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Pulmonary hypertension (PH) is a progressive disorder that causes significant morbidity and mortality despite existing therapies. PH pathogenesis is characterized by metabolic derangements that increase pulmonary artery smooth muscle cell (PASMC) proliferation and vascular remodeling. PH-associated decreases in peroxisome proliferator-activated receptor γ (PPARγ) stimulate PASMC proliferation, and PPARγ in coordination with PPARγ coactivator 1α (PGC1α) regulates mitochondrial gene expression and biogenesis. To further examine the impact of decreases in PPARγ expression on human PASMC (HPASMC) mitochondrial function, we hypothesized that depletion of either PPARγ or PGC1α perturbs mitochondrial structure and function to stimulate PASMC proliferation. To test this hypothesis, HPASMCs were exposed to hypoxia and treated pharmacologically with the PPARγ antagonist GW9662 or with siRNA against PPARγ or PGC1α for 72 hours. HPASMC proliferation (cell counting), target mRNA levels (qRT-PCR), target protein levels (Western blotting), mitochondria-derived H2O2 (confocal immunofluorescence), mitochondrial mass and fragmentation, and mitochondrial bioenergetic profiling were determined. Hypoxia or knockdown of either PPARγ or PGC1α increased HPASMC proliferation, enhanced mitochondria-derived H2O2, decreased mitochondrial mass, stimulated mitochondrial fragmentation, and impaired mitochondrial bioenergetics. Taken together, these findings provide novel evidence that loss of PPARγ diminishes PGC1α and stimulates derangements in mitochondrial structure and function that cause PASMC proliferation. Overexpression of PGC1α reversed hypoxia-induced HPASMC derangements. This study identifies additional mechanistic underpinnings of PH, and provides support for the notion of activating PPARγ as a novel therapeutic strategy in PH.

Background We previously demonstrated that chronic alcohol ingestion augments TGFβ1 expression in the lung fibroblast and increases the risk of fibroproliferative disrepair in a mouse model of acute lung injury. The effect of alcohol on TGFβ1 is mitigated by treatment with sulforaphane (SFP), which can activate nuclear factor (erythroid-derived 2)-like 2 (Nrf2). However, the mechanisms by which alcohol amplifies, or SFP attenuates, TGFβ1 expression in the fibroblast are not known. MicroRNA (miR)-21 has been shown to inhibit Smad7, a TGFβ1 signaling inhibitor. In this study, we hypothesized that alcohol augments TGFβ1 expression through up-regulation of miR-21, which subsequently inhibits Smad7. Methods Primary mouse lung fibroblasts were cultured ± alcohol ± SFP and assessed for gene expression of miR-21, and gene and/or protein expression of Nrf2, Nrf2-regulated antioxidant enzymes, Smad7, STAT3, and TGFβ1. NIH 3T3 fibroblasts were transfected with a miR-21 inhibitor and cultured ± alcohol. α-SMA, Smad7, and TGFβ1 protein expression were then assessed. In parallel, NIH 3T3 lung fibroblasts were transfected with Nrf2 silencing RNA (siRNA) and cultured ± alcohol ± SFP. Gene expression of miR-21, Nrf2, Smad7, and TGFβ1 was assessed. Results MiR-21 gene expression was increased by 12-fold at 48 hours, and Smad7 gene expression and protein expression were reduced by ~30% in alcohol-treated fibroblasts. In parallel, inhibition of miR-21 attenuated alcohol-mediated decrease in Smad7 and increase in TGFβ1 and α-SMA protein expression. Treatment with SFP mitigated the effect of alcohol on miR-21, Smad7 and total and phosphorylated STAT3, and restored Nrf2-regulated antioxidant gene expression. Silencing of Nrf2 prevented the effect of SFP on miR-21, Smad7, and TGFβ1 gene expression in alcohol-treated NIH 3T3 fibroblasts. Conclusions Alcohol treatment increases TGFβ1 in fibroblasts, at least in part, through augmentation of miR-21, which then inhibits Smad7 expression. These effects can be attenuated by activation of Nrf2 with SFP.

Pulmonary hypertension (PH) is characterized by increased pulmonary vascular remodeling, resistance, and pressures. Reactive oxygen species (ROS) contribute to PH-associated vascular dysfunction. NADPH oxidases (Nox) and mitochondria are major sources of superoxide (O(2)(•-)) and hydrogen peroxide (H(2)O(2)) in pulmonary vascular cells. Hypoxia, a common stimulus of PH, increases Nox expression and mitochondrial ROS (mtROS) production. The interactions between these two sources of ROS generation continue to be defined. We hypothesized that mitochondria-derived O(2)(•-) (mtO(2)(•-)) and H(2)O(2) (mtH(2)O(2)) increase Nox expression to promote PH pathogenesis and that mitochondria-targeted antioxidants can reduce mtROS, Nox expression, and hypoxia-induced PH. Exposure of human pulmonary artery endothelial cells to hypoxia for 72 h increased mtO(2)(•-) and mtH(2)O(2). To assess the contribution of mtO(2)(•-) and mtH(2)O(2) to hypoxia-induced PH, mice that overexpress superoxide dismutase 2 (Tg(hSOD2)) or mitochondria-targeted catalase (MCAT) were exposed to normoxia (21% O(2)) or hypoxia (10% O(2)) for three weeks. Compared with hypoxic control mice, MCAT mice developed smaller hypoxia-induced increases in RVSP, α-SMA staining, extracellular H(2)O(2) (Amplex Red), Nox2 and Nox4 (qRT-PCR and Western blot), or cyclinD1 and PCNA (Western blot). In contrast, Tg(hSOD2) mice experienced exacerbated responses to hypoxia. These studies demonstrate that hypoxia increases mtO(2)(•-) and mtH(2)O(2). Targeting mtH(2)O(2) attenuates PH pathogenesis, whereas targeting mtO(2)(•-) exacerbates PH. These differences in PH pathogenesis were mirrored by RVSP, vessel muscularization, levels of Nox2 and Nox4, proliferation, and H(2)O(2) release. These studies suggest that targeted reductions in mtH(2)O(2) generation may be particularly effective in preventing hypoxia-induced PH.

Hypoxia stimulates pulmonary hypertension (PH) in part by increasing the proliferation of pulmonary vascular wall cells. Recent evidence suggests that signaling events involved in hypoxia-induced cell proliferation include sustained nuclear factor-kappaB (NF-κB) activation, increased NADPH oxidase 4 (Nox4) expression, and downregulation of peroxisome proliferator-activated receptor gamma (PPARγ) levels. To further understand the role of reduced PPARγ levels associated with PH pathobiology, siRNA was employed to reduce PPARγ levels in human pulmonary artery smooth muscle cells (HPASMC) in vitro under normoxic conditions. PPARγ protein levels were reduced to levels comparable to those observed under hypoxic conditions. Depletion of PPARγ for 24-72 h activated mitogen-activated protein kinase, ERK 1/2, and NF-κB. Inhibition of ERK 1/2 prevented NF-κB activation caused by PPARγ depletion, indicating that ERK 1/2 lies upstream of NF-κB activation. Depletion of PPARγ for 72 h increased NF-κB-dependent Nox4 expression and H2O2 production. Inhibition of NF-κB or Nox4 attenuated PPARγ depletion-induced HPASMC proliferation. Degradation of PPARγ depletion-induced H2O2 by PEG-catalase prevented HPASMC proliferation and also ERK 1/2 and NF-κB activation and Nox4 expression, indicating that H2O2 participates in feed-forward activation of the above signaling events. Contrary to the effects of PPARγ depletion, HPASMC PPARγ overexpression reduced ERK 1/2 and NF-κB activation, Nox4 expression, and cell proliferation. Taken together these findings provide novel evidence that PPARγ plays a central role in the regulation of the ERK1/2-NF-κB-Nox4-H2O2 signaling axis in HPASMC. These results indicate that reductions in PPARγ caused by pathophysiological stimuli such as prolonged hypoxia exposure are sufficient to promote the proliferation of pulmonary vascular smooth muscle cells observed in PH pathobiology.

Pulmonary hypertension (PH) is a progressive disorder whose cellular pathogenesis involves enhanced smooth muscle cell (SMC) proliferation and resistance to apoptosis signals. Existing evidence demonstrates that the tumor suppressor programmed cell death 4 (PDCD4) affects patterns of cell growth and repair responses in the systemic vasculature following experimental injury. In the current study, the regulation PDCD4 and its functional effects on growth and apoptosis susceptibility in pulmonary artery smooth muscle cells were explored. We previously demonstrated that pharmacological activation of the nuclear transcription factor peroxisome proliferator-activated receptor-γ (PPARγ) attenuated hypoxia-induced proliferation of human pulmonary artery smooth muscle cells (HPASMCs) by inhibiting the expression and mitogenic functions of microRNA-21 (miR-21). In the current study, we hypothesize that PPARγ stimulates PDCD4 expression and HPASMC apoptosis by inhibiting miR-21. Our findings demonstrate that PDCD4 is reduced in the mouse lung upon exposure to chronic hypoxia (10% O2 for 3 wk) and in hypoxia-exposed HPASMCs (1% O2). HPASMC apoptosis was reduced by hypoxia, by miR-21 overexpression, or by siRNA-mediated PPARγ and PDCD4 depletion. Activation of PPARγ inhibited miR-21 expression and resultant proliferation, while restoring PDCD4 levels and apoptosis to baseline. Additionally, pharmacological activation of PPARγ with rosiglitazone enhanced PDCD4 protein expression and apoptosis in a dose-dependent manner as demonstrated by increased annexin V detection by flow cytometry. Collectively, these findings demonstrate that PPARγ confers growth-inhibitory signals in hypoxia-exposed HPASMCs through suppression of miR-21 and the accompanying derepression of PDCD4 that augments HPASMC susceptibility to undergo apoptosis.

The ligand-activated transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), regulates metabolism, cell proliferation, and inflammation. Pulmonary hypertension (PH) is associated with reduced PPARγ expression, and hypoxia exposure regimens that cause PH reduce PPARγ expression. The current study examines mechanisms of hypoxia-induced PPARγ downregulation in vitro and in vivo. Hypoxia reduced PPARγ mRNA and protein levels, PPARγ activity, and the expression of PPARγ regulated genes in human pulmonary artery smooth muscle cells (HPASMC) exposed to 1% oxygen for 72 hours. Similarly, exposure of mice to hypoxia (10% O2) for 3 weeks reduced PPARγ mRNA and protein in mouse lung. Inhibiting ERK1/2 with PD98059 or treatment with siRNA directed against either NF-κB p65 or Nox4 attenuated hypoxic reductions in PPARγ expression and activity. Furthermore, degradation of H2O2 using PEG-catalase prevented hypoxia-induced ERK 1/2 phosphorylation and Nox4 expression suggesting sustained ERK 1/2-mediated signaling and Nox4 expression in this response. Mammalian two hybrid assays demonstrated that PPARγ and p65 bind directly to each other in a mutually repressive fashion. Taken together, we conclude that hypoxic regimens that promote PH pathogenesis and HPASMC proliferation reduce PPARγ expression and activity through ERK1/2-, p65-, and Nox4-dependent pathways. These findings provide novel insights into mechanisms by which pathophysiological stimuli such as hypoxia cause loss of PPARγ activity and pulmonary vascular cell proliferation, pulmonary vascular remodeling, and PH. These results also indicate that restoration of PPARγ activity with pharmacological ligands may provide a novel therapeutic approach in selected forms of PH.

Peroxisome proliferator-activated receptor (PPAR) γ activation attenuates hypoxia-induced pulmonary hypertension (PH) in mice. The current study examined the hypothesis that PPARγ attenuates hypoxia-induced endothelin-1 (ET-1) signaling to mediate these therapeutic effects. To test this hypothesis, human pulmonary artery endothelial cells (HPAECs) were exposed to normoxia or hypoxia (1% O2) for 72 h and treated with or without the PPARγ ligand rosiglitazone (RSG, 10 μM) during the final 24 h of exposure. HPAEC proliferation was measured with MTT assays or cell counting, and mRNA and protein levels of ET-1 signaling components were determined. To explore the role of hypoxia-activated transcription factors, selected HPAECs were treated with inhibitors of hypoxia-inducible factor (HIF)-1α (chetomin) or nuclear factor (NF)-κB (caffeic acid phenethyl ester, CAPE). In parallel studies, male C57BL/6 mice were exposed to normoxia (21% O2) or hypoxia (10% O2) for 3 wk with or without gavage with RSG (10 mg·kg−1·day−1) for the final 10 days of exposure. Hypoxia increased ET-1, endothelin-converting enzyme-1, and endothelin receptor A and B levels in mouse lung and in HPAECs and increased HPAEC proliferation. Treatment with RSG attenuated hypoxia-induced activation of HIF-1α, NF-κB activation, and ET-1 signaling pathway components. Similarly, treatment with chetomin or CAPE prevented hypoxia-induced increases in HPAEC ET-1 mRNA and protein levels. These findings indicate that PPARγ activation attenuates a program of hypoxia-induced ET-1 signaling by inhibiting activation of hypoxia-responsive transcription factors. Targeting PPARγ represents a novel therapeutic strategy to inhibit enhanced ET-1 signaling in PH pathogenesis.

Pathogenesis of pulmonary hypertension is complex and involves activation of the transcription factor, hypoxia-inducible factor-1 (HIF-1) that shifts cellular metabolism from aerobic respiration to glycolysis, in part, by increasing the expression of its downstream target pyruvate dehydrogenase kinase-1 (PDK-1), thereby promoting a proliferative, apoptosis-resistant phenotype in pulmonary vascular cells. Activation of the nuclear hormone transcription factor, peroxisome proliferator-activated receptor gamma (PPARγ), attenuates pulmonary hypertension and pulmonary artery smooth muscle cell (PASMC) proliferation. In the current study, we determined whether PPARγ inhibits HIF-1α and PDK-1 expression in human PASMCs.HPASMCs were exposed to normoxia (21% O2) or hypoxia (1% O2) for 2-72 hours ± treatment with the PPARγ-ligand, rosiglitazone (RSG, 10μM).Compared to normoxia, HIF-1α mRNA levels were elevated in HPASMC at 2 hours hypoxia and reduced to baseline levels by 24-72 hours. HIF-1α protein levels increased following 4 and 8 hours of hypoxia and returned to baseline levels by 24 and 72 hours. PDK-1 protein levels increased following 24 hours hypoxia and remained elevated by 72 hours. RSG treatment at the onset of hypoxia attenuated HIF-1α protein and PDK-1 mRNA and protein levels at 4, 8 and 24 hours of hypoxia, respectively. However, RSG treatment during final 24 hours of 72-hour hypoxia, an intervention that inhibits HPASMC proliferation, failed to prevent hypoxia-induced PDK-1 expression.Hypoxia causes transient activation of HPASMC HIF-1α that is attenuated by RSG treatment initiated at hypoxia onset. These findings provide novel evidence that PPARγ modulates fundamental and acute cellular responses to hypoxia through both HIF-1-dependent and HIF-1-independent mechanisms.

Endothelin-1 (ET-1) plays a critical role in endothelial dysfunction and contributes to the pathogenesis of pulmonary hypertension (PH). We hypothesized that peroxisome proliferator–activated receptor γ (PPARγ) stimulates microRNAs that inhibit ET-1 and pulmonary artery endothelial cell (PAEC) proliferation. The objective of this study was to clarify molecular mechanisms by which PPARγ regulates ET-1 expression in vitro and in vivo. In PAECs isolated from patients with pulmonary arterial hypertension, microRNA (miR)-98 expression was reduced, and ET-1 protein levels and proliferation were increased. Similarly, hypoxia reduced miR-98 and increased ET-1 levels and PAEC proliferation in vitro. In vivo, hypoxia reduced miR-98 expression and increased ET-1 and proliferating cell nuclear antigen (PCNA) levels in mouse lung, derangements that were aggravated by treatment with the vascular endothelial growth factor receptor antagonist Sugen5416. Reporter assays confirmed that miR-98 binds directly to the ET-1 3′-untranslated region. Compared with littermate control mice, miR-98 levels were reduced and ET-1 and PCNA expression were increased in lungs from endothelial-targeted PPARγ knockout mice, whereas miR-98 levels were increased and ET-1 and PCNA expression was reduced in lungs from endothelial-targeted PPARγ–overexpression mice. Gain or loss of PPARγ function in PAECs in vitro confirmed that alterations in PPARγ were sufficient to regulate miR-98, ET-1, and PCNA expression. Finally, PPARγ activation with rosiglitazone regimens that attenuated hypoxia-induced PH in vivo and human PAEC proliferation in vitro restored miR-98 levels. The results of this study show that PPARγ regulates miR-98 to modulate ET-1 expression and PAEC proliferation. These results further clarify molecular mechanisms by which PPARγ participates in PH pathogenesis and therapy.

Activation of the nuclear hormone receptor, PPARγ, with pharmacological agonists promotes a contractile vascular smooth muscle cell phenotype and reduces oxidative stress and cell proliferation, particularly under pathological conditions including vascular injury, restenosis, and atherosclerosis. However, pharmacological agonists activate both PPARγ-dependent and -independent mechanisms in multiple cell types confounding efforts to clarify the precise role of PPARγ in smooth muscle cell structure and function in vivo. We, therefore, designed and characterized a mouse model with smooth muscle cell-targeted PPARγ overexpression (smPPARγOE). Our results demonstrate that smPPARγOE attenuated contractile responses in aortic rings, increased aortic compliance, caused aortic dilatation, and reduced mean arterial pressure. Molecular characterization revealed that compared to littermate control mice, aortas from smPPARγOE mice expressed lower levels of contractile proteins and increased levels of adipocyte-specific transcripts. Morphological analysis demonstrated increased lipid deposition in the vascular media and in smooth muscle of extravascular tissues. In vitro adenoviral-mediated PPARγ overexpression in human aortic smooth muscle cells similarly increased adipocyte markers and lipid uptake. The findings demonstrate that smooth muscle PPARγ overexpression disrupts vascular wall structure and function, emphasizing that balanced PPARγ activity is essential for vascular smooth muscle homeostasis.

Pulmonary hypertension (PH) is a progressive and often fatal disorder whose pathogenesis involves pulmonary artery smooth muscle cell (PASMC) proliferation. Although modern PH therapies have significantly improved survival, continued progress rests on the discovery of novel therapies and molecular targets. MicroRNA (miR)-21 has emerged as an important non-coding RNA that contributes to PH pathogenesis by enhancing vascular cell proliferation, however little is known about available therapies that modulate its expression. We previously demonstrated that peroxisome proliferator-activated receptor gamma (PPARγ) agonists attenuated hypoxia-induced HPASMC proliferation, vascular remodeling and PH through pleiotropic actions on multiple targets, including transforming growth factor (TGF)-β1 and phosphatase and tensin homolog deleted on chromosome 10 (PTEN). PTEN is a validated target of miR-21. We therefore hypothesized that antiproliferative effects conferred by PPARγ activation are mediated through inhibition of hypoxia-induced miR-21 expression. Human PASMC monolayers were exposed to hypoxia then treated with the PPARγ agonist, rosiglitazone (RSG,10 μM), or in parallel, C57Bl/6J mice were exposed to hypoxia then treated with RSG. RSG attenuated hypoxic increases in miR-21 expression in vitro and in vivo and abrogated reductions in PTEN and PASMC proliferation. Antiproliferative effects of RSG were lost following siRNA-mediated PTEN depletion. Furthermore, miR-21 mimic decreased PTEN and stimulated PASMC proliferation, whereas miR-21 inhibition increased PTEN and attenuated hypoxia-induced HPASMC proliferation. Collectively, these results demonstrate that PPARγ ligands regulate proliferative responses to hypoxia by preventing hypoxic increases in miR-21 and reductions in PTEN. These findings further clarify molecular mechanisms that support targeting PPARγ to attenuate pathogenic derangements in PH.

Rationale: Pulmonary hypertension (PH) is an important clinical problem that causes significant morbidity and mortality despite existing therapies. Recent evidence indicates that metabolic derangements in pulmonary vascular wall cells stimulate mitochondrial (MT) dysfunction that promotes proliferative vascular remodeling. We previously showed that expression of peroxisome proliferator-activated receptor gamma (PPARγ) was reduced in murine hypoxia-induced PH and that reductions in PPARγ were sufficient to stimulate Nox4 expression, H2O2 production, and human pulmonary artery smooth muscle cell (HPASMC) proliferation. Objective: We hypothesize that PPARγ coactivator 1 alpha (PGC1α, which stimulates MT biogenesis), is a critical PPARγ-regulated target that modulates MT function, reactive oxygen species (ROS) production, and the proliferative PASMC phenotype in PH. Methods and Results: To examine the molecular pathways stimulated by reductions in PPARγ, siPPARγ or GW9662 (PPARγ antagonist) were used to inhib...

Hypoxia stimulates pulmonary hypertension (PH) in part by increasing the proliferation of pulmonary vascular wall cells. Recent evidence suggests that hypoxia stimulates sustained nuclear factor-kappaB (NF-κB) activation, increased NADPH oxidase4 (Nox4) expression, and downregulation of peroxisome proliferator-activated receptor-gamma (PPARγ) to cause human pulmonary artery smooth muscle cell (HPASMC) proliferation. To further understand the role of reduced PPARγ in PH pathobiology, siRNA was employed to reduce PPARγ levels in HPASMC in vitro under normoxic conditions. PPARγ protein levels were reduced to levels comparable to those observed under hypoxic conditions. Knockdown of PPARγ for 24-72 hours activated mitogen-activated protein kinase, ERK 1/2, and NF-κB. Inhibition of ERK 1/2 prevented NF-κB activation caused by PPARγ knockdown indicating that ERK 1/2 lies upstream of NF-κB activation. Knockdown of PPARγ for 72 hours increased NF-κB-dependent Nox4 protein expression and H2O2 production. Degradati...

BACKGROUND IGF-I is an important growth and differentiative factor for osteoblasts and may have a role in defining prostate cancer risk and skeletal metastases. METHODS Conditioned media (CM) from human prostate cancer (PC), C4-2 and C4-2B, which produce osteoblastic lesions, and PC-3, which causes osteolysis, was added to MC3T3-E1 bone cultures. SCID mice were injected intratibially with these engineered cells. Tumor bearing tibiae were analyzed by microCT and pQCT. RESULTS CM from PC cells increased osteoblast proliferation and differentiation and was unaltered by the type of PC cell, IGF-I antibodies, or exogenous IGF-I and IGFBP2. Study of intratibial PC tumors in SCID mice showed that C4-2 cells grew slowly preserving bone structure, while PC-3 tumors caused rapid osteolysis. Overexpression of IGF-I did not change either tumor progression or skeletal response. CONCLUSIONS IGF-I is neither necessary nor sufficient for the osteoblastic response to PC metastases. Prostate © 2006 Wiley-Liss, Inc.

Receptor activator of NFkappa-B ligand (RANKL) is essential for osteoclast formation, function, and survival. Although RANKL mRNA and protein levels are modulated by 1,25(OH)2D3 and other osteoactive factors, regulatory mechanisms remain unclear. In this study, we show that 2 kb or 2 kb plus exon 1 of a RANKL promoter sequence conferred neither 1,25(OH)2D3 response nor tissue specificity. The histone deacetylase inhibitors trichostatin A (TSA) and sodium butyrate (SB), however, strongly increased RANKL promoter activity. A series of 5'-deleted RANKL promoter constructs from 2,020 to 110 bp showed fourfold increased activity after TSA treatment. TSA also dose dependently enhanced endogenous RANKL mRNA expression with 50 microM of TSA treatment causing equivalent RANKL expression to that seen with 1 nM 1,25(OH)2D3. Using a chromatin immunoprecipitation (ChIP) assay we showed that TSA significantly enhanced association of both acetylated histone H3 and H4 on the RANKL promoter, with H4 > H3. A similar increase in acetylated histone H4 on the RANKL gene locus was seen after 1,25(OH)2D3 treatment, but ChIP assay did not reveal localization of VDR/RXR heterodimers on the putative VDRE of the RANKL promoter. To explore the role of H4 acetylation of 1,25(OH)2D3 stimulated RANKL, we added both TSA and 1,25(OH)2D3 together. While the combination further increased acetylation of H4 on the RANKL locus, surprisingly, TSA inhibited 1,25(OH)2D3-induced RANKL mRNA expression by 70% at all doses of 1 ,25(OH)2D3 studied. These results suggest that TSA increases of endogenous expression of RANKL involve enhanced acetylation of histones on the proximal RANKL promoter. Preventing deacetylation, however, blocks 1,25(OH)2D3 action on this gene. Chromatin remodeling is therefore involved in RANKL expression.

Bone remodeling reflects an equilibrium between bone resorption and formation. The local expression of receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in bone determines the entry of monoblastic precursors into the osteoclast lineage and subsequent bone resorption. Nitric oxide (NO) inhibits osteoclastic bone resorption in vitro and regulates bone remodeling in vivo. An interaction of NO with RANKL and OPG has not been studied. Here, we show that treatment of ST-2 murine stromal cells with the NO donor sodium nitroprusside (100 μm) for 24 h inhibited 1,25 dihydroxyvitamin D3-induced RANKL mRNA to less than 33 ± 7% of control level, whereas OPG mRNA increased to 204 ± 19% of control. NOR-4 replicated these NO effects. The effects of NO were dose dependent and associated with changes in protein levels: RANKL protein decreased and OPG protein increased after treatment with NO. PTH-induced RANKL expression in primary stromal cells was inhibited by sodium nitroprusside, indicating that the NO effect did not require vitamin D. NO donor did not change the stability of RANKL or OPG mRNAs, suggesting that NO affected transcription. Finally, cGMP, which can function as a second messenger for NO, did not reproduce the NO effect, nor did inhibition of endogenous guanylate cyclase prevent the NO effect on these osteoactive genes. The effect of NO to decrease the RANKL/OPG equilibrium should lead to decreased recruitment of osteoclasts and positive bone formation. Thus, drugs and conditions that cause local increase in NO formation in bone may have positive effects on bone remodeling.

Objective: To explore the role of PPARγ in vascular regulation, smooth muscle VP16-PPARγ-overexpressing mice were subjected to studies of systemic vascular function, biomechanical testing, histological analysis, and molecular characterization. Methods: Mice with a flox-controlled VP16-PPARγ gene were crossed with mice containing a tamoxifen-inducible Cre recombinase driven by a smooth muscle myosin heavy chain promoter. All mice received tamoxifen injections at 6 weeks of age. Experiments were performed at 10-16 weeks of age. Results: Compared to littermate controls, transgenic mice displayed: 1) aortic dilation (by histology, echocardiography, and microCT), 2) increased aortic compliance, 3) reduced blood pressure, 4) 80% reduction in aortic ring contractile forces in response to KCl or phenylephrine, 5) lipid accumulation in the aortic media (Oil Red O staining), and 6) reduction in aortic contractile markers and extra-cellular matrix components with increased expression of adipose markers and selected ...

Hypoxia stimulates pulmonary hypertension in part by increasing the proliferation of pulmonary vascular wall cells via sustained activation of MAP kinase ERK 1/2, NF-κB, and downregulation of PPARγ levels. However, the upstream signaling events that mediate NF-κB upregulation and PPARγ downregulation remain unknown. We examined the role of Proline-rich tyrosine kinase 2 (Pyk2) in hypoxia-induced proliferation of human pulmonary artery smooth muscle cells (HPASMC). Exposure of HPASMC to hypoxia (1% O2) for 72 hrs resulted in the activation of Pyk2. siRNA-mediated Pyk2 depletion or its pharmacological inhibition attenuated hypoxia-induced HPASMC proliferation. Pyk2 inhibition attenuated hypoxia-induced: 1) ERK 1/2 activation, 2) NF-κB transcriptional activity and 3) reductions in PPARγ mRNA levels. Conversely, siRNA-mediated PPARγ depletion enhanced, whereas PPARγ overexpression attenuated HPASMC Pyk2 activation. Exposing C57BL/6 mice to 10% O2 for 3 weeks reduced lung PPARγ but increased Pyk2 levels compar...

The regulatory regions for transcriptional control of the MCSF gene are unknown. We examined regulatory control in a 774-bp murine MCSF promoter transfected into MC3T3-E1 osteoblast-like and COS-7 cells. Deletion of upstream sequence from -635 increased basal activity of the promoter by at least four-fold, an increase that was maintained when PU.1, NFkappaB and Egr1/Sp1 consensus sequences were subsequently removed. Mutagenesis identified a suppressor element between -635 and -642 from the transcriptional start site and an oligonucleotide representing this sequence was retarded by nuclear cell protein. TNFalpha (1 ng/ml), PTH (5x10(-8) M), and IL-1alpha (100 pg/ml), which increased MCSF protein secretion, failed to enhance the transcriptional rate of the full-length promoter. TNFalpha was able to stimulate transcription of a heterologous reporter transfected into COS-7 containing multiple copies of the murine MCSF NFkappaB site inserted before a minimal promoter. In contrast, deletion of the same NFkappaB response element increased basal activity in the native promoter. Thus, the NFkappaB sequence may act as a negative regulator in the context of the endogenous promoter. Our results indicate that constitutive transcriptional activity conferred by the MCSF promoter may be damped by a suppressor protein. Transcriptional regulation, however, does not appear to be a major stimulatory mechanism for MCSF secretion.

Expression of MCSF in bone is important to the regulation of osteoclastogenesis. We show here that tumor necrosis factor-alpha (TNFalpha) increases the production of both soluble (sMCSF) and membrane-bound (mMCSF) macrophage colony stimulating factor by ST2 bone stromal cells. Treatment of ST2 cells with TNFalpha caused sMCSF levels to increase by 394+/-5% from basal; mMCSF rose by 316+/-66% from 30+/-10 per 100,000 cells in the same time. These increases were consistent with increased expression of mRNAs encoding both isoforms. Increases in MCSF mRNA are also seen after stimulation with dexamethasone. To investigate the potential role of NFkappaB in this TNFalpha effect, we treated cells with sodium salicylate (NaS), an inhibitor of NFkappaB translocation. NaS decreased TNFalpha-stimulated NFkappaB activation by 50% as assessed by EMSA. Despite inhibition of NFkappaB signaling, NaS enhanced TNFalpha-stimulated MCSF secretion and did not prevent TNFalpha-stimulated increases in sMCSF mRNA, suggesting that NFkappaB was not involved in TNFalpha effect on the gene. TNFalpha failed to stimulate transcription of a 774 nucleotide MCSF promoter-luciferase reporter transfected into ST2 cells which contained the NFkappaB consensus sequence. Deletion of the seven nucleotides containing the NFkappaB homology response sequence from the MCSF promoter increased basal gene transcription by twofold. TNFalpha thus contributes to an osteoclastogenic environment through upregulation of bone expression of both MCSF isoforms. Our data suggests that NFkappaB is not the major signaling pathway through which this occurs.

Meiotic recombination between artificial repeats positioned on nonhomologous chromosomes occurs efficiently in the yeast Saccharomyces cerevisiae. Both gene conversion and crossover eventS have been observed, with crossovers yielding reciprocal translocations. In the current study, 5.5-kb ura3 repeats positioned on chromosomes V and XV were used to examine the effect of ectopic recombination on meiotic chromosome segregation. Ura+ random spores were selected and gene conversion vs. crossover events were distinguished by Southern blot analysis. Approximately 15% of the crossover events between chromosomes V and XV were associated with missegregation of one of these chromosomes. The missegregation was manifest as hyperploid spores containing either both translocations plus a normal chromosome, or both normal chromosomes plus one of the translocations. In those cases where it could be analyzed, missegregation occurred at the first meiotic division. These data are discussed in terms of a model in which ectopic crossovers compete efficiently with normal allelic crossovers in directing meiotic chromosome segregation.

Transforming growth factor-β1 (TGF- β1) and thrombospondin-1 (TSP-1) are hypoxia-responsive mitogens that promote vascular smooth muscle cell (SMC) proliferation, a critical event in the pathogenesis of pulmonary hypertension (PH). We previously demonstrated that hypoxia-induced human pulmonary artery smooth muscle (HPASMC) cell proliferation and expression of the NADPH oxidase subunit, Nox4, were attenuated by the peroxisome proliferator-activated receptor γ (PPARγ) agonist, rosiglitazone. The current study examines the hypothesis that rosiglitazone regulates Nox4 expression and HPASMC proliferation by attenuating TSP-1 signaling. Selected HPASMC were exposed to normoxic or hypoxic (1% O(2)) environments or TSP-1 (0-1 μg/ ml) for 72 hours ± administration of rosiglitazone (10 μM). Cellular proliferation, Nox4, TSP-1, and TGF-β1 expression and reactive oxygen species generation were measured. Mice exposed to hypoxia (10% O(2)) for three weeks were treated with rosiglitazone (10 mg/kg/day) for the final 10 days, and lung TSP-1 expression was examined. Hypoxia increased TSP-1 and TGF-β1 expression and HPASMC proliferation, and neutralizing antibodies to TSP-1 or TGF-β1 attenuated proliferation. Rosiglitazone attenuated hypoxia-induced HPASMC proliferation and increases in mouse lung and HPASMC TSP-1 expression, but failed to reduce increases in TGF-β1 expression or Nox4 expression and activity caused by direct TSP-1 stimulation. Transfecting HPASMC with siRNA to Nox4 attenuated hypoxia- or TSP-1-stimulated HPASMC proliferation. These findings provide novel evidence that TSP-1-mediated Nox4 expression plays a critical role in hypoxia-induced HPASMC proliferation. PPARγ activation with exogenous ligands attenuates TSP-1 expression to reduce Nox4 expression. These results clarify mechanisms of hypoxia-induced SMC proliferation and suggest additional pathways by which PPARγ agonists may regulate critical steps in the pathobiology of PH.

Increased NADP reduced (NADPH) oxidase 4 (Nox4) and reduced expression of the nuclear hormone receptor peroxisome proliferator-activated receptor γ (PPARγ) contribute to hypoxia-induced pulmonary hypertension (PH). To examine the role of Nox4 activity in pulmonary vascular cell proliferation and PH, the current study used a novel Nox4 inhibitor, GKT137831, in hypoxia-exposed human pulmonary artery endothelial or smooth muscle cells (HPAECs or HPASMCs) in vitro and in hypoxia-treated mice in vivo. HPAECs or HPASMCs were exposed to normoxia or hypoxia (1% O(2)) for 72 hours with or without GKT137831. Cell proliferation and Nox4, PPARγ, and transforming growth factor (TGF)β1 expression were measured. C57Bl/6 mice were exposed to normoxia or hypoxia (10% O(2)) for 3 weeks with or without GKT137831 treatment during the final 10 days of exposure. Lung PPARγ and TGF-β1 expression, right ventricular hypertrophy (RVH), right ventricular systolic pressure (RVSP), and pulmonary vascular remodeling were measured. GKT137831 attenuated hypoxia-induced H(2)O(2) release, proliferation, and TGF-β1 expression and blunted reductions in PPARγ in HPAECs and HPASMCs in vitro. In vivo GKT137831 inhibited hypoxia-induced increases in TGF-β1 and reductions in PPARγ expression and attenuated RVH and pulmonary artery wall thickness but not increases in RVSP or muscularization of small arterioles. This study shows that Nox4 plays a critical role in modulating proliferative responses of pulmonary vascular wall cells. Targeting Nox4 with GKT137831 provides a novel strategy to attenuate hypoxia-induced alterations in pulmonary vascular wall cells that contribute to vascular remodeling and RVH, key features involved in PH pathogenesis.

Both mechanical loading and nitric oxide (NO) have positive influences on bone mass. NO production is induced by mechanical strain via upregulation of eNOS mRNA and protein, the predominant NOS in adult bone. At the same time, strain causes decreased expression of RANKL, a factor critical for osteoclastogenesis. In this study, we harvested primary stromal cells from wild-type (WT) and eNOS(−/−) mice to test whether induction of NO by mechanical strain was necessary for transducing mechanical inhibition of RANKL. We found that strain inhibition of RANKL expression was prevented by NOS inhibitors (L-NAME and L-NMMA) in WT stromal cells. Surprisingly, stromal cells from eNOS(−/−) mice showed significant mechanical repression of RANKL expression (p

Mechanical strain inhibits osteoclastogenesis by regulating osteoblast functions: We have shown that strain inhibits receptor activator of NF-kappaB ligand (RANKL) expression and increases endothelial nitric oxide synthase (eNOS) and nitric oxide levels through ERK1/2 signaling in primary bone stromal cells. The primary stromal culture system, while contributing greatly to understanding of how the microenvironment regulates bone remodeling is limited in use for biochemical assays and studies of other osteoprogenitor cell responses to mechanical strain: Stromal cells proliferate poorly and lose aspects of the strain response after a relatively short time in culture. In this study, we used the established mouse osteoblast cell line, conditionally immortalized murine calvarial (CIMC-4), harvested from mouse calvariae conditionally immortalized by insertion of the gene coding for a temperature-sensitive mutant of SV40 large T antigen (TAg) and support osteoclastogenesis. Mechanical strain (0.5-2%, 10 cycles per min, equibiaxial) caused magnitude-dependent decreases in RANKL expression to less than 50% those of unstrained cultures. Overnight strains of 2% also increased osterix (OSX) and RUNX2 expression by nearly twofold as measured by RT-PCR. Importantly, the ERK1/2 inhibitor, PD98059, completely abrogated the strain effects bringing RANKL, OSX, and RUNX2 gene expression completely back to control levels. These data indicate that the strain effects on CIMC-4 cells require activation of ERK1/2 pathway. Therefore, the CIMC-4 cell line is a useful alternative in vitro model which effectively recapitulates aspects of the primary stromal cells and adds an extended capacity to study osteoblast control of bone remodeling in a mechanically active environment.

Mechanical input is known to regulate bone remodeling, yet the molecular events involved in mechanical signal transduction are poorly understood. We here investigate proximal events leading to the ERK1/2 activation that is required for mechanical repression of RANKL (receptor activator of NF-kappaB ligand) expression, the factor that controls local recruitment of osteoclasts. Using primary murine bone stromal cells we show that dynamic mechanical strain via substrate deformation activates Ras-GTPase, in particular the H-Ras isoform. Pharmacological inhibition of H-Ras function prevents strain activation of H-Ras as well as the downstream mechanical repression of RANKL. Furthermore, small interfering RNA silencing of H-Ras, but not K-Ras, abrogates mechanical strain repression of RANKL. H-Ras-specific inhibition of mechanorepression of RANKL was also demonstrated in a murine pre-osteoblast cell line (CIMC-4). The requirement of cholesterol for H-Ras activation was probed; cholesterol depletion of rafts using methyl-betacyclodextrin prevented mechanical H-Ras activation. That the mechanical repression of RANKL requires activation of H-Ras, a specific isoform of Ras-GTP that is known to reside in the lipid raft microdomain, suggests that spatial arrangements are critical for generation of specific downstream events in response to mechanical signals. By partitioning signals this way, cells may be able to generate different downstream responses through seemingly similar signaling cascades.

BACKGROUND Prostate cancer (PC) has a propensity to metastasize to the skeleton, inducing an osteoblastic response in the host. Recent epidemiological studies have suggested that circulating IGF-I may be important for both the pathogenesis and dissemination of PC. We have postulated that tumor secreted IGF-I in conjunction with endogenous IGF-I contributes to the osteoblastic phenotype characteristic of metastatic PC. METHODS To test this thesis we studied the established LNCaP PC progression model consisting of three genetically related human PC cell lines. RESULTS Using RIA, we found serum-free conditioned media (CM) of LNCaP and C4-2 had no measurable IGF-I, whereas IGF-I was easily detected in CM from C4-2B cells at 24 hr (i.e., 1.8 ± 0.53 ng/mg cell protein). Real-time PCR of IGF-I mRNA showed that C4-2B expressed 100-fold more IGF-I mRNA than LNCaP cells. In addition, C4-2B expression of IGF-I mRNA was substantially increased in the presence of exogenous IGF-I to nearly twofold. While IGFBP-3 and IGFBP-1 were not detectable in the CM of any PC line, all cells secreted IGFBP-2. C4-2B cells produced 40% more IGFBP-2 than LNCaP or C4-2 cells (C4-2B at 167 ± 43 ng/mg cell protein). RANKL, a product of bone stromal cells, was also differentially expressed: LNCaP had threefold higher RANKL mRNA compared to C4-2 and C4-2B and at least equivalent protein expression. CONCLUSIONS Our results suggest that PC cells that have metastasized to bone have an upregulated IGF-I regulatory system. This suggests an activated IGF-I axis contributes to the host–PC interaction in promoting osteoblastic metastases. © 2003 Wiley-Liss, Inc.

Exercise promotes positive bone remodeling through controlling cellular processes in bone. Nitric oxide (NO), generated from endothelial nitric-oxide synthase (eNOS), prevents resorption, whereas receptor activator of nuclear kappa B ligand (RANKL) promotes resorption through regulating osteoclast activity. Here we show that mechanical strain differentially regulates eNOS and RANKL expression from osteoprogenitor stromal cells in a magnitude-dependent fashion. Strain (0.25-2%) induction of eNOS expression was magnitude-dependent, reaching a plateau at 218 +/- 36% of control eNOS. This was accompanied by increases in eNOS protein and a doubling of NO production. Concurrently, 0.25% strain inhibited RANKL expression with increasing response up to 1% strain (44 +/- 3% of control RANKL). These differential responses to mechanical input were blocked when an ERK1/2 inhibitor was present during strain application. Inhibition of NO generation did not prevent strain-activated ERK1/2. To confirm the role of ERK1/2, cells were treated with an adenovirus encoding a constitutively activated MEK; Ad.caMEK significantly increased eNOS expression and NO production by more than 4-fold and decreased RANKL expression by half. In contrast, inhibition of strain-activated c-Jun kinase failed to prevent strain effects on either eNOS or RANKL. Our data suggest that physiologic levels of mechanical strain utilize ERK1/2 kinase to coordinately regulate eNOS and RANKL in a manner leading to positive bone remodeling.

Normal dynamic loading prevents bone resorption; however, the means whereby biophysical factors reduce osteoclast activity is not understood. The authors show here that mechanical strain applied to murine marrow cultures reduced osteoclast formation by 50%. The reduction in osteoclast formation was preceded by decreased stromal cell expression of osteoclast differentiation factor (ODF/TRANCE). Since osteoclast formation is critically dependent upon ODFT/TRANCE expression, a strain-induced reduction in this factor may contribute to reduced osteoclastogenesis.

Nitric oxide (NO) is an important molecule with diverse bio-messenger functions including regulation of gene expression. Transcriptional studies using sensitive luciferase reporter systems have suggested that NO inhibits the promoter activity of a variety of genes. Here we report that NO donors (sodium nitroprusside, 2',2'-(hydroxynitrosohydrazono)bis-ethanimine, and (+/-)-(E)-4-ethyl-2-[(Z)-hydroxyimino]-5-nitro-3-hexen-1-yl-nicotinamide) decrease luciferase activity in a promoter-independent fashion in both viral and eukaryotic promoters, with a reduction to nearly 50% in the presence of 100 microm NO donor. Addition of an SV40 enhancer downstream of the luciferase coding region shifted NO donor inhibition to the right, with inhibition at approximately 300 microm. In contrast, when studied in a chloramphenicol acetyltransferase reporter, two promoters indicating inhibition by NO were unaffected. The decrease in luciferase activity was not caused by NO suppression of the luciferase enzyme. Real-time PCR data showed that luciferase mRNA half-life decreased by nearly half in the presence of NO donor (from 75 to 45 min). The SV40 enhancer prolonged luciferase mRNA half-life and somewhat blunted the NO effect. Our data suggest that exogenous NO inhibits luciferase activity in a dose-dependent manner through decreasing luciferase mRNA stability. Thus, the use of luciferase reporter systems to study transcriptional regulation by NO should be attempted with caution.

IGF-I, a ubiquitous polypeptide, plays a key role in longitudinal bone growth and acquisition. The most predominant effect of skeletal IGF-I is acceleration of the differentiation program for osteoblasts. However, in vivo studies using recombinant human (rh) IGF-I and/or rhGH have demonstrated stimulation of both bone formation and resorption, thereby potentially limiting the usefulness of these peptides in the treatment of osteoporosis. In this study, we hypothesized that IGF-I modulates bone resorption by regulating expression of osteoprotegerin (OPG) and receptor activator of nuclear factor-kappaB (RANK) ligand (RANKL) in bone cells. Using Northern analysis in ST2 cells, we found that human IGF-I suppressed OPG mRNA in a time- and dose-dependent manner: 100 micro g/LIGF-I (13 nM) decreased OPG expression by 37.0 +/- 1.8% (P < 0.002). The half maximal inhibitory dose of IGF-I was reached at 50 micro g/liter ( approximately 6.5 nM) with no effect of IGF-I on OPG message stability. Conditioned media from ST2 cells confirmed that IGF-I decreased secreted OPG, reducing levels by 42%, from 12.1-7 ng/ml at 48 h (P < 0.05). Similarly, IGF-I at 100 micro g/liter (13 nM) increased RANKL mRNA expression to 353 +/- 74% above untreated cells as assessed by real-time PCR. In vivo, low doses of rhGH when administered to elderly postmenopausal women only modestly raised serum IGF-I (to concentrations of 18-26 nM) and did not affect circulating OPG concentrations; however, administration of rhIGF-I (30 micro g/kg.d) for 1 yr to older women resulted in a significant increase in serum IGF-I (to concentrations of 39-45 nM) and a 20% reduction in serum OPG (P < 0.05). In summary, we conclude that IGF-I in a dose- and time-dependent manner regulates OPG and RANKL in vitro and in vivo. These data suggest IGF-I may act as a coupling factor in bone remodeling by activating both bone formation and bone resorption; the latter effect appears to be mediated through the OPG/RANKL system in bone.

Mechanical input is known to regulate skeletal mass. In vitro, application of strain inhibits osteoclast formation by decreasing expression of the ligand RANKL in bone stromal cells, but the mechanism responsible for this down-regulation is unknown. In experiments here, application of 1.8% equibiaxial strain for 6 h reduced vitamin D-stimulated RANKL mRNA expression by nearly one-half in primary bone stromal cells. Application of strain caused a rapid activation of ERK1/2, which returned to baseline by 60 minutes. Adding the ERK1/2 inhibitor PD98059 30 minutes before strain delivery prevented the strain effect on RANKL mRNA expression, suggesting that activation of ERK1/2 was required for transduction of the mechanical force. Mechanical strain also activated N-terminal Jun kinase (JNK) that, in contrast, did not return to baseline during 24 h of continuous strain. This suggests that JNK may represent an accessory pathway for mechanical transduction in bone cells. Our data indicate that strain modulation of RANKL expression involves activation of MAPK pathways.

Normal dynamic loading prevents bone resorption; however, the means whereby biophysical factors reduce osteoclast activity are not understood. We show here that mechanical strain (2% at 10 cycles per minute) applied to murine marrow cultures reduced 1,25(OH)2D3-stimulated osteoclast formation by 50%. This was preceded by decreased expression of osteoclast differentiation factor (ODF/TRANCE). RT-PCR for ODF/TRANCE revealed that ODF/TRANCE mRNA in strained cultures was 59 ± 3% of that seen in control cultures. No significant effects on total cell count, thymidine uptake, or alkaline phosphatase activity were induced by strain. To isolate the cell targeted by strain, primary stromal cells were cultured from marrow. Mechanical strain also reduced mRNA for ODF/TRANCE to 60% that of control in these cells. In contrast, mRNA for membrane-bound macrophage colony-stimulating factor was not significantly affected. Soluble ODF (∼2 ng/ml) was able to reverse the effect of strain, returning osteoclast numbers to control. Because osteoclast formation is dependent upon ODF/TRANCE expression, strain-induced reductions in this factor may contribute to the accompanying reduction in osteoclastogenesis.