Osteogenic protein-1 and interleukin-6 with its soluble receptor synergistically stimulate rat osteoblastic cell differentiation

Osteogenic Protein-1 (OP-1, BMP-7), a member of the bone morphogenetic protein family, stimulates synthesis of biochemical markers characteristic of the osteoblastic and chondrocytic phenotypes and induces new bone formation. Interleukin-6 (IL-6), a cytokine produced by a wide variety of cells, appears to interact with other factors producing different biological effects. In the present study, we showed that OP-1 action in fetal rat calvaria (FRC) cells was enhanced by the combination of IL-6 and the soluble receptor IL-6sR. OP-1 alone induced alkaline phosphatase (AP) activity by 4- to 5-fold above the control. Exogenous IL-6 soluble receptor (IL-6sR) synergistically stimulated the OP-1-induced AP activity and mineralized bone nodule formation by an additional 3-fold. The stimulation was IL-6sR concentration-dependent. The combination of IL-6 and IL-6sR synergistically stimulated OP-1 action by an additional 6- to 7-fold. BMPR-II receptor mRNA expression in FRC cells treated with OP-1 and IL-6 plus IL-6sR was stimulated further, while BMPR-IA, -IB, and ActR-I expressions were not affected. The intracellular signaling molecules Smad2 and Smad5 mRNA expressions were not changed under these conditions. The expression of selected BMP family members (BMP-3, -4, and -6) was altered in FRC cells treated with OP-1 in combination with IL-6 and IL-6sR. The combination of IL-6 and IL-6sR reduced the OP-1-stimulated BMP-3 mRNA levels and enhanced the suppressive effect of OP-1 on BMP-4 and -6 mRNA expressions. In conclusion, the present results demonstrate that exogenous IL-6 and IL-6sR synergistically stimulate OP-1 action in primary cultures of rat osteoblastic cells. One possible mechanism of synergy involves differential regulation of the effects of OP-1 on the expression of the type II BMP receptor and several other BMPs.     

Endogenous production of tumor necrosis factor by primary cultures of murine calvarial cells: influence on IL-6 production and osteoclast development

We have previously shown that tumor necrosis factor (TNF) and interleukin-1 (IL-1) acted synergistically to stimulate the production of IL-6 by bone marrow stromal and osteoblastic cells; and that an antibody to IL-6 inhibited TNF-induced osteoclast development in murine calvarial cell cultures. Prompted by this evidence, we have now examined whether TNF and/or IL-1 are produced by murine calvarial cells, and whether these cytokines are involved in IL-6 production and osteoclast formation. When cultured under basal conditions, calvarial cells produced TNF and IL-6, and were able to form bone resorbing osteoclasts. A neutralizing antibody against TNF suppressed both basal IL-6 production and the formation of bone resorbing osteoclasts. The anti-TNF antibody also inhibited IL-6 production in response to exogenous IL-1 or parathyroid hormone (PTH). In contrast, a neutralizing anti-IL-1 receptor antibody had no effect on basal, TNF- or PTH-stimulated IL-6 production. These findings suggest that TNF, but not IL-1, is produced by murine bone cells and that endogenous TNF induces the IL-6 production, osteoclast formation, and bone resorption exhibited by these cultures under basal conditions. Furthermore, bone cell-derived TNF amplifies the stimulatory effect of exogenous IL-1 or PTH on IL-6 production by calvarial cells.     

Differential stimulation by PGE(2) and calcemic hormones of IL-6 in stromal/osteoblastic cells

Formation of osteoclast-like cells in mouse bone marrow cultures induced by either 1,25-dihydroxyvitamin D(3) (1,25-(OH)(2)D(3)), parathyroid hormone (PTH) or prostaglandin E(2) (PGE(2)), respectively, shows partial dependence on interleukin-6 receptor (IL-6R) activation. This suggests that locally produced IL-6 could be relevant for osteoclast formation. Therefore, we evaluated the effects of 1,25-(OH)(2)D(3), PTH, and PGE(2) on IL-6 production in stromal/osteoblastic cell lines. It appeared that these bone resorptive factors differed widely in their ability to modulate IL-6 mRNA expression and, consequently, protein synthesis in each of the cell lines studied. While 1,25-(OH)(2)D(3) was marginally effective only in ST2 cells, and PTH caused a 2- to 20-fold increase in IL-6 levels MC3T3-E1 and UMR-106 cells, PGE(2) enhanced IL-6 production in the ST2 and MC3T3-E1 cell line by two to three orders of magnitude, respectively, and also induced IL-6 in fibroblastic L929 cells. PGE(2)-stimulated IL-6 release from mesenchymal cells seems to be important for autocrine/paracrine control of osteoclast formation in health and disease.     

Nutritional status and postoperative cytokine response in colorectal cancer patients

The present study was designed to investigate the relationship between pre-operative nutritional status and peri-operative regulation of the cytokine network, and to clarify its relation to clinical outcome in colorectal cancer patients. Protein-energy malnutrition was assessed using the creatinine height index. Peripheral venous blood samples were obtained peri-operatively, and the serum concentrations of interleukin (IL-)6, IL-1 receptor antagonist (ra), IL-6 soluble receptor (sR) C-reactive protein (CRP) and the percentage of peripheral neutrophils were determined. Excessive operative blood loss was associated with postoperative morbidity. Pre-operative malnutrition was associated with postoperative mortality when excessive bleeding occurred. Postoperative IL-6 response was exaggerated and postoperative IL-1ra response was suppressed in nutritionally depleted patients. The postoperative serum concentrations of IL-6sR in malnourished patients remained at the lowest levels when excessive bleeding occurred. In these patients, the percentage of peripheral neutrophils remained at high levels even after resolution of the postoperative cytokine storm. A marked activation of the pro-inflammatory cytokine network associated with a decreased antagonistic reaction and an increased consumption of IL-6sR became prominent in malnourished patients when they underwent intense surgical stress. These immunological disturbances may be relevant to neutrophil activation and subsequent clinical outcome.     

Involvement of PKC-beta in PTH, TNF-alpha, and IL-1 beta effects on IL-6 promoter in osteoblastic cells and on PTH-stimulated bone resorption

Protein kinase C (PKC) has been shown to be activated by parathyroid hormone (PTH) in osteoblasts. Prior evidence suggests that this activation mediates responses leading to bone resorption, including production of the osteoclastogenic cytokine interleukin-6 (IL-6). However, the importance of specific PKC isozymes in this process has not been investigated. A selective antagonist of PKC-beta, LY379196, was used to determine the role of the PKC-beta isozyme in the expression of IL-6 in UMR-106 rat osteoblastic cells and in bone resorption in fetal rat limb bone organ cultures. PTH, tumor necrosis factor-alpha (TNF-alpha), and interleukin-1 beta (IL-1 beta) induced translocation of PKC-alpha and -beta(I) to the plasma membrane in UMR-106 cells within 5 min. The stimulation of PKC-beta(I) translocation by PTH, TNF-alpha or IL-1 beta was inhibited by LY379196. In contrast, LY379196 did not affect PTH, TNF-alpha-, or IL-1 beta-stimulated translocation of PKC-alpha. PTH, TNF-alpha, and IL-1 beta increased luciferase expression in UMR-106 cells transiently transfected with a -224/+11 bp IL-6 promoter-driven reporter construct. The IL-6 responses were also attenuated by treatment with LY379196. Furthermore, LY379196 inhibited bone resorption elicited by PTH in fetal rat bone organ cultures. These results indicate that PKC-beta(I) is a component of the signaling pathway that mediates PTH-, TNF-alpha-, and IL-1 beta-stimulated IL-6 expression and PTH-stimulated bone resorption.     

Suppression of IL-2-induced SAA gene expression in mice by the administration of an IL-1 receptor antagonist

The hepatic acute phase response induced by the administration of interleukin (IL)-2 is most likely mediated by secondary cytokines. In this investigation, we examined the role of endogenous IL-1 in the synthesis of the hepatic acute phase protein serum amyloid A (SAA) during IL-2 treatment. The injection of IL-2 induced SAA gene expression in the liver. The concurrent administration of an IL-1 receptor antagonist (IL-1RA) markedly reduced hepatic SAA mRNA levels and, to a lesser extent, SAA protein levels in the serum. Although IL-1 is an inducer of IL-6 production, the administration of the IL-1RA had no effect on circulating IL-6 levels in IL-2-treated mice. These findings suggest that the production of IL-1 is an important factor in the induction of SAA mRNA in mice undergoing immunotherapy with IL-2.     

Parathyroid hormone (PTH) and PTH-like protein (PLP) stimulate interleukin-6 production by osteogenic cells: a possible role of interleukin-6 in osteoclastogenesis

Osteogenic cells mediate PTH-stimulated osteoclastic bone resorption by a yet unidentified mechanism. We show that primairy rat osteoblast-like cells and the clonal osteogenic sarcoma cell line UMR-106 produce interleukin-6 (IL-6) and that bPTH(1-84) and synthetic hPLP(1-34) stimulate this production dose-dependently. With both peptides a close relation between IL-6 and cyclic-AMP production was found, though for PTH concentrations higher than 2.10(-8) M a clear dissociation was observed. Significant IL-6 activity was also detected in media of cultures of 17-day-old fetal mouse radii and metacarpals which was clearly stimulated by PTH. The source of IL-6 in these bone explants seems to be the osteogenic (cartilage) cells. Treatment of bone explants with IL-6 induced osteoclastic resorption which, however, depended on the bone resorption system used. This bone resorbing action of IL-6 is exerted probably through an effect on the formation of osteoclasts (osteoclastogenesis) rather than on the activation of already existing mature osteoclasts. We suggest that IL-6 produced by osteogenic cells may be a mediator in PTH-stimulated osteoclastic bone resorption.     

Mechanisms involved in prostaglandin-induced increase in bone resorption in neonatal mouse calvaria

Prostaglandins (PG) E1, E2 and F2alpha induce bone resorption in isolated neonatal parietal bone cultures, and an associated increase in interleukin-6 (IL-6) production. Indomethacin had little effect on the response to PGE2, or the relatively non-selective EP receptor agonists 11-deoxy PGE1 and misoprostol, but blocked the effects of PGF2alpha and the F receptor agonist fluprostenol, indicating an indirect action via release of other prostaglandins. It is more likely that there is positive autoregulation of prostaglandins production in this preparation mediated via stimulation of F receptors. The effects of selective EP receptor agonists sulprostone (EP1,3) and 17-phenyl trinor PGE2(EP1), indicated the involvement of EP2 and/or EP4 receptors, which signal via cAMP. The relatively weak increase in IL-6 production by misoprostol (with respect to resorption) suggests that these responses are controlled by different combination of EP2 and EP4 receptors. The PKA activator, forskolin, induced small increases in bone resorption at lower concentrations (50-500 ng/ml) but a reversal of this effect, and inhibition of resorption induced by other stimuli (PTH, PGE2), at higher concentrations (0.5-5 microg/ml). IL-6 production was markedly increased only at the higher concentrations. The inhibitory effect of forskolin may be a calcitonin-mimetic effect. PMA induced both resorption and IL-6 production which were both blocked by indomethacin, indicating a role for PKC in the control of prostaglandin production.     

Parathyroid hormone mediates hematopoietic cell expansion through interleukin-6

Parathyroid hormone (PTH) stimulates hematopoietic cells through mechanisms of action that remain elusive. Interleukin-6 (IL-6) is upregulated by PTH and stimulates hematopoiesis. The purpose of this investigation was to identify actions of PTH and IL-6 in hematopoietic cell expansion. Bone marrow cultures from C57B6 mice were treated with fms-like tyrosine kinase-3 ligand (Flt-3L), PTH, Flt-3L plus PTH, or vehicle control. Flt-3L alone increased adherent and non-adherent cells. PTH did not directly impact hematopoietic or osteoclastic cells but acted in concert with Flt-3L to further increase cell numbers. Flt-3L alone stimulated proliferation, while PTH combined with Flt-3L decreased apoptosis. Flt-3L increased blasts early in culture, and later increased CD45(+) and CD11b(+) cells. In parallel experiments, IL-6 acted additively with Flt-3L to increase cell numbers and IL-6-deficient bone marrow cultures (compared to wildtype controls) but failed to amplify in response to Flt-3L and PTH, suggesting that IL-6 mediated the PTH effect. In vivo, PTH increased Lin(-) Sca-1(+)c-Kit(+) (LSK) hematopoietic progenitor cells after PTH treatment in wildtype mice, but failed to increase LSKs in IL-6-deficient mice. In conclusion, PTH acts with Flt-3L to maintain hematopoietic cells by limiting apoptosis. IL-6 is a critical mediator of bone marrow cell expansion and is responsible for PTH actions in hematopoietic cell expansion.     

Pro-inflammatory cytokines downregulate platelet derived growth factor-α receptor gene expression in human osteoblastic cells

Platelet derived growth factor (PDGF) is thought to play a significant role in bone repair and regeneration. We previously demonstrated that PDGF-AA binding can be modulated by interleukin-1 (IL-1). We now report that TNF-α significantly reduces PDGF-AA binding by decreasing the number of PDGF-α receptor subunits on the surface of normal human osteoblastic cells. This inhibition is likely due to a decrease in synthesis of PDGF-α receptors since TNF-α causes a relatively rapid decrease in PDGF-α receptor mRNA levels as determined by Northern blot analysis. The physiologic importance of this inhibition is demonstrated by a TNF-α induced decrease in PDGF-AA stimulated tyrosine kinase activity. When saturating concentrations of TNF-α were used, the addition of IL-1 further inhibited PDGF-AA binding and further decreased surface expression of PDGF-α receptors. In contrast, other mediators such as IL-6, PTH, 1,25(OH)₂ vit D₃, hydrocortisone, PGE₂, bFGF, and IGF-1 had no effect. These results suggest that binding to the PDGF-α receptor is decreased by the strong pro-inflammatory cytokines such as IL-1β and TNF-α rather than as a general response to mediators important in bone resorption or bone formation. TNF-α and IL-1 are often co-expressed during destructive inflammatory processes. Thus, TNF-α and IL-1 may work in concert to limit the response of osteoblastic cells to PDGF-AA during periods of osseous inflammation. © 1996 Wiley-Liss, Inc.     

IL-6 is not required for parathyroid hormone stimulation of RANKL expression, osteoclast formation, and bone loss in mice

Continuous elevation of parathyroid hormone (PTH) increases osteoclast precursors, the number of osteoclasts on cancellous bone, and bone turnover. The essential molecular mediators of these effects are controversial, however, and both increased receptor activator of NF-kappaB ligand (RANKL) and IL-6 have been implicated. The goal of these studies was to determine whether continuous elevation of endogenous PTH alters IL-6 gene expression in vivo and whether IL-6 is required for PTH-induced bone loss. To accomplish this, we generated transgenic mice harboring a luciferase reporter gene under the control of IL-6 gene regulatory regions to allow accurate quantification of IL-6 gene activity in vivo. In these mice, induction of secondary hyperparathyroidism using a calcium-deficient diet did not alter IL-6-luciferase transgene expression, whereas RANKL mRNA expression was elevated in bone tissue. Moreover, secondary hyperparathyroidism induced an equivalent amount of bone loss in wild-type and IL-6-deficient mice, and PTH elevated RANKL mRNA and osteoclast formation to the same extent in bone marrow cultures derived from wild-type and IL-6-deficient mice. These results demonstrate that IL-6 is not required for the osteoclast formation and bone loss that accompanies continuous elevation of PTH.     

Involvement of PKC-β in PTH, TNF-α, and IL-1β Effects on IL-6 Promoter in Osteoblastic Cells and on PTH-Stimulated Bone Resorption

Protein kinase C (PKC) has been shown to be activated by parathyroid hormone (PTH) in osteoblasts. Prior evidence suggests that this activation mediates responses leading to bone resorption, including production of the osteoclastogenic cytokine interleukin-6 (IL-6). However, the importance of specific PKC isozymes in this process has not been investigated. A selective antagonist of PKC-β, LY379196, was used to determine the role of the PKC-β isozyme in the expression of IL-6 in UMR-106 rat osteoblastic cells and in bone resorption in fetal rat limb bone organ cultures. PTH, tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) induced translocation of PKC-α and -β_I to the plasma membrane in UMR-106 cells within 5 min. The stimulation of PKC-β_I translocation by PTH, TNF-α or IL-1β was inhibited by LY379196. In contrast, LY379196 did not affect PTH, TNF-α-, or IL-1β-stimulated translocation of PKC-α. PTH, TNF-α, and IL-1β increased luciferase expression in UMR-106 cells transiently transfected with a −224/+11 bp IL-6 promoter-driven reporter construct. The IL-6 responses were also attenuated by treatment with LY379196. Furthermore, LY379196 inhibited bone resorption elicited by PTH in fetal rat bone organ cultures. These results indicate that PKC-β_I is a component of the signaling pathway that mediates PTH-, TNF-α-, and IL-1β-stimulated IL-6 expression and PTH-stimulated bone resorption.     

Normal platelets possess the soluble form of IL-6 receptor

Interleukin 6 is a multifunctional cytokine that exerts its biological activity through binding to an 80 Kd specific receptor (IL-6Ralpha) and a 130 Kd signal-transducing unit (gp130). A 55 Kd soluble IL-6R (IL-6sR) has also been described which, after binding to IL-6 is also able to activate gp130. The presence of IL-6Ralpha was described in some megakaryoblastic cell lines but is still controversial in normal megakaryocytes. In this study we demonstrate the presence of intraplatelet IL-6sR by Western blot through the appearance of a 55 Kd protein and the finding of detectable amounts of IL-6sR in the platelet content by ELISA technique. Besides, we showed IL-6sR release during platelet activation induced by thrombin and a complex of ADP and epinephrine. IL-6Ralpha on platelet membrane could not be found neither by Western blot nor by flow cytometry. The IL-6sR released during platelet activation and complexed to IL-6 could act on cell types such as endothelial cells that do not possess IL-6Ralpha through binding to gp130.Besides, since we could not find IL-6R on platelet membrane, the potentiating effect of IL-6 on platelet function could be explained through binding of IL-6sR/IL-6 complex to platelet membrane gp130.     

Mechanisms underlying catabolic and anabolic functions of parathyroid hormone on bone by combination of culture systems of mouse cells

Since bone resorption and formation by continuous and intermittent parathyroid hormone (PTH) treatments involve various types of cells in bone, this study examined the underlying mechanism by combining culture systems using mouse primary calvarial osteoblasts and bone marrow cells. The PTH/PTHrP receptor (PTH1R) expression and the cAMP accumulation in response to PTH were increased in accordance with the differentiation of osteoblasts. Osteoclast formation was strongly induced by continuous PTH treatment in the monolayer co‐culture of osteoblasts and bone marrow cells, which was associated with RANKL expression in differentiated osteoblasts. Bone formation determined by ALP activity and the type I collagen mRNA expression was stimulated by intermittent PTH treatment in the monolayer co‐culture and in the bone marrow cell layer of the separated co‐culture in a double chamber dish, but not in the culture of bone marrow cells alone. The stimulation in the separated co‐culture, accompanied by IGF‐I production by osteoblasts, was abolished when bone marrow cells were derived from knockout mice of insulin‐receptor substrate‐1 (IRS‐1?/?) or when osteoblasts were from PTH1R?/? mice. We conclude that differentiated osteoblasts are most likely the direct target of both continuous and intermittent PTH, while bone marrow cells are likely the effector cells. The osteoblasts stimulated by continuous PTH express RANKL which causes osteoclastogenesis from the precursors in bone marrow via cell‐to‐cell contact, leading to bone resorption; while the osteoblasts stimulated by intermittent PTH secrete IGF‐I which activates IRS‐1 in osteoblast precursors in bone marrow via a paracrine mechanism, leading to bone formation. J. Cell. Biochem. 109: 755–763, 2010. © 2010 Wiley‐Liss, Inc.     

Repression of farnesoid X receptor during the acute phase response

The acute phase response is associated with changes in the hepatic expression of genes involved in lipid metabolism. Nuclear hormone receptors that heterodimerize with retinoid X receptor (RXR), such as thyroid receptors, peroxisome proliferator-activated receptors, and liver X receptors, modulate lipid metabolism. We recently demonstrated that these nuclear hormone receptors are repressed during the acute phase response induced by lipopolysaccharide (LPS), consistent with the known decreases in genes that they regulate. In the present study, we show that LPS significantly decreases farnesoid X receptor (FXR) mRNA in mouse liver as early as 8 h after LPS administration, and this decrease was dose-dependent with the half-maximal effect observed at 0.5 microg/100 g of body weight. Gel-shift experiments demonstrated that DNA binding activity to an FXR response element (IR1) is significantly reduced by LPS treatment. Supershift experiments demonstrated that the shifted protein-DNA complex contains FXR and RXR. Furthermore, the expression of FXR target genes, SHP and apoCII, were significantly reduced by LPS (70 and 60%, respectively). Also, LPS decreases hepatic LRH expression in mouse, which may explain the reduced expression of CYP7A1 in the face of SHP repression. In Hep3B human hepatoma cells, both tumor necrosis factor (TNF) and interleukin-1 (IL-1) significantly decreased FXR mRNA, whereas IL-6 did not have any effect. TNF and IL-1 also decreased the DNA binding activity to an IR1 response element and the expression of SHP and apoCII. Importantly, TNF and IL-1 almost completely blocked the expression of luciferase activity linked to a FXR response element promoter construct transfected into Hep3B cells. Together with our earlier studies on the repression of RXRs, peroxisome proliferator-activated receptors, LXRs, thyroid receptors, constitutive androstane receptor, and pregnane X receptor, these results suggest that decreases in nuclear hormone receptors are major contributors to the decreased gene expression that occurs in the negative acute phase response.     

Primary hyperparathyroidism is associated with increased circulating bone marrow-derived progenitor cells

Recently, parathyroid hormone (PTH) was shown to support survival of progenitor cells in bone marrow. The release of progenitor cells occurs in physiological and pathological conditions and was shown to contribute to neovascularization in tumors and ischemic tissues. In the present study we sought to investigate prospectively the effect of primary hyperparathyroidism (PHPT) on mobilization of bone marrow-derived progenitor cells. In 22 patients with PHPT and 10 controls, defined subpopulations of circulating bone marrow-derived progenitor cells (BMCs) were analyzed by flow cytometry (CD45(+)/CD34(+)/CD31(+) cells indicating endothelial progenitor cells, CD45(+)/CD34(+)/c-kit(+) cells indicating hematopoietic stem cells, and CD45(+)/CD34(+)/CXCR4(+) cells indicating progenitor cells with the homing receptor CXCR4). Cytokine serum levels (SCF, SDF-1, VEGF, EPO, and G-CSF) were assessed using ELISA. Levels of PTH and thyroid hormone as well as serum electrolytes, renal and liver parameters, and blood count were analyzed. Our data show for the first time a significant increase of circulating BMCs and an upregulation of SDF-1 and VEGF serum levels in patients with PHPT. The number of circulating BMCs returned to control levels measured 16.7 +/- 2.3 mo after surgery. There was a positive correlation of PTH levels with the number of CD45(+)/CD34(+)/CD31(+), CD45(+)/CD34(+)/c-kit(+), and CD45(+)/CD34(+)/CXCR4(+) cells. However, there was no correlation between cytokine serum concentrations (SDF-1, VEGF) and circulating BMCs. Serum levels of G-CSF, EPO, and SCF known to mobilize BMCs were even decreased or remained unchanged, suggesting a direct effect of PTH on stem cell mobilization. Our data suggest a new function of PTH mobilizing BMCs into peripheral blood.     

Atherogenic phospholipids attenuate osteogenic signaling by BMP-2 and parathyroid hormone in osteoblasts

Cardiovascular disease, such as atherosclerosis, has been associated with reduced bone mineral density and fracture risk. A major etiologic factor in atherogenesis is believed to be oxidized phospholipids. We previously found that these phospholipids inhibit spontaneous osteogenic differentiation of marrow stromal cells, suggesting that they may account for the clinical link between atherosclerosis and osteoporosis. Currently, anabolic agents that promote bone formation are increasingly used as a new treatment for osteoporosis. It is not known, however, whether atherogenic phospholipids alter the effects of bone anabolic agents, such as bone morphogenetic protein (BMP)-2 and parathyroid hormone (PTH). Therefore we investigated the effects of oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (ox-PAPC) on osteogenic signaling induced by BMP-2 and PTH in MC3T3-E1 cells. Results showed that ox-PAPC attenuated BMP-2 induction of osteogenic markers alkaline phosphatase and osteocalcin. Ox-PAPC also inhibited both spontaneous and BMP-induced expression of PTH receptor. Consistently, pretreatment of cells with ox-PAPC inhibited PTH-induced cAMP production and expression of immediate early genes Nurr1 and IL-6. Results from immunofluorescence and Western blot analyses showed that inhibitory effects of ox-PAPC on BMP-2 signaling were associated with inhibition of SMAD 1/5/8 but not p38-MAPK activation. These effects appear to be due to ox-PAPC activation of the ERK pathway, as the ERK inhibitor PD98059 reversed ox-PAPC inhibitory effects on BMP-2-induced alkaline phosphatase activity, osteocalcin expression, and SMAD activation. These results suggest that atherogenic lipids inhibit osteogenic signaling induced by BMP-2 and PTH, raising the possibility that hyperlipidemia and atherogenic phospholipids may interfere with anabolic therapy.