Cultured human skin fibroblasts: a model for the study of androgen action

Human skin may be considered as a target organ for androgens, as are male sex accessory organs, since all events involved in testosterone action have been observed in this tissue. As a corollary, the mechanism of androgen action can be studiedin vitro in cultured skin fibroblasts. The advantages of this system are that studies can be performed with intact human cells under carefully controlled conditions, differentiated genetic and biochemical characteristics of the cells are faithfully preserved and the biological material is renewable from a single biopsy specimen. The metabolism of androgens, in particular the 5α-reduction of testosterone to the active metabolite, dihydrotestosterone, the intracellular binding of androgen to its specific receptor protein and its subsequent translocation to the nucleus have been studied in skin fibroblasts. The intracellular androgen receptor content of genital skin fibroblasts is higher than that from nongenital skin sites. In addition, the androgen receptor has been characterized as a specific macromolecule with properties of high affinity and low capacity similar to that of other steroid hormone receptors. The pathophysiology of three genetic mutations which alter normal male sexual development and differentiation has been identified in the human skin fibroblast system. In 5α-reductase deficiency, an autosomal recessive disorder in which dihydrotestosterone formation is impaired, virilization of the Wolffian ducts is normal but the external genitalia and urogenital sinus derivatives are female in character. At least two types of X-linked disorders of the androgen receptor exist such that the actions of both testosterone and dihydrotestosterone are impaired and developmental abnormalities may involve both Wolffian derivatives and the external genitalia as well. These two forms of androgen insensitivity result from either the absence of androgen receptor binding activity (receptor(−)form) or apparently normal androgen receptor binding with absence of an appropriate biological response (receptor (+) form). In addition, studies with human skin fibroblasts may also be of value in defining the cellular mechanisms underlying the broad spectrum of partial defects in virilization. In summary, we have correlated our studies of the molecular mechanism of androgen action in human genital skin fibroblasts with those of other investigators as these studies contribute to our understanding of male sexual development and differentiation.     

Partial purification and characterisation of the human skin fibroblast androgen receptor: detection of abnormal receptor complexes in cells from patients with androgen insensitivity syndromes

After incubation of hGSF with [3H]5 alpha-dihydrotestosterone, 17 beta-hydroxy-7 alpha, 17 alpha-dimethyl-4-estrene-3-one, or 17 beta-hydroxy-17 alpha-methyl-4,9,11-estrien-3-one, androgen-receptor complexes were extracted with 0.5 M KCl and precipitated by 35% ammonium sulphate. Receptor complexes from control hGSF sedimented at approximately 4S on linear 5-20% sucrose gradients. The 4S peak was diminished or absent in cells from androgen insensitive patients exhibiting absent, deficient or unstable binding of androgens in intact hGSF. This procedure may be a useful means of distinguishing quantitative and qualitative defects in androgen binding to receptor, since one cell line found to have normal levels of androgen receptor complexes in whole cell assays had a profile resembling that of receptor negative cells on sucrose gradients. The complexes from one patient with complete androgen insensitivity having normal androgen binding in intact hGSF were indistinguishable from control complexes after sucrose gradient analysis and ADP-Sepharose chromatography. Receptor complexes were eluted from the ADP-Sepharose between 0.5-1.0 M KCl. HPLC-gel filtration of androgen receptor complexes at 22 degrees C revealed two peaks, the larger had a Mr of 60-65K, Stokes radius of 3.16 nm and a frictional ratio between 1.21 and 1.43. The second peak, Mr of 15K, was believed to represent a fragment of the receptor containing the steroid binding domain. On gel filtration at 22 degrees C the complexes from a patient with partial androgen insensitivity, who showed a diminished 4S receptor peak on sucrose gradients, revealed only the small "meroreceptor" fragment, suggesting that the mutation in this individual might render the androgen receptor more susceptible to proteolysis in vitro.     

Regulation of seminiferous tubular function by FSH and androgen.

Seminiferous tubules contain a cytoplasmic androgen receptor similar to the receptors in the epididymis and ventral prostate. The presence of a cytoplasmic receptor indicates that androgens maintain spermatogenesis by a direct action on certain types of cells within the seminiferous tubule. The Sertoli cell appears to be one of the cell types containing androgen receptors and the receptor might also be present in spermatogonia, primary spermatocytes, or peritubular cells. The Sertoli cell is stimulated by FSH to produce an androgen-binding protein which may serve to increase the accumulation of androgen in the seminiferous epithelium and make it available for binding by intracellular androgen receptors. This may be a way in which FSH enhances the action of androgen on spermatogenesis. Androgens act on the Sertoli cell to increase its response to FSH. This action of androgens on the Sertoli cell results in increased production of androgen-binding protein and may enhance the production of other substances which exert trophic effects on spermatogenesis.     

Functional characterisation of mutations in the ligand-binding domain of the androgen receptor gene in patients with androgen insensitivity syndrome

Five mutations in the ligand-binding domain of the androgen receptor gene were identified in patients with complete (A765T, C784Y, R831X and M895T) or partial (R840G) androgen insensitivity. A765T and R831X have been reported previously whereas the other three mutations are novel. Receptors carrying these mutations were transiently expressed in COS-1 cells, and androgen binding and capacity to transactivate an androgen-responsive reporter gene were assayed. C784Y led to abolished androgen binding and transactivating capacity, R840G and M895T showed reduced specific binding and partial transactivation. The in vitro functions of the R840G and M895T mutants were improved with supraphysiological concentrations of steroid. Received: 10 June 1998 / Accepted: 10 September 1998     

Genital fibroblasts from normal individuals and patients with androgen insensitivity: demonstration of reduced or absent levels of a specific protein (Mr approximately 41K, pI approximately 6) in receptor negative cells

Genital fibroblasts were obtained from normal individuals and from patients with a variety of syndromes of defective androgenization (complete androgen insensitivity, partial androgen insensitivity, microgenitalia, hypospadias, infertility). Cells were labelled with [35S]methionine, and patterns of protein synthesis compared by two-dimensional gel electrophoresis, with isoelectrofocusing electrophoresis gels or non-equilibrated pH gradient electrophoresis (NEPHGE) gels as the first dimension. A protein (mol. wt approximately 41K, pI approximately 6) was found on NEPHGE gels to be reduced or absent in fibroblasts in which androgen receptor levels were abnormal. The protein was unaltered by prior incubation with 1-100 nM dihydrotestosterone for 48 h, and was present in cells both from normal controls, and from patients with abnormal sexual differentiation showing normal androgen receptor levels. The coincidence of low or absent 41K with low or absent androgen receptors suggested the possibility that it may constitute a steroid-binding moiety of the androgen receptor. To test this possibility cytosols from normal foreskins or normal cultured fibroblasts were adsorbed with testosterone-sepharose affinity resin to remove androgen receptors. Cytosols so treated showed levels of 41K on NEPHGE indistinguishable from those in untreated cytosols, or in cytosols treated with underivatized sepharose. We therefore conclude that the 41K protein, while an accurate marker of the presence or absence of androgen receptors over a range of clinical disorders, is neither an androgen-induced protein nor an androgen-binding protein.     

Molecular defects of the androgen receptor

Defects of the androgen receptor cause a wide spectrum of abnormalities of phenotypic male development, ranging from individuals with mild defects of virilization to those with complete female phenotypes. In parallel with this phenotypic spectrum, a large number of different mutations have been identified that alter the synthesis or functional activity of the receptor protein. In many instances, the genetic mutations identified lead to an absence of the intact, full-length receptor protein. Such defects (splicing defects, termination codons, partial or complete gene deletions) invariably result in the phenotype of complete androgen insensitivity (complete testicular feminization). By contrast, single amino acid substitutions in the androgen receptor protein can result in the entire phenotypic spectrum of androgen resistant phenotypes and provide far more information on the functional organization of the receptor protein. Amino acid substitutions in different segments of the AR open-reading frame disturb AR function by distinct mechanisms. Substitutions in the DNA binding domain of the receptor appear to comprise a relatively homogeneous group. These substitutions impair the capacity of the receptor to bind to specific DNA sequence elements and to modulate the function of responsive genes. Amino acid substitutions in the hormone-binding domain of the receptor have a more varied effect on receptor function. In some instances, the resulting defect is obvious and causes an inability of the receptor to bind hormone. In other instances, the effect is subtler, and may result in the production of a receptor protein that displays qualitative abnormalities of hormone binding or from which hormone dissociates more rapidly. Often it is not possible to correlate the type of binding defect with the phenotype that is observed. Instead, it is necessary to measure the capacity of the receptor that is synthesized in functional assays in order to discern any type of correlation with phenotype. Finally, two types of androgen receptor mutation do not fit such a categorization. The first of these—the glutamine repeat expansion that is observed in spinal and bulbar muscular atrophy—leads to a reduction of receptor function that can be measured in heterologous cells or in fibroblasts established from such patients. The expression of ARs containing such expanded repeats in men is associated with a degeneration of motor neurons in the spinal cords of affected patients. Likewise, the alterations of androgen receptor structure that have been detected in advanced forms of prostate cancer also behave as gain-of-function mutations. In this latter type of mutation, the exquisite specificity of the normal androgen receptor is relaxed and the mutant receptors can be activated by a variety of steroidal and non-steroidal ligands.     

Assay of primate seminiferous tubule androgen receptors using [3H]mibolerone

The synthetic radiolabelled androgen mibolerone (7 alpha, 17 alpha-dimethyl-19-nortestosterone) was used to characterize androgen receptor binding in the seminiferous tubules from Cynomolgus monkey testis. Mibolerone binding was of high affinity (Kd = 0.6-5.4 nM) and limited capacity (37-50 fmol/mg protein), and was androgen specific. Sucrose density gradient centrifugation using a vertical tube rotor permitted the identification of a 9S molybdate-stabilized receptor under low salt conditions. The receptor bound to DEAE-cellulose. Methyltrienolone, but not mibolerone, also bound to a low affinity high capacity binding site in tubule cytosol, which probably represents glucocorticoid receptor binding, since it could be displaced by excess dexamethasone. However, occupancy of this low-affinity binding site by dexamethasone in an androgen receptor assay with [3H]methyltrienolone lead to a 33% underestimation of receptor binding, which appeared to relate to radioactive decomposition. Mibolerone, as well as methyltrienolone, bound to a progestin-binding protein in seminiferous tubule cytosol. These studies provide methods for the study of seminiferous tubule androgen receptors in subhuman primates and indicate that, due to its greater stability and lack of binding to glucocorticoid receptor, mibolerone is a useful new ligand in the study of androgen receptors in testis and its constituent cells.     

Binding of glucocorticoid antagonists to androgen and glucocorticoid hormone receptors in rat skeletal muscle

The binding of ten steroids possessing antiglucocorticoid activity has been studied in rat skeletal muscle cytosol. The affinity of these steroids for both the androgen and the glucocorticoid receptors was determined by competition with radioactive R1881 (methyltrienolone, metribolone) and dexamethasone, respectively. The antiglucocorticoid activity of these compounds was assessed in rat hepatoma (HTC) cells by measuring their inhibitory effect on the glucocorticoid-induced tyrosine aminotransferase activity. This led to identification of five novel in vitro glucocorticoid antagonists. All the steroids tested bound to both the glucocorticoid and the androgen receptors in muscle. Four steroids had an affinity for the glucocorticoid receptor higher than for the androgen receptor. The assumption is made that the steroids tested also behave as antagonists when binding to the glucocorticoid receptor in muscle and behave as agonists when binding to the androgen receptor. On this basis, the data allow one to compute a potential anticatabolic (PAG) and a potential anabolic (PAA) index for each compound. These indices might be of predictive value to determine whether these steroids exert their anabolic action in muscle through the glucocorticoid receptor or through the androgen receptor. The data also make it unlikely that satellite cells are a preferential target for anabolic steroids in muscle.     

Influence of gender and the endocrine environment on the distribution of androgen receptors in the lacrimal gland

Androgens are known to regulate both the structure and function of lacrimal tissue in a variety of species. To explore the endocrine basis for this hormone action, the following study was designed to: (1) determine the cellular distribution of androgen receptors in the lacrimal gland; and (2) examine the influence of gender and the endocrine environment on the glandular content of these binding sites. Lacrimal glands were obtained from intact, castrated, hypophysectomized, diabetic or sham-operated male or female adult rats, mice or hamsters, as well as from orchiectomized rats exposed to placebo compounds or physiological levels of testosterone. The cellular of androgen receptors was evaluated by utilizing an immunoperoxidase protocol, in which a purified rabbit polyclonal antibody to the rat androgen receptor was used as the first antibody. Our findings with lacrimal glands showed that: (1) androgen receptors are located almost exclusively in nuclei of epithelial cells; (2) the cellular distribution or intranuclear density of these binding sites is far more extensive in glands of males, as compared to females; (3) orchiectomy or hypophysectomy, but not sham-surgery or diabetes, lead to a dramatic reduction in the immunocytochemical expression of androgen receptors; and (4) testosterone administration to orchiectomized rats induces a marked increase in androgen receptor content, relative to that in placebo-exposed glands. Our results also reveal that a 10 kb androgen receptor mRNA exists in the rat lacrimal gland. Overall, these findings demonstrate that gender and the endocrine system may significantly influence the distribution of androgen binding sites in rat lacrimal tissue. Moreover, our results show that androgens up-regulate their own lacrimal gland receptors.     

Hormone-dependent androgen receptor phosphorylation is accompanied by receptor transformation in human lymph node carcinoma of the prostate cells

Phosphorylation of the androgen receptor was investigated in the absence of hormone as well as during and after transformation of the receptor to the tight nuclear binding form. Human prostate tumor cells (LNCaP) were labeled for 4 h with [32P]orthophosphate in the presence or absence of steroid. Subsequently, androgen receptors were immunoprecipitated either from total cell lysates or from nuclear extracts using a specific monoclonal antibody. The immunoprecipitated receptor preparations were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, Western blotting, using a polyclonal antiserum, and autoradiography. It was observed that the androgen receptor is already phosphorylated in the absence of hormone, but undergoes a hormone-induced additional phosphorylation. After administration of 10 nM R1881, a 1.8-fold increase in phosphorylation over nonstimulated control cells was reached. Moreover, the amount of nuclear extractable androgen receptor was increased; the acquisition of tight nuclear binding capacity was accompanied by hormone-induced receptor phosphorylation.     

Androgen and erythropoiesis: evidence for an androgen receptor in erythroblasts from human bone marrow cultures

The techniques for culturing erythroid precursors made possible the study of the effect of steroids on these cells, and it has been well established that androgens and 5 beta-steroids have a direct effect on erythroid precursor cells from animal or human bone marrow. By contrast, their mechanisms of intracellular action remain poorly understood. We used tritiated methyltrienolone (R1881), a synthetic androgen that binds strongly to the androgen receptor, to characterize the binding activity in nuclear extracts of erythroblasts from human bone marrow cultures. The nuclear extracts contained binding sites that were saturable at low concentrations of 3H-R1881 (8-12 nM). Scatchard analysis revealed that the dissociation constant of the hormone-receptor complexes (Kd) was 10-20 nM, and the number of binding sites was 64-103 fmol/mg of protein. On linear sucrose density gradient analysis (5-20%), the hormone-receptor complexes sedimented in the region of 3.9 S. Finally, 5 beta-dihydrotestosterone had also a strong affinity for the binding sites. The nuclear component binding has all the physicochemical characteristics usually attributed to androgen receptors. These data strongly suggest that androgen action on erythropoiesis is mediated by a nuclear androgen receptor.     

雄激素受体的作用机制

主要概述了雄激素受体的作用机制,特别对影响雄激素受体特异性的因素进行探讨.雄激素受体（AR）属于甾体激素受体超家族,能通过配体依赖方式与特异的DNA序列结合,调控基因转录.