William J. Kovacs

Alterations in the regulation of androgen-sensitive Cyp 4a monooxygenases cause hypertension

Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (−/−) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (−/−) mice and minimizes Cyp 4a12 expression and arachidonate ω-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (−/−) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 ω-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.

Effects of androgens on T and B lymphocyte development.

The sexually dimorphic nature of normal immune responses and the remarkably higher incidence of autoimmune diseases in females have suggested a role for gonadal steroid hormones as modulators of immune system function. We have investigated the effects of androgens on the development of lymphocytes in the thymus and bone marrow. Expression of the androgen receptor, the ligand-activated transcription factor that mediates hormone actions, has been documented in lymphoid and nonlymphoid cells of thymus and bone marrow, but not in mature peripheral lymphocytes. This expression pattern suggests that the major impact of androgens must be on the developmental maturation of T and B lymphocytes rather than on the mature effector cells. Recent experiments have explored whether developing lymphoid precursors are the direct targets of androgen action or whether supporting cells, such as thymic epithelial cells and bone marrow stromal cells, are required for the receptor-mediated effects of androgens on lymphoid cell development. Bone marrow transplantation techniques using an androgen-resistant mouse strain permit the creation of chimeric mice with androgen receptor-defective lymphoid or epithelial/stromal cellular compartments. Hormonalmanipulation experiments in these chimeric animals have suggested that thymic epithelial cells and bone marrow stromal cells are mediators of androgenic effects on immature lymphocytes. The long-range goal of these studies is to understand the basis for the disproportionate occurrence of autoimmune diseases in females.

Gonadal steroids and humoral immunity

Humoral immune responses are sexually dimorphic. Female individuals generally exhibit more-robust antibody responses to vaccines and, in the clinical setting as well as in experimental models, are more likely than male individuals to produce autoreactive antibodies of pathogenic potential. A number of differences between the sexes might account for these observations, including differences in the dosage of specific X-chromosome and Y-chromosomal genes, increased exposure of female individuals to antigenic stimulation in childbearing, and differences in circulating concentrations of gonadal steroid hormones. The role of gonadal steroids in modulating such humoral immune responses has been studied for nearly a century, but advances in our knowledge of B-lymphocyte development and function, the mechanisms of immune tolerance, and the molecular basis of gonadal steroid hormone action are now yielding new understanding of the influence of gonadal steroid hormones on the humoral immune system. This Review examines how oestrogens and androgens modulate B-lymphocyte development and function, focusing on the areas of B-cell production in the bone marrow, the maintenance of immune tolerance for self antigens, and the processes of immunoglobulin heavy chain gene somatic hypermutation and class switch recombination during maturation of cells involved in humoral immune responses.

Immunochemical and flow cytometric analysis of androgen receptor expression in thymocytes

A variety of evidence suggests that the cells of the immune system are targets for the actions of gonadal steroids. Experiments in both normal animals and in autoimmune disease models have established that androgens exert immunomodulatory effects at the level of the thymus. We have attempted to define precisely the potential target cells for androgen action in the thymus using recently developed antibodies to the androgen receptor. We report here that these antibodies reveal AR expression in all classes of thymocytes defined by surface markers CD4 and CD8. The highest levels of AR expression were observed in the CD4-CD8+ and CD4-CD8- subsets that include the most immature cells. These experiments establish that thymocytes are potential targets for direct actions of androgens. The data further suggest AR expression in thymocytes may be developmentally regulated in these cells, and that androgen effects early in the process of thymocyte selection may contribute to the sexual dimorphism of immune responsiveness.

A mutation that causes lability of the androgen receptor under conditions that normally promote transformation to the DNA-binding state.

Dihydrotestosterone-receptor complexes formed in human fibroblast cytosol prepared at 0 degrees C in the presence of sodium molybdate can be readily transformed to the DNA-binding state by heating at 25 degrees C. Under these conditions 50-70% of dihydrotestosterone-receptor complexes bind to DNA. We describe here studies of the transformation process in cytosols derived from normal cells and from fibroblasts propagated from subjects with syndromes of androgen resistance. In contrast to the situation with dihydrotestosterone, normal testosterone-receptor complexes are unstable under in vitro transforming conditions. Although equal amounts of hormone-receptor complex are formed at 0 degrees C, only 15% of testosterone-receptor complexes remain stable and acquire DNA-binding capacity after warming. This instability is not reversible upon lowering the temperature and is corrected by low concentrations (0.25 microM) of the protease inhibitor leupeptin. We have also identified two cousins with androgen resistance whose androgen-receptor complexes exhibit similar in vitro transformation lability with both dihydrotestosterone and testosterone. Phenotypic evidence in these subjects indicates that dihydrotestosterone-mediated processes are more completely impaired than are testosterone-mediated events. These findings suggest that dihydrotestosterone may amplify the androgenic signal at its targets not only by its higher affinity for the receptor but also by its more efficient conversion to the DNA-binding state and that such amplification may be less critical in target tissues in which testosterone suffices for androgenic effect. This offers one possible explanation of how a mutation that affects a single receptor protein may differentially impair the actions of two binding ligands of the receptor.

The inhibition of progesterone secretion and the regulation of cyclic nucleotides by atrial natriuretic factor in gonadotropin responsive murine Leydig tumor cells.

We have found that atrial natriuretic factor (ANF) has a profound effect on testicular cells in altering intracellular cyclic nucleotide levels as well as progesterone secretion. Using clonal cultured Leydig tumor cells we found that 1 X 10(-8)M ANF caused a two thousand-fold elevation in the accumulation of cellular cGMP and inhibited cAMP in treated cells by more than 90% as compared to the controls. ANF (1 X 10(-8)M) also significantly inhibited gonadotropin-stimulated accumulation of cAMP in response to bovine luteinizing hormone (bLH) or human chorionic gonadotropin (hCG). Gonadotropin-stimulated progesterone secretion was inhibited by ANF (1 X 10(-10) - 1 X 10(-9)M) in these cultured Leydig tumor cells. Approximately 50% inhibition of progesterone secretion was observed at the peptide concentration of 1 X 10(-9) M.

Case Report: Testosterone Treatment of Systemic Lupus Erythematosus in a Patient With Klinefelter’s Syndrome

Systemic lupus erythematosus occurs with much greater frequency in females than in males, and in some reports, researchers suggested that treatment with androgenic hormones might have therapeutic effects in this disease. The authors report a case of systemic lupus erythematosus in a hypogonadal male with Klinefelters syndrome who was treated with testosterone in doses sufficient to normalize the serum level of this hormone to the adult male range. Hematologic and serologic abnormalities, including elevated levels of anti-DNA antibodies and depressed complement levels, returned to normal within 9 months of increasing the testosterone dose. The findings in this patient indicate that androgenic steroids can exert significant effects on immune parameters, and suggest that effects of androgens on the immune system may contribute to the sexual dimorphism of autoimmune disease.

Bone marrow stromal cells mediate androgenic suppression of B lymphocyte development

Castration of normal male mice induces expansion of the bone marrow B cell population, an effect that can be reversed by androgen replacement. We employed in vitro cultures and two in vivo models to investigate whether androgens exert these effects directly on marrow lymphoid precursors or whether actions on marrow stromal elements are required. Immature B cells from normal mouse bone marrow were not responsive to the suppressive effect of androgens unless they were cocultured with marrow stromal cells or with supernatants from androgen-treated stromal cells, suggesting that the androgen effects are exerted through marrow stromal elements by production of a diffusible mediator. Further experiments revealed that bone marrow stromal cells produced TGF-beta in response to dihydrotestosterone (DHT), and neutralization of TGF-beta in the DHT-treated stromal cells reversed the suppressive effects. The stromal cell requirement for androgen-mediated effects was confirmed in vivo by experiments using chimeric animals created by bone marrow transplantation in which androgen receptor expression was restricted to either the stromal or lymphoid cells of the bone marrow. Androgens only affected B cell development in chimeric mice with androgen-sensitive stromal cells. These experiments suggest that effects of androgens on developing B cells are mediated through androgen receptors in bone marrow stromal cells. TGF-beta is a candidate mediator for these hormonal effects.

Atrial natriuretic factor regulates steroidogenic responsiveness and cyclic nucleotide levels in mouse Leydig cells in vitro.

The effects of synthetic atrial natriuretic factor (ANF) on the regulation of mouse Leydig cell steroidogenesis have been studied in vitro. ANF in nanomolar concentration increased testosterone production by more than 30-fold over basal levels. Concomitantly, cyclic guanosine monophosphate levels were increased 35-fold; cyclic adenosine monophosphate levels fell minimally (15-20%). ANF at low concentration (1 X 10(-11) M) inhibited testosterone production by luteinizing hormone-stimulated cells, while at higher concentration (greater than 2 X 10(-9) M) ANF stimulated steroidogenesis beyond the level attained by luteinizing hormone alone. These results indicate that ANF can exert stimulatory effects on testosterone steroidogenesis in vitro, and that the mechanism may involve an intracellular messenger other than cyclic adenosine monophosphate.

Testosterone induces expression of transforming growth factor-beta1 in the murine thymus

Castration of adult male mice results in enlargement of the thymus and diminution of peripheral suppressor T cell function. Replacement therapy with physiologic doses of androgens reverses these phenomena. Although the mediators involved are unknown, these effects of androgens on the thymus and peripheral immune system are reminiscent of those reported for transforming growth factor-beta (TGF-beta 1). We examined expression of TGF-beta 1 mRNA and bioactive protein in thymuses from castrate and androgen-replaced animals. Steady-state levels of thymic TGF-beta 1 mRNA fell slightly after castration, but rose 2.3-fold after testosterone replacement. Bioactive TGF-beta 1 production by cultured thymic explants also fell following castration to approx. 50% of the levels observed in intact animals. Following 1 week of testosterone replacement in castrate animals, TGF-beta 1 bioactivity produced in culture was restored to levels indistinguishable from those observed with explants from intact animals. Reverse transcription/polymerase chain reaction amplification of RNA revealed that thymocytes are a source of the androgen-modulated TGF-beta 1. These results suggest that TGF-beta 1 may mediate effects of androgens on the immune system.