Use of artificial androgen receptor coactivators to alter myoblast proliferation

Skeletal muscle has long been thought to be a target tissue for androgens, eliciting their effect through the androgen receptor. In order to better understand androgen receptor action, a series of mutated androgen receptors were developed and their degree of specificity and cellular responses determined. Specificity, as measured by a reporter assay using HeLa cells, indicated that mutation of the ligand-binding domain or the AR (mutation H865Y), in combination with the p65 transactivating domain, resulted in an increased response to androgens as well as decreased specificity. Transfection of the mutant AR into mouse and rat myoblast cell lines resulted in an increase in expression of the reporter gene consistent with the data from HeLa cells. Overexpression of the wild type or mutant AR into myoblasts and treatment with testosterone induced both greater proliferation and faster differentiation of the cells compared to those expressing endogenous AR. Additionally, when treated with estrogen, these cells were able to proliferate and differentiate to similar levels as cells treated with testosterone. The ability of the mutated AR to act as an artificial coactivator to up-regulate androgen responsive genes is a useful tool for understanding the interaction of androgens and muscle growth.     

雄激素受体信号转导途径及其在前列腺癌发展中的作用

前列腺癌是西方男性发病率最高的癌症之一,在采用雄激素阻断疗法后,大部分患者的病情可得到控制,但经过一段时间又会转变为雄激素非依赖型前列腺癌。雄激素受体(AR)在前列腺细胞中扮演重要的角色,它可调节大量基因的表达。在前列腺癌由雄激素依赖型向雄激素非依赖型的转变过程中,AR及其信号途径通过多种方式发挥作用,AR基因的扩增、AR的突变,以及与共激活子之间作用的改变都可能使细胞获得雄激素非依赖型的生长能力。此外,AR还受到多肽生长因子和细胞因子等的调节,表现激素非依赖型的转录激活活性。AR在前列腺癌中作用的阐明对前列腺癌的诊断与治疗有着重大的意义。     

Androgen receptor regulation of the versican gene through an androgen response element in the proximal promoter

Versican, one of the key components of prostatic stroma, plays a central role in tumor initiation and progression. Here, we investigated promoter elements and mechanisms of androgen receptor (AR)-mediated regulation of the versican gene in prostate cancer cells. Using transient transfection assays in prostate cancer LNCaP and cervical cancer HeLa cells engineered to express the AR, we demonstrate that the synthetic androgen R1881 and dihydrotestosterone stimulate expression of a versican promoter-driven luciferase reporter vector (versican-Luc). Further, both basal and androgen-stimulated versican-Luc activities were significantly diminished in LNCaP cells, when AR gene expression was knocked down using a short hairpin RNA. Methylation-protection footprinting analysis revealed an AR-protected element between positions +75 and +102 of the proximal versican promoter, which strongly resembled a consensus steroid receptor element. Electrophoretic mobility shift and supershift assays revealed strong and specific binding of the recombinant AR DNA binding domain to oligonucleotides corresponding to this protected DNA sequence. Site-directed mutagenesis of the steroid receptor element site markedly diminished R1881-stimulated versican-Luc activity. In contrast to the response seen using LNCaP cells, R1881 did not significantly induce versican promoter activity and mRNA levels in AR-positive prostate stromal fibroblasts. Interestingly, overexpression of beta-catenin in the presence of androgen augmented versican promoter activity 10- and 30-fold and enhanced versican mRNA levels 2.8-fold in fibroblasts. In conclusion, we demonstrate that AR transactivates versican expression, which may augment tumor-stromal interactions and may contribute to prostate cancer progression.     

Antiproliferative actions of the synthetic androgen, mibolerone, in breast cancer cells are mediated by both androgen and progesterone receptors

Androgen signaling, mediated by the androgen receptor (AR), is a critical factor influencing growth of normal and malignant breast cells. Given the increasing use of exogenous androgens in women, a better understanding of androgen action in the breast is essential. This study compared the effects of 5alpha-dihydrotestosterone (DHT) and a synthetic androgen, mibolerone, on estradiol (E(2))-induced proliferation of breast cancer cells. DHT modestly inhibited E(2)-induced proliferation and mibolerone significantly inhibited proliferation in T-47D cells. The effects of both androgens could be reversed by an AR antagonist, suggesting that their actions were mediated, in part, by AR. Whereas high physiological doses (10-100nM) of DHT reduced E(2)-mediated induction of the estrogen-regulated gene progesterone receptor (PR) to basal levels, mibolerone at lower doses (1nM) eliminated PR expression, suggesting that mibolerone may also act via the PR. In the AR positive, PR-negative MCF-7 cells, mibolerone had modest effects on E(2)-induced proliferation, but was a potent inhibitor of proliferation in the AR positive, PR positive MCF-7M11 PRA cells. The effects of mibolerone in breast cancer cells were similar to those of the progestin, medroxyprogesterone acetate. Our results demonstrate that mibolerone can have both androgenic and progestagenic actions in breast cancer cells.     

Extracellular correction of the androgen-receptor transformation defect in two families with complete androgen resistance

We have characterized the cellular and extracellular phenotype of the mutant androgen receptor (AR) from two families who have complete androgen resistance despite a normal androgen-binding capacity (Bmax) in their genital skin fibroblasts (GSF). The cellular receptors fail to up-regulate their basal AR activity in response to prolonged incubation with 5 alpha-dihydrotestosterone (DHT), or with two synthetic androgens, methyltrienolone (MT) and mibolerone (MB), and form A-R complexes with increased equilibrium (Kd) and non-equilibrium (k) dissociation constants. In addition, they are thermolabile when recently dissociated, but not in their native state. A-R complexes made in normal or mutant cytosol at 4 degrees C elute from DEAE-Sephacel at approximately 0.25 M KCl (untransformed), with or without prior passage through Sephadex G-25; when made in cells at 37 degrees C, extracted with 0.4 M KCl in a buffer containing 10 mM Na2MoO4, and desalted by G-25, they elute at less than or equal to 0.1 M KCl. Normal KCl-extracted DHT- and MB-R complexes dissociate (37 degrees C) at the same slow, linear rate as their in-cell counterparts (transformed); the mutant ones dissociated more slowly than their rapidly-dissociating in-cell counterparts and, to a variable extent, nonlinearly-an early faster phase, a later slower (transformed). Thus, as judged by two conventional criteria of steroid-R complex transformation, the mutant A-R complexes can transform, possibly in two steps, under certain cell-free conditions. This behavior differentiates a class of structural AR mutations whose molecular definition awaits application of recombinant DNA techniques to the X-linked AR locus.