Jeong-Hwa Baek

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.

Inactivation of the p53 gene by either mutation or HPV infection is extremely frequent in human oral squamous cell carcinoma cell lines

The state of p53 tumour suppressor and the frequency of high-risk human papillomavirus (HPV) infections were studied in nine human oral cancer cell lines. Three cancer cell lines (SCC-4, Tu-177 and FaDu) had similar amounts of p53 transcripts to normal cells, but contained significantly higher levels of p53 protein than the normal control cells. Sequencing highly conserved open reading frames of the p53 gene of these cancer cells showed point mutations in the SCC-4 and Tu-177 cell lines, a base transition from CCC to TCC occurred at codon 151; and in the line FaDu, a mutation of CGG to CTG occurred at codon 248. The HEp-2 and 1483 cancer lines contained significantly lower levels of p53 protein compared to the normal counterpart. Sequencing of p53 cDNA for HEp-2 and 1483 lines showed no mutations, but northern analysis revealed that these cell lines expressed HPV-18 E6/E7 messages. Four cell lines (SCC-9, SCC-15, SCC-25, and Tu-139) expressed negligible amounts of p53 transcripts compared to the normal counterpart and undetectable levels of p53 protein. These cell lines contained mutations in the highly conserved open reading frames of the p53 gene as follows: the SCC-9 had a deletion of 32 base pairs between codons 274 and 285; the line SCC-15 had an insertion of five base pairs between codons 224 and 225; the line SCC-25 had a deletion of two base pairs in codon 209; and the Tu-139 line had a deletion of 46 base pairs between codons 171 and 186.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

FGF2-activated ERK Mitogen-activated Protein Kinase Enhances Runx2 Acetylation and Stabilization

Runx2 is a key transcription factor regulating osteoblast differentiation and skeletal morphogenesis, and FGF2 is one of the most important regulators of skeletal development. The importance of the ERK mitogen-activated protein (MAP) kinase pathway in cranial suture development was demonstrated by the findings that the inhibition of FGF/FGF receptor (FGFR) signaling by a MEK blocker prevents the premature suture closure caused by an Fgfr2 mutation in mice. We previously demonstrated that ERK activation does not affect Runx2 gene expression but that it stimulates Runx2 transcriptional activity. However, the molecular mechanism underlying Runx2 activation by FGF/FGFR or ERK was still unclear. In this study, we found that FGF2 treatment increased the protein level of exogenously overexpressed Runx2 and that this increase is reversed by ERK inhibitors. In contrast, overexpression of constitutively active MEK strongly increased the Runx2 protein level, which paralleled an increase in Runx2 acetylation. As Runx2 protein phosphorylation mediated by ERK directly correlates with Runx2 protein stabilization, acetylation, and ubiquitination, we undertook to identify the ERK-dependent phosphorylation sites in Runx2. Analysis of two C-terminal Runx2 deletion constructs showed that the middle third of the protein is responsible for ERK-induced stabilization and activation. An in silico analysis of highly conserved ERK targets indicated that there are three relevant serine residues in this domain. Site-directed mutagenesis implicated Ser-301 in for ERK-mediated Runx2 stabilization and acetylation. In conclusion, the FGF2-induced ERK MAP kinase strongly increased the Runx2 protein level through an increase in acetylation and a decrease in ubiquitination, and these processes require the phosphorylation of Runx2 Ser-301 residue.

Tumor necrosis factor-α increases alkaline phosphatase expression in vascular smooth muscle cells via MSX2 induction

Vascular calcification is implicated in many diseases including atherosclerosis and diabetes. Tumor necrosis factor-alpha (TNF-alpha) has been shown to promote vascular calcification both in vitro and in vivo. However, the molecular mechanism of TNF-alpha-mediated vascular calcification has not yet been fully defined. Therefore, in this study, we aimed to investigate whether MSX2 acts as a crucial regulator in TNF-alpha-induced vascular calcification and to define the regulatory mechanism of MSX2 induction in human vascular smooth muscle cells (VSMCs). TNF-alpha increased the expression of osteogenic marker genes including RUNX2, osterix, alkaline phosphatase (ALP), and bone sialoprotein, and it also promoted matrix mineralization in VSMCs. In addition, TNF-alpha enhanced MSX2 expression in a dose- and time-dependent manner. MSX2 over-expression alone induced ALP expression, whereas knockdown of MSX2 with small interfering RNA completely blocked TNF-alpha-induced ALP expression. New protein synthesis was dispensable for MSX2 induction by TNF-alpha, and the inhibition of NF-kappaB by BAY-11-7082 or by dominant negative IkappaBalpha abolished the TNF-alpha-directed induction of MSX2 expression. However, inhibition of NADPH oxidase did not affect MSX2 expression. In conclusion, our study suggests that TNF-alpha directly induces MSX2 expression through the NF-kappaB pathway, which in turn induces expression of ALP, a key molecule in mineralization, in VSMCs.

Effect of Rehmannia glutinosa Libosch extracts on bone metabolism

BACKGROUND Rehmannia glutinosa Libosch extracts (RGX) were investigated to determine if they play roles in bone metabolism. METHODS The effects on osteoblasts were determined by measuring (1) cell proliferation, (2) alkaline phosphatase (ALP) activity, (3) mRNA expression of bone-related proteins, (4) transcriptional activity of Runx2, and (5) osteoprotegerin (OPG) secretion. The effects on the osteoclasts were investigated by measuring (1) tartrate-resistant acid phosphatase-positive [TRAP(+)] multinucleated cell (MNC) formation and (2) resorption areas after culturing osteoclast precursors. Bone mineral density (BMD) measurements and histological observations on rats were also carried out. RESULTS RGX treatment showed a significant increase in both the proliferation and ALP activity of osteoblasts. RGX increased the expression of the bone-related genes. OPG secretion was markedly increased after RGX treatment. In addition, RGX treatment decreased the number of TRAP(+) MNCs and the resorption areas. In vivo studies using ovariectomy-induced osteoporotic rats revealed that RGX alleviated the decrease in the trabecular BMD, and increased the cortical bone thickness and trabeculation of the bone marrow spaces. CONCLUSIONS RGX stimulates the proliferation and activities of osteoblasts, while inhibiting the generation and resorptive activities of osteoclasts. It also shows preventive effects on osteoporotic bone loss induced by an ovariectomy. Although the active substances have not yet been identified, it is believed that the RGX seems to contain active components that have a potential to enhance the bone metabolism in osteoporosis.

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.