Mark S. Nanes

Tumor necrosis factor-α: molecular and cellular mechanisms in skeletal pathology

Tumor necrosis factor-alpha (TNF) is one member of a large family of inflammatory cytokines that share common signal pathways, including activation of the transcription factor nuclear factor kappa B (Nf-kappa B) and stimulation of the apoptotic pathway. Data derived from early work supported a role for TNF as a skeletal catabolic agent that stimulates osteoclastogenesis while simultaneously inhibiting osteoblast function. The finding that estrogen deficiency was associated with increased production of cytokines led to a barrage of studies and lively debate on the relative contributions of TNF and other cytokines on bone loss, on the potential cell sources of TNF in the bone microenvironment, and on the mechanism of TNF action. TNF has a central role in bone pathophysiology. TNF is necessary for stimulation of osteoclastogenesis along with the receptor activator of Nf-kappa B ligand (RANKL). TNF also stimulates osteoblasts in a manner that hinders their bone-formative action. TNF suppresses recruitment of osteoblasts from progenitor cells, inhibits the expression of matrix protein genes, and stimulates expression of genes that amplify osteoclastogenesis. TNF may also affect skeletal metabolism by inducing resistance to 1,25-dihydroxyvitamin D(3) (1,25(OH)(2)D(3)) by a mechanism that extends to other members of the steroid hormone nuclear receptor family. Thus, TNF assails bone at many levels. This review will focus on the cellular and molecular mechanisms of TNF action in the skeleton that result in increased bone resorption and impaired formation. TNF and its signal pathway remains an important target for the development of new therapies for bone loss from osteoporosis and inflammatory arthritis.

Inhibition of Osteoblast Differentiation by Tumor Necrosis Factor-α1

Tumor necrosis factor-a (TNF-a) has a key role in skeletal disease in which it promotes reduced bone formation by mature osteoblasts and increased osteoclastic resorption. Here we show that TNF inhibits differentiation of osteoblasts from precursor cells. TNF-a treatment of fetal calvaria precursor cells, which spontaneously differentiate to the osteoblast phenotype over 21 days, inhibited differentiation as shown by reduced formation of multilayered, mineralizing nodules and decreased secretion of the skeletal-specific matrix protein osteocalcin. The effect of TNF was dose dependent with an IC50 of 0.6 ng/ml, indicating a high sensitivity of these precursor cells. Addition of TNF-a from days 2‐21, 2‐14, 7‐14, and 7‐10 inhibited nodule formation but addition of TNF after day 14 had no effect. Partial inhibition of differentiation was observed with addition of TNF on only days 7‐ 8, suggesting that TNF could act during a critical period of phenotype selection. Growth of cells on collagen-coated plates did not prevent TNF inhibition of differentiation, suggesting that inhibition of collagen deposition into matrix by proliferating cells could not, alone, explain the effect of TNF. Northern analysis revealed that TNF inhibited the expression of insulin-like growth factor I (IGF-I). TNF had no effect on expression of the osteogenic bone morphogenic proteins (BMPs-2, -4, and -6), or skeletal LIM protein (LMP-1), as determined by semiquantitative RT-PCR. Addition of IGF-I or BMP-6 to fetal calvaria precursor cell cultures enhanced differentiation but could not overcome TNF inhibition, suggesting that TNF acted downstream of these proteins in the differentiation pathway. The clonal osteoblastic cell line, MC3T3-E1‐14, which acquires the osteoblast phenotype spontaneously in postconfluent culture, was also studied. TNF inhibited differentiation of MC3T3-E1‐14 cells as shown by failure of mineralized matrix formation in the presence of calcium and phosphate. TNF was not cytotoxic to either cell type as shown by continued attachment and metabolism in culture, trypan blue exclusion, and Alamar Blue cytotoxicity assay. These results demonstrate that TNF-a is a potent inhibitor of osteoblast differentiation and suggest that TNF acts distal to IGF-I, BMPs, and LMP-1 in the progression toward the osteoblast phenotype. (Endocrinology 141: 3956 ‐3964, 2000)

Endogenous TNFα Lowers Maximum Peak Bone Mass and Inhibits Osteoblastic Smad Activation Through NF‐κB

Endogenous TNFα prevents the attainment of maximum achievable peak bone mass in vivo. In vitro, TNFα suppresses BMP‐2– and TGFβ‐mediated Smad activation through induction of NF‐κB. Consistently, pharmacological suppression of NF‐κB augments osteoblast differentiation and mineralization in vitro.

T lymphocytes amplify the anabolic activity of parathyroid hormone through Wnt10b signaling.

Intermittent administration of parathyroid hormone (iPTH) is used to treat osteoporosis because it improves bone architecture and strength, but the underlying cellular and molecular mechanisms are unclear. Here, we show that iPTH increases the production of Wnt10b by bone marrow CD8+ T cells and induces these lymphocytes to activate canonical Wnt signaling in preosteoblasts. Accordingly, in responses to iPTH, T cell null mice display diminished Wnt signaling in preosteoblasts and blunted osteoblastic commitment, proliferation, differentiation, and life span, which result in decreased trabecular bone anabolism and no increase in strength. Demonstrating the specific role of lymphocytic Wnt10b, iPTH has no anabolic activity in mice lacking T-cell-produced Wnt10b. Therefore, T-cell-mediated activation of Wnt signaling in osteoblastic cells plays a key permissive role in the mechanism by which iPTH increases bone strength, suggesting that T cell osteoblast crosstalk pathways may provide pharmacological targets for bone anabolism.

Mechanical Strain Differentially Regulates Endothelial Nitric-oxide Synthase and Receptor Activator of Nuclear κB Ligand Expression via ERK1/2 MAPK

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 κB 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.

Transcriptional Regulation of the Osterix (Osx, Sp7) Promoter by Tumor Necrosis Factor Identifies Disparate Effects of Mitogen-activated Protein Kinase and NFκB Pathways

Osteoblast (OB) differentiation is suppressed by tumor necrosis factor-α (TNF-α), an inflammatory stimulus that is elevated in arthritis and menopause. Because OB differentiation requires the expression of the transcription factor osterix (Osx), we investigated TNF effects on Osx. TNF inhibited Osx mRNA in pre-osteoblastic cells without affecting Osx mRNA half-life. Inhibition was independent of new protein synthesis. Analysis of the Osx promoter revealed two transcription start sites that direct the expression of an abundant mRNA (Osx1) and an alternatively spliced mRNA (Osx2). Promoter fragments driving the expression of luciferase were constructed to identify TNF regulatory sequences. Two independent promoters were identified upstream of each transcription start site. TNF potently inhibited transcription of both promoters. Deletion and mutational analysis identified a TNF-responsive region proximal to the Osx2 start site that retained responsiveness when inserted upstream of a heterologous promoter. The TNF response region was a major binding site for nuclear proteins, although TNF did not change binding at the site. The roles of MAPK and NFκB were investigated as signal mediators of TNF. Inhibitors of MEK1 and ERK1, but not of JNK or p38 kinase, abrogated TNF inhibition of Osx mRNA and promoter activity. TNF action was not prevented by blockade of NFκB nuclear entry. The forced expression of high levels of NFκB uncovered a proximal promoter enhancer; however, this site was not activated by TNF. The inhibitory effect of TNF on Osx expression may decrease OB differentiation in arthritis and osteoporosis.

PPARγ regulates hypoxia-induced Nox4 expression in human pulmonary artery smooth muscle cells through NF-κB

NADPH oxidases are a major source of superoxide production in the vasculature. The constitutively active Nox4 subunit, which is selectively upregulated in the lungs of human subjects and experimental animals with pulmonary hypertension, is highly expressed in vascular wall cells. We demonstrated that rosiglitazone, a synthetic agonist of the peroxisome proliferator-activated receptor-γ (PPARγ), attenuated hypoxia-induced pulmonary hypertension, vascular remodeling, Nox4 induction, and reactive oxygen species generation in the mouse lung. The current study examined the molecular mechanisms involved in PPARγ-regulated, hypoxia-induced Nox4 expression in human pulmonary artery smooth muscle cells (HPASMC). Exposing HPASMC to 1% oxygen for 72 h increased Nox4 gene expression and H(2)O(2) production, both of which were reduced by treatment with rosiglitazone during the last 24 h of hypoxia exposure or by treatment with small interfering RNA (siRNA) to Nox4. Hypoxia also increased HPASMC proliferation as well as the activity of a Nox4 promoter luciferase reporter, and these increases were attenuated by rosiglitazone. Chromatin immunoprecipitation assays demonstrated that hypoxia increased binding of the NF-κB subunit, p65, to the Nox4 promoter and that binding was attenuated by rosiglitazone treatment. The role of NF-κB in Nox4 regulation was further supported by demonstrating that overexpression of p65 stimulated Nox4 promoter activity, whereas siRNA to p50 or p65 attenuated hypoxic stimulation of Nox4 promoter activity. These results provide novel evidence for NF-κB-mediated stimulation of Nox4 expression in HPASMC that can be negatively regulated by PPARγ. These data provide new insights into potential mechanisms by which PPARγ activation inhibits Nox4 upregulation and the proliferation of cells in the pulmonary vascular wall to ameliorate pulmonary hypertension and vascular remodeling in response to hypoxia.

Transcriptional regulation of the Osterix (Osx, Sp7) promoter by TNF identifies disparate effects of mitogen activated protein kinase (MAPK) and NFκB pathways

Osteoblast (OB) differentiation is suppressed by tumor necrosis factor-α (TNF-α), an inflammatory stimulus that is elevated in arthritis and menopause. Because OB differentiation requires the expression of the transcription factor osterix (Osx), we investigated TNF effects on Osx. TNF inhibited Osx mRNA in pre-osteoblastic cells without affecting Osx mRNA half-life. Inhibition was independent of new protein synthesis. Analysis of the Osx promoter revealed two transcription start sites that direct the expression of an abundant mRNA (Osx1) and an alternatively spliced mRNA (Osx2). Promoter fragments driving the expression of luciferase were constructed to identify TNF regulatory sequences. Two independent promoters were identified upstream of each transcription start site. TNF potently inhibited transcription of both promoters. Deletion and mutational analysis identified a TNF-responsive region proximal to the Osx2 start site that retained responsiveness when inserted upstream of a heterologous promoter. The TNF response region was a major binding site for nuclear proteins, although TNF did not change binding at the site. The roles of MAPK and NFκB were investigated as signal mediators of TNF. Inhibitors of MEK1 and ERK1, but not of JNK or p38 kinase, abrogated TNF inhibition of Osx mRNA and promoter activity. TNF action was not prevented by blockade of NFκB nuclear entry. The forced expression of high levels of NFκB uncovered a proximal promoter enhancer; however, this site was not activated by TNF. The inhibitory effect of TNF on Osx expression may decrease OB differentiation in arthritis and osteoporosis.

Integration of the NfκB p65 subunit into the vitamin D receptor transcriptional complex: Identification of p65 domains that inhibit 1,25-dihydroxyvitamin D3-stimulated transcription

Resistance to the action of vitamin D (D) occurs in response to tumor necrosis factor‐α (TNF‐α), an effect mediated by nuclear factor kappa B (NfκB). To determine the mechanism of NfκB inhibition of D‐stimulated transcription, chromatin immunoprecipitation assays (CHIP) were done in osteoblastic ROS 17/2.8 cells that had been treated with TNF‐α or transfected with the p65 subunit of NfκB. These treatments caused stable incorporation of p65 into the transcription complex bound to the vitamin D response element (VDRE) of the osteocalcin promoter. Deletion analysis of p65 functional domains revealed that the p65 N‐terminus and a midmolecular region were both required for the inhibitory action of p65. Pull‐down assays were done using an immobilized glutathione S‐transferase (GST)‐VDR fusion protein to study the effect of p65 on VDR binding to steroid coactivator‐1 (SRC‐1), a major D‐dependent coactivator. p65 inhibited VDR‐SRC‐1 binding in a dose‐dependent manner. Mutations of p65 that abrogated the inhibitory effect on D‐stimulated transcription also failed to inhibit VDR‐SRC‐1 binding. The inhibitory effect of p65 on VDR transactivation was not due to recruitment of a histone deacetylase (HDAC), since inhibition was not relieved by the HDAC inhibitors sodium butyrate or trichostatin A. Overexpression of SRC‐1 or the general coactivators, Creb binding protein or SRC‐3, also failed to relieve p65 inhibition of transcription. In addition, Chip assays revealed that TNF‐α treatment prevented D recruitment of SRC‐1 to the transcription complex. These results show that TNF‐α inhibition of vitamin D‐action includes stable integration of p65 in the VDR transcription complex. Once anchored to proteins within the complex, p65 disrupts VDR binding to SRC‐1, thus decreasing the efficiency of D‐stimulated gene transcription.

Evaluation of vitamin D repletion regimens to correct vitamin D status in adults.

OBJECTIVE To determine the efficacy and safety of commonly prescribed regimens for the treatment of vitamin D insufficiency. METHODS We performed a retrospective analysis of 306 consecutive patients who were prescribed ergocalciferol (vitamin D2) for correction of vitamin D insufficiency at the Atlanta Veterans Affairs Medical Center between February 2003 and May 2006. Serum levels of parathyroid hormone, 25-hydroxyvitamin D (25-OHD), and calcium were compared before and after treatment with ergocalciferol. Patients who did not have a 25-OHD determination (n = 41) were excluded from analysis. Vitamin D deficiency, insufficiency, and sufficiency were defined as a serum 25-OHD level of <20 ng/mL, 21 to 29 ng/mL, and > or =30 ng/mL, respectively. RESULTS We identified 36 discrete prescribing regimens. The 3 most common regimens were ergocalciferol 50,000 IU once weekly for 4 weeks followed by 50,000 IU once monthly for 5 months (n = 48); ergocalciferol 50,000 IU once monthly for 6 months (n = 80); and ergocalciferol 50,000 IU 3 times weekly for 6 weeks (n = 27). Each of these 3 treatments significantly increased serum 25-OHD (P<.01), but vitamin D sufficiency was achieved in only 38%, 42%, and 82% of study subjects, respectively. Regimens with >600,000 IU of ergocalciferol given for a mean of 60 +/- 40 days achieved sufficiency in 64% of cases, without vitamin D toxicity. CONCLUSION In this study, regimens that contained at least 600,000 IU of ergocalciferol appeared to be the most effective in achieving vitamin D sufficiency. Guidelines for the treatment of vitamin D insufficiency in healthy adults should be developed.