CCAAT/enhancer binding protein-mediated role of thyroid hormone in the developmental expression of the kidney androgen-regulated protein gene in proximal convoluted tubules

The kidney androgen-regulated protein (KAP) gene is exclusively expressed in proximal tubules of mouse kidney and in the uterus of pregnant females before they give birth. It displays an exquisite and differential regulation of expression by steroid and thyroid hormones (THs) in different proximal tubule segments. Whereas the pars recta (PR cells) responds to thyroid and sexual hormones, the pars convoluta (PCT cells) represents a truly androgen-dependent compartment because expression occurs only in the presence of androgens and functional androgen receptors. Nevertheless, different hypothyroidism models have indicated that TH might also contribute to the androgenic response in PCT cells. In the present study, we aimed to determine the molecular mechanisms that ultimately control KAP expression in these cells. Using several genetically deficient mouse models and different pharmacologic and hormonal treatments, we determined that thyroid and GH modulate CCAAT/enhancer binding protein alpha and beta levels that, in turn, control KAP expression in PCT cells in a developmentally dependent manner. We demonstrated that these factors bind to sites in the proximal KAP promoter, thereby collaborating with androgens for full KAP expression. Finally, we propose that TH and GH, acting through CCAAT/enhancer binding protein, may constitute a general regulatory mechanism of androgen-dependent genes in mouse kidney.     

The presence of a hitherto undefined high-capacity androgen binding macromolecule in human ovarian cancer tissue

Sex hormone binding globulin (SHBG) is known to interfere in the quantitation of androgen receptors (AR) if dihydrotestosterone (DHT) is used. We used a monoclonal antibody to remove SHBG from cytosol. In cytosol of benign prostatic hyperplastic (BPH) tissue low capacity binding for DHT, but not for R1881, was found after removal of SHBG. AR were detected in 18 of 20 ovarian cancer cytosols. In the two AR-negative cases, non-saturable binding for DHT, testosterone and R1881 was observed. Incubation with anti-SHBG did not change this. An hitherto undefined androgen binding macromolecule(s), with high-capacity binding for natural and synthetic androgens, but not for estrogen and progesterone, seems to be present in these ovarian cancer tissues. The functionality of these androgen binding macromolecules in ovarian cancer is yet to be demonstrated.     

Differential androgen sensitivity is associated with clonal heterogeneity in steroid metabolism, ornithine decarboxylase regulation and IL-1alpha action in mouse mammary tumor cells

Upon androgen deprivation, Shionogi (SC-115) mouse mammary tumors undergo phenotypic changes enabling their escape from growth dependence on androgens. Even within androgen-responsive cell populations, marked clonal heterogeneity is observed in the trophic effects of androgens. The present study compares several parameters of androgen action between three SC-115 cell clonal subpopulations exhibiting high (clone 107), low (clone S1A2) and no trophic response (clone 415) to androgens. These parameters pertain to (1) kinetics of androgen binding, (2) metabolism of 5alpha-dihydrotestosterone (DHT), 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) and 5alpha-androstane-3beta,17beta-diol (3beta-diol), (3) ornithine decarboxylase (ODC) activity and (4) interleukin-1alpha (IL-1alpha) action on cell proliferation. Only marginal differences in the affinity and abundance of androgen-specific binding sites were detected between the three clones. While clone S1A2 degraded DHT to 3alpha-diol at a much faster rate than the highly androgen-sensitive 107 cells and androgen-insensitive 415 cells, differences in the rates of intracrine conversion of 3alpha-diol and 3beta-diol to DHT did not correlate with the ability of these steroids to stimulate cell proliferation. Induction of ODC activity at the onset of exponential growth was strongly DHT-dependent in 107 cells, whereas this dependence was markedly attenuated in androgen-hyposensitive cells. Unexpectedly, DHT strongly repressed the marked ODC induction resulting from fresh medium addition in 415 cells which show no growth response to androgens. Low IL-1alpha concentrations were mitogenic in all three SC-115 clones. Whereas the mitogenic action of IL-1alpha was completely androgen-dependent in 107 cells, this dependence was relieved in S1A2 cells, which responded to DHT and IL-1alpha in an additive fashion. Thus, clonal heterogeneity in the pattern of steroid metabolism within Shionogi tumors cannot solely account for loss of androgen dependence, which may rather correlate with the constitutive activation of transduction pathways controlling the expression of growth-associated genes (e.g. ODC) by serum growth factors, including IL-1alpha.     

Regional differences in the testosterone to dihydrotestosterone ratio in the epididymis and vas deferens of adult mice

The concentrations of testosterone and dihydrotestosterone (DHT) were measured in the testis and in different segments of the epididymis and vas deferens of adult mice. There were marked regional variations in the concentrations of testosterone and DHT from the testis to the caudal part of the vas deferens. In the testis, testosterone was the predominant androgen (364 +/- 90 ng/g) while DHT was weakly represented (8 +/- 2 ng/g). Qualitative and quantitative changes occurred in epididymis: DHT was the main steroid in the caput (29.3 +/- 2.7 ng/g) and corpus (33.1 +/- 4.4 ng/g) while testosterone and DHT were in similar quantities in the cauda (18.6 +/- 2.6 and 19.0 +/- 2.7 ng/g, respectively). The proximal region of the vas deferens contained higher amounts (71.4 +/- 8.0 ng/g) of androgens (testosterone + DHT) than did the caput epididymidis (39.1 +/- 3.3 ng/g). Testosterone was the predominant androgen in each part of the vas deferens and its concentrations decreased from the proximal (64.5 +/- 7.5 ng/g) to the caudal (26.9 +/- 4.3 ng/g) region. Castration and section of the efferent ducts of the testis showed that the epididymis received testosterone essentially via the blood supply and that epididymal DHT was produced locally from circulating testosterone.     

Estrogen and androgen regulation of sex hormone binding globulin secretion by a human liver cell line

Both estrogens and androgens have been shown to stimulate sex hormone binding globulin (SHBG) secretion in vitro in the hepatocellular carcinoma cell line, Hep G2, in contrast to the expected inhibition by androgens from in vivo studies. However, such in vitro stimulation was only demonstrated at high steroid doses, generally in serum-containing medium, with added Phenol Red. In the present study, Hep G2 cells were grown in serum-free medium, without Phenol Red, under the influence of testosterone (T) (0, 0.5-500 nM) and ethinyl estradiol (EE2) (0, 50 pM-500 nM). Levels of secreted SHBG and albumin were correlated with androgen receptors in cytosolic (ARc) and nuclear (ARn) fractions and with DNA levels. In the presence of increasing T levels, SHBG levels fell to 39% of control values at 5 nM T (P = 0.047), rising to 97% of control at 500 nM. Conversely, incubation with EE2 produced a rise in SHBG secretion of more than 100% at 0.5 nM (P less than 0.02) which was sustained to 50 nM (P less than 0.005). DNA levels did not change with the addition of testosterone or EE2, with the exception of a 15% reduction at 5 nM EE2 (P less than 0.05). Albumin levels in the medium were not significantly altered by either steroid. However, in response to T, androgen receptor (AR) levels were reduced in cytosolic (42% of control) and nuclear (22%) fractions at 5 nM, and these changes in ARc and ARn correlated with SHBG levels over the range of T concentrations (P = 0.04 and P = 0.017, respectively). Nuclear estrogen receptor (ER) increased over 10-fold at 5 and 50 pM EE2 (P less than 0.001) and maintained 50 nM (P less than 0.001). Cytosolic ER was reduced at 0.5 and 5 nM but recovered at 50 nM, correlating with SHBG levels (P less than 0.001). These findings are consistent with the hypothesis that estrogens and androgens regulate SHBG synthesis in man by direct, specific, probably receptor-mediated effects on hepatocytes. Hep G2 cells grown in serum-free medium are a suitable experimental system for further study of this phenomenon.     

Testosterone and dihydrotestosterone, but not estradiol, enhance survival of new hippocampal neurons in adult male rats

Past research suggested that androgens may play a role in the regulation of adult neurogenesis within the dentate gyrus. We tested this hypothesis by manipulating androgen levels in male rats. Castrated or sham castrated male rats were injected with 5-Bromo-2'deoxyuridine (BrdU). BrdU-labeled cells in the dentate gryus were visualized and phenotyped (neural or glial) using immunohistochemistry. Castrated males showed a significant decrease in 30-day cell survival within the dentate gyrus but there was no significant change in cell proliferation relative to control males, indicating that androgens positively affect cell survival, but not cell proliferation. To examine the role of testosterone on hippocampal cell survival, males were injected with testosterone s.c. for 30 days starting the day after BrdU injection. Higher doses (0.5 and 1.0 mg/kg) but not a lower dose (0.25 mg/kg) of testosterone resulted in a significant increase in neurogenesis relative to controls. We next tested the role of testosterone's two major metabolites, dihydrotestosterone (DHT), and estradiol, upon neurogenesis. Thirty days of injections of DHT (0.25 and 0.50 mg/kg) but not estradiol (0.010 and 0.020 mg/kg) resulted in a significant increase in hippocampal neurogenesis. These results suggest that testosterone enhances hippocampal neurogenesis via increased cell survival in the dentate gyrus through an androgen-dependent mechanism.     

Differential regulation of gonadotropin-releasing hormone secretion and gene expression by androgen: membrane versus nuclear receptor activation

Steroid hormones induce rapid membrane receptor-mediated effects that appear to be separate from long-term genomic events. The membrane receptor-mediated effects of androgens on GT1-7 GnRH-secreting neurons were examined. We observed androgen binding activity with a cell-impermeable BSA-conjugated testosterone [testosterone 3-(O-carboxymethyl)oxime (T-3-BSA)] and were able to detect a 110-kDa protein recognized by the androgen receptor (AR) monoclonal MA1-150 antibody in the plasma membrane fraction of the GT1-7 cells by Western analysis. Further, a transfected green fluorescent protein-tagged AR translocates and colocalizes to the plasma membrane of the GT1-7 neuron. Treatment with 10 nM 5alpha-dihydrotestosterone (DHT) inhibits forskolin-stimulated accumulation of cAMP, through a pertussis toxin-sensitive G protein, but has no effect on basal cAMP levels. The inhibition of forskolin-stimulated cAMP accumulation by DHT was blocked by hydroxyflutamide, a specific inhibitor of the nuclear AR. DHT, testosterone (T), and T-3-BSA, all caused significant elevations in intracellular calcium concentrations ([Ca(2+)](i)). T-3-BSA stimulates GnRH secretion 2-fold in the GT1-7 neuron, as did DHT or T. Interestingly GnRH mRNA levels were down-regulated by DHT and T as has been reported, but not by treatment with T-3-BSA or testosterone 17beta-hemisuccinate BSA. These studies indicate that androgen can differentially regulate GnRH secretion and gene expression through specific membrane-mediated or nuclear mechanisms.     

Reexamination of testosterone, dihydrotestosterone, estradiol and estrone levels across the menstrual cycle and in postmenopausal women measured by liquid chromatography-tandem mass spectrometry

Measuring serum androgen levels in women has been challenging due to limitations in method accuracy, precision sensitivity and specificity at low hormone levels. The clinical significance of changes in sex steroids across the menstrual cycle and lifespan has remained controversial, in part due to these limitations. We used validated liquid chromatography-tandem mass spectrometry (LC-MS/MS) assays to determine testosterone (T) and dihydrotestosterone (DHT) along with estradiol (E2) and estrone (E1) levels across the menstrual cycle of 31 healthy premenopausal females and in 19 postmenopausal females. Samples were obtained in ovulatory women in the early follicular phase (EFP), midcycle and mid luteal phase (MLP). Overall, the levels of T, DHT, E2 and E1 in premenopausal women measured by LC-MS/MS were lower overall than previously reported with immunoassays. In premenopausal women, serum T, free T, E2, E1 and SHBG levels peaked at midcycle and remained higher in the MLP, whereas DHT did not change. In postmenopausal women, T, free T, SHBG and DHT were significantly lower than in premenopausal women, concomitant with declines in E2 and E1. These data support the hypothesis that the changes in T and DHT that occur across the cycle may reflect changes in SHBG and estrogen, whereas in menopause, androgen levels decrease. LC-MS/MS may provide more accurate and precise measurement of sex steroid hormones than prior immunoassay methods and can be useful to assess the clinical significance of changes in T, DHT, E2 and E1 levels in females.     

Androgen-induced neurite outgrowth is mediated by neuritin in motor neurones

In the brain, the spinal cord motor neurones express the highest levels of the androgen receptor (AR). Experimental data have suggested that neurite outgrowth in these neurones may be regulated by testosterone or its derivative 5alpha-dihydrotestosterone (DHT), formed by the 5alpha-reductase type 2 enzyme. In this study we have produced and characterized a model of immortalized motor neuronal cells expressing the mouse AR (mAR) [neuroblastoma-spinal cord (NSC) 34/mAR] and analysed the role of androgens in motor neurones. Androgens either activated or repressed several genes; one has been identified as the mouse neuritin, a protein responsible for neurite elongation. Real-time PCR analysis has shown that the neuritin gene is expressed in the basal condition in immortalized motor neurones and is selectively up-regulated by androgens in NSC34/mAR cells; the DHT effect is counteracted by the anti-androgen Casodex. Moreover, DHT induced neurite outgrowth in NSC34/mAR, while testosterone was less effective and its action was counteracted by the 5alpha-reductase type 2 enzyme inhibitor finasteride. Finally, the androgenic effect on neurite outgrowth was abolished by silencing neuritin with siRNA. Therefore, the trophic effects of androgens in motor neurones may be explained by the androgenic regulation of neuritin, a protein linked to neurone development, elongation and regeneration.     

Androgen and estrogen dynamics in the female baboon (Papio anubis)

Androgen and estrogen dynamics were studied in 5 female baboons (Papio anubis) using constant infusions of [3H]androstenedione/[14C]estrone and [3H]testosterone/[14C]estradiol. Blood samples were obtained prior to the infusions and both blood and plasma was used for measurements of androstenedione (A), testosterone (T), dihydrotestosterone (DHT), estrone (E1), estradiol (E2). Plasma was used for measurements of sex-hormone binding globulin (SHBG), and the percents of T and E2 free, bound to SHBG, and to albumin. Blood samples obtained during the infusions were analyzed for radioactivity as purified androgens and estrogens. Metabolic clearance rates (MCR), and transfer factors ([rho]BB; fraction of steroid infused which is converted to and measured in blood as product) and blood production rates were calculated from whole blood data. All urine was collected for 96 h and an aliquot analyzed for radioactivity as the glucuronides of estrone and estradiol and the % peripheral aromatization calculated. The MCR's, calculated in whole blood, of A, E1, E2 and T were 53 +/- 6 1/day/kg, 39.3 +/- 3 1/day/kg, 29.9 +/- 5.2 1/day/kg and 10.1 +/- 2.3 1/day/kg, respectively. Each MCR was different (P less than 0.05) from the others. The PB of E1 was 15 +/- 2 micrograms/day and was not different from that of E2 (12 +/- 3 micrograms/day). The PB of A, 231 +/- 55 micrograms/day, was greater than that of T, 13 +/- 5 micrograms/day. The interconversions of both the androgens (18.9 +/- 3.4% vs 3.9 +/- 1.0%) and the estrogens (48.8 +/- 10.7% vs 4.0 +/- 0.8%) favored the oxidative pathway, i.e. conversion of 17-OH to 17-oxo steroids. The conversion ratio of A to DHT was greater than that of T to DHT (16.4 +/- 2.1% vs 5.3 +/- 0.7%), and A is a more important source of DHT than is T. The percent of T bound to SHBG (80.7 +/- 0.9%) was greater than percent of E2 (36.9 +/- 9.8%) and inversely the percents of T bound to albumin and free (17.5 +/- 0.8% and 1.65 +/- 0.16%) were less than the respective percents for estradiol (60.5 +/- 9.5% and 2.40 +/- 0.27%). The mean SHBG concentration was 54 +/- 6 nM. The peripheral aromatization of androstenedione, 1.36 +/- 0.05%, was greater than of testosterone, 0.18 +/- 0.02%. This difference is, in part, due to the lack of SHBG-binding of androstenedione. The general pattern of androgen and estrogen dynamics is similar to that in women. This similarity is due, in part, to the presence of SHBG in both baboons and women.     

Intracellular Ca2+ stimulates the binding to androgen receptors in platelets

In this study, we demonstrated that ADP-induced platelet aggregation activates the binding of testosterone (T) to its receptor. It is well known that binding of ADP to its receptors induced the release of Ca2+ ions from dense bodies into the cytosol of platelets. In this work, we compared the binding of testosterone or dihydrotestosterone to their receptors using cytosol obtained from ADP-treated and non-treated platelets. These experiments were repeated using EGTA (a calcium chelator) or U73122 (a phospholipase C enzymatic activity inhibitor) to the ADP-treated platelets. In addition, we also developed a competition analysis for the androgen receptors (AR) using [3H]DHT, non-radioactive T, DHT or cyproterone acetate from ADP-treated platelets cytosol. The results from this study indicate that the cytosol obtained from non-ADP-treated platelets did not show any binding to [3H]T or [3H]DHT, whereas cytosol from ADP-treated platelets binds to the radio-labeled androgens. Furthermore cytosol from ADP plus U73122-treated platelets did not show binding to [3H]T or [3H]DHT. These data suggest that intracellular Ca2+ ions stimulates the binding of androgens to their receptors in platelets cytosol. The competition analysis shows that T and DHT have high affinities for the androgen receptors with similar IC50 values, whereas cyproterone acetate shows a lower affinity. The results from these data clearly indicate the presence of androgen receptors in platelets.     

The role of transforming growth factor-β1, -β2, and -β3 in androgen-responsive growth of NRP-152 rat prostatic epithelial cells

We have investigated the role of autocrine/paracrine TGF-β secretion in the regulation of cell growth by androgens as demonstrated by its inhibition by two androgen response modifiers; the nonsteroidal antiandrogen hydroxyflutamide (OHF), believed to act by inhibiting androgen binding to androgen receptors, or finasteride, an inhibitor of 5α-reductase, the enzyme necessary for the conversion of testosterone to 5α-dihydrotestosterone (DHT), using the nontumorigenic rat prostatic epithelial cell line NRP-152. Growth of these cells was stimulated three- to sixfold over control by either testosterone or DHT under serum-free culture conditions. This was accompanied by a two- to threefold decrease in the secretion rate of TGF-β1, -β2, and -β3. Finasteride reversed the ability of testosterone but not DHT to stimulate growth and downregulate expression of TGF-β1, -β2, and -β3 in a dose-dependent fashion, suggesting that this activity of testosterone required its conversion to DHT. OHF antagonized the stimulatory effects of DHT on NRP-152 cell growth but could reverse the inhibitory effects of DHT only on TGF-β2 and TGF-β3 and not TGF-β1 secretion. This suggests that either TGF-β1 regulation by DHT or the androgen antagonism of OHF occurs independent of androgen receptor binding. Neutralizing antibodies to TGF-β (pantropic and isoform-specific) were able to block the ability of finasteride to antagonize the effects of testosterone nearly completely while only partially inhibiting the antiandrogenic effects of OHF. Thus, the ability of androgens to stimulate growth of NRP-152 cells involves the downregulation of the production of TGF-β1, -β2, and -β3 in addition to other growth-stimulatory mechanisms. J. Cell. Physiol. 175:184–192, 1998. Published 1998 Wiley-Liss, Inc.

1 This article is a US Government work and, as such, is in the public domain in the United States of America.

     

Physiological effects of androgens on human vascular endothelial and smooth muscle cells in culture

Androgenic hormones are associated with atherosclerotic cardiovascular disease, although the underlying cellular and molecular mechanisms remain unclear. This study examines the impact of androgens on the physiology of human vascular endothelial cells (EC) and smooth muscle cells (SMC) in culture. Cells were incubated with testosterone, dihydrotestosterone (DHT) or dehydroepiandrosterone (DHEA) at various physiological concentrations (5-50 nM) in the present or absence of an androgen receptor (AR) blocker flutamide (100 nM). Cell growth and death, DNA and collagen synthesis, and gene protein expression were assessed. It was shown that: (1) DHEA protected EC from superoxide injury via AR-independent mechanisms; (2) testosterone induced DNA synthesis and growth in EC via an AR-independent manner with activation of ERK1/2 activity; (3) DHT inhibited DNA synthesis and growth in EC in an AR-dependent manner; (4) testosterone and DHT enhanced ERK1/2 activation and proliferation in SMC via AR-independent and -dependent pathways, respectively; and (5) these androgens did not significantly affect collagen synthesis in SMC. We conclude that androgens possess multiple effects on vascular cells via either AR-dependent or -independent mechanisms. Testosterone and DHEA may be “beneficial” in preventing atherosclerosis by improving EC growth and survival; in contrast, stimulation of VSMC proliferation by testosterone and DHT is potentially “harmful”. The relationship of these in vitro effects by androgens to in vivo vascular function and atherogenesis needs to be further clarified.     

Rapid androgen actions on calcium signaling in rat sertoli cells and two human prostatic cell lines: similar biphasic responses between 1 picomolar and 100 nanomolar concentrations

Androgen-induced calcium fluxes and gap junctional intercellular communication (GJIC) were studied in three different cell types. A transient (2-3 min duration) increase in intracellular calcium levels was observed within 20-30 sec of androgen addition, which was followed by a plateau phase with steroid concentrations higher than 1 nM. The kinetics of the calcium responses were similar in immature rat Sertoli cells, which contain normal nuclear receptors; the human prostatic tumor cell line, LNCaP, which contains a mutated nuclear receptor; and the human prostatic cell line, PC3, which does not contain a nuclear receptor. The human A431 tumor cell line did not respond to androgens. Concentrations of testosterone and the synthetic androgen, R1881, between 1-1000 pM induced transient calcium increases with ED(50) values near 1 pM and 1 nM, whereas dihydrotestosterone (DHT) was not active at these concentrations. At concentrations higher than 1 nM, testosterone, R1881, and DHT were equipotent in stimulating an increase in calcium that lasted for more than 10 min, with ED(50) values between 5 and 20 nM. Testosterone covalently bound to albumin was also active, whereas 11 related androstane compounds as well as progesterone and estradiol-17beta were inactive at 1000 nM. The calcium response induced by the three androgens (10 nM) was abolished in all cell types by hydroxyflutamide (1000 nM) and finasteride (1000 nM), but not by cyproterone acetate (1000 nM). The calcium response was also abolished in the absence of extracellular calcium and strongly inhibited by the presence of verapamil. Exposure of the responsive cells to brief (150-sec) pulses of androgens generated calcium responses that were similar to those after continuous exposure. After exposure of Sertoli cells for only 30 sec to 100 nM testosterone, the calcium response lasted for at least 50 min. Although nuclear binding of androgens could be demonstrated, there was no evidence for tight binding to the plasma membrane under similar conditions. When protein synthesis was inhibited, an enhancement of GJIC between rat Sertoli cells, but not between LNCaP cells or PC3 cells, was observed within 15 min of the addition of 10 nM testosterone. Because nuclear androgens are not present in PC3 cells and many functional properties of the responsive system are different from the nuclear receptor in all three cell types, we postulate the existence of an alternative cell surface receptor system with biphasic response characteristics (high and low affinity). The calcium signals are probably coupled to the regulation of gap junctional efficiency between Sertoli cells. The low-affinity receptors may convey complementary androgen signals at elevated local levels such as in the testis, when nuclear receptors are (over)saturated.     

Testosterone and dihydrotestosterone radioimmunoassays in Müllerian ducts of control and testosterone propionate injected quail embryos (author's transl)

Testosterone (T) and dihydrotestosterone (DHT) have been quantitated by means of radioimmunoassay in Müllerian ducts (CM) from control quail embryos (6 to 8-day male and 6 to 15-day female) and from female embryos injected with 50 nanograms of testosterone propionate (PT) on the 8th day. These hormones are demonstrable in undifferentiated CM from 6-day control embryos. In control males although a highly significant decrease of the CM weight occurs during the CM involution, the detected amounts of androgens remain at a constant level. In control females, the right CM shows a slight increase of the androgen content during the rudimentation, i.e. from day 8 on; in the left CM: the highest steroid levels are found on the 6th day; while the CM differentiate, concentrations decrease and become similar to those found in neutral tissues. Given testosterone propionate on day 8 female embryos: right CM: both testosterone and DHT levels highly increase until day 14; the CM of treated embryos contain 8 times as much steroid as control; left CM: testosterone and DHT increase after injection until day 9,5; they slightly decrease between days 9,5 and 12, and then remain constant on day 14. Differences in concentrations are highly significant between CM of control and of PT - injected embryos. It seems that the binding sites of these androgens are more numerous during the involution and that bound testosterone or DHT could take a part in CM regression in male embryos and right CM rudimentation in female embryos.     

Modulatory effects of indomethacin on androgen metabolism in human gingival and oral periosteal fibroblasts.

The non-steroidal anti-inflammatory agent indomethacin (I) suppresses gingival inflammation and alveolar bone resorption. Androgens particularly 5 alpha-dihydrotestosterone (DHT) have anabolic effects on connective tissue and bone matrices. Human oral periosteal fibroblasts (HPF) and gingival fibroblasts (HGF) instigate healing in inflammatory periodontal lesions. The aim of this investigation was to compare the modulatory effects of I on the metabolism of two androgen substrates in human oral periosteal and gingival fibroblasts in culture. Monolayer cultures of both cell types (5(th)-9(th) passage) were established in Eagle's MEM and incubated with 14C-testosterone/14C-4-androstenedione and serial concentrations of I (0.5-50 microg/ml) for 24 h. The steroid metabolites were solvent extracted from the medium, separated by TLC and quantified using a radioisotope scanner. Both androgen substrates were metabolized mainly to DHT and 4-androstenedione/testosterone respectively, expressing 5 alpha-reductase and 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) activity in both HPF and HGF. There were 51% and 73% increases in the levels of DHT over controls, with HGF and HPF respectively (n = 6; n = 4, P < 0.01) in response to I at 1-5 microg/ml, often reaching control values at 50 microg/ml. The expression of 17 beta-HSD activity showed less stimulation than the levels of DHT. Both androgen substrates were effective in this metabolic conversion, which is applicable to healing responses in both males and females in vivo. There were 57% increases (n = 4; P < 0.01) over controls, in the formation of androstanediol from 14C-4-androstenedione at 10 microg of I, in HPF. This transformation may regulate androgen action in androgen-dependent tissue. In addition to its anti-inflammatory properties, indomethacin can contribute to anabolic reparatory responses, by increasing the expression of steroid metabolizing enzymes in gingival and periosteal fibroblasts, in the inflammatory periodontal lesion.     

Steroid metabolism and binding activity in a murine renal tumor cell line

The purpose of this study was to partially characterize the steroid binding activity of murine renal tumor cells in continuous culture. The steroid receptor content of a cloned renal tumor cell line (RAG) and a subline RAG-2 was examined by sucrose gradient analysis, hydroxylapatite and dextran-coated charcoal methods. The RAG cells lacked estrogen- and progestin-binding activity, whereas specific 5 alpha-dihydrotestosterone (DHT) and dexamethasone (Dx) binding activities were detected as 8S peaks on low salt gradients. The specificity of DHT binding was examined by sucrose gradient analysis: DHT, R1881 and ORG2058 all completely inhibited [3H]DHT binding whereas diethylstilbestrol and Dx were ineffective. The androgen receptor content of the RAG cells was approx. 15 fmol/mg cytosol protein by the hydroxylapatite-filter assay, with an estimated Kd for methyltrienolone (R1881) of 5 nM at 0 degrees C. Scatchard analysis of [3H]Dx binding by RAG cytosol showed a Kd of 6 nM for Dx and 44 nM for corticosterone at 0 degrees C. Glucocorticoid receptor levels were estimated to be 182 fmol/mg cytosol protein by dextran-coated charcoal assay. Metabolism of [3H]testosterone and [3H]DHT by RAG cells was examined 1, 4 and 6 h after exposure to labeled hormone. Radioactive DHT was the primary intracellular metabolite recovered after exposure to [3H]testosterone. There was little conversion of DHT to androstanediol.     

Serum androgens and sex hormone binding globulin in obese Pima Indian females

Levels of serum androgens and sex hormone binding globulin (SHBG) were measured in 20 obese Pima Indian females aged 19–44 and compared with those of normal-weight Caucasians aged 20–46. The Pima exhibited significantly decreased SHBG compared to Caucasians, but a strong effect of age on androgen levels rendered mean comparisons useless. Androstenedione (A) and dehydroepiandrosterone-sulfate (DHEA-S) decreased significantly, and testosterone (T) declined slightly with age in the Pima, whereas these androgens showed no significant decreases in Caucasians for this age range. A possible relationship of androgens to the Pima female's propensity for android obesity as well as possible effects of obesity on SHBG, androgens, and aging is discussed.