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Leydig cell tumors are usually benign tumors of the male gonad. However, if the tumor is malignant, no effective treatments are currently available. Leydig cell tumors express platelet-derived growth factor (PDGF), kit ligand and their respective receptors, PDGFR and c-kit. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the c-kit and PDGFR tyrosine kinases, on the growth of rodent Leydig tumor cell lines in vivo and in vitro, and examined, in human Leydig cell tumor samples, the expression of activated PDGFR and c-kit and the mutations in exons of the c-kit gene commonly associated with solid tumors. Imatinib caused concentration-dependent decreases in the viability of Leydig tumor cell lines, which coincided with apoptosis and inhibition of proliferation and ligand-stimulated phosphorylation of c-kit and PDGFRs. Mice bearing s.c. allografts of a Leydig tumor cell line treated with imatinib p.o., had an almost complete inhibition of tumor growth, less tumor cell proliferation, increased apoptosis, and a lesser amount of tumor-associated mean vessel density compared with controls. No drug-resistant tumors appeared during imatinib treatment but tumors regrew after drug withdrawal. Human Leydig cell tumors showed an intense expression of the phosphorylated form of c-kit and a less intense expression of phosphorylated PDGFRs. No activating mutations in common regions of mutation of the c-kit gene were found. Our studies suggest that Leydig cell tumors might be a potential target for imatinib therapy.

Supplementary Table 1 from Imatinib Mesylate Inhibits Leydig Cell Tumor Growth: Evidence for In vitro and In vivo Activity

Supplementary Table 2 from Imatinib Mesylate Inhibits Leydig Cell Tumor Growth: Evidence for In vitro and In vivo Activity

Silvia Migliaccio1, Marina Brama1, Nazzarena Malavolta21Dipartimento di Fisiopatologia Medica, Policlinico Umberto I, Università degli Studi Sapienza di Roma, Italy; 2Dipartimento di Medicina Interna, Policlinico S Orsola Malpighi, Bologna, ItalyAbstract: Glucocorticoids (GC)-induced osteoporosis (GIOP) is the most common cause of secondary osteoporosis, which leads to an increased fracture risk in patients. The normal bone turnover depends on a balance between osteoblasts and osteoclasts activity and GC can cause a rapid bone loss, decreasing bone formation and increasing bone resorption. The decreased bone formation is mainly due to the GC-induced apoptosis of both osteoblasts and osteocytes, while the increased bone resorption is due to the increased life-span of pre-existing osteoclasts. Bisphosphonates are clearly effective in preventing and treating GIOP but anabolic therapeutic strategies are the new promising therapeutic alternative. Experimental and clinical studies indicate that teriparatide, the active (1–34) parathyroid hormone (PTH) molecule, is efficacious for the treatment of GIOP, being able to induce an increase in bone mass in these patients. Intermittent administration of human PTH (1–34) stimulates bone formation by increasing osteoblast number. Additionally, human PTH (1–34) modulates the level and/or activity of locally produced growth factors and cytokines. Teriparatide has been demonstrated in several clinical studies to significantly decrease the incidence of fractures in patients affected by GIOP. It has recently received an indication for GIOP and its label indication has also been expanded.Keywords: glucocorticoids, osteoblasts, osteoclasts, osteoporosis, teriparatide

Osteogenesis imperfecta (OI) is a rare inherited connective disorder causing increased bone fragility and low bone mass. OI includes severe bone fragility, impaired dentinogenesis, with less common alterations in the joints, blood vessels, heart valves, skin. Interestingly, description of left ventricular rupture, aortic dissection and heart valves incompetence has been previously described. Death may occur in OI patients for cardiac disease in asyntomatic subjects. Aim of our study has been to evaluate the presence of potential subclinical cardiac disorders and to characterize cardiac functional parameters by echocardiography in adults with OI in absence of cardiac symptoms. Forty patients (21 females and 19 males) affected by type I, III, IV OI and 40 control subjects (20 females and 20 males) were evaluated in the study. Patients and controls underwent clinical examination, screening for endocrine and metabolic disorders, 12-lead electrocardiogram and echocardiogram. In particular, all subjects were evaluated by two-dimensional echocardiography with continuous- and pulse-wave Doppler. Patients and controls belonged to NYHA class I and no significant electrocardiographic alteration was documented in both groups. Thirty-eight patients (95%) showed valvular regurgitation compared to one control subject (2.5%; P0.001). As regards the diastolic function parameters, in OI patients E wave velocity was reduced by 23% (95% CI: 9% to 29%; P0.001), E/A ratio was reduced by 17% (95% CI: 15% to 26%; P0.001) while isovolumetric relaxation time (IRT) was increased by 47% (95% CI: 26% to 53%; P0.001) and E wave deceleration time (DT) was increased by 18% (95% CI: 13% to 26%; P0.001) compared to controls. In conclusion, our data indicate that adult patients affected by OI have an altered diastolic function in absence of other metabolic alterations. These diastolic echocardiographic parameters might worsen over time, especially if other cardiovascular risk factors (e.g., smoking, hypertension, metabolic and endocrine alterations) are not carefully checked, monitored and treated. In the context of a multidisciplinary evaluation of OI patients, our data suggest that a careful cardiological evaluation of these patients is indicated beside skeletal evaluation and therapeutical skeletal options.

Background: Cross-sex hormone treatment in male-to-female (M2F) transsexuals appears reasonably safe. Little is known about its long-term use. The aim of our study was to evaluate the effect of long-term high dose estrogens, plus the antiandrogen cyproterone acetate, on bone composition and on biochemical/hormonal parameters in M2F transsexuals. Methods: A retrospective analysis was performed on 45 young M2Fs (mean age 39.5 years; body mass index (BMI) = 22) receiving estrogens (previously 100 μg ethinyl estradiol, now 2–4 mg oral estradiol valerate/day or 100 μg transdermal estradiol/day) plus the antiandrogen cyproterone acetate 100 mg/day. Data were retrieved from 20 subjects after reassignment surgery (mean hormonal treatment duration 15.6 years). A complete hormonal and biochemical assessment, as well as bone biochemical markers (parathyroid hormone (PTH), calcium, phosphorus, alkaline phosphatase and plasma pyridinoline crosslinks), were evaluated. Bone mineral density (BMD) was measured us...

Titanium has limitations in its clinical performance in dental and orthopaedic applications. This study describes a coating process using pulsed laser deposition (PLD) technology to produce surfaces of titanium carbide (TiC) on titanium substrates and evaluates the biological response both in vitro and in vivo. X-ray photoelectron spectroscopy (XPS) analysis revealed the presence of 18.6-21.5% TiC in the surface layer, accompanied by oxides of titanium 78.5-81.4% in the following concentrations: 11.1-13.0% Ti(2)O(3), 50.8-55.8% TiO(2), 14.5-14.7% TiO. Expression of genes central to osteoblast differentiation (alkaline phosphatase, A2 pro-collagen type 1, osteocalcin, BMP-4, TGFbeta and Cbfa-1) were up-regulated in all cell lines (primary human osteoblasts, hFOB1.19 and ROS.MER#14) grown on TiC compared with uncoated titanium when measured by semiquantitative PCR and real time-PCR, whilst genes involved in modulation of osteoclastogenesis and osteoclast activity (IL-6 and M-CSF) were unchanged. Bone density was shown to be greater around TiC-coated implants after 2 and 4 weeks in sheep and both 4 and 8 weeks in rabbits compared to uncoated titanium. Rapid bone deposition was demonstrated after only 2 weeks in the rabbit model when visualized with intravital staining. It is concluded that coating with TiC will, in comparison to uncoated titanium, improve implant hardness, biocompatibility through surface stability and osseointegration through improved bone growth.

The platelet-derived growth factor (PDGF) family of ligands and receptors play a pivotal role in the development of various organs. The critical importance of the PDGF-mediated signaling during embryonic development and adult physiology of the kidney and the common mesonephric origin of the epididymis and kidney prompted us to investigate the immunohistochemical localization of PDGF A- and B-chain and PDGF receptor (PDGFR) alpha- and beta-subunit in rat and mouse epididymis, the expression profiles of the corresponding mRNAs, and the consequences of a loss-of-function mutation at the PDGF-A, PDGF-B, and PDGFR-beta loci on mouse epididymis phenotypic appearance. Prenatally, PDGF-A and PDGFR-alpha immunohistochemical staining was seen in both species, whereas PDGF-B and PDGFR-beta were absent. The cellular localization of PDGF-A within the epithelium and the alpha-receptor in the mesenchyme in either mouse or rat before birth suggests that the PDGF-A/PDGFR-alpha system might be involved in the epididymal epithelial-mesenchymal interaction during the fetal period of life. Postnatally, PDGF A- and B-ligand and PDGFR alpha- and beta-subunit were confined in the epithelium. The identity of PDGF and PDGFR proteins were further confirmed by immunoblotting. In line with the immunohistochemical studies, PDGF-A and PDGFR-alpha mRNAs were seen by reverse transcription-polymerase chain reaction in rat and mouse tissue before birth, whereas PDGF-B and PDGFR-beta were almost not detectable. During the first days of life, PDGF-B and PDGFR-beta genes started to appear, and the overall trend in mRNA expression throughout postnatal development showed that the transcripts levels for PDGF-A, PDGF-B, PDGFR-beta, and PDGFR-alpha were constant with the only exception of a progressive decrease of PDGFR-alpha in adult rats. The PDGF-A null mutation strongly influenced the epididymal phenotype starting from puberty; only fetal PDGF-B and PDGFR-beta -/- mice were available, and no differences were seen in the epididymis of these animals, compared with wild-type littermates. Taken together, these data indicate that the PDGF system is highly expressed in the epididymis and suggest that PDGF could be involved in the maintenance of morphological structure and functional control of this organ.

Background Nano-hydroxyapatite (nHA) is a potential ideal biomaterial for bone regeneration. However, studies have yet to characterize the behavior of human osteoblasts derived from alveolar bone on nHA. Thus, the aim of the present study was to evaluate the influence of nHA on the adhesion, proliferation and differentiation of these alveolar bone-derived cells. Methods Primary human alveolar osteoblasts were collected from the alveolar ridge of a male periodontal patient during osseous resective surgery and grown on culture plates coated with either polylysine or polylysine with nano-hydroxyapatite (POL/nHA) composite. The cells were grown and observed for 14 days, and then assessed for potential modifications to osteoblasts homeostasis as evaluated by quantitative reverse transcriptase-polymerase chain reaction (real time RT-PCR), scanning electron microscopy and atomic force microscopy. Results Real time PCR revealed a significant increase in the expression of the selected markers of osteoblast differentiation (bone morphogenetic protein (BMP)-2,-5,-7, ALP, COLL-1A2, OC, ON) in cells grown on the POL/nHA substrate. In addition, as compared with the POL surface, cells grown on the POL/nHA substrate demonstrated better osteoconductive properties, as demonstrated by the increase in adhesion and spreading, likely as a result of the increased surface roughness of the composite. Conclusions The increased expression of BMPs and osteoinductive biomarkers suggest that nano-hydroxyapatite may stimulate the proliferation and differentiation of local alveolar osteoblasts and thus encourage bone regeneration at sites of alveolar bone regeneration.

Raloxifene (RAL), a selective estrogen receptor modulator, is indicated for the prevention and treatment of postmenopausal osteoporosis. RAL, by decreasing bone turnover, prevents bone loss and microarchitecture damage, reducing the incidence of osteoporotic fractures. Our previous in vitro data demonstrated that RAL modulates osteoclast activity by, at least in part, an IL-6- and TNF-alpha-dependent mechanism. In this study we evaluated the effects of RAL treatment (60 mg/d) on circulating levels of these cytokines in 14 postmenopausal women with osteoporosis. Lumbar bone density (determined by dual energy x-ray absorptiometry) and IL-6 and TNF-alpha levels were measured before and after 6 and 24 months of therapy. After 24 months, RAL increased bone density. IL-6 and TNF-alpha expression, elevated before treatment, significantly decreased (50% and 30%, respectively) after 6 months. This effect was sustained up to the end of the treatment (75% and 35%, respectively). Thus, our data show that RAL can modulate circulating levels of cytokines involved in osteoclastogenesis and bone resorption, suggesting that modulation of soluble factors could play a pivotal role in the mechanisms of the osteoprotective effect of RAL.

Background and aims: Bone density and quality alterations worsen the ability of osteoporotic bone to support prosthetic implants. The aim of our study was to evaluate potential differences in bone quality and bone turnover markers in aged individuals undergoing surgery for hip fragility fracture or hip osteoarthritis. Methods: Eighteen subjects with hip fragility fractures (Hip Fracture Group), 35 subjects with osteoarthritis of the hip (Hip Osteoarthritis Group) and 19 subjects with normal femoral bone mineral density (Control Group) were evaluated. Serum and urinary bone markers were assayed preoperatively in all surgical patients, and within 48 hours after fracture in the Hip Fracture, Osteoarthritis and Control groups. Histomorphometric analysis was performed on surgical samples. Results: A significant alteration in calcium and PTH serum levels with hyperparathyroidism was observed in the Hip Fracture Group compared with Hip Osteoarthritis and Control Groups. C-Terminal telopeptides of type I-collagen (CTx) and tartrate resistant-acid phosphatase (TRAP), markers of bone resorption, were increased in the Hip Fracture Group compared with both Osteoarthritis and Control Groups (CTx: p

Prolonged tadalafil administration in men with erectile dysfunction is associated with increased testosterone (T): estradiol (E(2)) ratio mainly related to reduction of E(2) levels.To investigate the presence of phosphodiesterase type 5 (PDE5) isoenzyme in primary human visceral adipocytes and whether different PDE5 inhibitors (PDE5i) could directly modulate aromatase (ARO) expression in differentiated human visceral adipocytes in culture.PDE5 mRNA and protein expression in primary human visceral adipocytes as well as mRNA and protein expression of ARO, with functional activity after selective PDE5 blockade by tadalafil and sildenafil.Purified primary human visceral pre-adipocytes were differentiated ex vivo and were exposed to tadalafil or sildenafil (1 µM) for different intervals of time (6-12-24-96 hours). ARO mRNA content and expression were measured by Western Blot and quantitative reverse transcription-polymerase chain reaction (qRT-PCR), respectively. T and E(2) in supernatants were measured by ELISA also in the presence of letrozole.Differentiated adipocytes were found to express detectable levels of PDE5 transcripts. Acute exposure (6 hours) to both PDE5i tadalafil and sildenafil increased ARO mRNA expression by 4.7- and 2.8-fold, respectively (P0.001). ARO mRNA and protein levels were increased by the treatment with PDE5i in a time- and dose-dependent manner. Such effect was mimicked by 8-bromo-cGMP but was lost after 24 and 96 hours; differently, the PDE3B specific inhibitor milrinone (1 µM), displayed no effect. Accordingly, long-term exposure (24 and 96 hours) to PDE5i caused a significant increase in E(2) concentrations in the supernatant (1.7 and 2 fold, respectively; P0.001), with a parallel reduction of T (15% and 30%, respectively; P0.001). Such effect was reversed by the co-incubation with the specific ARO-inhibitor letrozole.Our results demonstrate that PDE5 is expressed in human visceral adipocytes and that acute exposure to PDE5i selectively stimulates ARO expression, which is related to a specific PDE5 blockade. We speculate that modulation of ARO activity by PDE5i could be one of the mechanisms responsible, at least in part, for the beneficial effects of PDE5i on endothelial and metabolic functions.

SUMMARY Background: Obese individuals often present comorbidities while they appear protected against the development of osteoporosis. However, few and contradictory data are now available on skeletal modifications in obese patients. The aim of this study was to characterise bone mineral density (BMD) in overweight (BMI > 25 30) patients. Methods: We selected 398 patients (291 women, 107 men, age 44.1 + 14.2 years, BMI 35.8 + 5.9 kg ⁄m 2 ) who underwent clinical examination, blood tests and examination of body composition. Subjects with chronic conditions or taking medications interfering with bone metabolism, hormonal and nutritional status and recent weight loss were excluded. Results: Interestingly, 37% (n = 146) of this population showed a significantly lower than expected lumbar BMD: 33% (n = 98) of women showed a T-score )1.84 ± 0.71, and 45% (n = 48) of men showed a T-score )1.88 ± 0.64. When the population was divided into subgroups based on different BMI, it was noted that overweight (BMI > 25 30) was associated with a low bone mass, compatible with a diagnosis of osteoporosis. No differences were observed in hormones and lipid profiles among subgroups. Conclusions: Our results indicate that a subpopulation of obese patients has a significant low lumbar BMD than expected for age. Thus, a careful characterisation of skeletal metabolism might be useful in all obese individuals to avoid fragility fractures later in life. What’s known Women with high body mass index seem to be protected against osteoporosis. However, merging data on this issue are conflicting.

Ghrelin (Ghr), the natural ligand of growth hormone secretagogue receptor, is principally produced by the stomach. An interesting aspect in Ghr cardiovascular effects was elicited by the identification of ghrelin and GHS (growth hormone secretagogue) receptor mRNA expression in several cardiovascular tissues and cell types. In man, Ghr administration induced lowering of blood pressure, and decreased plasma levels were reported in several pathological conditions. The present investigation was performed to elucidate ghrelin effect on contraction and proliferation of human aortic smooth muscle cells (HASMC). Ghrelin receptor expression in HASMC was evaluated by RT-PCR, and binding studies were performed to elucidate the receptor kinetics. Ghr effect on angiotensin II-induced HASMC contraction and proliferation was evaluated in vitro. In addition, involvement of cAMP, ERK, and Akt pathways was investigated. PCR documented GHS-R1a expression. Binding of [(125)I-His(9)]-Ghrelin to HASMC was saturable in a dose-dependent manner. Scatchard analysis showed a single class of binding sites (Kd 1.58+/-0.23nM, B(max) 5848+/-291fmol/10(5) cells). In competition binding, (d-Lys(3))-GHRP-6 showed a capacity to compete with [(125)I-His(9)]-Ghrelin with Ki of 4.25nM. Ghrelin was able to inhibit angiotensin II-induced proliferation and contraction in a dose-response fashion via the cAMP/PKA pathway. Our data document that Ghr affects several HASMC functions, opening the way to consider ghrelin as a possible therapeutic target in many pathological conditions associated with vascular damage and remodelling.

Proliferation and migration of gonocytes, the precursors of spermatogonial stem cells, to the germline niche in the basal membrane of the seminiferous tubules, are two crucial events that take place between postnatal d 0.5 (P0.5) and P5.0 in the mouse and involve a selection of the cells that are committed to the germline stem cells lineage. Here we show that from embryonic d 18.0 (E18) and up to P5, the gonocytes express platelet-derived growth factor (PDGF) receptor beta-subtype (PDGFR-beta) and that during the same time period, the Sertoli cells express PDGF-B and PDGF-D, both ligands for PDGFR-beta. Inhibition of the PDGFR-beta tyrosine kinase activity during the first five postnatal days provokes a profound reduction of gonocyte number through inhibition of their proliferation and induction of apoptosis. Moreover, we found that PDGFR-beta ligands are chemotactic for gonocytes. These data suggest that PDGFR-beta activation has the remarkable capability to drive the selection, survival, and migration of the gonocytes from the center of the seminiferous tubules to the testicular germline niche on the basal membrane.

The direct ghrelin (Ghr) involvement in cardiovascular (CV) system homeostasis has been suggested by the expression of its receptor in CV tissues and by evidence that ghrelin mediates CV activities in animals and in humans. Moreover, low Ghr plasma levels have been reported in pathological conditions characterized by high cardiovascular risk. In the present study, we investigated Ghr effect on proliferation of human aortic endothelial cell (HAEC) and involved transduction pathways. Our results indicate that ghrelin elicited proliferation in a dose-dependent manner (EC(50) about of 5nmol/L) in cultured HAEC, and that this effect was inhibited by the receptor antagonist (D-Lys3)-GHRP-6. Western blot experiments documented an activation of external receptor activated kinases (ERK1/2) and Akt in a dose-dependent fashion, as well as involvement of the cAMP pathway in ERK1/2 phosphorylation. Experiments conducted with appropriate pharmacological inhibitors to investigate Ghr-induced HAEC proliferation confirmed the involvement of ERK1/2 and I3P/Akt pathways, as well as the role of AMP cyclase/PKA pathway in ERK1/2 phosphorylation. Our results indicate that Ghr promotes HAEC proliferation, and thus may be a protective factor against vascular damage. The low ghrelin serum levels reported in insulin-resistant states may not be able to effectively counteract endothelial cell injury.

It is well-known that glucocorticoid-induced osteoporosis (GIO) is the most common form of secondary osteoporosis. It is estimated that 30% to 50% of patients receiving chronic glucocorticoid therapy suffer vertebral or hip fractures, which are often asymptomatic. Vertebral fractures occur early after exposure to glucocorticoids, at a time when bone mineral density (BMD) declines rapidly. Glucocorticoids impair osteoblast homeostasis and induce apoptosis of both osteoblasts and osteocytes, leading to suppression of bone formation. Glucocorticoids also favor osteoclastogenesis and, as a consequence, increase bone resorption. The end-point of these alterations is a net decrease in BMD and alterations in bone quality. Bisphosphonates have been approved for both prevention and treatment of GIO. At the present time, anabolic therapeutic agents are still under investigation.

Inhibition of platelet-derived growth factor receptor (PDGFR) signaling restricts the growth of human breast cancer in the bone of nude mice. We hypothesized that osteoblast-secreted substances may alter the response capacity of breast cancer cells to the PDGFRs tyrosine kinase inhibitor imatinib mesylate. We found that osteoblast-conditioned medium (OCM) increases the proliferation rate of the estrogen receptor negative (ER−) MDA-MB-231 and of the ER+ MCF-7 human breast cancer cell lines and the growth-promoting effect on ER+ cells is independent from estrogen. OCM significantly improved the dose- and the time-dependent sensitivity of the tumor cells to the anti-proliferative effect of imatinib. We also found that MDA-MB-231 and MCF-7 cells express the two PDGFRs subtypes, PDGFR-α and PDGFR-β, and OCM treatment increases the expression of the PDGFRs. Furthermore, imatinib inhibited the phosphorylation rate of its target tyrosine kinase receptors. We conclude that bone microenvironment, through osteoblast-secreted substances may cause estrogen-independent proliferation of breast cancer cells by a mechanism mediated by the induction of PDGFRs expression. The enhanced sensitivity of OCM-treated breast cancer cells to imatinib would justify investigation on the efficacy of imatinib in bone breast cancer metastasis.

Breast cancer (BC) is linked to estrogen exposure. Estradiol (E2) stimulates BC cells proliferation by binding the estrogen receptor (ER). Hormone-related cancers have been linked to estrogenic environmental contaminants. Cadmium (Cd) a toxic pollutant, acts as estrogens in BC cells. Purpose of our study was to evaluate whether Cd regulates MCF-7 cell proliferation by activating ERK1/2, Akt and PDGFRalpha kinases. Cd increased cell proliferation and the ER-antagonist ICI 182,780 blunted it. To characterize an ER-dependent mechanism, ERalpha/beta expression was evaluated. Cd decreased ERalpha expression, but not ERbeta. Cd also increased ERK1/2, Akt and PDGFRalpha phosphorylation while ICI blocked it. Since stimulation of phosphorylation was slower than expected, c-fos and c-jun proto-oncogenes, and PDGFA were analyzed. Cd rapidly increased c-jun, c-fos and PDGFA expression. Cells were also co-incubated with the Cd and specific kinases inhibitors, which blocked the Cd-stimulated proliferation. In conclusion, our results indicate that Cd increases BC cell proliferation in vitro by stimulating Akt, ERK1/2 and PDGFRalpha kinases activity likely by activating c-fos, c-jun and PDGFA by an ERalpha-dependent mechanism.

Leydig cell tumors are usually benign tumors of the male gonad. However, if the tumor is malignant, no effective treatments are currently available. Leydig cell tumors express platelet-derived growth factor (PDGF), kit ligand and their respective receptors, PDGFR and c-kit. We therefore evaluated the effects of imatinib mesylate (imatinib), a selective inhibitor of the c-kit and PDGFR tyrosine kinases, on the growth of rodent Leydig tumor cell lines in vivo and in vitro, and examined, in human Leydig cell tumor samples, the expression of activated PDGFR and c-kit and the mutations in exons of the c-kit gene commonly associated with solid tumors. Imatinib caused concentration-dependent decreases in the viability of Leydig tumor cell lines, which coincided with apoptosis and inhibition of proliferation and ligand-stimulated phosphorylation of c-kit and PDGFRs. Mice bearing s.c. allografts of a Leydig tumor cell line treated with imatinib p.o., had an almost complete inhibition of tumor growth, less tumor cell proliferation, increased apoptosis, and a lesser amount of tumor-associated mean vessel density compared with controls. No drug-resistant tumors appeared during imatinib treatment but tumors regrew after drug withdrawal. Human Leydig cell tumors showed an intense expression of the phosphorylated form of c-kit and a less intense expression of phosphorylated PDGFRs. No activating mutations in common regions of mutation of the c-kit gene were found. Our studies suggest that Leydig cell tumors might be a potential target for imatinib therapy.

In cultured osteoblasts, protein kinase C (PKC) activity increases and estrogen receptor alpha (ERalpha) binding capacity decreases upon confluence. We investigated potential interactions between ERalpha and PKC isoforms and their confluence-induced modulations in clonal ROS.SMER#14 cells and primary osteoblasts. In sub-confluent ROS.SMER#14 cells, which express an exogenous plus small amounts of the endogenous ERalpha gene, the receptor appeared as two main bands of approximately 66 and approximately 46 kDa. In over-confluent, more differentiated cells, the cytosolic approximately 66 kDa ERalpha appeared decreased and the approximately 46 kDa variant increased. Enhanced expression and/or membrane translocation of PKCalpha and PKCepsilon, but not PKCzeta, was evidenced at over-confluence, along with transient increases in expression and kinase activity of c-Src, accompanied by membrane translocation of the kinase-activated enzyme. In contrast, negligible membrane translocation of PKCalpha and/or activated c-Src was observed in parental ROS 17/2.8 cells, which express low levels of full-length ERalpha. PKCalpha from over-confluent cells phosphorylated p60c-Src in vitro, suggesting functional interaction between the two kinases. ERalpha co-immunoprecipitated c-Src and PKCalpha, mostly in its cleaved form (PKMalpha). An analogous interaction was observed in primary osteoblasts. However, in these cells, much more PKCalpha/PKMalpha was ERalpha-co-immunoprecipitated at over-confluence, a condition in which the shorter, approximately 46 kDa ERalpha variant is increased. This interaction was enhanced by estradiol treatment or PKC down-regulation, but was unaffected by c-Src inhibition. These data highlight direct PKCalpha-c-Src-ERalpha interactions, which may be crucial in the modulation of estrogen responsiveness and the differentiation process in osteoblasts.

Raloxifene is a selective estrogen receptor modulator (SERM) that prevents bone loss. Although it is largely used for the treatment of osteoporosis, the mechanisms by which this compound modulates the activity of bone cells are still poorly understood. In this study we investigate whether raloxifene affects osteoclast and osteoblast activity in vitro. Bone marrow cultures were established from neonatal mice and treated with 1,25(OH)(2) vitamin D(3) (VitD(3), 10(-8) mol/L) to induce osteoclast generation. Similar to 17beta-estradiol, raloxifene significantly reduced the number of osteoclasts in a concentration-dependent manner, with maximal inhibition at 10(-11) mol/L (-48%). However, as for 17beta-estradiol, at a high concentration (10(-7) mol/L), the inhibitory effect of raloxifene was abolished. In a pit assay, raloxifene inhibited bone resorption. A maximal effect was observed at 10(-9) mol/L, and maintained at a high concentration, indicating that inhibition of osteoclast formation and inhibition of bone resorption may be due to activation of, at least in part, different pathways. Osteoblasts from neonatal mice calvariae were also exposed to raloxifene. In these cells, this compound induced a concentration-dependent increase of proliferation, which was blocked by the estrogen-receptor antagonist ICI 164,384. Raloxifene also increased the osteoblast-specific transcription factor Cbfa1/Runx2 and alpha2 procollagen type I chain mRNAs, with a pattern that only partially coincided with that of 17beta-estradiol. Consistent with decreased osteoclastogenesis, raloxifene inhibited the mRNA expression of interleukin (IL)-1beta and IL-6 at a low concentration, but not at a high concentration, whereas 17beta-estradiol had similar effects on IL-6 and inhibited IL-1beta at both concentrations. Furthermore, both compounds were able to inhibit tumor necrosis factor (TNF)-alpha-induced IL-1beta, but not IL-6, increase. In conclusion, these data show that raloxifene negatively modulates osteoclasts, and positively affects osteoblasts, suggesting not only an antiresorptive role, but also an osteoblast stimulatory role.