prevents reproductive behavior in rats and unmasks genetic adaptation in knockout mice. Mol Endocrinol 2002;16:1511–1523. 33 Bubulya A, Chen SY, Fisher CJ, Zheng Z, Shen XQ, Shemshedini L: c-Jun potentiates the functional interaction between the amino and carboxyl termini of the androgen receptor. J Biol Chem 2001;276:44704–44711. 34 O’Bryant EL, Jordan CL: SRC-1 and p300 immunoreactivity in rat spinal cord and brain (abstract 73.16). 32nd Annu Meet Soc Neurosci, 2002. 35 Breedlove SM, Arnold AP: Hormonal control of a developing neuromuscular system. II. Sensitive periods for the androgen-induced masculinization of the rat spinal nucleus of the bulbocavernosus. J Neurosci 1983;3:424–432.


36 Kang HY,Yeh S, Fujimoto N, Chang C: Cloning and characterization of human prostate coactivator ARA54, a novel protein that associates with the androgen receptor. J Biol Chem 1999;274:8570–8576. 37 Lee YH, Koh SS, Zhang X, Cheng X, Stallcup MR: Synergy among nuclear receptor coactivators: Selective requirement for protein methyltransferase and acetyltransferase activities. Mol Cell Biol 2002;22:3621–3632. 38 Nazareth LV, Stenoien DL, Bingman WE 3rd, James AJ, Wu C, Zhang Y, Edwards DP, Mancini M, Marcelli M, Lamb DJ, Weigel NL: A C619Y mutation in the human androgen receptor causes inactivation and mislocalization of the receptor with concomitant sequestration of SRC-1 (steroid receptor coactivator 1). Mol Endocrinol 1999;13:2065–2075. 39 Needham M, Raines S, McPheat J, Stacey C, Ellston J, Hoare S, Parker M: Differential interaction of steroid hormone receptors with LXXLL motifs in SRC-1a depends on residues flanking the motif. J Steroid Biochem Mol Biol 2000;72:35–46. 40 Ogawa H, Nishi M, Kawata M: Localization of nuclear coactivators p300 and steroid receptor coactivator 1 in the rat hippocampus. Brain Res 2001;890:197–202.

41 Saitoh M, Takayanagi R, Goto K, Fukamizu A, Tomura A, Yanase T, Nawata H: The presence of both the amino- and carboxyl-terminal domains in the AR is essential for the completion of a transcriptionally active form with coactivators and intranuclear compartmentalization common to the steroid hormone receptors: A three-dimensional imaging study. Mol Endocrinol 2002;16:694–706. 42 Stenoien DL, Cummings CJ, Adams HP, Mancini MG, Patel K, DeMartino GN, Marcelli M, Weigel NL, Mancini MA: Polyglutamine-expanded androgen receptors form aggregates that sequester heat shock proteins, proteasome components and SRC-1, and are suppressed by the HDJ-2 chaperone. Hum Mol Genet 1999;8: 731–741. 43 Ting HJ, Yeh S, Nishimura K, Chang C: Supervillin associates with androgen receptor and modulates its transcriptional activity. Proc Natl Acad Sci USA 2002;99:661–666. 44 Ueda T, Mawji NR, Bruchovsky N, Sadar MD: Ligand-independent activation of the androgen receptor by IL-6 and the role of the coactivator SRC-1 in prostate cancer cells. J Biol Chem 2002;278:5929–5940.


51