Tomoko Miyoshi

Simvastatin antagonizes tumor necrosis factor-α inhibition of bone morphogenetic proteins-2-induced osteoblast differentiation by regulating Smad signaling and Ras/Rho-mitogen-activated protein kinase pathway

Recent studies have shown that the mevalonate pathway plays an important role in skeletal metabolism. Statins stimulate bone morphogenetic proteins-2 (BMP-2) production in osteoblasts, implicating a possible beneficial role for statins in promoting anabolic effects on bone. Here, we investigated the effects of a lipophilic simvastatin on osteoblast differentiation using mouse myoblast C2C12 cells, in the presence of tumor necrosis factor-alpha (TNF-alpha), an inflammatory cytokine that inhibits osteogenesis. The addition of TNF-alpha to C2C12 cells suppressed the BMP-2-induced expression of key osteoblastic markers including Runx2 and alkaline phosphatase (ALP) activity. Simvastatin had no independent effects on Runx2 and alkaline phosphatase activity; however, it reversed the suppressive effects of TNF-alpha. The ability of simvastatin to reverse TNF-alpha inhibition of BMP-induced Smad1,5,8 phosphorylation and Id-1 promoter activity suggests the involvement of Smad signaling pathway in simvastatin action. In addition, cDNA array analysis revealed that simvastatin increased expression levels of Smads in C2C12 cells exposed to TNF-alpha that also activated mitogen-activated protein kinase (MAPK) signaling pathways, including extracellular signal-regulated kinase 1/2 (ERK1/2), P38, and stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK). Simvastatin potently suppressed TNF-alpha-induced phosphorylation of ERK1/2 and SAPK/JNK by inhibiting TNF-alpha-induced membrane localization of Ras and RhoA. Farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) reversed the simvastatin effects on TNF-alpha-induced activation of Ras/Rho/MAPK pathways. FPP and GGPP also restored the simvastatin effects on TNF-alpha-induced suppression of Runx2 and ALP activity. In addition, simvastatin decreased the expression levels of TNF type-1 and -2 receptor mRNAs. Collectively, simvastatin supports BMP-induced osteoblast differentiation through antagonizing TNF-alpha-to-Ras/Rho/MAPK pathway and augmenting BMP-Smad signaling, suggesting a potential usage of statins to ameliorate inflammatory bone damage.

Mutual Regulation of Follicle-Stimulating Hormone Signaling and Bone Morphogenetic Protein System in Human Granulosa Cells

Abstract Bone morphogenetic proteins (BMPs) play critical roles in folliculogenesis by modulating the actions of follicle-stimulating hormone (FSH) in the ovary. However, the effects of FSH on the BMP system remain unknown. Here, we have investigated the effects of FSH on BMP signaling using the human granulosa-like tumor cell line KGN. KGN cells express BMP type I and type II receptors and the BMP signaling molecules SMADs. FSH administration upregulated BMP type IA (BMPR1A) and IB (BMPR1B) receptors, activin type II receptor (ACVR2), and BMP type II receptor (BMPR2). FSH also augmented SMAD1 and SMAD5 expression, and conversely, FSH suppressed the expression of the inhibitory SMADs, SMAD6 and SMAD7. Bioassays revealed that FSH enhances BMP-induced SMAD1/5/8 phosphorylation and cellular DNA synthesis induced by BMP6 and BMP7. Since overexpression of BMPR1A and BMPR1B, but not SMADs, significantly enhanced the BMP responses, these type I receptors were revealed to be limiting factors for BMP signaling in KGN cells. BMPs significantly suppressed progesterone synthesis induced by forskolin and dibutyryl-cAMP (BtcAMP) but had no effect on estradiol induced by the same factors. KGN cAMP levels induced by forskolin were not altered by BMPs, suggesting that BMPs regulate steroidogenesis at a level downstream of cAMP synthesis in KGN cells. In this regard, BMPs specifically reduced the STAR transcription, whereas the levels of CYP11A, HSD3B2, and CYP19 stimulated by forskolin as well as BtcAMP were not altered. Collectively, the two major factors, FSH-cAMP pathway and BMP system, are reciprocally and functionally linked. Given that BMPs downregulate FSH receptors in KGN cells, this interaction may contribute to fine-tuning of the mutual sensitivity toward BMP ligands and FSH.

Bone morphogenetic protein 6 (BMP6) and BMP7 inhibit estrogen-induced proliferation of breast cancer cells by suppressing p38 mitogen-activated protein kinase activation

Estrogen is involved in the development and progression of breast cancer. Here, we investigated the effects of bone morphogenetic proteins (BMPs) on breast cancer cell proliferation caused by estrogen using human breast cancer MCF-7 cells. MCF-7 cells express estrogen receptors (ESR1 and ESR2), BMP receptors, and SMAD signaling molecules. Estradiol and membrane-impermeable estradiol stimulated MCF-7 cell proliferation. Estradiol also reduced mRNA levels of ESR1, aromatase, and steroid sulfatase. Treatment with BMPs and activin had no effects on MCF-7 cell proliferation. However, BMP2, BMP4, BMP6, BMP7, and activin suppressed estradiol-induced cell mitosis, with the effects of BMP6, BMP7, and activin being more prominent than those of BMP2 and BMP4. Activin decreased ESR1 mRNA expression, while BMP6 and BMP7 impaired steroid sulfatase expression in MCF-7 cells. Interestingly, SMAD1,5,8 activation elicited by BMP6 and BMP7, but not by BMP2 and BMP4, was preserved even under the exposure of a high concentration of estradiol. The difference of BMP responsiveness was likely due to the differential modulation of BMP receptor expression induced by estradiol. In this regard, estradiol decreased the expression levels of BMPR1A, BMPR1B, ACVR2A, and ACVR2B but did not affect ACVR1 and BMPRII, leading to the sustained effects of BMP6 and BMP7 in estrogen-treated MCF-7 cells. Estradiol rapidly activated MAPK phosphorylation including extracellular signal-regulated kinase 1/2, p38, and stress-activated protein kinase/c-Jun NH2-terminal kinase pathways and BMP6, BMP7, and activin preferentially inhibited estradiol-induced p38 phosphorylation. SB203580, a selective p38 MAPK inhibitor effectively suppressed estradiol-induced cell mitosis, suggesting that p38 MAPK plays a key role in estrogen-sensitive breast cancer cell proliferation. Thus, a novel interrelationship between estrogen and the breast cancer BMP system was uncovered, in which inhibitory effects of BMP6 and BMP7 on p38 signaling and steroid sulfatase expression were functionally involved in the suppression of estrogen-induced mitosis of breast cancer cells.

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.

p38-Mitogen-Activated Protein Kinase Stimulated Steroidogenesis in Granulosa Cell-Oocyte Cocultures: Role of Bone Morphogenetic Proteins 2 and 4

Roles of the p38-MAPK pathway in steroidogenesis were investigated using coculture of rat granulosa cells with oocytes. Activin and FSH readily phosphorylated p38 in granulosa cells. Activin effect on p38 phosphorylation was abolished by a selective activin receptor-like kinase-4, -5, and -7 inhibitor, SB431542. SB431542 decreased FSH-induced estradiol but had no effect on progesterone production with a marginal cAMP reduction, suggesting that endogenous activin is primarily involved in estradiol synthesis. FSH-induced p38 activation was not affected either by SB431542 or follistatin, suggesting that FSH activates p38 not through the endogenous activin. Bone morphogenetic protein (BMP)-2 and BMP-4 also enhanced FSH-induced p38 phosphorylation, which was augmented by oocyte action. A specific p38 inhibitor, SB203580, decreased FSH-induced estradiol production. However, FSH-induced cAMP accumulation was not changed by SB203580, suggesting that p38 activation is linked to estradiol synthesis independently of cAMP. BMP-2 and BMP-4 inhibited FSH- and forskolin (FSK)-induced progesterone and cAMP synthesis regardless of oocyte action. BMP-2, BMP-4, and activin increased FSH-induced estradiol production, which was enhanced in the presence of oocytes. In contrast to activin that enhanced FSK-induced estradiol, BMP-2 and BMP-4 had no effects on FSK-induced estradiol production, suggesting that BMP-2 and BMP-4 directly activate FSH-receptor signaling. Given that activin increased, but BMP-2 and BMP-4 decreased, FSH-induced cAMP, the effects of BMP-2 and BMP-4 on estradiol enhancement appeared to be diverged from the cAMP-protein kinase A pathway. Thus, BMP-2 and BMP-4 differentially regulate steroidogenesis by stimulating FSH-induced p38 and suppressing cAMP. The former is involved in estradiol production and enhanced by oocyte action, whereas the latter leads to reduction of progesterone synthesis.

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.

Aldosterone Breakthrough Caused by Chronic Blockage of Angiotensin II Type 1 Receptors in Human Adrenocortical Cells: Possible Involvement of Bone Morphogenetic Protein-6 Actions

Circulating aldosterone concentrations occasionally increase after initial suppression with angiotensin II (Ang II) converting enzyme inhibitors or Ang II type 1 receptor blockers (ARBs), a phenomenon referred to as aldosterone breakthrough. However, the underlying mechanism causing the aldosterone breakthrough remains unknown. Here we investigated whether aldosterone breakthrough occurs in human adrenocortical H295R cells in vitro. We recently reported that bone morphogenetic protein (BMP)-6, which is expressed in adrenocortical cells, enhances Ang II- but not potassium-induced aldosterone production in human adrenocortical cells. Accordingly, we examined the roles of BMP-6 in aldosterone breakthrough induced by long-term treatment with ARB. Ang II stimulated aldosterone production by adrenocortical cells. This Ang II stimulation was blocked by an ARB, candesartan. Interestingly, the candesartan effects on Ang II-induced aldosterone synthesis and CYP11B2 expression were attenuated in a course of candesartan treatment for 15 d. The impairment of candesartan effects on Ang II-induced aldosterone production was also observed in Ang II- or candesartan-pretreated cells. Levels of Ang II type 1 receptor mRNA were not changed by chronic candesartan treatment. However, BMP-6 enhancement of Ang II-induced ERK1/2 signaling was resistant to candesartan. The BMP-6-induced Smad1, -5, and -8 phosphorylation, and BRE-Luc activity was augmented in the presence of Ang II and candesartan in the chronic phase. Chronic Ang II exposure decreased cellular expression levels of BMP-6 and its receptors activin receptor-like kinase-2 and activin type II receptor mRNAs. Cotreatment with candesartan reversed the inhibitory effects of Ang II on the expression levels of these mRNAs. The breakthrough phenomenon was attenuated by neutralization of endogenous BMP-6 and activin receptor-like kinase-2. Collectively, these data suggest that changes in BMP-6 availability and response may be involved in the occurrence of cellular escape from aldosterone suppression under chronic treatment with ARB.

Involvement of bone morphogenetic protein-4 in GH regulation by octreotide and bromocriptine in rat pituitary GH3 cells

Here we investigated roles of the pituitary bone morphogenetic protein (BMP) system in modulating GH production regulated by a somatostatin analog, octreotide (OCT) and a dopamine agonist, bromocriptine (BRC) in rat pituitary somatolactotrope tumor GH3 cells. The GH3 cells were found to express BMP ligands, including BMP-4 and BMP-6; BMP type-1 and type-2 receptors (except the type-1 receptor, activin receptor-like kinase (ALK)-6); and Smad signaling molecules. Forskolin stimulated GH production in accordance with cAMP synthesis. BRC, but not OCT, suppressed forskolin-induced cAMP synthesis by GH3 cells. Individual treatment with OCT and BRC reduced forskolin-induced GH secretion. A low concentration (0.1 microM) of OCT in combination with BRC (1-100 microM) exhibited additive effects on reducing GH and cAMP production induced by forskolin. However, a high concentration (10 microM) of OCT in combination with BRC failed to suppress GH and cAMP production. BMP-4 specifically enhanced GH secretion and cAMP production induced by forskolin in GH3 cells. BRC, but not OCT, inhibited BMP-4-induced activation of Smad1,5,8 phosphorylation and Id-1 transcription and decreased ALK-3 expression. Of note, in the presence of a high concentration of OCT, the BRC effects suppressing BMP-4-Smad1,5,8 signaling were significantly impaired. In the presence of BMP-4, a high concentration of OCT also attenuated the BRC effects suppressing forskolin-induced GH and cAMP production. Collectively, a high concentration of OCT interferes with BRC effects by reducing cAMP production and suppressing BMP-4 signaling in GH3 cells. These findings may explain the mechanism of resistance of GH reduction to a combination therapy with OCT and BRC for GH-producing pituitary adenomas.

Functional relationship between fibroblast growth factor-8 and bone morphogenetic proteins in regulating steroidogenesis by rat granulosa cells

Bone morphogenetic proteins (BMPs) have been recognized as crucial molecules in regulating ovarian physiology, with different BMPs having differential actions in FSH-induced estradiol production. To identify the roles of oocyte factors that modulate steroidogenesis controlled by BMPs, we here investigated the effects of FGF-8 in rat granulosa/oocyte co-cultures. FGF-8 potently suppressed FSH-induced estradiol production, but did not affect cAMP-induced estradiol produced by rat granulosa cells. FGF-8 had no effects on progesterone and cAMP production induced by FSH and forskolin. The inhibitory effects of FGF-8 on FSH-induced estradiol production were not altered by BMP-2, -4, -6 or -7. In the presence of FGF-8, BMPs suppressed FSH-induced progesterone by reducing cAMP, suggesting that FGF-8 and BMP independently regulate FSH receptor signaling. Notably, FGF-8-induced ERK and SAPK/JNK phosphorylation in granulosa cells, in which ERK activation was further enhanced by FSH and oocytes. Inhibition of ERK and SAPK/JNK reduced FSH-induced progesterone and cAMP levels, suggesting that the activation of these pathways enhances FSH-induced cAMP signaling. In addition, ERK inhibition upregulated FSH-induced estradiol synthesis, indicating that ERK pathway is also involved in suppressing aromatase activity in granulosa cells. Interestingly, FGF-8 enhanced BMP-induced Smad1/5/8 and Id-1-promoter activities with decreased expression of Smad6/7. Since the SAPK/JNK inhibitor inhibited FGF-8 effects in upregulating Id-1 transcription, SAPK/JNK appears to be involved in the mechanism by which FGF-8 enhances BMP-Smad signaling. Furthermore, in the presence of oocytes, the inhibition of endogenous FGF receptor signaling suppressed FSH- and forskolin-induced progesterone and cAMP, showing that endogenous FGF system is involved in activation of FSH-induced cAMP-PKA signaling via ERK and SAPK/JNK. Thus, the oocyte factor, FGF-8, not only suppresses FSH-induced estradiol production by activating ERK, but also enhances BMP-Smad signaling in granulosa cells. This interaction between FGF-8 and BMPs may play a key role in regulating steroidogenesis through oocyte-granulosa cell communication.

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.