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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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Related in: MedlinePlus

PI3K is involved in HGF-induced OPN expression.Cells were pretreated for 30 min with Ly294002 or transfected with p85 mutant and siRNA followed by stimulation with HGF for 24 h. Media and total RNA were collected, and the expression of OPN was analyzed with qPCR and ELISA (n = 4) (A-D). Primary osteoblasts were incubated with HGF for indicated time intervals, and p85 phosphorylation was examined by Western blotting (E). Primary osteoblasts were pretreated with c-Met inhibitor for 30 min or transfected with c-Met siRNA for 24 h followed by stimulation with HGF for 30 min, and p85 phosphorylation was determined by Western blotting (n = 5) (F). *: p<0.05 as compared with HGF-treated group.
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pone-0038378-g002: PI3K is involved in HGF-induced OPN expression.Cells were pretreated for 30 min with Ly294002 or transfected with p85 mutant and siRNA followed by stimulation with HGF for 24 h. Media and total RNA were collected, and the expression of OPN was analyzed with qPCR and ELISA (n = 4) (A-D). Primary osteoblasts were incubated with HGF for indicated time intervals, and p85 phosphorylation was examined by Western blotting (E). Primary osteoblasts were pretreated with c-Met inhibitor for 30 min or transfected with c-Met siRNA for 24 h followed by stimulation with HGF for 30 min, and p85 phosphorylation was determined by Western blotting (n = 5) (F). *: p<0.05 as compared with HGF-treated group.

Mentions: PI3K/Akt signaling pathway can be activated by a variety of growth factors, such as insulin and HGF [22], [23]. To determine whether PI3K was involved in HGF triggered OPN expression, osteoblasts were pretreated with Ly294002, a PI3K inhibitor for 30 min and then incubated with HGF for 24 h. As shown in Fig. 2A–D, pretreatment with Ly294002 reduced HGF-induced OPN expression. Transfection of cells with p85 mutant or siRNA also reduced HGF-induced OPN expression (Fig. 2A–D). We then directly measured p85 phosphorylation in response to HGF. Stimulation of primary osteoblasts led to a significant increase in phosphorylation of PI3K (Fig. 2E). In addition, treatment with c-Met inhibitor or transfection with c-Met siRNA reduced HGF-mediated p85 phosphorylation (Fig. 2F). Furthermore, pretreatment of cells with Akt inhibitor or transfection with Akt mutant and siRNA for 24 h markedly attenuated the HGF-induced OPN production (Fig. 3A–D). Akt phosphorylation in response to HGF was then measured. As shown in Fig. 3E, treatment of primary osteoblasts with HGF resulted in a time-dependent phosphorylation of Akt. Pretreatment of cells with c-Met inhibitor and Ly294002 or transfection with c-Met siRNA inhibited the HGF-induced Akt phosphorylation (Fig. 3F). Taken together, these results indicate that the PI3K and Akt pathways are involved in HGF-induced OPN production.


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)

PI3K is involved in HGF-induced OPN expression.Cells were pretreated for 30 min with Ly294002 or transfected with p85 mutant and siRNA followed by stimulation with HGF for 24 h. Media and total RNA were collected, and the expression of OPN was analyzed with qPCR and ELISA (n = 4) (A-D). Primary osteoblasts were incubated with HGF for indicated time intervals, and p85 phosphorylation was examined by Western blotting (E). Primary osteoblasts were pretreated with c-Met inhibitor for 30 min or transfected with c-Met siRNA for 24 h followed by stimulation with HGF for 30 min, and p85 phosphorylation was determined by Western blotting (n = 5) (F). *: p<0.05 as compared with HGF-treated group.
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Related In: Results  -  Collection

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

pone-0038378-g002: PI3K is involved in HGF-induced OPN expression.Cells were pretreated for 30 min with Ly294002 or transfected with p85 mutant and siRNA followed by stimulation with HGF for 24 h. Media and total RNA were collected, and the expression of OPN was analyzed with qPCR and ELISA (n = 4) (A-D). Primary osteoblasts were incubated with HGF for indicated time intervals, and p85 phosphorylation was examined by Western blotting (E). Primary osteoblasts were pretreated with c-Met inhibitor for 30 min or transfected with c-Met siRNA for 24 h followed by stimulation with HGF for 30 min, and p85 phosphorylation was determined by Western blotting (n = 5) (F). *: p<0.05 as compared with HGF-treated group.
Mentions: PI3K/Akt signaling pathway can be activated by a variety of growth factors, such as insulin and HGF [22], [23]. To determine whether PI3K was involved in HGF triggered OPN expression, osteoblasts were pretreated with Ly294002, a PI3K inhibitor for 30 min and then incubated with HGF for 24 h. As shown in Fig. 2A–D, pretreatment with Ly294002 reduced HGF-induced OPN expression. Transfection of cells with p85 mutant or siRNA also reduced HGF-induced OPN expression (Fig. 2A–D). We then directly measured p85 phosphorylation in response to HGF. Stimulation of primary osteoblasts led to a significant increase in phosphorylation of PI3K (Fig. 2E). In addition, treatment with c-Met inhibitor or transfection with c-Met siRNA reduced HGF-mediated p85 phosphorylation (Fig. 2F). Furthermore, pretreatment of cells with Akt inhibitor or transfection with Akt mutant and siRNA for 24 h markedly attenuated the HGF-induced OPN production (Fig. 3A–D). Akt phosphorylation in response to HGF was then measured. As shown in Fig. 3E, treatment of primary osteoblasts with HGF resulted in a time-dependent phosphorylation of Akt. Pretreatment of cells with c-Met inhibitor and Ly294002 or transfection with c-Met siRNA inhibited the HGF-induced Akt phosphorylation (Fig. 3F). Taken together, these results indicate that the PI3K and Akt pathways are involved in HGF-induced OPN production.

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