Gwan-Shik Kim

BMP2-activated Erk/MAP Kinase Stabilizes Runx2 by Increasing p300 Levels and Histone Acetyltransferase Activity

Runx2 is a critical transcription factor for osteoblast differentiation. Regulation of Runx2 expression levels and transcriptional activity is important for bone morphogenetic protein (BMP)-induced osteoblast differentiation. Previous studies have shown that extracellular signal-regulated kinase (Erk) activation enhances the transcriptional activity of Runx2 and that BMP-induced Runx2 acetylation increases Runx2 stability and transcriptional activity. Because BMP signaling induces Erk activation in osteoblasts, we sought to investigate whether BMP-induced Erk signaling regulates Runx2 acetylation and stability. Erk activation by overexpression of constitutively active MEK1 increased Runx2 transcriptional activity, whereas U0126, an inhibitor of MEK1/2, suppressed basal Runx2 transcriptional activity and BMP-induced Runx2 acetylation and stabilization. Overexpression of constitutively active MEK1 stabilized Runx2 protein via up-regulation of acetylation and down-regulation of ubiquitination. Erk activation increased p300 protein levels and histone acetyltransferase activity. Knockdown of p300 using siRNA diminished Erk-induced Runx2 stabilization. Overexpression of Smad5 increased Runx2 acetylation and stabilization. Erk activation further increased Smad-induced Runx2 acetylation and stabilization, whereas U0126 suppressed these functions. On the other hand, knockdown of Smad1 and Smad5 by siRNA suppressed both basal and Erk-induced Runx2 protein levels. Erk activation enhanced the association of Runx2 with p300 and Smad1. Taken together these results indicate that Erk signaling increases Runx2 stability and transcriptional activity, partly via increasing p300 protein levels and histone acetyltransferase activity and subsequently increasing Runx2 acetylation by p300. In addition to the canonical Smad pathway, a BMP-induced non-Smad Erk signaling pathway cooperatively regulates osteoblast differentiation partly via increasing the stability and transcriptional activity of Runx2.

Molecular Consequences of the ACVR1R206H Mutation of Fibrodysplasia Ossificans Progressiva

Fibrodysplasia ossificans progressiva (FOP), a rare genetic and catastrophic disorder characterized by progressive heterotopic ossification, is caused by a point mutation, c.617G>A; p.R206H, in the activin A receptor type 1 (ACVR1) gene, one of the bone morphogenetic protein type I receptors (BMPR-Is). Although altered BMP signaling has been suggested to explain the pathogenesis, the molecular consequences of this mutation are still elusive. Here we studied the impact of ACVR1 R206H mutation on BMP signaling and its downstream signaling cascades in murine myogenic C2C12 cells and HEK 293 cells. We found that ACVR1 was the most abundant of the BMPR-Is expressed in mesenchymal cells but its contribution to osteogenic BMP signal transduction was minor. The R206H mutant caused weak activation of the BMP signaling pathway, unlike the Q207D mutant, a strong and constitutively active form. The R206H mutant showed a decreased binding affinity for FKBP1A/FKBP12, a known safeguard molecule against the leakage of transforming growth factor (TGF)-β or BMP signaling. The decreased binding affinity of FKBP1A to the mutant R206H ACVR1 resulted in leaky activation of the BMP signal, and moreover, it decreased steady-state R206H ACVR1 protein levels. Interestingly, while WT ACVR1 and FKBP1A were broadly distributed in plasma membrane and cytoplasm without BMP-2 stimulation and then localized in plasma membrane on BMP-2 stimulation, R206H ACVR1 and FKBP1A were mainly distributed in plasma membrane regardless of BMP-2 stimulation. The impaired binding to FKBP1A and an altered subcellular distribution by R206H ACVR1 mutation may result in mild activation of osteogenic BMP-signaling in extraskeletal sites such as muscle, which eventually lead to delayed and progressive ectopic bone formation in FOP patients.

Inhibitory action of bisphosphonates on bone resorption does not involve the regulation of RANKL and OPG expression

The mechanism of inhibitory action of bisphosphonates on bone resorption is not fully elucidated. Osteoclast formation and activity are regulated by osteoblast-derived factors such as the osteoclast differentiating factor, receptor activator of NF-κB ligand (RANKL) and the inhibitor, osteoprotegerin (OPG). To investigate in vitro effects of bisphosphonates on mouse osteoblastic cells, we examined the expression levels of RANKL and OPG in the cells treated with alendronate or pamidronate (10(-8) ∼10(-5)M) alone, or combined with 10 nM of 1,25-(OH)2VitD3 for 24 or 48 h. Various concentrations of alendronate and pamidronate did not change the mRNA expression of RANKL and OPG consistently irrespective of 1,25-(OH)2VitD3 presence. When added into cocultures of mouse osteoblastic cells and bone marrow cells, both alendronate and pamidronate inhibited osteoclast formation and bone resorption but failed to alter the RANKL and OPG mRNA expression. These results indicate that the inhibition of bone resorption by bisphosphonates is not mediated by the regulation of RANKL and OPG expression.

Epidermal growth factor receptor regulates osteoclast differentiation and survival through cross-talking with RANK signaling

The epidermal growth factor receptor (EGFR) functions in various cellular physiological processes such as proliferation, differentiation, and motility. Although recent studies have reported that EGFR signaling is involved in osteoclast recruitment and formation, the molecular mechanism of EGFR signaling for the regulation of osteoclastogenesis remains unclear. We investigated the role of the EGFR in osteoclast differentiation and survival and show that the expression of the EGFR was highly up‐regulated by receptor activator of nuclear factor‐κB ligand (RANKL) during osteoclast differentiation. EGFR‐specific tyrosine kinase inhibitors and EGFR knockdown blocked RANKL‐dependent osteoclast formation, suggesting that EGFR signaling plays an important role in osteoclastogenesis. EGFR inhibition impaired the RANKL‐mediated activation of osteoclastogenic signaling pathways, including c‐Jun N‐terminal kinase (JNK), NF‐κB, and Akt/protein kinase B (PKB). In addition, EGFR inhibition in differentiated osteoclasts caused apoptosis through caspase activation. Inhibition of the phosphoinositide‐3 kinase (PI3K)‐Akt/PKB pathway and subsequent activation of BAD and caspases‐9 and ‐3 may be responsible for the EGFR inhibition‐induced apoptosis. The EGFR physically associated with receptor activator of nuclear factor‐κB (RANK) and Grb2‐associated binder 2 (Gab2). Moreover, RANKL transactivated EGFR. These data indicate that EGFR regulates RANKL‐activated signaling pathways by cross‐talking with RANK, suggesting that the EGFR may play a crucial role as a RANK downstream signal and/or a novel type of RANK co‐receptor in osteoclast differentiation and survival. J. Cell. Physiol. 217: 409–422, 2008.

Trichostatin A-mediated upregulation of p21 WAF1 contributes to osteoclast apoptosis

Histone deacetylase inhibitors (HDIs), a new class of anti-cancer agents, have been reported to suppress formation of osteoclast precursors and their fusion into multinucleated cells. However, little is known about the effect of HDIs on mature osteoclasts, which may have significance for their therapeutic use. Here, we demonstrate a novel action of HDIs on osteoclast apoptosis. Primary multinucleated mature osteoclasts were prepared from mouse bone marrow cells. Treatment of osteoclasts with the HDI trichostatin A (TSA) caused apoptosis, as confirmed by annexin V staining and caspase activation. TSA caused the upregulation of p21WAF1 in osteoclasts. To understand the role of p21WAF1 upregulation in TSA-treated osteoclasts, shRNA against p21WAF1-containing lentivirus was introduced into osteoclasts. The suppression of p21WAF1 decreased TSA-directed osteoclast apoptosis. Collectively, our results provide evidence that TSA causes osteoclast apoptosis, which involves, in part, TSA-induced upregulation of p21WAF1, and strongly supports HDIs as potential therapeutic agents for excessive bone resorption.

Msx2 mediates the inhibitory action of TNF-α on osteoblast differentiation

TNF-α, a proinflammatory cytokine, inhibits osteoblast differentiation under diverse inflammatory conditions; however, the underlying mechanisms in terms of the TNF-α signaling pathway remain unclear. In this study, we examined the role of Msx2 in TNF-α-mediated inhibition of alkaline phosphatase (ALP) expression and the signaling pathways involved. TNF-α down-regulated ALP expression induced by bone morphogenetic protein 2 (BMP2) in C2C12 and Runx2-/- calvarial cells. Over-expression of Msx2 suppressed BMP2-induced ALP expression. Furthermore, TNF-α induced Msx2 expression, and the knockdown of Msx2 by small interfering RNAs rescued ALP expression, which was inhibited by TNF-α. TNF-α activated the NF-κB and the JNK pathways. Inhibition of NF-κB or JNK activation reduced the inhibitory effect of TNF-α on ALP expression, whereas TNF-α-induced Msx2 expression was only suppressed by the inhibition of the NF-κB pathway. Taken together, these results indicate that Msx2 mediates the inhibitory action of TNF-α on BMP2-regulated osteoblast differentiation and that the TNF-α-activated NF-κB pathway is responsible for Msx2 induction.

N-acetylcysteine prevents LPS-induced pro-inflammatory cytokines and MMP2 production in gingival fibroblasts.

Periodontitis is an inflammatory process that ultimately results in tooth loss. Although the primary etiologic agent for periodontitis is bacteria, the majority of periodontal tissue destruction is thought to be caused by an inappropriate host response. Reactive oxygen species (ROS) have been known to be involved in periodontal tissue destruction. We treated human gingival fibroblasts with lipopolysaccharide (LPS) obtained fromE. coli and the periodontopathogensActinobacillus actinomycetemcomitans andPorphyromonas gingivalis, and examined their inflammatory responses in the presence and absence of the antioxidant N-acetylcysteine (NAC). LPS enhanced ROS production, as well as, expression of pro-inflammatory cytokines such as interleukin-1β, interleukin-6, interleukin-8 and tumor necrosis factor-α, and the production and activation of MMP2. NAC suppressed all LPS-induced inflammatory responses examined, suggesting that LPS-induced ROS may play a major regulatory role in these responses in gingival fibroblasts. In addition, NAC prevented LPS-induced activation of p38 MAPK and JNK but not phosphorylation and subsequent degradation of IkB. These results indicate that NAC exerts anti-inflammatory effects in LPS-stimulated gingival fibroblasts, functioning at least in part via down-regulation of JNK and p38 MAPK activation. Furthermore, this work suggests that antioxidants may be useful in adjunctive therapies that complement conventional periodontal treatments.

N‐acetylcysteine stimulates osteoblastic differentiation of mouse calvarial cells

Estrogen deficiency causes osteoporosis via increased generation of reactive oxygen species (ROS), and thus, antioxidants may prove to be the effective therapeutic candidates. We examined the effects of the antioxidant N‐acetylcysteine (NAC) on osteoblastic differentiation in mouse calvarial cells. NAC (10–30 mM) enhanced alkaline phosphatase activity, mRNA expression of osteoblast differentiation‐associated genes and mineralized nodule formation. It also increased expression of bone morphogenetic proteins‐2, ‐4, and ‐7. The osteogenic activity of NAC was partially reduced by inhibition of glutathione synthesis. Since caffeic acid phenethyl ester did not stimulate osteoblast differentiation, it is unlikely that ROS scavenging activity of NAC is sufficient for osteogenic activity. We observed that NAC suppressed small GTPase RhoA activity and activation of RhoA by Pasteurella multocida toxin suppressed the osteogenic activity of NAC. These results suggest that NAC might exert its osteogenic activity via increased glutathione synthesis and inhibition of RhoA activation. J. Cell. Biochem. 103: 1246–1255, 2008.

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.

Modulation of the resorption and osteoconductivity of α-calcium sulfate by histone deacetylase inhibitors

Calcium sulfate (CS) is an osteoconductive material with a long history of clinical use. However, its resorptive properties are not optimal for bone regeneration. Recently, histone deacetylase inhibitors (HDIs) have been suggested as bone regeneration tools. In this study, we investigated the effects of the HDIs sodium butyrate and trichostatin A on alpha-form CS (alphaCS) performance. MC3T3-E1 pre-osteoblasts cultured on alphaCS containing either HDI (alphaCS/HDI) showed higher levels of alkaline phosphatase activity than those cultured on alphaCS alone. The expression of genes characteristic of the osteoblast phenotype, including Runx2, osteocalcin, and bone sialoprotein, was strongly promoted by alphaCS/HDI. When cultured on alphaCS/HDIs, the osteoclastic differentiation of RAW264.7 monocytes was substantially suppressed, as measured by tartrate-resistant acid phosphatase (TRAP) activity and the expression levels of calcitonin receptor and TRAP. Neither HDI affected the CS setting time, compressive strength, or dissolution in a simulated body fluid. In a rat calvarial model of critical size bone defects, alphaCS/HDIs enhanced osteoblast differentiation, led to new bone formation, and delayed resorption, as confirmed by micro-computed tomography and histological analyses.