No abstract

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.

We concluded that our new finding with the mechanical osteotomy suggests a biological explanation to the expected difference in subsequent bone healing. Our hypothesis is that the difference of surface characteristic yields to different bleeding pattern and subsequently results in different bone healing. The analyses of bone healing will support our hypothesis.

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.

Mechanisms underlying cancer cell death caused by inhibitors of subcellular Hsp70 proteins have been elucidated. An inhibitor of Hsp70, apoptozole (Az), is mainly translocated into lysosomes of cancer cells where it induces lysosomal membrane permeabilization, thereby promoting lysosome-mediated apoptosis. Additionally, Az impairs autophagy in cancer cells owing to its ability to disrupt the lysosomal function. However, the Az-triphenylphosphonium conjugate, Az-TPP-O3, localizes mainly to mitochondria of cancer cells where it inhibits the mortalin-p53 interaction and induces mitochondrial outer membrane permeabilization, consequently leading to mitochondria-mediated apoptosis. Unlike Az, Az-TPP-O3 does not have an effect on autophagy in cancer cells. Collectively, the findings indicate that inhibitors of lysosomal Hsp70 and mitochondrial mortalin enhance cancer cell death via distinctively different mechanisms. Additionally, the findings arising from this effort demonstrate that studies aimed at determining subcellular locations and functions of small-molecule modulators provide a deeper understanding of their modes of action in cells.

Our objective was to test effects of b-adrenergic blockade on hindlimb unloading (HU)-induced bone loss and serum leptin and to compare these responses with those observed with leptin replacement. Adult male rats were randomized into six groups (n = 10 each): HU rats treated with vehicle (VEHHU), leptin analog (LEPHU), or b-blocker (BBHU) during a 28-day HU and cage activity controls (CC) treated with the same three agents and pair-fed to HU rats. On days 0 and 28, pQCT scans of proximal tibia and serum collections for leptin assays were performed, and histomorphometric measures of proximal tibia cancellous bone were assessed. The 20% decrease in cancellous vBMD observed in the VEHHU group was halved in BBHU rats and LEPHU rats. Bone formation rate (BFR) in BBHU rats, but not in LEPHU rats, was preserved. The 3-fold increase in resorption surface with HU was abolished by BB and LEP treatments. The decrease in serum leptin after a 28-day HU was attenuated in BBHU and LEPHU rats and was predictive of the decrease in BFR with HU. Blocking sympathetic adrenergic signaling by peripheral administration of a b-blocker during HU mitigates disuse-induced decreases in cancellous bone mass through stimulation of osteoblastic activity and suppression of osteoclastic activity. A direct effect of b-adrenergic blockade on bone cells during HU may be enhanced by an indirect effect mitigating reductions in circulating leptin, possibly through disinhibition of leptin release from adipocytes.

To understand the osteogenic effect of the middle layer of the silk cocoon, sericin was examined for its cellular effects associated with tumor necrosis factor-α (TNF-α) signaling in this study. The fragmented sericin proteins in the silk mat were evaluated for the TNF-α expression level in murine macrophages. The concentration of protein released from silk mats was higher in the outermost and the innermost layers than in the middle layers, and the protein released from the silk mat was identified as sericin. The level of TNF-α in murine macrophages was dependent on the applied concentration of sericin, and the expression of genes associated with osteogenesis in osteoblast-like cells was dependent on the applied concentration of TNF-α. In animal experiments, silk mats from the middle layers led to a higher regenerated bone volume than silk mats from the innermost layer or the outermost layer. If TNF-α protein was incorporated into the silk mats from the middle layers, bone regeneration was suppressed compared with unloaded silk mats from the middle layers. Accordingly, silk mats from the silk cocoon can be considered to be a fragmented sericin-secreting carrier, and the level of sericin secretion is associated with TNF-α induction and bone regeneration.

Parathyroid hormone-related protein (PTHrP) is known to induce the expression of receptor activator of NF-κB ligand (RANKL) in stromal cells/osteoblasts. However, the signaling pathways involved remain controversial. In the present study, we investigated the role of cAMP/protein kinase A (PKA) and calcineurin/NFAT pathways in PTHrP-induced RANKL expression in C2C12 and primary cultured mouse calvarial cells. PTHrP-mediated induction of RANKL expression was significantly inhibited by H89 and FK506, an inhibitor of PKA and calcineurin, respectively. PTHrP upregulated CREB phosphorylation and the transcriptional activity of NFAT. Knockdown of CREB or NFATc1 blocked PTHrP-induced RANKL expression. PTHrP increased the activity of the RANKL promoter reporter that contains approximately 2 kb mouse RANKL promoter DNA sequences. Insertions of mutations in CRE-like element or in NFAT-binding element abrogated PTHrP-induced RANKL promoter activity. Chromatin immunoprecipitation assays showed that PTHrP increased the binding of CREB and NFATc1/NFATc3 to their cognate binding elements in the RANKL promoter. Inhibition of cAMP/PKA and its downstream ERK activity suppressed PTHrP-induced expression and transcriptional activity of NFATc1. CREB knockdown prevented PTHrP induction of NFATc1 expression. Furthermore, NFATc1 and CREB were co-immunoprecipitated. Mutations in CRE-like element completely blocked NFATc1-induced transactivation of the RANKL promoter reporter; however, mutations in NFAT-binding element partially suppressed CREB-induced RANKL promoter activity. Overexpression of CREB increased NFATc1 binding to the RANKL promoter and vice versa. These results suggest that PTHrP-induced RANKL expression depends on the activation of both cAMP/PKA and calcineurin/NFAT pathways, and subsequently, CREB and NFAT cooperate to transactivate the mouse RANKL gene.