Caspase-2 deficiency protects mice from diabetes-induced marrow adiposity

Type I (T1) diabetes is an autoimmune and metabolic disease associated with bone loss. Bone formation and density are decreased in T1-diabetic mice. Correspondingly, the number of TUNEL positive, dying osteoblasts increases in bones of T1-diabetic mice. Moreover, two known mediators of osteoblast death, TNFα and ROS, are increased in T1-diabetic bone. TNFα and oxidative stress are known to activate caspase-2, a factor involved in the extrinsic apoptotic pathway. Therefore, we investigated the requirement of caspase-2 for diabetes-induced osteoblast death and bone loss. Diabetes was induced in 16-week old C57BL/6 caspase-2 deficient mice and their wild type littermates and markers of osteoblast death, bone formation and resorption, and marrow adiposity were examined. Despite its involvement in extrinsic cell death, deficiency of caspase-2 did not prevent or reduce diabetes-induced osteoblast death as evidenced by a twofold increase in TUNEL positive osteoblasts in both mouse genotypes. Similarly, deficiency of caspase-2 did not prevent T1-diabetes induced bone loss in trabecular bone (BV/TV decreased by 30 and 50%, respectively) and cortical bone (decreased cortical thickness and area with increased marrow area). Interestingly, at this age, differences in bone parameters were not seen between genotypes. However, caspase-2 deficiency attenuated diabetes-induced bone marrow adiposity and adipocyte gene expression. Taken together, our data suggest that caspase-2 deficiency may play a role in promoting marrow adiposity under stress or disease conditions, but it is not required for T1-diabetes induced bone loss.     

Inhibition of PPARgamma prevents type I diabetic bone marrow adiposity but not bone loss

Diabetes type I is associated with bone loss and increased bone adiposity. Osteoblasts and adipocytes are both derived from mesenchymal stem cells located in the bone marrow, therefore we hypothesized that if we could block adipocyte differentiation we might prevent bone loss in diabetic mice. Control and insulin-deficient diabetic BALB/c mice were chronically treated with a peroxisomal proliferator-activated receptor gamma (PPARgamma) antagonist, bisphenol-A-diglycidyl ether (BADGE), to block adipocyte differentiation. Effects on bone density, adiposity, and gene expression were measured. BADGE treatment did not prevent diabetes-associated hyperglycemia or weight loss, but did prevent diabetes-induced hyperlipidemia and effectively blocked diabetes type I-induced bone adiposity. Despite this, BADGE treatment did not prevent diabetes type I suppression of osteoblast markers (runx2 and osteocalcin) and bone loss (as determined by micro-computed tomography). BADGE did not suppress osteoblast gene expression or bone mineral density in control mice, however, chronic (but not acute) BADGE treatment did suppress osteocalcin expression in osteoblasts in vitro. Taken together, our findings suggest that BADGE treatment is an effective approach to reduce serum triglyceride and free fatty acid levels as well as bone adiposity associated with type I diabetes. The inability of BADGE treatment to prevent bone loss in diabetic mice suggests that marrow adiposity is not linked to bone density status in type I diabetes, but we cannot exclude the possibility of additional BADGE effects on osteoblasts or other bone cells, which could contribute to preventing the rescue of the bone phenotype.     

Chronic hyperglycemia modulates osteoblast gene expression through osmotic and non-osmotic pathways

Insulin dependent diabetes mellitus (IDDM; type I) is a chronic disease stemming from little or no insulin production and elevated blood glucose levels. IDDM is associated with osteoporosis and increased fracture rates. The mechanisms underlying IDDM associated bone loss are not known. Previously we demonstrated that osteoblasts exhibit a response to acute (1 and 24 h) hyperglycemia and hyperosmolality. Here we examined the influence of chronic hyperglycemia (30 mM) and its associated hyperosmolality on osteoblast phenotype. Our findings demonstrate that osteoblasts respond to chronic hyperglycemia through modulated gene expression. Specifically, chronic hyperglycemia increases alkaline phosphatase activity and expression and decreases osteocalcin, MMP-13, VEGF and GAPDH expression. Of these genes, only MMP-13 mRNA levels exhibit a similar suppression in response to hyperosmotic conditions (mannitol treatment). Acute hyperglycemia for a 48-h period was also capable of inducing alkaline phosphatase and suppressing osteocalcin, MMP-13, VEGF, and GAPDH expression in differentiated osteoblasts. This suggests that acute responses in differentiated cells are maintained chronically. In addition, hyperglycemic and hyperosmotic conditions increased PPARgamma2 expression, although this increase reached significance only in 21 days chronic glucose treated cultures. Given that osteocalcin is suppressed and PPARgamma2 expression is increased in type I diabetic mouse model bones, these findings suggest that diabetes-associated hyperglycemia may modulate osteoblast gene expression, function and bone formation and thereby contribute to type I diabetic bone loss.     

Amelioration of type I diabetes-induced osteoporosis by parathyroid hormone is associated with improved osteoblast survival

Type 1 diabetic osteoporosis results from impaired osteoblast activity and death. Therefore, anti-resorptive treatments may not effectively treat bone loss in this patient population. Intermittent parathyroid hormone (PTH) treatment stimulates bone remodeling and increases bone density in healthy subjects. However, PTH effects may be limited in patients with diseases that interfere with its signaling. Here, we examined the ability of 8 and 40 μg/kg intermittent PTH to counteract diabetic bone loss. PTH treatment reduced fat pad mass and blood glucose levels in non-diabetic PTH-treated mice, consistent with PTH-affecting glucose homeostasis. However, PTH treatment did not significantly affect general body parameters, including the blood glucose levels, of type 1 diabetic mice. We found that the high dose of PTH significantly increased tibial trabecular bone density parameters in control and diabetic mice, and the lower dose elevated trabecular bone parameters in diabetic mice. The increased bone density was due to increased mineral apposition and osteoblast surface, all of which are defective in type 1 diabetes. PTH treatment suppressed osteoblast apoptosis in diabetic bone, which could further contribute to the bone-enhancing effects. In addition, PTH treatment (40 μg/kg) reversed preexisting bone loss from diabetes. We conclude that intermittent PTH may increase type 1 diabetic trabecular bone volume through its anabolic effects on osteoblasts.     

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.     

CCAAT/enhancer binding protein β-deficiency enhances type 1 diabetic bone phenotype by increasing marrow adiposity and bone resorption

Bone loss in type 1 diabetes is accompanied by increased marrow fat, which could directly reduce osteoblast activity or result from altered bone marrow mesenchymal cell lineage selection (adipocyte vs. osteoblast). CCAAT/enhancer binding protein beta (C/EBPβ) is an important regulator of both adipocyte and osteoblast differentiation. C/EBPβ-null mice have delayed bone formation and defective lipid accumulation in brown adipose tissue. To examine the balance of C/EBPβ functions in the diabetic context, we induced type 1 diabetes in C/EBPβ-null (knockout, KO) mice. We found that C/EBPβ deficiency actually enhanced the diabetic bone phenotype. While KO mice had reduced peripheral fat mass compared with wild-type mice, they had 5-fold more marrow adipocytes than diabetic wild-type mice. The enhanced marrow adiposity may be attributed to compensation by C/EBPδ, peroxisome proliferator-activated receptor-γ2, and C/EBPα. Concurrently, we observed reduced bone density. Relative to genotype controls, trabecular bone volume fraction loss was escalated in diabetic KO mice (-48%) compared with changes in diabetic wild-type mice (-22%). Despite greater bone loss, osteoblast markers were not further suppressed in diabetic KO mice. Instead, osteoclast markers were increased in the KO diabetic mice. Thus, C/EBPβ deficiency increases diabetes-induced bone marrow (not peripheral) adipose depot mass, and promotes additional bone loss through stimulating bone resorption. C/EBPβ-deficiency also reduced bone stiffness and diabetes exacerbated this (two-way ANOVA P < 0.02). We conclude that C/EBPβ alone is not responsible for the bone vs. fat phenotype switch observed in T1 diabetes and that suppression of CEBPβ levels may further bone loss and decrease bone stiffness by increasing bone resorption.     

Augmented LPS responsiveness in type 1 diabetes‐derived osteoclasts

Bone abnormalities are frequent co‐morbidities of type 1 diabetes (T1D) and are principally mediated by osteoblasts and osteoclasts which in turn are regulated by immunologic mediators. While decreased skeletal health in T1D involves alterations in osteoblast maturation and function, the effect of altered immune function on osteoclasts in T1D‐associated bone and joint pathologies is less understood. Here T1D‐associated osteoclast‐specific differentiation and function in the presence and absence of inflammatory mediators was characterized utilizing bone marrow‐derived osteoclasts (BM‐OCs) isolated from non‐obese diabetic (NOD) mice, a model for spontaneous autoimmune diabetes with pathology similar to individuals with T1D. Differentiation and osteoclast‐mediated bone resorption were evaluated along with cathepsin K, MMP‐9, and immune soluble mediator expression. The effect of lipopolysaccharide (LPS), a pro‐inflammatory cytokine cocktail, and NOD‐derived conditioned supernatants on BM‐OC function was also determined. Although NOD BM‐OCs cultures contained smaller osteoclasts, they resorbed more bone concomitant with increased cathepsin K, MMP‐9, and pro‐osteoclastogenic mediator expression. NOD BM‐OCs also displayed an inhibition of LPS‐induced deactivation that was not a result of soluble mediators produced by NOD BM‐OCs, although a pro‐inflammatory milieu did enhance NOD BM‐OCs bone resorption. Together these data indicate that osteoclasts from a T1D mouse model hyper‐respond to RANK‐L resulting in excessive bone degradation via enhanced cathepsin K and MMP‐9 secretion concomitant with an increased expression of pro‐osteoclastic soluble mediators. Our data also suggest that inhibition of LPS‐induced deactivation in NOD‐derived BM‐OC cultures is most likely due to NOD osteoclast responsiveness rather than LPS‐induced expression of soluble mediators. J. Cell. Physiol. 228: 349–361, 2013. © 2012 Wiley Periodicals, Inc.     

Gene array profile identifies collagen type XV as a novel human osteoblast‐secreted matrix protein

Bone marrow stromal cells (MSCs) and osteoblasts are the two main non‐haematopoietic cellular components of human bone tissue. To identify novel osteoblast‐related molecules, we performed a gene expression profiling analysis comparing MSCs and osteoblasts isolated from the same donors. Genes differentially overexpressed in osteoblasts were mainly related to the negative control of cell proliferation, pro‐apoptotic processes, protein metabolism and bone remodelling. Notably, we also identified the collagen XV (COL15A1) gene as the most up‐regulated gene in osteoblasts compared with MSCs, previously described as being expressed in the basement membrane in other cell types. The expression of collagen type XV was confirmed at the protein level on isolated osteoblasts and we demonstrated that it significantly increases during the osteogenic differentiation of MSCs in vitro and that free ionised extracellular calcium significantly down‐modulates its expression. Moreover, light and electron microscopy showed that collagen type XV is expressed in bone tissue biopsies mainly by working osteoblasts forming new bone tissue or lining bone trabeculae. To our knowledge, these data represent the first evidence of the expression of collagen type XV in human osteoblasts, a calcium‐regulated protein which correlates to a specific functional state of these cells. J. Cell. Physiol. 220: 401–409, 2009. © 2009 Wiley‐Liss, Inc.     

Bone inflammation and altered gene expression with type I diabetes early onset

Type I diabetes is associated with bone loss and marrow adiposity. To identify early events involved in the etiology of diabetic bone loss, diabetes was induced in mice by multiple low dose streptozotocin injections. Serum markers of bone metabolism and inflammation as well as tibial gene expression were examined between 1 and 17 days post‐injection (dpi). At 3 dpi, when blood glucose levels were significantly elevated, body, fat pad and muscle mass were decreased. Serum markers of bone resorption and formation significantly decreased at 5 dpi in diabetic mice and remained suppressed throughout the time course. An osteoclast gene, TRAP5 mRNA, was suppressed at early and late time points. Suppression of osteogenic genes (runx2 and osteocalcin) and induction of adipogenic genes (PPARγ2 and aP2) were evident as early as 5 dpi. These changes were associated with an elevation of serum cytokines, but more importantly we observed an increase in the expression of cytokines in bone, supporting the idea that bone, itself, exhibits an inflammatory response during diabetes induction. This inflammation could in turn contribute to diabetic bone pathology. IFN‐γ (one of the key cytokines elevated in bone and known to be involved in bone regulation) deficiency did not prevent diabetic bone pathology. Taken together, our findings indicate that bone becomes inflamed with the onset of T1‐diabetes and during this time bone phenotype markers become altered. However, inhibition of one cytokine, IFN‐γ was not sufficient to prevent the rapid bone phenotype changes. J. Cell. Physiol. 218: 575–583, 2009. © 2008 Wiley‐Liss, Inc.     

PPAR agonists stimulate adipogenesis at the expense of osteoblast differentiation while inhibiting osteoclast formation and activity

Drugs used in the treatment of type 2 diabetes and cardiovascular disease, specifically peroxisome proliferator‐activated receptor (PPAR) agonists, have been reported to affect bone cell function and fracture risk. In this study, we assessed the direct effects of PPAR‐γ agonists (rosiglitazone and troglitazone), used in the treatment of diabetes, and a PPAR‐α agonist (fenofibrate), used to treat hyperlipidaemia, on the function of primary osteoblasts and osteoclasts. Formation of ‘trabecular’ bone structures by rat calvarial osteoblasts was reduced by up to 85% in cultures treated with rosiglitazone and by 45% in troglitazone‐treated or fenofibrate‐treated cultures; at the same time, lipid droplet formation was increased by 40–70%. The expression of key osteogenic markers was similarly downregulated in cultures treated with PPAR agonists, whereas adipogenesis markers were upregulated. Formation of osteoclasts in cultures derived from mouse marrow diminished with fenofibrate treatment, whereas both glitazones reduced resorptive activity without affecting osteoclast number. Metformin, although not a PPAR agonist, is also commonly used in the treatment of type 2 diabetes. Here, metformin was found to have no effect on bone cell function. Taken together, these data suggest that PPAR‐γ agonists may enhance bone loss via increased adipogenesis at the expense of osteoblast formation. In contrast, PPAR‐α agonists may prevent bone loss. Given that the prevalence of diabetes and cardiovascular disease is expected to rise significantly, greater attention may need to be paid to the effects of PPAR agonists on bone homeostasis. Copyright © 2014 John Wiley & Sons, Ltd.     

DNA damage and senescence in osteoprogenitors expressing Osx1 may cause their decrease with age

Age‐related bone loss in mice results from a decrease in bone formation and an increase in cortical bone resorption. The former is accounted by a decrease in the number of postmitotic osteoblasts which synthesize the bone matrix and is thought to be the consequence of age‐dependent changes in mesenchymal osteoblast progenitors. However, there are no specific markers for these progenitors, and conclusions rely on results from in vitro cultures of mixed cell populations. Moreover, the culprits of such changes remain unknown. Here, we have used Osx1‐Cre;TdRFP mice in which osteoprogenitors express the TdRFP fluorescent protein. We report that the number of TdRFP‐Osx1 cells, freshly isolated from the bone marrow, declines by more than 50% between 6 and 24 months of age in both female and male mice. Moreover, TdRFP‐Osx1 cells from old mice exhibited markers of DNA damage and senescence, such as γH2AX foci, G1 cell cycle arrest, phosphorylation of p53, increased p21^CIP1 levels, as well as increased levels of GATA4 and activation of NF‐κB – two major stimulators of the senescence‐associated secretory phenotype (SASP). Bone marrow stromal cells from old mice also exhibited elevated expression of SASP genes, including several pro‐osteoclastogenic cytokines, and increased capacity to support osteoclast formation. These changes were greatly attenuated by the senolytic drug ABT263. Together, these findings suggest that the decline in bone mass with age is the result of intrinsic defects in osteoprogenitor cells, leading to decreased osteoblast numbers and increased support of osteoclast formation.     

Hypoxia‐inducible factors 1α and 2α exert both distinct and overlapping functions in long bone development

The hypoxia‐inducible factors have recently been identified as critical regulators of angiogenic–osteogenic coupling. Mice overexpressing HIFα subunits in osteoblasts produce abundant VEGF and develop extremely dense, highly vascularized long bones. In this study, we investigated the individual contributions of Hif‐1α and Hif‐2α in angiogenesis and osteogenesis by individually disrupting each Hifα gene in osteoblasts using the Cre‐loxP method. Mice lacking Hif‐1α demonstrated markedly decreased trabecular bone volume, reduced bone formation rate, and altered cortical bone architecture. By contrast, mice lacking Hif‐2α had only a modest decrease in trabecular bone volume. Interestingly, long bone blood vessel development measured by angiography was decreased by a similar degree in both ΔHif‐1α and ΔHif‐2α mice suggesting a common role for these Hifα subunits in skeletal angiogenesis. In agreement with this idea, osteoblasts lacking either Hif‐1α or Hif‐2α had profound reductions in VEGF mRNA expression but only the loss of Hif‐1α impaired osteoblast proliferation. These findings indicate that expression of both Hif‐1α and Hif‐2α by osteoblasts is required for long bone development. We propose that both Hif‐1α and Hif‐2α function through cell non‐autonomous modes to promote vascularization of bone and that Hif‐1α also promotes bone formation by exerting direct actions on the osteoblast. J. Cell. Biochem. 109: 196–204, 2010. © 2009 Wiley‐Liss, Inc.     

PERK is essential for neonatal skeletal development to regulate osteoblast proliferation and differentiation

Loss of function mutations of Perk (eukaryotic translation initiation factor 2 alpha kinase 3) in humans and mice cause severe neonatal developmental defects, including diabetes, growth retardation and multiple skeletal dysplasias. Comprehensive analyses on bone tissue, at the cellular and molecular level in PERK-deficient mice demonstrated that neonatal Perk-/- mice are severely osteopenic, which is caused by a deficiency in the number of mature osteoblasts, impaired osteoblast differentiation, and reduced type I collagen secretion. Impaired differentiation of osteoblasts in Perk KO mice was associated with decreased expression of Runx2 and Osterix, key regulators of osteoblast development. Reduced cell proliferation and reduced expression of key cell cycle factors including cyclin D, cyclin E, cyclin A, Cdc2, and CDK2 occur in parallel with the differentiation defect in mutant osteoblasts. In addition, the trafficking and secretion of type I collagen is compromised as manifested by abnormal retention of procollagen I in the endoplasmic reticulum, and reduced mature collagen production and mineralization. Taken together, these studies identify PERK as a novel regulator of skeletal development and osteoblast biology.     

A novel purification method for multipotential skeletal stem cells

At least some cells within bone marrow stromal populations are multipotential (i.e., differentiate in vitro into osteoblasts, chondrocytes, and adipocytes) and thus designated skeletal stem cells (SSCs) or mesenchymal stem cells (MSCs) amongst other names. Recently, a subpopulation of stromal cells, notably osteoblasts or their progenitors, has been identified as a definitive regulatory component of the hematopoietic stem cell (HSC) niche. Thus, the development of methods for purifying not only SSCs but cells comprising the HSC niche is of interest. Here, we report a method for purifying a novel bone marrow‐derived population with a high frequency of osteoprogenitors and high expression levels of osteoblast differentiation markers (highly purified osteoprogenitors (HipOPs)) as well as markers of the bone niche for HSCs. In vivo transplantation experiments demonstrated that donor HipOPs differentiated into not only osteoblasts, osteocytes and cells around sinusoids but also hematopoietic cells. Thus, HipOPs represent a novel population for simultaneous reconstruction of bone and bone marrow microenvironments. J. Cell. Biochem. 108: 368–377, 2009. © 2009 Wiley‐Liss, Inc.     

Ubiquitin ligase Smurf1 mediates tumor necrosis factor-induced systemic bone loss by promoting proteasomal degradation of bone morphogenetic signaling proteins

Chronic inflammatory disorders, such as rheumatoid arthritis, are often accompanied by systemic bone loss, which is thought to occur through inflammatory cytokine-mediated stimulation of osteoclast resorption and inhibition of osteoblast function. However, the mechanisms involved in osteoblast inhibition remain poorly understood. Here we test the hypothesis that increased Smad ubiquitin regulatory factor 1 (Smurf1)-mediated degradation of the bone morphogenetic protein pathway signaling proteins mediates reduced bone formation in inflammatory disorders. Osteoblasts derived from bone marrow or long bone samples of adult tumor necrosis factor (TNF) transgenic (TNF-Tg) mice were used in this study. TNF decreased the steady-state levels of Smad1 and Runx2 protein similarly to those in long bones of TNF-Tg mice. In the presence of the proteasome inhibitor MG132, TNF increased accumulation of ubiquitinated Smad1 protein. TNF administration over calvarial bones caused decreases in Smad1 and Runx2 protein levels and mRNA expression of osteoblast marker genes in wild-type, but not in Smurf1(-/-) mice. Vertebral bone volume and strength of TNF-Tg/Smurf1(-/-) mice were examined by a combination of micro-CT, bone histomorphometry, and biomechanical testing and compared with those from TNF-Tg littermates. TNF-Tg mice had significantly decreased bone volume and biomechanical properties, which were partially rescued in TNF-Tg/Smurf1(-/-) mice. We conclude that in chronic inflammatory disorders where TNF is increased, TNF induces the expression of ubiquitin ligase Smurf1 and promotes ubiquitination and proteasomal degradation of Smad1 and Runx2, leading to systemic bone loss. Inhibition of ubiquitin-mediated Smad1 and Runx2 degradation in osteoblasts could help to treat inflammation-induced osteoporosis.     

Leptin treatment prevents type I diabetic marrow adiposity but not bone loss in mice

Leptin is a hormone secreted by adipocytes that is implicated in the regulation of bone density. Serum leptin levels are decreased in rodent models of type 1 (T1-) diabetes and in diabetic patients. Whether leptin mediates diabetic bone changes is unclear. Therefore, we treated control and T1-diabetic mice with chronic (28 days) subcutaneous infusion of leptin or saline to elucidate the therapeutic potential of leptin for diabetic osteoporosis. Leptin prevented the increase of marrow adipocytes and the increased aP2 expression that we observed in vehicle-treated diabetic mice. However, leptin did not prevent T1-diabetic decreases in trabecular bone volume fraction or bone mineral density in tibia or vertebrae. Consistent with this finding, markers of bone formation (osteocalcin RNA and serum levels) in diabetic mice were not restored to normal levels with leptin treatment. Interestingly, markers of bone resorption (TRAP5 RNA and serum levels) were decreased in diabetic mice by leptin treatment. In summary, we have demonstrated a link between low leptin levels in T1-diabetes and marrow adiposity. However, leptin treatment alone was not successful in preventing bone loss.     

Identification of C‐terminal Hsp70‐interacting protein as a mediator of tumour necrosis factor action in osteoblast differentiation by targeting osterix for degradation

In patients with inflammatory arthritis, tumour necrosis factor (TNF)‐α are overproduced in inflamed joints. This leads to local erosion of cartilage and bone, periarticular osteopenia, as well as osteoporosis. But less is known regarding the molecular mechanisms that mediate the effect of TNF‐α on osteoblast function. The purpose of this study was to test that C terminus of Hsc70‐interacting protein (CHIP) has a specific role in suppressing the osteogenic activity of osteoblasts under inflammatory conditions. C2C12, MC3T3‐E1 and HEK293T cell lines were cultured and cotransfected with related plasmids. After transfection, the cells were cultured further in the presence or absence of murine TNF‐α and subjected to real time RT‐PCR, Western blot, Ubiquitination assay, Co‐immunoprecipitation, Luciferase reporter assay, Small interfering RNAs and Mineralization assay. The expression levels of TNF‐α‐induced CHIP and Osx were examined by RT‐PCR and Western blot analysis. Co‐immunoprecipitation and ubiquitination assays revealed ubiquitinated Osx, confirmed that CHIP indeed interacted with Osx and identified K55 and K386 residues as the ubiquitination sites in Osx, Luciferase reporter assay and Small interfering RNAs examined whether TNF‐α target the bone morphogenetic protein signalling through CHIP. We established stable cell lines with the overexpression of HA‐CHIP, Mineralization assay and CHIP siRNA demonstrated the important roles of CHIP on osteoblast function in conditions in which TNF‐α is overexpressed. We found that the K55 and K386 residues are ubiquitination site(s) in Osx, and that TNF‐α inhibits osteoblast differentiation by promoting Osx degradation through up‐regulation of E3 ubiquitin ligase CHIP in osteoblast. Thus, CHIP targets Osx for ubiquitination and degradation in osteoblasts after chronic exposure to TNF‐α, and inhibition of CHIP expression in osteoblasts may be a new mechanism to limit inflammation‐mediated osteoporosis by promoting their differentiation into osteoblasts.     

Upregulation of heme oxygenase‐1 inhibits the maturation and mineralization of osteoblasts

Heme‐oxygenase‐1 (HO‐1), an important enzyme involved in vascular disease, transplantation, and inflammation, catalyzes the degradation of heme into carbon monoxide and biliverdin. It has been reported that overexpression of HO‐1 inhibits osteoclastogenesis. However, the effect of HO‐1 on osteoblast differentiation is still not clear. We here used adenoviral vector expressing recombinant human HO‐1 and HO‐1 inducer hemin to study the effects of HO‐1 in primary cultured osteoblasts. The results showed that induction of HO‐1 inhibited the maturation of osteoblasts including mineralized bone nodule formation, alkaline phosphatase activity and decreased mRNA expression of several differentiation markers such as alkaline phosphatase, osteocalcin, and RUNX2. Furthermore, downstream products of HO‐1, bilirubin, carbon monoxide, and iron, are involved in the inhibitory action of HO‐1. HO‐1 can be induced by H₂O₂, lipopolysaccharide and inflammatory cytokines such as TNF‐α and IL‐1β in osteoblasts and also in STZ‐induced diabetic mice. In addition, endogenous PPARγ ligand, 15‐deoxy‐Δ^12,14‐prostaglandin‐J2 (15d‐PGJ2) markedly increased both mRNA and protein levels of HO‐1 in osteoblasts via PI3K‐Akt and MAPK pathways. Blockade of HO activity by ZnPP IX antagonized the inhibitory action on osteocalcin expression by hemin and 15d‐PGJ2. Our results indicate that upregulation of HO‐1 inhibits the maturation of osteoblasts and HO‐1 may be involved in oxidative‐ or inflammation‐induced bone loss. J. Cell. Physiol. 222: 757–768, 2010. © 2009 Wiley‐Liss, Inc.