Apelin receptor (APJ) deficiency has been reported to be preventive against atherosclerosis. However, the mechanism of this effect remains unknown. In this study, quantitative real-time RT-PCR, Western blotting and ELISA analyses revealed a significant increase in the expression of intercellular adhesion molecule-1(ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1) in human umbilical vein endothelial cells (HUVECs) treated with apelin. Inhibitors of cellular signal transduction molecules were used to demonstrate involvement of nuclear factor kappa-B (NF-κB) and c-Jun N-terminal kinase (JNK) pathways in apelin-APJ-induced activation of adhesion molecules and chemokines. Inhibition of APJ expression by RNA interference abrogated apelin-induced expression of adhesion molecules and chemokines and apelin-stimulated cellular signal transduction in HUVECs. The apelin-APJ system in endothelial cells is involved in the expression of adhesion molecules and chemokines, which are important for the initiation of endothelial inflammation-related atherosclerosis. Therefore, apelin-APJ and the cell signaling pathways activated by this system in endothelial cells may represent targets for therapy of atherosclerosis.

Vascular calcification results from osteoblastic differentiation of vascular smooth muscle cells (VSMCs) and is a major risk factor for cardiovascular events. Ghrelin is a newly discovered bioactive peptide that acts as a natural endogenous ligand of the growth hormone secretagog receptor (GHSR). Several studies have identified the protective effects of ghrelin on the cardiovascular system, however research on the effects and mechanisms of ghrelin on vascular calcification is still quite rare. In this study, we determined the effect of ghrelin on osteoblastic differentiation of VSMCs and investigated the mechanism involved using the two universally accepted calcifying models of calcifying vascular smooth muscle cells (CVSMCs) and beta-glycerophosphate (beta-GP)-induced VSMCs. Our data demonstrated that ghrelin inhibits osteoblastic differentiation and mineralization of VSMCs due to decreased alkaline phosphatase (ALP) activity, Runx2 expression, bone morphogenetic protein-2 (BMP-2) expression and calcium content. Further study demonstrated that ghrelin exerted this suppression effect via an extracellular signal-related kinase (ERK)-dependent pathway and that the suppression effect of ghrelin was time dependent and dose dependent. Furthermore, inhibition of the growth hormone secretagog receptor (GHSR), the ghrelin receptor, by siRNA significantly reversed the activation of ERK by ghrelin. In conclusion, our study suggests that ghrelin may inhibit osteoblastic differentiation of VSMCs through the GHSR/ERK pathway.

One of the key drivers for squamous cell carcinoma (SCC) proliferation is activation of the epidermal growth factor receptor (EGFR), a known proto-oncogene. However, the mechanism of EGFR-dependent SCC proliferation remains unclear. Our previous studies indicate that epidermal growth factor (EGF)-induced SCC cell proliferation requires the SH3 domain of phospholipase C-γ1 (PLC-γ1), but not its catalytic activity. The SH3 domain of PLC-γ1 is known to activate the short form of nuclear phosphatidylinositol 3-kinase enhancer (PIKE) that enhances the activity of nuclear class Ia phosphatidylinositol 3-kinase (PI3K) required for proliferation. However, PIKE has been described for more than a decade to be present exclusively in neuronal cells. In the present study, we found that PIKE was highly expressed in malignant human keratinocytes (SCC4 and SCC12B2) but had low expression in normal human keratinocytes. Immunohistochemical analysis showed strong nuclear staining of PIKE in human epidermal and tongue SCC specimens but little staining in the adjacent non-cancerous epithelium. Treatment of SCC4 cells with EGF-induced translocation of PLC-γ1 to the nucleus and binding of PLC-γ1 to the nuclear PIKE. Knockdown of PLC-γ1 or PIKE blocked EGF-induced activation of class Ia PI3K and protein kinase C-ζ and phosphorylation of nucleolin in the nucleus as well as EGF-induced SCC cell proliferation. However, inhibition of the catalytic activity of PLC-γ1 had little effect. These data suggest that PIKE has a critical role in EGF-induced SCC cell proliferation and may function as a proto-oncogene in SCC.Oncogene advance online publication, 20 February 2012; doi:10.1038/onc.2012.10.

Twenty-one 3.5-month-old female Sprague-Dawley rats were randomly assigned to three groups: BTX group, in which each rat received a single intramuscular injection of 2 U of Clostridium botulinum toxin (BTX) in the quadriceps femoris muscle of the right hind limb; BTX + SR group, in which each rat received a BTX injection and a dose of strontium ranelate (dose level of 625 mg/kg/day); and the control group. All the rats were killed at 9 weeks post-treatment. It was showed that BTX-induced rats a rapid loss of body weight in the first 3 weeks, after which their body weight showed a slow increase similar to that observed in the control rats. The net body weight loss was mainly attributed to muscle atrophy. BTX caused remarkable bone degradation in either the trabecular bone or the cortical bone of the disuse femur. The deteriorations in the bone mass and bone microstructure were locally limited and could be prevented by strontium ranelate treatment. Biomechanical analysis showed that strontium ranelate treatment improved the mechanical performance of the tibia in BTX-treated rats. It was showed that a clinical-corresponding dose of strontium ranelate could prevent bone loss in long-term immobilized rats.

Several reports have shown that circulating insulin level is positively correlated with arterial calcification; however, the relationship between insulin and arterial calcification remains controversial and the mechanism involved is still unclear. We used calcifying vascular smooth muscle cells (CVSMCs), a specific subpopulation of vascular smooth muscle cells that could spontaneously express osteoblastic phenotype genes and form calcification nodules, to investigate the effect of insulin on osteoblastic differentiation of CVSMCs and the cell signals involved. Our experiments demonstrated that insulin could promote alkaline phosphatase (ALP) activity, osteocalcin expression and the formation of mineralized nodules in CVSMCs. Suppression of receptor activator of nuclear factor κB ligand (RANKL) with small interfering RNA (siRNA) abolished the insulin-induced ALP activity. Insulin induced the activation of extracellular signal-regulated kinase (ERK)1/2, mitogen-activated protein kinase (MAPK) and RAC-alpha serine/threonine-protein kinase (Akt). Furthermore, pretreatment of human osteoblasts with the ERK1/2 inhibitor PD98059, but not the phosphoinositide 3-kinase (PI3K) inhibitor, LY294002, or the Akt inhibitor, 1L-6-hydroxymethyl-chiro-inositol 2-(R)-2-O-methyl-3-O-octadecylcarbonate (HIMO), abolished the insulin-induced RANKL secretion and blocked the promoting effect of insulin on ALP activities of CVSMCs. Recombinant RANKL protein recovered the ALP activities decreased by RANKL siRNA in insulin-stimulated CVSMCs. These data demonstrated that insulin could promote osteoblastic differentiation of CVSMCs by increased RANKL expression through ERK1/2 activation, but not PI3K/Akt activation.

To explore the possible role for connective tissue growth factor (CTGF) during tooth movement, we evaluated CTGF gene and protein expression in MG-63 cells subjected to cyclic stretch. Cyclic stretch caused a time-dependent increase in CTGF mRNA and protein levels.Inhibition of p38 MAP kinase or ERK activation did not affect cyclic stretch-induced CTGF expression. Specific inhibitors of PI3K suppressed stretch -induced CTGF expression in a time-dependent manner. cyclic stretch activated JNK and ERK, but not p38 MAP kinase in osteoblast-like cells. PI3K inhibitors suppressed cyclic stretch-induced JNK, but not p38 MAP kinase activation. Finally, SP600125, a Specific Inhibitor of JNK, suppressed stretch -induced CTGF Expression. These results suggest that stretch-induced CTGF expression is mediated through the PI3K-JNK -dependent pathway, not by p38 MAP kinase and ERK pathways.

UNLABELLED Omentin-1 inhibited osteoblast differentiation in vitro. In co-culture systems of osteoblasts and osteoclast precursors, omentin-1 reduced osteoclast formation by stimulating osteoprotegerin (OPG) and inhibiting receptor activator for nuclear factor κB ligand (RANKL) production in osteoblasts. In vivo, adenovirus-mediated overexpression of omentin-1 suppressed bone turnover and restored bone mineral density (BMD) and bone strength in ovariectomized mice. INTRODUCTION Omentin-1 (also intelectin-1) is a recently identified visceral adipose tissue-derived cytokine that is highly abundant in plasma. This study was undertaken to investigate the effects of omentin-1 on bone metabolism. METHODS Osteoblast differentiation was assessed by measuring alkaline phosphatase activity, osteocalcin production and matrix mineralization. OPG and RANKL protein expression and secretion in osteoblasts were detected by Western blot and ELISA, respectively. The effect of recombinant omentin-1 on osteoclast formation was examined in co-culture systems of osteoblasts and osteoclast precursors. The effects of intravenous administration of adenoviral-delivered omentin-1 on bone mass, bone strength, and bone turnover were also examined in ovariectomized mice. RESULTS In vitro, omentin-1 inhibited osteoblast differentiation, while it had no direct effect on osteoclast differentiation; it also reduced osteoclast formation in the co-culture systems through stimulating OPG and inhibiting RANKL production in osteoblasts. In vivo, adenovirus-mediated overexpression of omentin-1 partially restored BMD and bone strength in ovariectomized mice, accompanied by decreased levels of plasma osteocalcin and tartrate-resistant acid phosphatase-5b and lower serum RANKL/OPG ratios. CONCLUSION The present study suggests that omentin-1 ameliorates bone loss induced by estrogen deficiency via downregulating the RANKL/OPG ratio.

AIMS Omentin-1 (also known as intelectin-1) is a recently identified visceral adipose tissue-derived cytokine that is inversely related to obesity. Our previous study showed that omentin-1 inhibits osteoblastic differentiation of calcifying vascular smooth muscle cells (CVSMCs) in vitro. This study was undertaken to investigate the effects of omentin-1 on arterial calcification and bone metabolism in vivo. METHODS AND RESULTS In vitro, omentin-1 stimulated production of osteoprotegerin (OPG) and inhibited production of receptor activator for nuclear factor κB ligand (RANKL) in both CVSMCs and osteoblasts. In vivo, adenovirus-mediated over-expression of omentin-1 attenuated arterial calcification and bone loss in OPG(-/-) mice. All these in vitro and in vivo actions were abrogated by blockade of the PI3K-Akt signalling pathway. Furthermore, omentin-1 reduced serum levels of RANKL, tartarate-resistant acid phosphatase-5b and osteocalcin, all of which are increased dramatically in OPG(-/-) mice. CONCLUSION These data suggest that omentin-1 ameliorates arterial calcification and bone loss in vivo through the regulation of the RANK signalling pathway.

BACKGROUND The relationship between bone turnover markers (BTMs) and BMD decreasing rate (BDR) in Chinese women is unclear. Wu investigated the relationship between (BTMs) and BDR at various skeletal sites in Chinese middle-aged women. METHODS A cross-section study of 555 healthy Chinese women over 35-60years of age. BMD at posteroanterior spine, the left hip, and the left forearm were measured with a DXA. Levels of serum osteocalcin (OC), bone-specific alkaline phosphatase (BAP), cross-linked N-terminal telopeptides of type I collagen (sNTX) and total urinary deoxypyridinoline (uDPD) were determined. RESULTS BDR at various skeletal sites had significant negative correlation with serum OC(r=-0.395 to -0.530), BAP(r=-0.297 to -0.486), and sNTX(r=-0.207 to -0.272). After adjustment of age and weight, serum OC, BAP, and sNTX rather than total uDPD still exhibited significant correlations with BDR. Stepwise regression analyses showed that, serum OC and BAP were the significantly negative determinants of BDR. Between 4.7-27.7% and 1.2-16.1% of the changes in BDR were determined by serum OC and BAP, respectively. However, sNTX and total uDPD had no significant effect on BDR at various skeletal sites. CONCLUSIONS This study indicated the correlation between BTMs and early-stage BDR in Chinese middle-aged women and suggested that serum OC and BAP, rather than sNTX and total uDPD, are the key determining factors of early BMD decreases.

Vascular calcification, which results from a process osteoblastic differentiation of vascular smooth muscle cells (VSMCs), is a major risk factor for cardiovascular morbidity and mortality. Apelin is a recently discovered peptide that is the endogenous ligand for the orphan G-protein-coupled receptor, APJ. Several studies have identified the protective effects of apelin on the cardiovascular system. However, the effects and mechanisms of apelin on the osteoblastic differentiation of VSMCs have not been elucidated. Using a culture of calcifying vascular smooth muscle cells (CVMSCs) as a model for the study of vascular calcification, the relationship between apelin and the osteoblastic differentiation of VSMCs and the signal pathway involved were investigated. Alkaline phosphatase (ALP) activity and osteocalcin secretion were examined in CVSMCs. The involved signal pathway was studied using the extracellular signal-regulated kinase (ERK) inhibitor, PD98059, the phosphatidylinositol 3-kinase (PI3-K) inhibitor, LY294002, and APJ siRNA. The results showed that apelin inhibited ALP activity, osteocalcin secretion, and the formation of mineralized nodules. APJ protein was detected in CVSMCs, and apelin activated ERK and AKT (a downstream effector of PI3-K). Suppression of APJ with siRNA abolished the apelin-induced activation of ERK and Akt. Furthermore, inhibition of APJ expression, and the activation of ERK or PI3-K, reversed the effects of apelin on ALP activity. These results showed that apelin inhibited the osteoblastic differentiation of CVSMCs through the APJ/ERK and APJ/PI3-K/AKT signaling pathway. Apelin appears to play a protective role against arterial calcification.