Monitoring the HIV continuum of care in key populations across Europe and Central Asia.

OBJECTIVES: The aim of the study was to measure and compare national continuum of HIV care estimates in Europe and Central Asia in three key subpopulations: men who have sex with men (MSM), people who inject drugs (PWID) and migrants. METHODS: Responses to a 2016 European Centre for Disease Prevention and Control (ECDC) survey of 55 European and Central Asian countries were used to describe continuums of HIV care for the subpopulations. Data were analysed using three frameworks: Joint United Nations Programme on HIV/AIDS (UNAIDS) 90-90-90 targets; breakpoint analysis identifying reductions between adjacent continuum stages; quadrant analysis categorizing countries using 90% cut-offs for continuum stages. RESULTS: Overall, 29 of 48 countries reported national data for all HIV continuum stages (numbers living with HIV, diagnosed, receiving treatment and virally suppressed). Six countries reported all stages for MSM, seven for PWID and two for migrants. Thirty-one countries did not report data for MSM (34 for PWID and 41 for migrants). In countries that provided key-population data, overall, 63%, 40% and 41% of MSM, PWID and migrants living with HIV were virally suppressed, respectively (compared with 68%, 65% and 68% nationally, for countries reporting key-population data). Variation was observed between countries, with higher outcomes in subpopulations in Western Europe compared with Eastern Europe and Central Asia. CONCLUSIONS: Few reporting countries can produce the continuum of HIV care for the three key populations. Where data are available, differences exist in outcomes between the general and key populations. While MSM broadly mirror national outcomes (in the West), PWID and migrants experience poorer treatment and viral suppression. Countries must develop continuum measures for key populations to identify and address inequalities.

HIV continuum of care in Europe and Central Asia.

OBJECTIVES: The European Centre for Disease Prevention and Control (ECDC) supports countries to monitor progress in their response to the HIV epidemic. In line with these monitoring responsibilities, we assess how, and to what extent, the continuum of care is being measured across countries. METHODS: The ECDC sent out questionnaires to 55 countries in Europe and Central Asia in 2014. Nominated country representatives were questioned on how they defined and measured six elements of the continuum. We present our results using three previously described frameworks [breakpoints; Joint United Nations Programme on HIV/AIDS (UNAIDS) 90-90-90 targets; diagnosis and treatment quadrant]. RESULTS: Forty countries provided data for at least one element of the continuum. Countries reported most frequently on the number of people diagnosed with HIV infection (37; 93%), and on the number in receipt of antiretroviral therapy (ART) (35; 88%). There was little consensus across countries in their approach to defining linkage to, and retention in, care. The most common breakpoint (>19% reduction between two adjacent elements) related to the estimated number of people living with HIV who were diagnosed (18 of 23; 78%). CONCLUSIONS: We present continuum data from multiple countries that provide both a snapshot of care provision and a baseline against which changes over time in care provision across Europe and Central Asia may be measured. To better inform HIV testing and treatment programmes, standard data collection approaches and definitions across the HIV continuum of care are needed. If countries wish to ensure an unbroken HIV continuum of care, people living with HIV need to be diagnosed promptly, and ART needs to be offered to all those diagnosed.

The mare as a model for luteinized unruptured follicle syndrome: intrafollicular endocrine milieu.

Luteinized unruptured follicle (LUF) syndrome is a recurrent anovulatory dysfunction that affects up to 23% of women with normal menstrual cycles and up to 73% with endometriosis. Mechanisms underlying the development of LUF syndrome in mares were studied to provide a potential model for human anovulation. The effect of extended increase in circulating LH achieved by administration of recombinant equine LH (reLH) or a short surge of LH and decrease in progesterone induced by prostaglandin F2α (PGF2α) on LUF formation (Experiment 1), identification of an optimal dose of COX-2 inhibitor (flunixin meglumine, FM; to block the effect of prostaglandins) for inducing LUFs (Experiment 2), and evaluation of intrafollicular endocrine milieu in LUFs (Experiment 3) were investigated. In Experiment 1, mares were treated with reLH from Day 7 to Day 15 (Day 0=ovulation), PGF2α on Day 7, or in combination. In Experiment 2, FM at doses of 2.0 or 3.0 mg/kg every 12 h and human chorionic gonadotropin (hCG) (1500 IU) were administered after a follicle ≥32 mm was detected. In Experiment 3, FM at a dose of 2.0 mg/kg every 12 h plus hCG was used to induce LUFs and investigate the intrafollicular endocrine milieu. No LUFs were induced by reLH or PGF2α treatment; however, LUFs were induced in 100% of mares using FM. Intrafollicular PGF2α metabolite, PGF2α, and PGE2 were lower and the ratio of PGE2:PGF2α was higher in the induced LUF group. Higher levels of intrafollicular E2 and total primary sex steroids were observed in the induced LUF group along with a tendency for higher levels of GH, cortisol, and T; however, LH, PRL, VEGF-A, and NO did not differ between groups. In conclusion, this study reveals part of the intrafollicular endocrine milieu and the association of prostaglandins in LUF formation, and indicates that the mare might be an appropriate model for studying the poorly understood LUF syndrome.

Preventing childhood obesity in early care and education settings: lessons from two intervention studies.

BACKGROUND: Obesity prevention in young children is a public health priority. In the USA, nearly 10% of children less than 5 years of age are obese, and most attend some form of out-of-home child care. While a number of interventions have been conducted in early care and education settings, few have targeted the youngest children in care or the less formal types of child care like family child care homes. Additionally, only two previous studies provided recommendations to help inform future interventions. METHODS: This paper presents lessons learned from two distinct intervention studies in early care and education settings to help guide researchers and public health professionals interested in implementing and evaluating similar interventions. We highlight two studies: one targeting children ages 4 to 24 months in child care centres and the other intervening in children 18 months to 4 years in family child care homes. We include lessons from our pilot studies and the ongoing larger trials. RESULTS: To date, our experiences suggest that an intervention should have a firm basis in behaviour change theory; an advisory group should help evaluate intervention materials and plan for delivery; and realistic recruitment goals should recognize economic challenges of the business of child care. A flexible data collection approach and realistic sample size calculations are needed because of high rates of child (and sometimes facility) turnover. An intervention that is relatively easy to implement is more likely to appeal to a wide variety of early care and education providers. CONCLUSIONS: Interventions to prevent obesity in early care and education have the potential to reach large numbers of children. It is important to consider the unique features and similarities of centres and family child care homes and take advantage of lessons learned from current studies in order to develop effective, evidence-based interventions.

Hormone-induced 14-3-3γ adaptor protein regulates steroidogenic acute regulatory protein activity and steroid biosynthesis in MA-10 Leydig cells.

Cholesterol is the sole precursor of steroid hormones in the body. The import of cholesterol to the inner mitochondrial membrane, the rate-limiting step in steroid biosynthesis, relies on the formation of a protein complex that assembles at the outer mitochondrial membrane called the transduceosome. The transduceosome contains several mitochondrial and cytosolic components, including the steroidogenic acute regulatory protein (STAR). Human chorionic gonadotropin (hCG) induces de novo synthesis of STAR, a process shown to parallel maximal steroid production. In the hCG-dependent steroidogenic MA-10 mouse Leydig cell line, the 14-3-3γ protein was identified in native mitochondrial complexes by mass spectrometry and immunoblotting, and its levels increased in response to hCG treatment. The 14-3-3 proteins bind and regulate the activity of many proteins, acting via target protein activation, modification and localization. In MA-10 cells, cAMP induces 14-3-3γ expression parallel to STAR expression. Silencing of 14-3-3γ expression potentiates hormone-induced steroidogenesis. Binding motifs of 14-3-3γ were identified in components of the transduceosome, including STAR. Immunoprecipitation studies demonstrate a hormone-dependent interaction between 14-3-3γ and STAR that coincides with reduced 14-3-3γ homodimerization. The binding site of 14-3-3γ on STAR was identified to be Ser-194 in the STAR-related sterol binding lipid transfer (START) domain, the site phosphorylated in response to hCG. Taken together, these results demonstrate that 14-3-3γ negatively regulates steroidogenesis by binding to Ser-194 of STAR, thus keeping STAR in an unfolded state, unable to induce maximal steroidogenesis. Over time 14-3-3γ homodimerizes and dissociates from STAR, allowing this protein to induce maximal mitochondrial steroid formation.

Validation of the aging hen (Gallus gallus domesticus) as an animal model for uterine leiomyomas.

Uterine leiomyomas, or fibroids, are the most frequent gynecological tumors in premenopausal women with as many as 65% of women becoming clinically symptomatic. Uterine fibroids are benign myometrial tumors that produce large quantities of extracellular matrix proteins. Despite its high morbidity, the molecular basis underlying the development of uterine leiomyomas is not well understood. Domestic hens of Gallus gallus domesticus develop oviductal leiomyomas similar to those found in humans. We investigated the natural history of chicken leiomyomas, in vivo expression of protein biomarkers, and in vitro expression of ovarian steroid receptors. Based on the analysis of 263 hens, tumor prevalence, tumor number per hen, and tumor size increased as the hens aged. Immunohistochemistry for alpha-smooth muscle actin (SMA) and desmin confirmed the smooth muscle phenotype of the chicken leiomyomas. Intense collagen expression was detected in these oviductal leiomyomas by Mason's trichrome, and the tumors also showed increased expression of TGFB3 and collagen type I mRNAs. Consistent with human leiomyomas, chicken fibroids displayed increased BCL2 and estrogen (E) and progesterone (P) receptor expression. Chicken leiomyomas were dissociated for in vitro culture. Cells from explants were positive for SMA, desmin, and E and P receptors until the fourth passage. These cells also displayed a response similar to human cells when challenged with halofuginone, an antifibrotic agent. Our findings indicate that the chicken is an excellent complementary model for studies involving the pathophysiology of human uterine leiomyomas.

Therapeutic effect of flax-based diets on fatty liver in aged laying hens.

This study examined the ability of flax-based ingredients to attenuate nonalcoholic fatty liver disease ( NAFLD: ) in aged laying hens-a novel and more physiologically relevant model of human disease. Our results showed only hens supplemented with whole flaxseed ( WFX: ) reduced steatosis and hepatocellular ballooning. Serum AST was also reduced in hens provided WFX and defatted flaxseed meal ( DFM: ). Hepatic ω-3 PUFA enrichment was improved with supplementation of WFX, DFM, and flaxseed oil ( FXO: ). However, this effect was more evident in the WFX group. In contrast, transcript abundance of genes linked to NAFLD were predominantly modified with FXO supplementation. Taken together, our data indicate a potential synergistic relationship between the fatty acid and lignan content in flaxseed which attenuated the progression of NAFLD in aged laying hens. Although more research is necessary, these findings demonstrate the potential use of whole flaxseed for the treatment and prevention of NAFLD in humans.

Microsatellite and chromosome evolution of parthenogenetic sitobion aphids in Australia.

Single-locus microsatellite variation correlated perfectly with chromosome number in Sitobion miscanthi aphids. The microsatellites were highly heterozygous, with up to 10 alleles per locus in this species. Despite this considerable allelic variation, only seven different S. miscanthi genotypes were discovered in 555 individuals collected from a wide range of locations, hosts and sampling periods. Relatedness between genotypes suggests only two successful colonizations of Australia. There was no evidence for genetic recombination in 555 S. miscanthi so the occurrence of recent sexual reproduction must be near zero. Thus diversification is by mutation and chromosomal rearrangement alone. Since the aphids showed no sexual recombination, microsatellites can mutate without meiosis. Five of seven microsatellite differences were a single repeat unit, and one larger jump is likely. The minimum numbers of changes between karyotypes corresponded roughly one-to-one with microsatellite allele changes, which suggests very rapid chromosomal evolution. A chromosomal fission occurred in a cultured line, and a previously unknown chromosomal race was detected. All 121 diverse S. near fragariae were heterozygous but revealed only one genotype. This species too must have a low rate of sexual reproduction and few colonizations of Australia.

Interleukin-1 inhibits Leydig cell steroidogenesis primarily by decreasing 17 alpha-hydroxylase/C17-20 lyase cytochrome P450 expression.

Macrophage-secreted cytokines, such as interleukin-1 (IL-1), have been shown to modulate Leydig cell function. The present study examined the effect of recombinant murine IL-1 alpha on Leydig cell steroidogenesis and its mechanism of action. Addition of IL-1 to macrophage-depleted primary cultures of mouse Leydig cells caused a dose-dependent decrease in cAMP-dependent testosterone production and 17 alpha-hydroxylase/C17-20 lyase (P450c17) mRNA levels. Chronic treatment (48 h) of Leydig cells in culture with 50 microM 8-bromo-cAMP (8-Br-cAMP) resulted in a 60-fold increase in testosterone production. Treatment with 8-Br-cAMP plus 0.2 and 2 U/ml IL-1 decreased testosterone production to 63 +/- 14% and 41 +/- 19%, respectively, while 5, 10, and 20 U/ml IL-1 decreased testosterone production to less than 5% that of cells treated with 8-Br-cAMP alone. Chronic treatment with IL-1 plus 8-Br-cAMP caused a shift in steroid production from testosterone to progesterone, but total steroid production (the sum of testosterone plus progesterone) was unaffected by IL-1 treatment. Treatment with 8-Br-cAMP alone caused a marked increase in P450c17 mRNA levels compared to that in control cultures, where P450c17 mRNA was undetectable. IL-1 caused a dose-dependent decrease in 8-Br-cAMP-stimulated P450c17 levels (0.2 U/ml by 31 +/- 9%, 2 U/ml by 82 +/- 12%, and 10 or 20 U/ml by 100% compared to that in cells treated with 8-Br-cAMP alone). In contrast to the effect on P450c17 mRNA, only the highest concentrations of IL-1 (10 and 20 U/ml) had any effect on cholesterol side-chain cleavage enzyme (P450scc) mRNA levels (53 +/- 16% and 38 +/- 20% decreases, respectively). The inhibitory effect of IL-1 on 8-Br-cAMP-stimulated P450c17 expression was reversible. Within 12 h after the removal of IL-1, P450c17 mRNA was restored to 24%; after 24 h, to 36%; after 36 h, to 65%; and after 48 h, to 84% of that with 8-Br-cAMP alone. P450c17 expression was more sensitive to IL-1-mediated inhibition than P450scc; therefore, inhibition of P450c17 is most likely primarily responsible for the observed inhibitory effects of IL-1 on Leydig cell testosterone production.

Reconstitution of steroid 17,20-lyase activity after separation and purification of cytochrome P-450 and its reductase from rat testis microsomes.

The testicular enzyme, 17,20-lyase, catalyzes the removal of the C-17 side chain from steroids in the synthesis of androgens. This activity employs cytochrome P-450 as an oxygen donor. Attempts to purify the cytochrome and its reductase from testis microsomes have previously been unsuccessful due to the low concentrations of these components (2--5% that of liver). The cytochrome and reductase were solubilized from rat testis microsomes using a mixture of sodium cholate and Emulgen 913. The components were then separated by DEAE chromatography. The cytochrome was further purified by chromatography using hydroxylapatite for an 8.5-fold enrichment. The reductase was further purified by hydroxylapatite and affinity chromatography. An 84-fold enrichment was achieved. 17,20-Lyase activity could be partially restored by mixing the cytochrome and reductase in the presence of phospholipid.

NADPH-cytochrome P-450 reductase from untreated rat testicular and liver microsomes: isolation and comparison.

NADPH-cytochrome P-450 reductase (EC 1.6.2.4), the enzyme involved in progesterone 17-hydroxylation, was purified to apparent homogeneity by detergent solubilization of the microsomal fractions of liver and testis from untreated rats. The enzymes from these two tissues were then compared with regard to several parameters. The liver and testicular reductases have the same subunit mol wt (79,000), determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and have similar specific activity for cytochrome c reduction (48.1 and 58.2 mumol min-1 mg-1, respectively). The absolute flavin absorption spectra of liver and testicular reductase were very similar. The spectra of native, reduced, and oxidized reductases were characteristic of flavoprotein with two flavin groups. The Km values for NADPH during cytochrome reduction were shown to be the same, within experimental error (5.29 +/- 0.46 microM-1 for liver and 4.37 +/- 0.53 microM-1 for testis). Monospecific antiserum was prepared against both rat liver and testicular reductases. There were no antigenic differences demonstrated by immunological tests using either antibody preparation. Antiliver reductase antiserum was shown to inhibit testicular microsomal progesterone 17-hydroxylase activity by 70%. Testicular cytochrome c reductase activity was inhibited by 94%, and liver cytochrome c reductase activity was inhibited by 85%. Peptide maps of both forms of reductase isolated from immunoprecipitates showed very similar patterns after enzymatic proteolysis. These results indicate that microsomal NADPH-cytochrome P-450 reductase involved in steroid hydroxylations in untreated rat liver and testis could not be distinguished from each other by these methods and, therefore, are probably the same protein.

Expression, regulation, and production of tumor necrosis factor-alpha in mouse testicular interstitial macrophages in vitro.

Tumor necrosis factor-alpha (TNF alpha) is a cytokine principally secreted from macrophages and monocytes activated by agents such as lipopolysaccharide (LPS). We have recently shown that TNF alpha inhibited mouse Leydig cell steroidogenesis in vitro. LPS injection has also been shown to repress Leydig cell function and induce TNF alpha messenger RNA (mRNA) expression in testicular interstitial macrophages in vivo. A paracrine regulation of Leydig cell testosterone synthesis by testicular interstitial macrophages via TNF alpha has been proposed. To further support this possibility, we examined whether LPS can induce TNF alpha mRNA expression and protein production in testicular interstitial macrophages in vitro. The regulation of LPS-stimulated TNF alpha mRNA expression in vitro was also investigated by employing the protein synthesis inhibitor cycloheximide (CHX). TNF alpha secretion into culture supernatants was examined by both bioassay and enzyme-linked immunosorbent assay. Isolated testicular interstitial macrophages were cultured for 24 h before the initiation of treatments. Cells were treated with or without LPS (1.0 micrograms/ml) and in the presence or absence of CHX (5.0 micrograms/ml) at different time points. Northern blot analysis showed that TNF alpha mRNA was rapidly and significantly induced by LPS in testicular interstitial macrophages. The peak expression was at 2 h after the treatment, which was 8.3 +/- 2.6-fold over the control (P < 0.05). TNF alpha mRNA then declined quickly and completely disappeared by 8 h after LPS treatment. In contrast to this rapid and transient induction of TNF alpha message by LPS alone, CHX extended the induction and caused a marked increase in LPS-induced TNF alpha mRNA at 2 and 6 h. CHX induced more LPS-stimulated TNF alpha mRNA at 6 h than that at 2 h. At 3 h after LPS treatment, TNF alpha secretion was significantly stimulated (5.6 +/- 1.2 U/micrograms macrophage DNA) measured by L929 tumor fibroblast cytotoxicity. TNF alpha was also detected by enzyme-linked immunosorbent assay in culture media of testicular interstitial macrophages treated with control medium or LPS for 1, 2, and 6 h. TNF alpha secretion was increased in a time-dependent way. There are significantly higher LPS-induced TNF alpha levels in culture media at 2 h (35.4 +/- 2.2 pg/micrograms macrophage DNA) and 6 h (85.5 +/- 11.1 pg/micrograms macrophage DNA) than those in control groups. The current study demonstrates that LPS activates testicular interstitial macrophages to express TNF alpha mRNA and secrete TNF alpha protein in vitro.

Glucocorticoid-mediated repression of P450scc mRNA and de novo synthesis in cultured Leydig cells.

The regulation of cholesterol side-chain cleavage enzyme (P450scc) by glucocorticoids was investigated in mouse Leydig cell cultures. We recently demonstrated that P450scc is constitutively synthesized in Leydig cells and that the rate of P450scc synthesis is increased by chronic treatment of the cultures with 8-bromo-cAMP. We now report that glucocorticoids, specifically, decrease the constitutive and cAMP-induced synthesis of P450scc protein as well as the accumulation of P450scc mRNA. The treatment of cultures with as little as 10 nM dexamethasone resulted in a 50-60% decrease in the rate of synthesis of P450scc protein and mRNA content. The glucocorticoid-mediated decrease in P450scc synthesis was prevented when cultures were treated with the antiglucocorticoid RU-486. RU-486 alone had no effect on the rate of protein synthesis. The effect was specific for glucocorticoids; corticosterone (100 nM) or cortisol (100 nM) brought about a similar decrease as dexamethasone. Treatment of cultures with the progesterone agonist R5020 (100 nM), testosterone (2 microM), or estradiol (50 nM) had no effect on the rate of specific protein synthesis. The synthesis of iron sulfur protein reductase (ISP-reductase) and F1-ATPase were not affected by dexamethasone, indicating that the effect was specific for P450scc. The amount of P450scc mRNA was decreased 61% by dexamethasone and increased 144% by treatment with 8-bromo-cAMP. These data together with our previous finding on the negative regulation of P450(17 alpha) protein synthesis by testosterone suggest that the steroidogenic P450 enzymes in Leydig cells are negatively regulated by steroid hormones acting via their cognate receptors.

The role of tumor necrosis factor-alpha in the regulation of mouse Leydig cell steroidogenesis.

Tumor necrosis factor-alpha (TNF alpha), a cytokine secreted by activated macrophages, has been shown to modulate Leydig cell steroidogenesis. The present study examined the regulation of mouse Leydig cell function by TNF alpha at the molecular level. The effects of TNF alpha on both basal and 8-Br-cAMP-stimulated testosterone production, as well as cholesterol side-chain cleavage enzyme (P450scc) and 17 alpha-hydroxylase/C17,20 lyase (P450c17), were investigated. Treatment of Leydig cells with 0.1, 1.0, and 10.0 ng/ml TNF alpha inhibited basal testosterone secretion by 20 +/- 5.0%, 61.1 +/- 6.6%, and 60.7 +/- 5.8% of control, respectively, but had no effects on basal P450scc messenger RNA (mRNA) or protein levels. Treatment of Leydig cells with 8-Br-cAMP caused a 150.7 +/- 32.9-fold increase in testosterone production and marked stimulation of P450scc and P450c17 mRNA and protein accumulation. TNF alpha caused a significant and dose-dependent inhibition of 8-Br-cAMP-stimulated testosterone secretion by 35.9 +/- 9.9%, 90.9 +/- 1.7%, and 96.9 +/- 1.4% with 0.1, 1.0, and 10.0 ng/ml TNF alpha, respectively. TNF alpha also caused a decrease in P450scc and P450c17 mRNA and protein. Treatment with 0.1, 1.0, and 10.0 ng/ml TNF alpha decreased 8-Br-cAMP-stimulated P450scc mRNA by 11.5 +/- 6.9%, 29.3 +/- 2.7%, and 59.2 +/- 8.7%, and decreased 8-Br-cAMP-induced P450c17 mRNA 41.9 +/- 13.5%, 95.7 +/- 2.3%, and 98.5 +/- 1.2%, respectively. The inhibitory effects of TNF alpha on 8-Br-cAMP-stimulated P450 enzyme protein accumulation were also dose dependent, 35.6 +/- 11.4%, 52.9 +/- 14.1%, and 56.0 +/- 7.9% inhibition of P450scc protein levels, and 65.8 +/- 9.4%, 95.5 +/- 1.9%, and 96.9 +/- 2.1% suppression on P450c17 protein levels were observed with 0.1, 1.0, and 10.0 ng/ml TNF alpha, respectively. The inhibitory effect of TNF alpha on 8-Br-cAMP-induced P450c17 mRNA expression was reversible. Within 48 h after the removal of TNF alpha from culture, P450c17 mRNA was restored to 80.6 +/- 3.1% of the level in cultures treated with 8-Br-cAMP alone for 4 days. TNF alpha-mediated inhibition of 8-Br-cAMP-stimulated testosterone secretion from Leydig cells was also reversible. In addition, no significant cell mortality was noted in TNF alpha-treated cells. These data demonstrate that TNF alpha inhibits both basal and 8-Br-cAMP-stimulated testosterone secretion from Leydig cells in a dose-dependent manner.(ABSTRACT TRUNCATED AT 400 WORDS)

Glucocorticoid and cyclic adenosine 3'5'-monophosphate-mediated induction of cholesterol side-chain cleavage cytochrome P450 (P450scc) in MA-10 tumor Leydig cells. Increases in mRNA are cycloheximide sensitive.

The regulation of cholesterol side-chain cleavage enzyme (P450scc) was investigated in MA-10 tumor Leydig cells. We recently demonstrated that the constitutive and cAMP-stimulated expression of P450scc in normal mouse Leydig cells is negatively regulated by glucocorticoids. We now report that glucocorticoids have the opposite effect in MA-10 cells causing a 1.7-fold increase in the rate of P450scc synthesis and a 2.1-fold increase in the amount of P450scc mRNA. Treatment of MA-10 cells with 10 microM 8-bromo-cAMP (8-Br-cAMP) (cAMP) resulted in a 1.7-fold increase in P450scc synthesis and a 3-fold increase in P450scc mRNA. Combined treatment with dexamethasone and cAMP resulted in additive increases in synthesis (2.8-fold) and mRNA (5.3-fold). Increases in de novo synthesis and mRNA levels were reflected by modest increases in the amount of immunoreactive P450scc enzyme protein. Dexamethasone-mediated stimulation in synthesis and accumulation of P450scc mRNA were blocked by the antiglucocorticoid RU-486. Cycloheximide blocked both cAMP- and dexamethasone-induced increases but had no effect on constitutive levels of P450scc mRNA. Treatment of MA-10 cells with 10 microM 8-Br-cAMP had no effect on cell morphology and stimulated progesterone accumulation to a minor degree. Treatment of MA-10 cells with 1 mM 8-Br-cAMP resulted in cell rounding and loss of cells from culture dishes. The results of this study demonstrate that: 1) dexamethasone increases P450scc de novo synthesis and mRNA levels in MA-10 tumor Leydig cells, opposite to the effect in normal Leydig cells; 2) dexamethasone- and cAMP-stimulated increases occur via distinct mechanisms; 3) and synthesis of protein factor(s) is required to mediate the action of both dexamethasone and cAMP.

Effect of transforming growth factor beta-1 on the cholesterol side-chain cleavage system in the adrenal gland of sheep fetuses and newborns.

The present work examined the effect of transforming growth factor-beta1 (TGFbeta1) on the cholesterol side-chain cleavage system of sheep fetal and neonatal adrenal glands. Freshly isolated fetal adrenal cells produced 3-fold less pregnenolone from 22R- hydroxycholesterol than neonatal cells. Also, the relative amounts of immunoreactive cytochrome P450 side-chain cleavage (P450scc), adrenodoxin, and adrenodoxin reductase were 1.5- to 2-fold lower in mitochondria from fetal than neonatal cells. However, during culture under control conditions, the cholesterol side-chain cleavage activity and the amounts of P450scc, adrenodoxin, and adrenodoxin reductase of fetal cells increased after 48 h to reach neonatal values. A 5-day treatment with TGFbeta1 (1ng/ml) decreased significantly both the cholesterol side-chain cleavage activity and the amounts of immunoreactive P450scc, adrenodoxin, and adrenodoxin reductase in sheep fetal and neonatal adrenal cells in culture. Immunoreactive TGFbeta1- like material was present in freshly isolated adrenal cells from both fetuses and newborn lambs. After 2 days of culture, the amount of TGFbeta1-like protein was 2-fold lower than in freshly isolated fetal cells. No change was observed for neonatal cells. Finally, TGFbeta1 encoding messenger RNAs and TGFbeta-like immunoreactive protein were much higher in adrenal cortices from fetuses than from neonates. Taken together, these results have made TGFbeta1 a potentially attractive candidate for being an auto/paracrine negative regulator of the cholesterol side-chain cleavage system in the sheep fetal adrenal gland.

Tumor necrosis factor-alpha inhibition of 17 alpha-hydroxylase/C17-20 lyase gene (Cyp17) expression.

Testosterone biosynthesis in Leydig cells is dependent on the action of 17 alpha-hydroxylase/C17-20 lyase cytochrome P450 (P450c17), which is encoded by the Cyp17 gene. Tumor necrosis factor-alpha (TNF alpha), a proinflammatory cytokine, inhibits cAMP-stimulated testosterone production in mouse Leydig cells. The inhibition of testosterone production is parallel to the inhibition of P450c17 messenger RNA and protein levels. To examine the mechanism of TNF alpha-mediated inhibition of steroidogenesis, the effect of TNF alpha on cAMP-stimulated induction of Cyp17 expression was investigated. To determine whether the protein kinase C (PKC) signaling pathway is involved in TNF alpha inhibition of steroidogenesis, the effects of the PKC activator, phorbol 12-myristate 13-acetate (PMA), and the PKC inhibitor, calphostin C, were examined. Treatment of normal mouse Leydig cells in primary culture with 50 microM 8-bromo-cAMP (cAMP) plus 1 ng/ml TNF alpha or 10 nM PMA caused a similar (approximately 90%) decrease in testosterone accumulation and cAMP-stimulated P450c17 messenger RNA levels compared to those after treatment with cAMP alone. To determine whether TNF alpha inhibits the cAMP-induced expression of the Cyp17 gene, plasmids containing two different size fragments of the 5'-flanking region of the Cyp17 gene upstream of the chloramphenicol acetyltransferase (CAT) reporter gene were transiently transfected into MA-10 tumor Leydig cells, and the effect of TNF alpha on cAMP-induced CAT activity was determined. Treatment of cells, transfected with either plasmid, with 500 microM cAMP plus increasing concentrations (0.1, 1.0, and 10 ng/ml) of TNF alpha resulted in a dose-dependent repression of cAMP-stimulated CAT activity. Higher concentrations of TNF alpha (up to 100 ng/ml) did not result in greater inhibition. Treatment of transfected cells with 10 nM PMA resulted in a 51 +/- 6.6% inhibition of cAMP-stimulated CAT activity. Calphostin C (1 microM) completely reversed the inhibitory effect of TNF alpha or PMA. Calphostin C alone had no effect on promoter activity. TNF alpha-stimulated PKC alpha translocation was quantitated by Western blot. After treatment for 3 h, the distribution of immunoreactive PKC alpha in cytosol vs. nucleus was 55%/45%, 60%/40%, and 29%/71% in control, cAMP-treated, and TNF alpha-treated cells, respectively. TNF alpha-stimulated PKC alpha translocation was further demonstrated by indirect immunofluorescence assay. PMA, a known activator of PKC, and TNF alpha had a similar inhibitory effect on P450c17 expression, testosterone production, and Cyp17-CAT activity.(ABSTRACT TRUNCATED AT 400 WORDS)

Acute in vivo inhibition of testosterone by endotoxin parallels loss of steroidogenic acute regulatory (StAR) protein in Leydig cells.

In an experimental endotoxemia model utilizing mice, serum testosterone was found to be decreased 90% two h post ip injection of 200 micrograms of lipopolysaccharide (LPS). This decrease was sustained for 9 days. The early depression of serum testosterone was shown to be associated with a decrease in Steroidogenic Acute Regulatory (StAR) protein levels while the prolonged decrease corresponded to decreased protein and transcript levels of steroidogenic enzymes in Leydig cells. This acute and prolonged depression of testosterone production could lead to impaired spermatogenesis, accessory duct failure, and contribute to decreased male fertility following acute inflammation.

Inhibition of transcription affects synthesis of steroidogenic acute regulatory protein and steroidogenesis in MA-10 mouse Leydig tumor cells.

Hormonal induction of steroidogenesis in the adrenal and gonads is dependent on the synthesis and function of the steroidogenic acute regulatory protein (StAR). As a first approach to investigate the role of translation in the control of StAR expression, we examined StAR protein synthesis and steroid production in MA-10 mouse Leydig tumor cells in the presence of the transcriptional inhibitor, actinomycin D. We show that human CG (hCG)-induced StAR synthesis, as determined by radiolabeling MA-10 cells with [35S]methionine and immunoprecipitation of StAR, is blocked by actinomycin D. The rate of hCG-stimulated progesterone production is also decreased, but not completely blocked, suggesting a possible StAR-independent mechanism that may contribute approximately 10-20% of the acute steroidogenic potential of the cells. When MA-10 cells were pretreated with hCG to increase StAR messenger RNA levels and then the proteins radiolabeled in the presence of hCG or hCG plus actinomycin D, no difference was observed in the amount of the 30-kDa StAR protein synthesized. However, a 50% increase in the precursor form of StAR protein was detected with hCG treatment alone. These data suggest that ongoing StAR protein synthesis is not inhibited by actinomycin D, but that continued synthesis requires transcriptional activity. Progesterone production was inhibited by actinomycin D in the hCG-pretreated cells, supporting the proposal that maintaining StAR protein synthesis is required for optimal steroid production in MA-10 mouse Leydig tumor cells.

Transcriptional regulation of the rat steroidogenic acute regulatory protein gene by steroidogenic factor 1.

Steroidogenic acute regulatory (StAR) protein is synthesized in response to tropic hormones to facilitate cholesterol transport to the inner mitochondrial membrane-bound P450 side-chain cleavage enzyme (P450scc), the first enzymatic step in the steroid hormone biosynthetic pathway. Gonadotropins activate expression of their target genes via the cAMP second messenger system. We have demonstrated that cAMP administration to rat luteal cells stimulates expression of both StAR messenger RNA and protein. Because cholesterol delivery is the first regulated step in steroidogenesis, and because StAR messenger RNA levels are increased in response to tropic hormone and cAMP stimulation, the mechanism by which tropic hormones/cAMP stimulate transcription needs to be elucidated. To this end, approximately 2.7 kb of the rat StAR promoter was isolated and sequenced. Sequence analysis revealed the presence of a TATA-like element as well as multiple regulatory motifs including steroidogenic factor 1 (SF-1) binding sites, an estrogen receptor half-site, and two AP-1 sites within the promoter region. 5'-RACE experiments determined the transcription start site to be located 82 bp upstream of the ATG translation start codon. Electrophoretic mobility shift assays and supershift analysis demonstrated SF-1 binding to three SF-1 binding sites in the rat StAR promoter with high affinity and two SF-1 binding sites with low affinity. Transfection of mouse Y1 adrenal tumor cells and human bladder carcinoma cells (HTB9s) with the rat StAR promoter demonstrated that SF-1 was able to activate transcription of the luciferase reporter gene and that the rat StAR promoter was responsive to cAMP. Nested deletions of the rat StAR promoter (1.9 kb) identified a region between -1413 and -998 that is essential for maximal activation of the rat StAR gene in HTB9 cells; however, deletion of this region does not affect responsiveness to cAMP. 5'-Deletion and site-directed mutagenesis experiments demonstrated that the SF-1 motifs identified within the rat StAR promoter (located at positions -764, -455, and -106) were sufficient to activate transcription as well as confer cAMP responsiveness to the rat StAR gene. Site-directed mutagenesis studies using the smallest promoter fragment demonstrated that the two proximal SF-1 binding sites are crucial for StAR gene transcription, both at a basal level and in response to cAMP stimulation. These studies provide novel insights into the regulation of the rat StAR gene at the transcriptional level by SF-1.