Abdul S. Qadir

TNF-α Upregulates Sclerostin Expression in Obese Mice Fed a High-Fat Diet

Sclerostin decreases bone mass by antagonizing the Wnt signaling pathway. We examined whether obesity‐induced bone loss is associated with the expression of sclerostin. Five‐week‐old male mice were assigned to one of two groups (n = 10 each) and fed either a control diet (10% kcal from fat; CON) or a high‐fat diet (60% kcal from fat; HF) for 12 weeks. Thex final body weight and whole body fat mass of the HF mice were higher than those of the CON mice. The distal femur cancellous bone mineral density and bone formation rate was lower in HF mice than in CON mice. The percent erosion surface was higher in the HF mice than the CON mice. The serum levels and femoral osteocytic protein expression levels of tumor necrosis factor‐α (TNF‐α) were significantly higher in HF mice than in CON mice. Sclerostin mRNA levels and osteocytic sclerostin protein levels in femoral cortex were also higher in HF mice than in CON mice. Sclerostin expression in MLO‐Y4 osteocytes increased with TNF‐α treatment, and TNF‐α‐induced sclerostin expression was blocked by the inhibition of NF‐κB activation. Chromatin immunoprecipitation and a luciferase reporter assay demonstrated that NF‐κB directly binds to the NF‐κB binding elements on the mouse sost promoter and stimulates sclerostin expression. These results support a model in which, in the context of obesity or other inflammatory diseases that increase the production of TNF‐α, TNF‐α upregulates the expression of sclerostin through NF‐κB signaling pathway, thus contributing to bone loss. J. Cell. Physiol. 229: 640–650, 2014.

High extracellular calcium-induced NFATc3 regulates the expression of receptor activator of NF-κB ligand in osteoblasts

Nuclear factor of activated T cell (NFAT) is a key transcription factor for receptor activator of NF-κB ligand (RANKL)-induced osteoclast differentiation. However, it is unclear whether NFAT plays a role in the expression of RANKL in osteoblasts. High extracellular calcium ([Ca(2+)](o)) increases intracellular calcium, enhances RANKL expression in osteoblasts/stromal cells, and induces osteoclastogenesis in a coculture of osteoblasts and hematopoietic bone marrow cells. Because intracellular calcium signaling activates the calcineurin/NFAT pathway, we examined the role of NFAT activation on high [Ca(2+)](o)-induced RANKL expression in MC3T3-E1 subclone 4 (MC4) cells. Among the family of NFAT transcription factors, expression of NFATc1 and NFATc3, but not NFATc2, NFATc4 or NFAT5, was observed in MC4 cells. High [Ca(2+)](o) increased the expression levels of NFATc1, NFATc3 and RANKL. Cyclosporin A and FK506, inhibitors of calcineurin phosphatase, blocked high [Ca(2+)](o)-induced expression of NFAT and RANKL. Knockdown of NFATc1 and NFATc3 by siRNA prevented high [Ca(2+)](o)-induced RANKL expression, whereas overexpression of NFATc1 and NFATc3 induced RANKL expression. Furthermore, overexpressed NFATc1 upregulated NFATc3 expression, but NFATc1 knockdown decreased NFATc3 expression. Chromatin immunoprecipitation and reporter assay results showed that NFATc3, but not NFATc1, directly binds to the RANKL promoter and stimulates RANKL expression. In summary, these results demonstrate that high [Ca(2+)](o) increases expression of RANKL via activation of the calcineurin/NFAT pathway in osteoblasts. In addition, high [Ca(2+)](o) induces the activation and expression of NFATc1; NFATc3 expression and activity are subsequently increased; and NFATc3 directly binds to the RANKL promoter to increase its expression.

miR‐124 Negatively Regulates Osteogenic Differentiation and In vivo Bone Formation of Mesenchymal Stem Cells

MicroRNAs are novel key regulators of cellular differentiation. Dlx transcription factors play an important role in osteoblast differentiation, and Dlx5 and Dlx2 are known targets of miR‐124. Therefore, in the present study, we investigated the regulatory effects of miR‐124 on the osteogenic differentiation and in vivo bone formation of mesenchymal stem cells (MSCs). During osteogenic induction by BMP2, the expression levels of miR‐124 were inversely correlated with those of osteogenic differentiation marker genes in human and mouse bone marrow‐derived MSCs, MC3T3‐E1 cells and C2C12 cells. The overexpression of a miR‐124 mimic significantly decreased the expression levels of Dlx5, Dlx3, and Dlx2, whereas the silencing of miR‐124 with hairpin inhibitors significantly increased the expression of these Dlx genes. Luciferase reporter assays demonstrated that miR‐124 directly targets the 3′UTRs of Dlx3, Dlx5, and Dlx2. The overexpression of a miR‐124 mimic suppressed the osteogenic marker gene expression levels, alkaline phosphatase activity and matrix mineralization, which were all significantly increased by the overexpression of a miR‐124 inhibitor. When ectopic bone formation was induced by the subcutaneous transplantation of human bone marrow‐derived MSCs in nude mice, MSCs overexpressing a miR‐124 inhibitor significantly enhanced woven bone formation compared with control MSCs. However, MSCs overexpressing a miR‐124 mimic exhibited increased adipocyte differentiation at the expense of ectopic bone formation. These results suggest that miR‐124 is a negative regulator of osteogenic differentiation and in vivo bone formation and that the targeting of Dlx5, Dlx3, and Dlx2 genes partly contributes to this inhibitory effect exerted by miR‐124. J. Cell. Biochem. 116: 730–742, 2015.

Hypoxia inducible factor-1α directly induces the expression of receptor activator of nuclear factor-κB ligand in periodontal ligament fibroblasts

During orthodontic tooth movement, local hypoxia and enhanced osteoclastogenesis are observed in the compression side of periodontal tissues. The receptor activator of nuclear factor-κB ligand (RANKL) is an osteoblast/stromal cell-derived factor that is essential for osteoclastogenesis. In this study, we examined the effect of hypoxia on RANKL expression in human periodontal ligament fibroblasts (PDLFs) to investigate the relationship between local hypoxia and enhanced osteoclastogenesis in the compression side of periodontal tissues. Hypoxia significantly enhanced the levels of RANKL mRNA and protein as well as hypoxia inducible factor-1α (HIF-1α) protein in PDLFs. Constitutively active HIF-1α alone significantly increased the levels of RANKL expression in PDLFs under normoxic conditions, whereas dominant negative HIF-1α blocked hypoxia-induced RANKL expression. To investigate further whether HIF-1α directly regulates RANKL transcription, a luciferase reporter assay was performed using the reporter vector containing the RANKL promoter sequence. Exposure to hypoxia or overexpression of constitutively active HIF-1α significantly increased RANKL promoter activity, whereas dominant negative HIF-1α blocked hypoxia-induced RANKL promoter activity. Furthermore, mutations of putative HIF-1α binding elements in RANKL promoter prevented hypoxia-induced RANKL promoter activity. The results of chromatin immunoprecipitation showed that hypoxia or constitutively active HIF-1α increased the DNA binding of HIF-1α to RANKL promoter. These results suggest that HIF-1α mediates hypoxia-induced up-regulation of RANKL expression and that in compression side periodontal ligament, hypoxia enhances osteoclastogenesis, at least in part, via an increased RANKL expression in PDLFs.

Tumor necrosis factor-α enhances the transcription of smad ubiquitination regulatory factor 1 in an activating protein-1- and runx2-dependent manner†

Smad ubiquitination regulatory factor 1 (Smurf1) is an E3 ubiquitin ligase that negatively regulates osteoblast differentiation. Although tumor necrosis factor‐α (TNF‐α) has been shown to increase Smurf1 expression, the details of the regulatory mechanisms remain unclear. Here, we investigated the molecular mechanism by which TNF‐α stimulates Smurf1 expression in C2C12 and primary cultured mouse calvarial cells. TNF‐α treatment rapidly induced the activation of NF‐κB and MAPKs. Smurf1 induction by TNF‐α was blocked by the inhibition of JNK or ERK, while the inhibition of NF‐κB and p38 MAPK had no effect on Smurf1 induction. TNF‐α treatment or c‐Jun overexpression enhanced the activity of a luciferase reporter that contained a 2.7 kb mouse Smurf1 promoter sequence. Site‐directed mutagenesis of the Smurf1 reporter and chromatin immunoprecipitation analysis demonstrated that the activating protein‐1 (AP‐1) binding motif at −922 bp on the mouse Smurf1 promoter mediated TNF‐α/JNK/AP‐1‐stimulated Smurf1 transcription. Interestingly, Smurf1 expression was not observed in Runx2‐null mouse calvarial cells. When Runx2 was ectopically expressed in these cells, the basal and TNF‐α‐induced expression of Smurf1 was restored. Overexpression of Runx2 transactivated the Smurf1 promoter in a dose‐dependent manner. Reporter and chromatin immunoprecipitation assays demonstrated that the Runx2‐binding motif at −202 bp functioned in Runx2‐mediated Smurf1 expression. ERK activation by TNF‐α treatment or constitutively active MEK1 overexpression increased Smurf1 expression in a Runx2‐dependent manner. These results suggest that the JNK/AP‐1 and ERK/Runx2 signaling pathways mediate TNF‐α‐dependent Smurf1 transcription. J. Cell. Physiol.

Msx2 is required for TNF-α-induced canonical Wnt signaling in 3T3-L1 preadipocytes.

Tumor necrosis factor-α (TNF-α) is known to suppress adipocyte differentiation via a β-catenin-dependent pathway. However, the mechanisms by which TNF-α induces Wnt/β-catenin signaling pathway in adipocytes is unclear. Msx2, a homeobox transcription factor, is known to increase osteoblast differentiation through activation of the Wnt/β-catenin pathway. Therefore, in the present study, we investigated whether TNF-α activates the Wnt/β-catenin signaling pathway via the induction of Msx2 expression in 3T3-L1 preadipocytes. We found that TNF-α transiently increased Msx2 expression as well as the expression of canonical Wnt signaling molecules, including Wnt3a, Wnt7a, Wnt7b, Wnt10b, low-density lipoprotein receptor-related protein 5 (LRP5) and T-cell factor 1 (TCF1). Furthermore, TNF-α increased β-catenin/TCF-dependent transcriptional activity. To better understand the role of Msx2 in Wnt signaling, we examined the effects of Msx2 overexpression and knockdown on Wnt/β-catenin signaling. Msx2 overexpression alone significantly increased the levels of Wnt3a, Wnt7a, Wnt7b, Wnt10b, LRP5 and TCF1 expression, whereas knockdown of Msx2 using small interfering RNA prevented TNF-α-induced expression of Wnt signaling molecules. Taken together, the results of this study indicate that TNF-α enhances the Wnt/β-catenin signaling pathway by inducing Msx2 expression, which in turn suppresses adipocytic differentiation.

Propranolol, a β-adrenergic antagonist, attenuates the decrease in trabecular bone mass in high calorie diet fed growing mice

We investigated the effects of high calorie and low calorie diets on skeletal integrity, and whether β-adrenergic blockade (BB) attenuates bone loss induced by dietary calorie alteration. Male 6-week-old C57BL/6 mice were assigned to either an ad-lib fed control diet (CON), a high calorie diet (HIGH), or a low calorie diet (LOW) group. In each diet group, mice were treated with either vehicle (VEH) or propranolol, a β-adrenergic antagonist. Over 12-weeks, β-blockade mitigated body weight and fat mass increases induced by the high calorie diet. Femoral trabecular bone mineral density and the expression levels of osteogenic marker genes in bone marrow cells were reduced in HIGHVEH and LOWVEH mice, and BB significantly attenuated this decline only in HIGH mice. In summary, the magnitude of bone loss induced by low calorie diet was greater than that caused by high calorie diet in growing mice, and β-blockade mitigated high calorie diet-induced bone loss. [BMB Reports 2014; 47(9): 506-511]

Myeloid Elf-1-like factor stimulates adipogenic differentiation through the induction of peroxisome proliferator-activated receptor γ expression in bone marrow†

Myeloid Elf‐1 like factor (MEF) is one of the Ets transcription factors known to regulate cell proliferation and differentiation. A previous report has shown that osteoblast‐specific MEF transgenic mice (Col1a1‐MEF TG mice) have low bone mass but high bone marrow adiposity. In the present study, we explored a previously unappreciated mechanism whereby MEF promotes adipogenesis in bone marrow. An adipogenic colony‐forming unit assay showed that bone marrow cells derived from Col1a1‐MEF TG mice had a higher adipogenic differentiation potential compared to those from wild‐type. The levels of adipogenic marker genes expression in 3T3L1 cells were higher when co‐cultured with Col1a1‐MEF TG bone marrow cells than with wild‐type cells. MC3T3‐E1 preosteoblasts transfected with MEF secreted higher levels of 15‐deoxy‐delta (12, 14)‐prostaglandin J2, a potent endogenous ligand of peroxisome proliferator‐activated receptor γ (PPARγ), under adipogenic conditions. MEF overexpression increased the adipogenic marker genes expression including PPARγ and lipid droplet accumulation in MC3T3‐E1 preosteoblasts and 3T3L1 preadipocytes. Endogenous MEF expression levels increased as adipocyte differentiation proceeded. Knockdown of MEF by siRNA suppressed expression levels of adipogenic marker genes including PPARγ. MEF directly bound to the MEF binding element on the mouse PPARγ promoter, transactivating promoter activity. Immunohistochemical staining of tibia sections demonstrated that bone lining cells and bone marrow cells express higher levels of PPARγ protein in Col1a1‐MEF TG mice than in wild‐type mice. These results suggest that MEF transactivates PPARγ expression, which, in turn, enhances adipogenic differentiation. Furthermore, MEF overexpressing osteoblasts secrete higher levels of adipogenic factors, creating a marrow microenvironment that favors adipogenesis. J. Cell. Physiol. 227: 3603–3612, 2012.

cAMP/Protein Kinase A Signaling Inhibits Dlx5 Expression via Activation of CREB and Subsequent C/EBPβ Induction in 3T3-L1 Preadipocytes

Distal-less homeobox 5 (Dlx5) is a negative regulator of adipogenesis. Dlx5 expression is decreased by adipogenic stimuli, but the mechanisms of Dlx5 downregulation by adipogenic stimuli have not yet been determined. Here, we tested the impact of cAMP/PKA (protein kinase A) signaling induced by 3-isobutyl-1 methyl xanthine (IBMX), forskolin, and 8-CPT-cAMP on the expression of Dlx5 in 3T3-L1 preadipocytes. Significant downregulation of Dlx5 mRNA expression and protein production levels were observed via cAMP/PKA-dependent signaling. Forced expression of cAMP-responsive element-binding protein (CREB) and CCAAT/enhancer-binding protein β (C/EBPβ) was sufficient for downregulation of Dlx5 expression and revealed that CREB functions upstream of C/EBPβ. In addition, C/EBPβ knockdown by siRNA rescued Dlx5 expression in IBMX-treated 3T3-L1 preadipocytes. Luciferase assays using a Dlx5-luc-2935 reporter construct demonstrated the requirement of the Dlx5 promoter region, ranging from −774 to −95 bp that contains two putative C/EBPβ binding elements (site-1: −517 to −510 bp and site-2: −164 to −157 bp), in the suppression of Dlx5 transcription. Consequently, chromatin immunoprecipitation analysis confirmed the importance of site-1, but not site-2, in C/EBPβ binding and transcriptional suppression of Dlx5. In conclusion, we elucidated the underling mechanism of Dlx5 downregulation in IBMX-induced adipogenesis. IBMX activated cAMP/PKA/CREB signaling and subsequently upregulated C/EBPβ, which binds to the Dlx5 promoter to suppress Dlx5 transcription.