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Hepatocyte growth factor increases osteopontin expression in human osteoblasts through PI3K, Akt, c-Src, and AP-1 signaling pathway.

Chen HT, Tsou HK, Chang CH, Tang CH - PLoS ONE (2012)

Bottom Line: Here we found that HGF induced OPN expression in human osteoblasts dose-dependently.HGF-mediated AP-1-luciferase activity and c-Jun binding to the AP-1 element was reduced by c-Met inhibitor, Ly294002, Akt inhibitor, and PP2.Our results suggest that the interaction between HGF and c-Met increases OPN expression in human osteoblasts via the PI3K, Akt, c-Src, c-Jun, and AP-1 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan.

ABSTRACT

Background: Hepatocyte growth factor (HGF) has been demonstrated to stimulate osteoblast proliferation and participated bone remodeling. Osteopontin (OPN) is a secreted phosphoglycoprotein that belongs to the SIBLING family and is present during bone mineralization. However, the effects of HGF on OPN expression in human osteoblasts are large unknown.

Methodology/principal findings: Here we found that HGF induced OPN expression in human osteoblasts dose-dependently. HGF-mediated OPN production was attenuated by c-Met inhibitor and siRNA. Pretreatment of osteoblasts with PI3K inhibitor (Ly294002), Akt inhibitor, c-Src inhibitor (PP2), or AP-1 inhibitor (curcumin) blocked the potentiating action of HGF. Stimulation of osteoblasts with HGF enhanced PI3K, Akt, and c-Src activation. In addition, incubation of cells with HGF also increased c-Jun phosphorylation, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the OPN promoter. HGF-mediated AP-1-luciferase activity and c-Jun binding to the AP-1 element was reduced by c-Met inhibitor, Ly294002, Akt inhibitor, and PP2.

Conclusions/significance: Our results suggest that the interaction between HGF and c-Met increases OPN expression in human osteoblasts via the PI3K, Akt, c-Src, c-Jun, and AP-1 signaling pathway.

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HGF increases OPN expression through c-Met receptor.(A&B) Cells were incubated with HGF for 24 h, and OPN mRNA was examined by qPCR (n = 4). (C-E) Osteoblasts were incubated with HGF for 24 h, and OPN protein was examined by ELISA and Western blotting (n = 4). (F-I) Cells were pretreated for 30 min with c-Met inhibitor or transfected with c-Met siRNA for 24 h followed by stimulation with HGF for 24 h, and OPN expression was examined by qPCR and ELISA (n = 4). *: p<0.05 as compared with basal level (A-D) or HGF-treated group (F-I).
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pone-0038378-g001: HGF increases OPN expression through c-Met receptor.(A&B) Cells were incubated with HGF for 24 h, and OPN mRNA was examined by qPCR (n = 4). (C-E) Osteoblasts were incubated with HGF for 24 h, and OPN protein was examined by ELISA and Western blotting (n = 4). (F-I) Cells were pretreated for 30 min with c-Met inhibitor or transfected with c-Met siRNA for 24 h followed by stimulation with HGF for 24 h, and OPN expression was examined by qPCR and ELISA (n = 4). *: p<0.05 as compared with basal level (A-D) or HGF-treated group (F-I).

Mentions: HGF has been reported to stimulate proliferation and differentiation of osteoblasts [13], [14]. To examine the effects of HGF on OPN expression, osteoblasts were exposed to HGF, and the mRNA levels of OPN was determined. Treatment of osteoblasts with HGF (3–30 ng/ml) for 24 h induced OPN mRNA levels (Fig. 1A&B). In addition, stimulation of osteoblasts with HGF also led to increased protein expression of OPN in a concentration-dependent manner by using ELISA and Western blotting (Fig. 1C–E). It has been reported that HGF exerts its effects through interaction with a specific receptor c-Met [21]. Pretreatment of osteoblasts with c-Met inhibitor reduced HGF-increased OPN expression (Fig. 1F–I). In addition, transfection of cells with c-Met siRNA also reduced HGF-increased OPN expression (Fig. 1F–I). Therefore, an interaction between HGF and c-Met is very important for OPN production in human osteoblasts.


Hepatocyte growth factor increases osteopontin expression in human osteoblasts through PI3K, Akt, c-Src, and AP-1 signaling pathway.

Chen HT, Tsou HK, Chang CH, Tang CH - PLoS ONE (2012)

HGF increases OPN expression through c-Met receptor.(A&B) Cells were incubated with HGF for 24 h, and OPN mRNA was examined by qPCR (n = 4). (C-E) Osteoblasts were incubated with HGF for 24 h, and OPN protein was examined by ELISA and Western blotting (n = 4). (F-I) Cells were pretreated for 30 min with c-Met inhibitor or transfected with c-Met siRNA for 24 h followed by stimulation with HGF for 24 h, and OPN expression was examined by qPCR and ELISA (n = 4). *: p<0.05 as compared with basal level (A-D) or HGF-treated group (F-I).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3366938&req=5

pone-0038378-g001: HGF increases OPN expression through c-Met receptor.(A&B) Cells were incubated with HGF for 24 h, and OPN mRNA was examined by qPCR (n = 4). (C-E) Osteoblasts were incubated with HGF for 24 h, and OPN protein was examined by ELISA and Western blotting (n = 4). (F-I) Cells were pretreated for 30 min with c-Met inhibitor or transfected with c-Met siRNA for 24 h followed by stimulation with HGF for 24 h, and OPN expression was examined by qPCR and ELISA (n = 4). *: p<0.05 as compared with basal level (A-D) or HGF-treated group (F-I).
Mentions: HGF has been reported to stimulate proliferation and differentiation of osteoblasts [13], [14]. To examine the effects of HGF on OPN expression, osteoblasts were exposed to HGF, and the mRNA levels of OPN was determined. Treatment of osteoblasts with HGF (3–30 ng/ml) for 24 h induced OPN mRNA levels (Fig. 1A&B). In addition, stimulation of osteoblasts with HGF also led to increased protein expression of OPN in a concentration-dependent manner by using ELISA and Western blotting (Fig. 1C–E). It has been reported that HGF exerts its effects through interaction with a specific receptor c-Met [21]. Pretreatment of osteoblasts with c-Met inhibitor reduced HGF-increased OPN expression (Fig. 1F–I). In addition, transfection of cells with c-Met siRNA also reduced HGF-increased OPN expression (Fig. 1F–I). Therefore, an interaction between HGF and c-Met is very important for OPN production in human osteoblasts.

Bottom Line: Here we found that HGF induced OPN expression in human osteoblasts dose-dependently.HGF-mediated AP-1-luciferase activity and c-Jun binding to the AP-1 element was reduced by c-Met inhibitor, Ly294002, Akt inhibitor, and PP2.Our results suggest that the interaction between HGF and c-Met increases OPN expression in human osteoblasts via the PI3K, Akt, c-Src, c-Jun, and AP-1 signaling pathway.

View Article: PubMed Central - PubMed

Affiliation: School of Chinese Medicine, College of Chinese Medicine, China Medical University, Taichung, Taiwan.

ABSTRACT

Background: Hepatocyte growth factor (HGF) has been demonstrated to stimulate osteoblast proliferation and participated bone remodeling. Osteopontin (OPN) is a secreted phosphoglycoprotein that belongs to the SIBLING family and is present during bone mineralization. However, the effects of HGF on OPN expression in human osteoblasts are large unknown.

Methodology/principal findings: Here we found that HGF induced OPN expression in human osteoblasts dose-dependently. HGF-mediated OPN production was attenuated by c-Met inhibitor and siRNA. Pretreatment of osteoblasts with PI3K inhibitor (Ly294002), Akt inhibitor, c-Src inhibitor (PP2), or AP-1 inhibitor (curcumin) blocked the potentiating action of HGF. Stimulation of osteoblasts with HGF enhanced PI3K, Akt, and c-Src activation. In addition, incubation of cells with HGF also increased c-Jun phosphorylation, AP-1-luciferase activity, and c-Jun binding to the AP-1 element on the OPN promoter. HGF-mediated AP-1-luciferase activity and c-Jun binding to the AP-1 element was reduced by c-Met inhibitor, Ly294002, Akt inhibitor, and PP2.

Conclusions/significance: Our results suggest that the interaction between HGF and c-Met increases OPN expression in human osteoblasts via the PI3K, Akt, c-Src, c-Jun, and AP-1 signaling pathway.

Show MeSH
Related in: MedlinePlus