Human cell-based anti-inflammatory effects of rosiglitazone.

PURPOSE: The C-X-C motif chemokine ligand 10 (CXCL10) participates in diabetes and diabetic cardiomyopathy development from the early stages. Rosiglitazone (RGZ) exhibits anti-inflammatory properties and can target cardiomyocytes secreting CXCL10, under interferon (IFN)γ and tumor necrosis factor (TNF)α challenge. Cardiomyocyte remodeling, CD4 + T cells and dendritic cells (DCs) significantly contribute to the inflammatory milieu underlying and promoting disease development. We aimed to study the effect of RGZ onto inflammation-induced secretion of CXCL10, IFNγ, TNFα, interleukin (IL)-6 and IL-8 by human CD4 + T and DCs, and onto IFNγ/TNFα-dependent signaling in human cardiomyocytes associated with chemokine release. METHODS: Cells maintained within an inflammatory-like microenvironment were exposed to RGZ at near therapy dose (5 µM). ELISA quantified cytokine secretion; qPCR measured mRNA expression; Western blot analyzed protein expression and activation; immunofluorescent analysis detected intracellular IFNγ/TNFα-dependent trafficking. RESULTS: In human CD4 + T cells and DCs, RGZ inhibited CXCL10 release likely with a transcriptional mechanism, and reduced TNFα only in CD4 + T cells. In human cardiomyocytes, RGZ impaired IFNγ/TNFα signal transduction, blocking the phosphorylation/nuclear translocation of signal transducer and activator of transcription 1 (Stat1) and nuclear factor-kB (NF-kB), in association with a significant decrease in CXCL10 expression, IL-6 and IL-8 release. CONCLUSION: As the combination of Th1 biomarkers like CXCL10, IL-8, IL-6 with classical cardiovascular risk factors seems to improve the accuracy in predicting T2D and coronary events, future studies might be desirable to further investigate the anti-Th1 effect of RGZ.

Circulating levels of fetuin-A are associated with moderate-severe hepatic steatosis in young adults.

PURPOSE: The hepatokine fetuin-A might have a role as molecular link between non-alcoholic fatty liver disease (NAFLD) and type 2 diabetes mellitus (T2DM). The aim of this study was to evaluate the association between fetuin-A and the prevalence and severity of NAFLD in a population of young adults. METHODS: Ninety-seven adults (age 35.7 ± 12.4 years, female 64.9%), enrolled in a previous study evaluating NAFLD prevalence in the presence or absence of family history of T2DM, were included. Serum levels of fetuin-A (ELISA BioVendor, Czech Republic) and the main biochemical parameters were assessed. Presence and severity of NAFLD were evaluated by ultrasonography (Toshiba, Japan). A linear regression was run to predict fetuin-A levels and a logistic regression was performed to predict moderate-severe steatosis. RESULTS: Fetuin-A associated inversely with age (ß - 0.12, p = 0.03) and directly with body mass index (BMI) (ß 0.5, p = 0.048), waist circumference (WC) (ß 0.3, p = 0.027), triglycerides (TG) (ß 0.1, p = 0.001) and uric acid (ß 1.7, p = 0.018), after adjustment for age and sex. In a model including age, BMI, WC, TG and uric acid, age (ß - 0.2, p = 0.002) and TG (ß 0.04, p = 0.02) were independent predictors of fetuin-A. Prevalence of steatosis was 66%. The rates of mild and moderate-severe steatosis were 50.5% and 15.5%, respectively. In the logistic model, the independent predictors of moderate-severe steatosis were fetuin-A (OR 1.22, p = 0.036), age (OR 1.17, p = 0.01) and BMI (OR 2.75, p = 0.011). CONCLUSION: In a sample of young adults, circulating levels of fetuin-A correlated with moderate-severe NAFLD, independent of confounders, and with some metabolic parameters. Fetuin-A might be a useful marker to predict NAFLD and metabolic disorders.

Comparative study of testosterone and vitamin D analogue, elocalcitol, on insulin-controlled signal transduction pathway regulation in human skeletal muscle cells.

PURPOSE: Skeletal muscle (Skm) plays a key role in regulating energetic metabolism through glucose homeostasis. Several hormones such as Testosterone (T) and Vitamin D (VD) have been shown to affect energy-dependent cell trafficking by determining Insulin (I)-like effects. AIM: To elucidate possible hormone-related differences on muscular metabolic control, we analyzed and compared the effects of T and elocalcitol (elo), a VD analogue, on the activation of energy-dependent cell trafficking, metabolism-related-signal transduction pathways and transcription of gene downstream targets. METHODS: Human fetal skeletal muscle cells (Hfsmc) treated with T or elo were analyzed for GLUT4 localization, phosphorylation/activation status of AKT, ERK1/2, IRS-1 signaling and c-MYC protein expression. RESULTS: T, similar to elo, induced GLUT4 protein translocation likely in lipid raft microdomains. While both T and elo induced a rapid IRS-1 phosphorylation, the following dynamic in phosphorylation/activation of AKT and ERK1/2 signaling was different. Moreover, T but not elo increased c-MYC protein expression. CONCLUSIONS: All together, our evidence indicates that whether both T and elo are able to affect upstream I-like pathway, they differently determine downstream effects in I-dependent cascade, suggesting diverse physiological roles in mediating I-like response in human skeletal muscle.

The phosphodiesterase 5 inhibitor sildenafil decreases the proinflammatory chemokine IL-8 in diabetic cardiomyopathy: in vivo and in vitro evidence.

PURPOSE: Interleukin (IL)-8 is a proinflammatory C-X-C chemokine involved in inflammation underling cardiac diseases, primary or in comorbid condition, such diabetic cardiomyopathy (DCM). The phosphodiesterase type 5 inhibitor sildenafil can ameliorate cardiac conditions by counteracting inflammation. The study aim is to evaluate the effect of sildenafil on serum IL-8 in DCM subjects vs. placebo, and on IL-8 release in human endothelial cells (Hfaec) and peripheral blood mononuclear cells (PBMC) under inflammatory stimuli. METHODS: IL-8 was quantified: in sera of (30) DCM subjects before (baseline) and after sildenafil (100 mg/day, 3-months) vs. (16) placebo and (15) healthy subjects, by multiplatform array; in supernatants from inflammation-challenged cells after sildenafil (1 µM), by ELISA. RESULTS: Baseline IL-8 was higher in DCM vs. healthy subjects (149.14 ± 46.89 vs. 16.17 ± 5.38 pg/ml, p < 0.01). Sildenafil, not placebo, significantly reduced serum IL-8 (23.7 ± 5.9 pg/ml, p < 0.05 vs. baseline). Receiver operating characteristic (ROC) curve for IL-8 was 0.945 (95% confidence interval of 0.772 to 1.0, p < 0.01), showing good capacity of discriminating the response in terms of drug-induced IL-8 decrease (sensitivity of 0.93, specificity of 0.90). Sildenafil significantly decreased IL-8 protein release by inflammation-induced Hfaec and PBMC and downregulated IL-8 mRNA in PBMC, without affecting cell number or PDE5 expression. CONCLUSION: Sildenafil might be suggested as potential novel pharmacological tool to control DCM progression through IL-8 targeting at systemic and cellular level.

Testosterone-mediated activation of androgenic signalling sustains in vitro the transformed and radioresistant phenotype of rhabdomyosarcoma cell lines.

PURPOSE: Rhabdomyosarcoma (RMS), the most common soft-tissue sarcoma in childhood, rarely affects adults, preferring male. RMS expresses the receptor for androgen (AR) and responds to androgen; however, the molecular action of androgens on RMS is unknown. METHODS: Herein, testosterone (T) effects were tested in embryonal (ERMS) and alveolar (ARMS) RMS cell lines, by performing luciferase reporter assay, RT-PCR, and western blotting experiments. RNA interference experiments or bicalutamide treatment was performed to assess the specific role of AR. Radiation treatment was delivered to characterise the effects of T treatment on RMS intrinsic radioresistance. RESULTS: Our study showed that RMS cells respond to sub-physiological levels of T stimulation, finally promoting AR-dependent genomic and non-genomic effects, such as the transcriptional regulation of several oncogenes, the phosphorylation-mediated post-transductional modifications of AR and the activation of ERK, p38 and AKT signal transduction pathway mediators that, by physically complexing or not with AR, participate in regulating its transcriptional activity and the expression of T-targeted genes. T chronic daily treatment, performed as for the hormone circadian rhythm, did not significantly affect RMS cell growth, but improved RMS clonogenic and radioresistant potential and increased AR mRNA both in ERMS and ARMS. AR protein accumulation was evident in ERMS, this further developing an intrinsic T-independent AR activity. CONCLUSIONS: Our results suggest that androgens sustain and improve RMS transformed and radioresistant phenotype, and therefore, their therapeutic application should be avoided in RMS post puberal patients.

Testosterone insulin-like effects: an in vitro study on the short-term metabolic effects of testosterone in human skeletal muscle cells.

PURPOSE: Testosterone by promoting different metabolic pathways contributes to short-term homeostasis of skeletal muscle, the largest insulin-sensitive tissue and the primary site for insulin-stimulated glucose utilization. Despite evidences indicate a close relationship between testosterone and glucose metabolism, the molecular mechanisms responsible for a possible testosterone-mediated insulin-like effects on skeletal muscle are still unknown. METHODS: Here we used undifferentiated proliferating or differentiated human fetal skeletal muscle cells (Hfsmc) to investigate the short-term effects of testosterone on the insulin-mediated biomolecular metabolic machinery. GLUT4 cell expression, localization and the phosphorylation/activation of AKT, ERK, mTOR and GSK3ß insulin-related pathways at different time points after treatment with testosterone were analyzed. RESULTS: Independently from cells differentiation status, testosterone, with an insulin-like effect, induced Glut4-mRNA expression, GLUT4 protein translocation to the cytoplasmic membrane, while no effect was observed on GLUT4 protein expression levels. Furthermore, testosterone treatment modulated the insulin-dependent signal transduction pathways inducing a rapid and persistent activation of AKT, ERK and mTOR, and a transient inhibition of GSK3ß. T-related effects were shown to be androgen receptor dependent. CONCLUSION: All together our data indicate that testosterone through the activation of non-genomic pathways, participates in skeletal muscle glucose metabolism by inducing insulin-related effects.

Phosphodiesterase type 5 inhibitors: back and forward from cardiac indications.

PDE5 inhibitors (PDE5i) are widely known as treatment for erectile dysfunction (ED). This favorable action has emerged as a "side effect" from pioneering studies when PDE5i have been originally proposed as treatment for coronary artery disease (CAD). PDE5i showed marginal benefits for CAD treatment; although disappointing for that indication, they improved systemic and pulmonary vasodilation and ameliorated general endothelial function. Therefore, PDE5i have been approved and licensed also for pulmonary artery hypertension (PAH), besides ED. Nowadays, fine-tuned biomolecular mechanisms of PDE5i are well recognized to be beneficial onto myocardial contractility and geometry, to reduce tissue fibrosis, hypertrophy and apoptosis. PDE5i consistently exert benefits on heart failure, infarct, cardiomyopathy. The concept that PDE5i likely blunt Th1-driven inflammatory processes, which shift the homeostatic balance from health to disease, has emerged; PDE5i seem to decrease the release of active biomolecules from cells to tissues interested by inflammation. In this view, following clinical and basic research progresses, PDE5i can be undoubtedly "re-allocated" for cardiac indications and, hopefully, they could be approved as therapeutic tools to treat and prevent heart disease. This review aims to summarize PDE5i different clinical applications, from past to present and future, focusing on their potential power as treatment for cardiac diseases.

The endocrine disruptor cadmium alters human osteoblast-like Saos-2 cells homeostasis in vitro by alteration of Wnt/ß-catenin pathway and activation of caspases.

PURPOSE: The pollutant Cadmium (Cd) is widespread in the environment and causes alterations of human health by acting as an endocrine disruptor. Bone tissue seems to be a crucial target of Cd contamination. Indeed, we have previously demonstrated that this endocrine disruptor induces osteoblast apoptosis and necrosis. Thus, aim of this study was to further evaluate the effect of Cd on osteoblasts homeostasis, investigating potential modification of the Wnt/ß-catenin intracellular pathway, the intracellular process involved in programmed cellular death and the cytoskeletal alterations. MATERIAL AND METHODS: To this purpose, human osteoblastic Saos-2 cells, a human osteosarcoma osteoblast-like cell line, were cultured and treated with Cd. RESULTS: Osteoblastic cells were treated for 6 h with 10µM Cd, which induced nuclear translocation of ß-catenin and increased expression of Wnt/ß-catenin target genes. Longer exposure to the same Cd concentration induced osteoblastic cell apoptosis. To better characterize the intracellular events involved in these Cd-induced alterations, we evaluated the effect of Cd exposure on actin filaments and proteins associated to cytoskeletal actin, characterized by the presence of LIM domains. Long (15, 24 h) exposure of osteoblasts to Cd reduced LIM proteins expression and induced actin filaments destruction and a significant caspase-3 activation after 24 h. In addition, to prove that Cd induces osteoblastic cells apoptosis after long exposure, we performed TUNEL assay which demonstrated increase of cell apoptosis after 24 h. CONCLUSION: In conclusion, our study shows that osteoblasts exposed to Cd for short intervals of time demonstrated an increase in cell proliferation through a Wnt/ß-catenin dependent mechanism, likely as a compensatory mechanism in response to cell injury. Longer exposure to the same Cd concentration induced cells apoptosis through cytoskeleton disruption-mediated mechanisms and caspase activation.

The environmental pollutant cadmium induces homeostasis alteration in muscle cells in vitro.

BACKGROUND: Cadmium (Cd) is a heavy metal widely distributed throughout the environment as a result of contamination from a variety of sources. It exerts toxic effects in many tissues but scarce data are present as yet on potential effects on skeletal muscle tissue. AIM: To evaluate the potential alteration induced by Cd in skeletal muscle cells. MATERIALS AND METHODS: C2C12 skeletal muscle cells were treated with Cd at different times of cellular differentiation and gene expression was evaluated. RESULTS: Exposure to Cd decreased significantly p21 mRNA expression and strongly up-regulated cyclin D1 mRNA expression in committed cells and in differentiated myotubes. Moreover, myogenin, fast MyHC-IIb and slow MyHC-I mRNAs expression were also significantly decreased both in committed cells and in myotubes. Moreover, Cd exposure induced a strong increase of Pax3, Pax7 and Myf5 mRNAs expression and stimulated an up-regulation of IL6 and TNF-α proinflammatory cytokines. CONCLUSION: These data lead to hypothesize that environmental Cd exposure might trigger an injury-like event in muscle tissue, possibly by an estrogen receptor-mediated mechanism.

Insulin-like effect of the phosphodiesterase type 5 inhibitor tadalafil onto male human skeletal muscle cells.

BACKGROUND: Phosphodiesterase type 5 inhibitors (PDE5i), widely used to treat male erectile dysfunction, seem to counteract insulin resistance (IR) in animals and humans. IR, primarily manifest in peripheral tissues and particularly in skeletal muscle, is due to impaired insulin signal transduction. Investigators have been focusing onto intracellular defects responsible for IR to identify suitable pharmacological tools targeted toward the specific defects. Albeit some effects of PDE5i have been reported onto animal muscular tissues or cells, whether and how they might affect metabolic processes directly in human skeletal muscle still remains unclear. AIM: We aimed to investigate in human fetal skeletal muscle cells (Hfsmc) the effect of tadalafil, one of PDE5i, onto some intracellular factors involved in response to insulin, such as ras-raf mitogen activated protein kinase (MAPK), phosphatidylinositol 3-kinase/protein kinase B (PKB/Akt), glycogen synthase kinase 3ß (GSK-3ß), and the transcriptional factor c-Myc; proliferation rate; lactate (lact) and free fatty acid (ffa) release; activity of citrate synthase (CS) and succinate dehydrogenase (SDH), both enzymes of Kreb's cycle; PDE5 gene expression. MATERIALS AND METHODS: Western blot analysis, enzyme-linked immunosorbent assay, enzymatic assays, cell count, MTT assay and Real Time PCR were performed in Hfsmc with and without tadalafil. RESULTS: In Hfsmc tadalafil affected the insulin-related intracellular cascade, by increasing MAPK, PKB/Akt, GSK-3ß phosphorylation and c-Myc expression. ffa release and CS activity also significantly increased, with no changes in SDH activity and lact release. CONCLUSIONS: Tadalafil, like insulin, targeted part of the machinery dedicated to energy management and metabolic control in human skeletal muscle cells.

Immunomodulatory effects of BXL-01-0029, a less hypercalcemic vitamin D analogue, in human cardiomyocytes and T cells.

Current immunosuppressive protocols have reduced rejection occurrence in heart transplantation; nevertheless, management of heart transplant recipients is accompanied by major adverse effects, due to drug doses close to toxic range. In allograft rejection, characterized by T-helper 1 (Th1) cell-mediated response, the CXCL10-CXCR3 axis plays a pivotal role in triggering a self-promoting inflammatory loop. Indeed, CXCL10 intragraft production, required for initiation and development of graft failure, supports organ infiltration by Th1 cells. Thus, targeting the CXCL10-CXCR3 axis while avoiding generalized immunosuppression, may be of therapeutic significance. Based on preclinical evidence for immunoregulatory properties of vitamin D receptor agonists, we propose that a less hypercalcemic vitamin D analogue, BXL-01-0029, might have the potential to contribute to rejection management. We investigated the effect of BXL-01-0029 on CXCL10 secretion induced by proinflammatory stimuli, both in human isolated cardiomyocytes (Hfcm) and purified CD4+ T cells. Mycophenolic acid (MPA), the active agent of mycophenolate mofetil, was used for comparison. BXL-01-0029 inhibited IFNgamma and TNFalpha-induced CXCL10 secretion by Hfcm more potently than MPA, impairing cytokine synergy and pathways. BXL-01-0029 reduced also CXCL10 protein secretion and gene expression by CD4+ T cells. Furthermore, BXL-01-0029 did not exert any toxic effect onto both cell types, suggesting its possible use as a dose-reducing agent for conventional immunosuppressive drugs in clinical transplantation.

Elocalcitol inhibits inflammatory responses in human thyroid cells and T cells.

T-helper 1 (Th1) cell-mediated inflammatory responses predominate in the early pathogenesis of Graves' disease (GD), whereas Th2 cell-mediated immunity may play a role in later stages. The chemokine CXCL10 and its receptor CXCR3 are expressed in most thyroid glands of early GD patients. Circulating CXCL10 levels inversely correlate with disease duration; CXCL10 maximal expression also correlates with interferon (IFN)gamma levels in recent GD onset. Methimazole (MMI) reduces CXCL10 secretion by isolated thyrocytes, decreases serum CXCL10 levels, and promotes a transition from Th1 to Th2 dominance in patients in GD active phase. Vitamin D receptor agonists exhibit antiinflammatory properties and promote tolerance induction. We investigated the effects and the mechanism of action of a nonhypercalcemic vitamin D receptor agonist, elocalcitol (BXL-628), compared with MMI on CXCL10 secretion induced by proinflammatory cytokines. Furthermore, we studied the effects of both drugs on Th1, Th17, and Th2 cytokine secretion in CD4+ T cells. ELISA, cytometry, immunocytochemistry, Western blot, and quantitative real-time PCR were used for protein and gene analysis. In human thyrocytes, elocalcitol inhibited IFNgamma and TNFalpha-induced CXCL10 protein secretion more potently than MMI. Elocalcitol impaired both cytokine intracellular pathways, whereas MMI was effective only on the IFNgamma pathway. In CD4+ T cells, elocalcitol decreased Th1- and Th17-type cytokines, and promoted Th2-type cytokine secretion. Elocalcitol and MMI inhibited Th1 cytokine-mediated responses in thyrocytes and CD4+ T cells. In addition, elocalcitol promoted a shift toward a Th2 response. In conclusion, elocalcitol could represent a novel pharmacological tool in the treatment of autoimmune thyroid diseases.

The phosphodiesterase 5 inhibitor tadalafil regulates lipidic homeostasis in human skeletal muscle cell metabolism.

PURPOSE: Tadalafil seems to ameliorate insulin resistance and glucose homeostasis in humans. We have previously reported that tadalafil targets human skeletal muscle cells with an insulin (I)-like effect. We aim to evaluate in human fetal skeletal muscle cells after tadalafil or I: (i) expression profile of I-regulated genes dedicated to cellular energy control, glycolitic activity or microtubule formation/vesicle transport, as GLUT4, PPARγ, HK2, IRS-1, KIF1C, and KIFAP3; (ii) GLUT4, Flotillin-1, and Caveolin-1 localization, all proteins involved in energy-dependent cell trafficking; (iii) activation of I-targeted paths, as IRS-1, PKB/AKT, mTOR, P70/S6K. Free fatty acids intracellular level was measured. Sildenafil or a cGMP synthetic analog were used for comparison; PDE5 and PDE11 gene expression was evaluated in human fetal skeletal muscle cells. METHODS: RTq-PCR, PCR, western blot, free fatty acid assay commercial kit, and lipid stain non-fluorescent assay were used. RESULTS: Tadalafil upregulated I-targeted investigated genes with the same temporal pattern as I (GLUT4, PPARγ, and IRS-1 at 3 h; HK2, KIF1C, KIFAP3 at 12 h), re-localized GLUT4 in cell sites positively immune-decorated for Caveolin-1 and Flotillin-1, suggesting the involvement of lipid rafts, induced specific residue phosphorylation of IRS-1/AKT/mTOR complex in association with free fatty acid de novo synthesis. Sildenafil or GMP analog did not affect GLUT4 trafficking or free fatty acid levels. CONCLUSION: In human fetal skeletal muscle cells tadalafil likely favors energy storage by modulating lipid homeostasis via IRS-1-mediated mechanisms, involving activation of I-targeted genes and intracellular cascade related to metabolic control. Those data provide some biomolecular evidences explaining, in part, tadalafil-induced favorable control of human metabolism shown by clinical studies.

Methimazole inhibits CXC chemokine ligand 10 secretion in human thyrocytes.

CXC chemokine ligand 10 (CXCL10) plays a pivotal role in the self-perpetuation of the inflammatory processes in patients with autoimmune thyroid disease. Treatment with methimazole (MMI) reduces serum CXCL10 in patients with Graves' disease. In isolated human thyrocytes, tumor necrosis factor (TNF)alpha demonstrates a potent synergistic effect on interferon (IFN)gamma-induced CXCL10 secretion. We investigated the mechanism underlying the synergism between IFNgamma and TNFalpha and the effect of MMI on CXCL10 secretion in human thyrocytes. A peroxisome proliferator-activated receptor gamma agonist, rosiglitazone (RGZ), a known inhibitor of T helper 1 (Th1)-mediated responses, was also studied for comparison. Experiments were carried out in human thyrocytes isolated from internodular parenchyma of thyroid tissues derived from patients who had undergone surgery for multinodular goiter. ELISA was used to measure CXCL10 levels in culture supernatant. Flow cytometry was used to assess IFNgamma membrane receptor expression. Specific mRNA analysis was performed by Taqman real-time PCR. Immunofluorescence was performed to detect nuclear translocation of nuclear factor-kappaB (NF-kappaB). In human thyrocytes, the synergistic effect of TNFalpha with IFNgamma on CXCL10 secretion is due to the upregulation of IFNgamma receptor expression. MMI decreased cytokine-induced CXCL10 secretion by reducing TNFalpha-induced upregulation of the IFNgamma receptor. RGZ decreased the cytokine-induced CXCL10 secretion by impairing NF-kappaB translocation, without affecting IFNgamma receptor. MMI and RGZ targeted thyrocytes with the same pharmacological potency, likely acting throughout different mechanisms. Targeting T helper 1-mediated autoimmune thyroid disease with drugs that impair different intracellular pathways could be a novel pharmacological tool.

Biosynthesis of bone sialoprotein by a human osteoclast-like cell line (FLG 29.1).

Biosynthesis of bone sialoprotein (BSP) by a human osteoclastic cell line (FLG 29.1) during its differentiation induced by phorbol 12-myristate 13-acetate (TPA) was studied using metabolic radiolabeling experiments. The FLG 29.1 cells were metabolically radiolabeled with [3H]glucosamine and [35S]sulfate, and the labeled glycoproteins were analyzed by anion exchange chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation experiments. One of the major glycoproteins synthesized by the TPA-treated FLG 29.1 cells was sulfated, had an identical electrophoretic mobility to purified BSP, and could be immunoprecipitated with a specific antibody against human BSP (LF 6). Thus, this glycoprotein was tentatively identified as the BSP. Furthermore, mRNA for BSP was also detected in TPA-treated FLG 29.1 cells by RNA-polymerase chain reaction. Most BSP synthesized by FLG 29.1 cells remained cell-associated, and this is in contrast with those synthesized by osteoblasts, where the protein is rapidly released into the extracellular matrix. Immunocytochemistry using an anti-BSP antibody showed a prominent paranuclear (suggestive of Golgi apparatus) localization of BSP in the TPA-treated FLG 29.1 cells after permeabilization, while untreated cells were not significantly immunostained. Localization of BSP at the plasma membrane was also demonstrated in the TPA-treated FLG 29.1 cells by the fluorescence-activated cell sorting analysis. Since TPA has been demonstrated to induce expression of various osteoclastic characteristics in FLG 29.1 cells, induction of BSP expression by TPA suggests that the protein may play a role during the differentiation process of osteoclasts or in functions of differentiated osteoclasts.

Natriuretic hormone receptors and actions on bone endothelial cells.

[125I]Atrial natriuretic peptide (ANP) was used to identify ANP receptors on a clonal line of bovine bone endothelial (BBE) cells. Specific binding of [125I]ANP was saturable and of high affinity. Computer analysis of the equilibrium binding data indicated that the Scatchard plots are best fit by a straight line (Kd = 69.3 +/- 20.9 pM; binding capacity = 37.9 fmol/10(6) cells). The order of potency for competing with [125I]ANP binding was human ANP (hANP) > rat atriopeptin-1 (rAP-1) > porcine brain natriuretic peptide (pBNP) > porcine C-type natriuretic peptide. Affinity cross-linking studies indicated the presence of two major 130- and 70-kilodalton bands that specifically bound to hANP, rAP-1, pBNP, and porcine C-type natriuretic peptide. The binding of natriuretic peptides to BBE cells resulted in an increase in cGMP production and a significant decrease in Na+/K+/Cl- cotransport, without effects on cAMP intracellular accumulation. hANP, rAP-1, and pBNP at 100-nM concentrations, significantly inhibited PTH-induced cAMP production. Treatment with natriuretic hormones was also associated with an increase in 6-keto-prostaglandin F1 alpha levels in the culture medium of BBE cells and a higher cell growth rate. These studies demonstrate that bone endothelial cells bear receptors for natriuretic hormones associated with changes in PTH-induced cAMP production, prostaglandin production, and cell proliferation.

Inhibition of spontaneous and androgen-induced prostate growth by a nonhypercalcemic calcitriol analog.

We have recently found that analog V (BXL-353, a calcitriol analog) inhibits growth factor (GF)-stimulated human benign prostate hyperplasia (BPH) cell proliferation by disrupting signal transduction, reducing Bcl-2 expression, and inducing apoptosis. We now report that BXL-353 blocks in vitro and in vivo testosterone (T) activity. BPH cells responded to T and dihydrotestosterone (DHT) with dose-dependent growth and reduced apoptosis. Exposure of BPH cells to BXL-353 significantly antagonized both T- and DHT-induced proliferation and induced apoptosis, even in the presence of T. To verify whether BXL-353 reduced prostate growth in vivo, we administered it orally to either intact or castrated rats, supplemented with T enanthate. Nonhypercalcemic doses of BXL-353 time- and dose-dependently reduced the androgen effect on ventral prostate weight, similarly to finasteride. Comparable results were obtained after chronic administration of BXL-353 to intact rats. Clusterin (an atrophy marker) gene and protein were up-regulated by BXL-353 in rat prostate, and nuclear fragmentation was widely present. The antiandrogenic properties of BXL-353 did not interfere with pituitary and testis function, as assessed by serum determination of rat LH and T. BXL-353 did not compete for androgen binding to BPH homogenates and failed to inhibit 5alpha-reductase type 1 and type 2 activities. In conclusion, BXL-353 blocks in vitro and in vivo androgen-stimulated prostate cell growth, probably acting downstream from the androgen receptor, without affecting calcemia or sex hormone secretion. BXL-353 and other vitamin D(3) analogs might thus represent an interesting class of compounds for treating patients with BPH.

Expression and cellular localization of keratinocyte growth factor and its receptor in human hyperplastic prostate tissue.

It is well recognized that the actions of androgens alone do not explain the hyperplastic development of the gland that occurs in elderly men. The increasing number of reports confirming the lack of mitogenic activity of androgens coupled with the powerful mitogenic activity of growth factors and the discovery of growth factor receptors led to an increased interest in the putative role of growth factors in prostate physiopathology. We have previously demonstrated the presence and the cellular localization of epidermal growth factor and of the related peptide, transforming growth factor-alpha, together with their common receptor in the epithelial compartment of the human hyperplastic prostate tissue (BPH). In the present study we examined the expression and cellular localization of messenger ribonucleic acid (RNA) encoding keratinocyte growth factor (KGF) and its receptor in human hyperplastic prostate tissue. RT-PCR of total RNA extracted from BPH tissues documented the presence of transcripts for KGF and its receptor. In situ hybridization with specific RNA probes synthesized from the respective complementary DNA demonstrated that KGF mRNA was mainly localized in the stromal cells, whereas its receptor was mainly localized in the prostate epithelium. Moreover, the mitogenic activity of KGF on cultured BPH cells compared to that of other growth factors has been tested. Our findings clearly indicate that KGF has the ability to function as a potent mitogen in BPH cells. Our data support the hypothesis that KGF plays an important role in prostate growth and that in human prostate it seems to act in a paracrine fashion.

Expression and biological effects of endothelin-1 in human gonadotropin-releasing hormone-secreting neurons.

In a previous report, we demonstrated that in FNC-B4 cells, derived and characterized from a human fetal olfactory epithelium, both sex steroids and odorants regulate GnRH secretion. We now report the presence and biological activity of endothelin (ET)-1 in this GnRH-secreting neuronal cell. By in situ hybridization and immunohistochemistry, we found gene and protein expression of ET-1 and its converting enzyme ECE-1 in both fetal olfactory mucosa and FNC-B4 cells. The presence of authentic ET-1 in the conditioned media of FNC-B4 cells was further supported by combined RIAs and high-performance liquid chromatography studies. Experiments with radiolabeled ET-1 and ET-3 strongly indicated the presence of two classes of binding sites, corresponding to the ETA (16,500 sites/cell) and the ETB receptors (8,700 sites/cell). Functional studies, using selective analogs, indicated that these two classes of receptors subserve distinct functions in human GnRH-secreting cells. The ETA receptor subtype mediated an increase in intracellular calcium and GnRH secretion. Conversely, stimulation of the ETB subtype induced DNA synthesis and mitogen-activated protein kinase p44ERK1 expression. This is the first demonstration, in a human in vitro model, of a neuroendocrine role for ET-1 as regulator of GnRH-secreting neuron activity.

Expression of functional estrogen receptors in human fetal male external genitalia.

It is generally assumed that male genital development is determined by androgens on a default program leading to female genitalia. Female genitalia virilization is due to high levels of androgens, whereas feminization is linked to reduction or lack of fetal androgen. Excess androgen determines sex reversion in female, whereas excess estrogen does not cause male feminization. In the present study, we investigate the presence of androgen receptors (AR) and estrogen receptors (ER) in human fetal penile tissue and in a cellular model of human fetal penile smooth muscle cells (hfPSMC). By immunohistochemistry, we showed the presence of ER and AR in the developing penile tissue of male fetuses. Besides the presence of AR, hfPSMC showed ERalpha/beta as demonstrated by RT-PCR, Western blot, and binding techniques. These receptors are functionally active because cell stimulation with 17beta-estradiol increased progesterone receptor B expression and inhibited hfPSMC growth, both effects being reversed by tamoxifen. Conversely, cell proliferation was stimulated by R1881 and testosterone, an effect enhanced by letrozole. These findings are the first demonstration of the presence of functional ER in differentiating male external genitalia and indicate a possible novel inhibitory role of estrogens in the regulation of the development of these sex structures.