Hyun-Jung Park

βPix-enhanced p38 Activation by Cdc42/Rac/PAK/MKK3/6-mediated Pathway IMPLICATION IN THE REGULATION OF MEMBRANE RUFFLING

βPix (PAK-interacting exchange factor) is a recently identified guanine nucleotide exchange factor for Rho family small G protein Cdc42/Rac. The protein interacts with p21-activated protein kinase (PAK) through its SH3 domain. We examined the effect of βPix on MAP kinase signaling and cytoskeletal rearrangement in NIH3T3 fibroblast cells. Overexpression of βPix enhanced the activation of p38 in the absence of other stimuli and also induced translocation of p38 to the nucleus. This βPix-induced p38 activation was blocked by coexpression of dominant-negative Cdc42/Rac or kinase-inactive PAK, indicating that the effect of βPix on p38 is exerted through the Cdc42/Rac-PAK pathway and requires PAK kinase activity. The essential role of βPix in growth factor-stimulated p38 activation was evidenced by the blocking of platelet-derived growth factor-induced p38 activation in the cells expressing βPix SH3m (W43K) and βPix DHm (L238R,L239R). In addition, SB203580, a p38 inhibitor, and kinase-inactive p38 (T180A,Y182F) blocked membrane ruffling induced by βPix, suggesting that p38 might be involved in mediating βPix-induced membrane ruffling. The results in this study suggest that βPix might have a role in nuclear signaling, as well as in actin cytoskeleton regulation, and that some part of these cellular functions is possibly mediated by p38 MAP kinase.

Improvement of Cognitive Deficit in Alzheimer's Disease Patients by Long Term Treatment with Korean Red Ginseng

A 24-week randomized open-label study with Korean red ginseng (KRG) showed cognitive benefits in patients with Alzheimer’s disease. To further determine long-term effect of KRG, the subjects were recruited to be followed up to 2 yr. Cognitive function was evaluated every 12 wk using the Alzheimer’s Disease Assessment Scale (ADAS) and the Korean version of the Mini Mental Status Examination (K-MMSE) with the maintaining dose of 4.5 g or 9.0 g KRG per d. At 24 wk, there had been a significant improvement in KRG-treated groups. In the long-term evaluation of the efficacy of KRG after 24 wk, the improved MMSE score remained without significant decline at the 48th and 96th wk. ADAS-cog showed similar findings. Maximum improvement was found around week 24. In conclusion, the effect of KRG on cognitive functions was sustained for 2 yr follow-up, indicating feasible efficacies of long-term follow-up for Alzheimer’s disease.

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.

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.

The cooperation of CREB and NFAT is required for PTHrP-induced RANKL expression in mouse osteoblastic cells.

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. J. Cell. Physiol. 230: 667–679, 2015.

The Effect of Antioxidants on the Production of Pro-Inflammatory Cytokines and Orthodontic Tooth Movement

Orthodontic force causes gradual compression of the periodontal ligament tissues, which leads to local hypoxia in the compression side of the tissues. In this study, we investigated whether antioxidants exert a regulatory effect on two factors: the expression of pro-inflammatory cytokines in human periodontal ligament fibroblasts (PDLFs) that were exposed to mechanical compression and hypoxia and the rate of orthodontic tooth movement in rats. Exposure of PDLFs to mechanical compression (0.5–3.0 g/cm2) or hypoxic conditions increased the production of intracellular reactive oxygen species. Hypoxic treatment for 24 h increased the mRNA levels of IL-1β, IL-6 and IL-8 as well as vascular endothelial growth factor (VEGF) in PDLFs. Resveratrol (10 nM) or N-acetylcysteine (NAC, 20 mM) diminished the transcriptional activity of hypoxiainducible factor-1 and hypoxia-induced expression of VEGF. Combined treatment with mechanical compression and hypoxia significantly increased the expression levels of IL-1β, IL-6, IL-8, TNF-α and VEGF in PDLFs. These levels were suppressed by NAC and resveratrol. The maxillary first molars of rats were moved mesially for seven days using an orthodontic appliance. NAC decreased the amount of orthodontic tooth movement compared to the vehicle-treated group. The results from immunohistochemical staining demonstrated that NAC suppressed the expression of IL-1β and TNF-α in the periodontal ligament tissues compared to the vehicle-treated group. These results suggest that antioxidants have the potential to negatively regulate the rate of orthodontic tooth movement through the down-regulation of pro-inflammatory cytokines in the compression sides of periodontal ligament tissues.

Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression, which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation.