Ryutaro Yamanaka

Simvastatin inhibits osteoclast differentiation induced by bone morphogenetic protein-2 and RANKL through regulating MAPK, AKT and Src signaling

The mevalonate pathway plays a crucial role in bone metabolism. Here we examined roles of simvastatin in osteoclast function and differentiation induced by RANKL and BMP-2 using mouse macrophage-like MLC-6 cells and human osteoclast precursor cells. MLC-6 cells expressed BMP type-I and -II receptors and Smads as well as osteoclast markers including TRAP, RANK, cathepsin-K, M-CSF receptor, MMP-9 and calcitonin receptor. Treatment with RANKL and BMP-2 acted synergistically to stimulate RANK, TRAP and cathepsin-K expression in MLC-6 cells. Simvastatin suppressed osteoclastic activity shown by increases in RANK, TRAP and cathepsin-K expression induced by RANKL and BMP-2. In contrast simvastatin alone had no effects on the osteoclastic markers in MLC-6 cells. Simvastatin activated ERK, SAPK/JNK and AKT pathways and inactivated Ras in MLC-6 cells. Simvastatin had no effect on BMP-induced Smad1/5/8 phosphorylation regardless of RANKL stimulation. Since chemical inhibition of ERK, SAPK/JNK and AKT increased TRAP and cathepsin-K expression induced by BMP-2 and RANKL, these pathways are functionally involved in inhibition of osteoclastic activity. In addition, Src phosphorylation induced by RANKL, which is involved in osteoclast differentiation, was suppressed by simvastatin. We further confirmed an inhibitory mechanism of simvastatin on osteoclast differentiation using human osteoclast precursor cells which express BMP receptor and Smad signaling machinery. Simvastatin also activated ERK pathways and inactivated Src phosphorylation in human osteoclasts differentiated by M-CSF and RANKL treatments. The inhibition of TRAP and RANK expression by simvastatin was reversed by ERK inhibition, whereas Src inhibitor enhanced simvastatin-induced suppression of osteoclast markers. Collectively, our data show that simvastatin inhibits osteoclastic differentiation through inhibiting Src as well as enhancing MAPK/AKT pathways.

Estrogen and glucocorticoid regulate osteoblast differentiation through the interaction of bone morphogenetic protein-2 and tumor necrosis factor-α in C2C12 cells

Imbalanced functions between osteoclasts and osteoblasts are involved in inflammatory bone damage. The clinical effectiveness of blocking TNF-alpha in treatment of active rheumatoid arthritis established the significance of TNF-alpha in the pathogenesis. In the present study, we investigated the cellular mechanism by which estrogen and glucocorticoid interact in osteoblastic differentiation regulated by BMP and TNF-alpha using mouse myoblastic C2C12 cells. The expression of estrogen receptors, (ER)alpha and ERbeta, and glucocorticoid receptor (GCR) was significantly increased by BMP-2 treatment regardless of the presence of estradiol and dexamethasone. Estradiol, but not dexamethasone, enhanced BMP-induced Runx2 and osteocalcin expression in C2C12 cells. In addition, TNF-alpha suppressed BMP-2-induced Runx2 and osteocalcin expression, and estradiol and dexamethasone reversed the TNF-alpha effects on BMP-2-induced Runx2 expression. Dexamethasone also abolished osteocalcin expression induced by BMP-2. Interestingly, BMP-2-induced Smad1/5/8 phosphorylation and Id-1 promoter activity were enhanced by estradiol pretreatment. On the other hand, dexamethasone suppressed BMP-2-induced Smad1/5/8 activation. TNF-alpha-induced SAPK/JNK activity was suppressed by estradiol, while NFkappaB phosphorylation was inhibited by dexamethasone. Of note, the inhibitory effects of TNF- on BMP-2-induced Runx2 and osteocalcin expression were reversed by SAPK/JNK inhibition regardless of the presence of estradiol. The estradiol effects that enhance BMP-2-induced Runx2 and osteocalcin mRNA expression were restored by antagonizing ER, and moreover, membrane-impermeable estradiol-BSA failed to enhance the BMP-2-induced osteoblastic differentiation. Thus, estrogen and glucocorticoid are functionally involved in the process of osteoblast differentiation regulated by BMPs and TNF-alpha. BMP-2 increases the sensitivities of ERs and GCR, whereas estrogen and glucocorticoid differentially regulate BMP-Smad and TNF-alpha signaling.

Pro-apoptotic effects of imatinib on PDGF-stimulated pulmonary artery smooth muscle cells from patients with idiopathic pulmonary arterial hypertension☆

BACKGROUND Remodeling of the pulmonary artery by an inappropriate increase of pulmonary artery smooth muscle cells (PASMCs) is problematic in the treatment of idiopathic pulmonary arterial hypertension (IPAH). Effective treatment that achieves reverse remodeling is required. The aim of this study was to assess the pro-apoptotic effects of imatinib, a platelet-derived growth factor (PDGF)-receptor tyrosine kinase inhibitor, on PASMCs obtained from patients with IPAH. METHODS PASMCs were obtained from 8 patients with IPAH undergoing lung transplantation. Cellular proliferation was assessed by (3)H-thymidine incorporation. Pro-apoptotic effects of imatinib were examined using TUNEL and caspase-3,7 assays and using transmission electron microscopy. RESULTS Treatment with imatinib (0.1 to 10 μg/mL) significantly inhibited PDGF-BB (10 ng/mL)-induced proliferation of PASMCs from IPAH patients. Imatinib (1 μg/mL) did not induce apoptosis in quiescent IPAH-PASMCs, but it had a pro-apoptotic effect on IPAH-PASMCs stimulated with PDGF-BB. Imatinib did not induce apoptosis in normal control PASMCs with or without PDGF-BB stimulation. PDGF-BB induced phosphorylation of Akt at 15 min, and Akt phosphorylation was inhibited by imatinib in IPAH-PASMCs. Akt-I-1/2 (1 μmol/L), an Akt inhibitor, in the presence of PDGF-BB significantly increased apoptotic cells compared with the control condition. Thus, Akt-I-1/2 could mimic the effects of imatinib on PASMCs. CONCLUSION Imatinib has anti-proliferative and pro-apoptotic effects on IPAH-PASMCs stimulated with PDGF. The inhibitory effect of imatinib on Akt phosphorylation induced by PDGF plays an important role in the pro-apoptotic effect.

Effects of bone morphogenetic protein (BMP) on adrenocorticotropin production by pituitary corticotrope cells: Involvement of up-regulation of bmp receptor signaling by somatostatin analogs

The mechanism by which somatostatin analogs suppress ACTH production by corticotropinomas has yet to be fully elucidated. We here studied the effects of somatostatin analogs on ACTH secretion using mouse corticotrope AtT20 cells focusing on the biological activity of bone morphogenetic proteins (BMPs). BMP ligands, receptors and Smads, and somatostatin receptors (SSTRs)-2, -3, and -5 were expressed in AtT20 cells. BMP-2, -4, -6, and -7 decreased basal ACTH production with BMP-4 effects being the most prominent. BMP-4 also inhibited CRH-induced ACTH production and proopiomelanocortin (POMC) transcription. However, the decrease in CRH-induced cAMP accumulation caused by BMP-4 was not sufficient to completely account for BMP-4 actions, indicating that ACTH suppression by BMPs was not directly linked to cAMP inhibition. CRH-activated ERK1/ERK2, p38-MAPK, stress-activated protein kinase/c-Jun NH(2)-terminal kinase, protein kinase C, and Akt pathways and CRH-induced ACTH synthesis was significantly decreased in the presence of U0126 or SB203580. Because BMPs attenuated CRH-induced ERK and p38 phosphorylation, it was suggested that BMP-4 suppresses ACTH production by inhibiting CRH-induced ERK and p38 phosphorylation. Somatostatin analogs octreotide and pasireotide (SOM230) significantly suppressed CRH-induced ACTH and cAMP production in AtT20 cells and reduced ERK and p38 phosphorylation. Notably, CRH-induced ACTH production was enhanced in the presence of noggin, a BMP-binding protein. The inhibitory effects of octreotide and SOM230 on CRH-induced ACTH production were also attenuated by noggin, implying that the endogenous BMP system plays a key role in inhibiting CRH-induced ACTH production by AtT20 cells. The findings that OCT and SOM230 up-regulated BMP-Smad1/Smad5/Smad8 signaling and ALK-3 and BMPRII and down-regulated inhibitory Smad6/7 establish that the activation of endogenous BMP system is functionally involved in the mechanism by which somatostatin analogs suppress CRH-induced ACTH production.

Involvement of the bone morphogenetic protein system in endothelin-and aldosterone-induced cell proliferation of pulmonary arterial smooth muscle cells isolated from human patients with pulmonary arterial hypertension

Recent genetic studies have uncovered a link between familial and idiopathic pulmonary arterial hypertension (PAH) and germline mutations in the bone morphogenetic protein type-II receptor (BMPRII). The pathology of PAH is characterized by remodeling of the pulmonary arteries due to pulmonary artery smooth muscle cell (PASMC) hyperproliferation. Although increased endothelial injury and impaired suppression of PASMC proliferation are both critical for the cellular pathogenesis of PAH, a detailed molecular mechanism underlying PAH has yet to be elucidated. In the present study, we investigated the roles of the BMP system and other vasoactive factors associated with PAH (including endothelin (ET), angiotensin II (Ang II) and aldosterone) in the mitotic actions of PASMCs isolated from idiopathic and secondary PAH lungs. ET1 and aldosterone stimulated PASMC proliferation of idiopathic PAH more effectively than secondary PAH, whereas Ang II and ET3 failed to activate mitosis in either of the PASMC cell type. The effects of ET1 and aldosterone were blocked by bosentan, an ET type-A/B receptor (ETA/BR) antagonist, and eplerenone, a selective mineralocorticoid receptor (MR) blocker, respectively. Among the BMP ligands examined, BMP-2 and BMP-7, but not BMP-4 or BMP-6, significantly increased cell mitosis in both PASMC cell types. Notably, ET1- and aldosterone-induced mitosis and mitogen-activated protein kinase phosphorylation were significantly increased in the presence of BMP-2 and BMP-7 in PASMCs isolated from idiopathic PAH, although additive effects were not observed in PASMCs isolated from secondary PAH. Inhibition of extracellular signal-regulated kinase 1 (ERK1)/ERK2 signaling suppressed basal-, ET1- and aldosterone-induced PASMC mitosis more potently than that of stress-activated protein kinase/c-Jun NH2-terminal kinase inhibition. Given the fact that BMP-2 and BMP-7 upregulated ETA/BR and MR expression and that BMP-2 decreased 11βHSD2 (11β-hydroxysteroid dehydrogenase type 2) levels in PASMCs isolated from idiopathic PAH, BMPR-Smad signaling may have a key role in amplifying the ETA/BR and/or MR-ERK signaling in PASMCs of the PAH lung. Collectively, the functional link between BMP and ET and/or the MR system may be involved in the progress of PASMC mitosis, ultimately leading to the development of clinical PAH.

A Novel Antagonistic Effect of the Bone Morphogenetic Protein System on Prolactin Actions in Regulating Steroidogenesis by Granulosa Cells

To investigate the mechanism by which prolactin (PRL) regulates follicular steroidogenesis in the ovary, we examined the functional roles of PRL in steroidogenesis using rat oocyte/granulosa cell coculture and focusing on the bone morphogenetic protein (BMP) system. The expression of long and short forms of PRL receptor (PRLR) were detected in both oocytes and granulosa cells, and PRL effectively up-regulated PRLR expression in granulosa cells in the presence of FSH. PRL suppressed FSH-induced estradiol production and increased FSH-induced progesterone production in granulosa cells. The PRL effects on FSH-induced progesterone were blocked by coculture with oocytes, implying roles of oocyte-derived factors in suppression of progesterone production in PRL-exposed granulosa cells. In accordance with the data for steroids, FSH-induced aromatase expression was suppressed by PRL, whereas FSH-induced steroidogenic acute regulatory protein, P450scc (P450 side-chain cleavage enzyme), and 3β-hydroxysteroid dehydrogenase type 2 levels were amplified by PRL. However, forskolin- and N(6),O(2)-dibutyryl cAMP-induced steroid levels and FSH- and forskolin-induced cAMP were not affected by PRL, suggesting that PRL action on FSH-induced steroidogenesis was not due to cAMP-protein kinase A regulation. Treatment with a BMP-binding protein, noggin, facilitated PRL-induced estradiol reduction, and noggin increased PRL-induced progesterone production in FSH-treated granulosa cells cocultured with oocytes, suggesting that endogenous BMPs reduce progesterone but increase estradiol when exposed to high concentrations of PRL. PRL increased the expression of BMP ligands in oocyte/granulosa cell coculture and augmented BMP-induced phosphorylated mothers against decapentaplegic 1/5/8 signaling by reducing inhibitory phosphorylated mothers against decapentaplegic 6 expression through the Janus kinase/signal transducer and activator of transcription (STAT) pathway. In addition to STAT activation, PRL enhanced FSH-induced MAPK phosphorylation in granulosa cells, in which ERK activation was preferentially involved in suppression of FSH-induced estradiol. Furthermore, noggin treatment enhanced PRLR signaling including MAPK and STAT. Considering that BMPs suppressed PRLR in granulosa cells, it is likely that the BMP system in growing follicles plays a key role in antagonizing PRLR signaling actions in the ovary exposed to high concentrations of PRL.

Inhibitory Effects of Simvastatin on Platelet-derived Growth Factor Signaling in Pulmonary Artery Smooth Muscle Cells From Patients With Idiopathic Pulmonary Arterial Hypertension

Idiopathic pulmonary arterial hypertension (IPAH) is a progressive disease characterized by inappropriate increase of pulmonary artery smooth muscle cells (PASMCs) leading to occlusion of pulmonary arterioles. Inhibition of platelet-derived growth factor (PDGF) signaling is starting to garner attention as a targeted therapy for IPAH. We assessed the inhibitory effects of simvastatin, a 3-hydroxy-3-methylglutanyl coenzyme A reductase inhibitor, on PDGF-induced proliferation and migration of PASMCs obtained from 6 patients with IPAH who underwent lung transplantation. PDGF stimulation caused a significantly higher growth rate of PASMCs from patients with IPAH than that of normal control PASMCs as assessed by 3H-thymidine incorporation. Simvastatin (0.1 μmol/L) significantly inhibited PDGF-induced cell proliferation of PASMCs from patients with IPAH but did not inhibit proliferation of normal control cells at the same concentration. Western blot analysis revealed that simvastatin significantly increased the expression of cell cycle inhibitor p27. PDGF significantly increased the migration distance of IPAH-PASMCs compared with that of normal PASMCs, and simvastatin (1 μmol/L) significantly inhibited PDGF-induced migration. Immunofluorescence staining revealed that simvastatin (1 μmol/L) inhibited translocation of Rho A from the cytoplasm to membrane and disorganized actin fibers in PASMCs from patients with IPAH. In conclusion, simvastatin had inhibitory effects on inappropriate PDGF signaling in PASMCs from patients with IPAH.

The efficacy of add-on tacrolimus for minor flare in patients with systemic lupus erythematosus: a retrospective study

Objective We have assessed the effectiveness of tacrolimus for minor flares in systemic lupus erythematosus (SLE) patients. Methods The medical records of 313 patients were retrospectively reviewed over a period of seven years, from 2006 to 2013. We enrolled patients with minor flare treated with add-on tacrolimus, without glucocorticoid (GC) intensification (tacrolimus group). Minor flare was defined as a ≥1-point increase in a total score between 3 and 11 in the SLE Disease Activity Index (SLEDAI). We enrolled as controls patients who were administered increased doses of GC for minor flare (GC group). All patients were followed for one year. The primary outcome measure was the proportion of responders. Results There were 14 eligible patients in the tacrolimus group and 20 eligible patients in the GC group. The mean SLEDAI at flare tended to be higher in the tacrolimus group than in the GC group (7.5 vs. 6.2, p = 0.085). A mean dose of 1.6 mg tacrolimus/day was administered for flare, while the mean GC dose was 13.7 mg/day in the GC group. The proportion of responders was 86% (12/14) in the tacrolimus group and 75% (15/20) in the GC group (p = 0.67). The mean dose of GC at 12 months was higher in the GC group than in the tacrolimus group (9.7 mg/day vs. 7.1 mg/day, p < 0.05). Only one patient discontinued tacrolimus because of fatigue after three months. Conclusion Adding tacrolimus without increasing the GC dose may provide an effective treatment option for minor flares in patients with SLE.

Unexpected occurrence of adrenal Cushing’s syndrome in a patient with systemic lupus erythematosus

Unexpected occurrence of adrenal Cushing’s syndrome in a patient with systemic lupus erythematosus