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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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AP-1 is involved in the potentiation of OPN production by HGF.Cells were pretreated for 30 min with curcumin or transfected with c-Jun 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 = 5) (A–D). Primary osteoblasts were incubated with HGF for indicated time intervals, and c-Jun phosphorylation was determined by Western blotting (E). Primary osteoblasts were pretreated with c-Met inhibitor, Ly294002, Akt inhibitor, and PP2 or transfected with c-Met and Akt siRNA, and then stimulated with HGF for 120 min. A chromatin immunoprecipitation assay was then performed. The chromatin was immunoprecipitated with anti-c-Jun. One percent of the precipitated chromatin was assayed to verify equal loading (input) (F). MG63 cells were pretreated with c-Met inhibitor, Ly294002, Akt inhibitor, or PP2, and then stimulated with HGF for 120 min, and c-Jun immunofluorescence staining was examined (G). *: p<0.05 as compared with HGF-treated group.
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pone-0038378-g005: AP-1 is involved in the potentiation of OPN production by HGF.Cells were pretreated for 30 min with curcumin or transfected with c-Jun 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 = 5) (A–D). Primary osteoblasts were incubated with HGF for indicated time intervals, and c-Jun phosphorylation was determined by Western blotting (E). Primary osteoblasts were pretreated with c-Met inhibitor, Ly294002, Akt inhibitor, and PP2 or transfected with c-Met and Akt siRNA, and then stimulated with HGF for 120 min. A chromatin immunoprecipitation assay was then performed. The chromatin was immunoprecipitated with anti-c-Jun. One percent of the precipitated chromatin was assayed to verify equal loading (input) (F). MG63 cells were pretreated with c-Met inhibitor, Ly294002, Akt inhibitor, or PP2, and then stimulated with HGF for 120 min, and c-Jun immunofluorescence staining was examined (G). *: p<0.05 as compared with HGF-treated group.

Mentions: The promoter region of human OPN contains AP-1 binding site [20]. To examine the role of the AP-1 binding site in HGF-mediated OPN expression, the AP-1 inhibitor curcumin was used. Pretreatment of cells with curcumin reduced HGF-enhanced OPN expression (Fig. 5A–D). AP-1 activation was further evaluated by analyzing the phosphorylation of c-Jun as well as by a chromatin immunoprecipitation assay. Treatment of primary osteoblasts with HGF increased c-Jun phosphorylation (Fig. 5E). In addition, transfection of cells with c-Jun siRNA suppressed HGF-induced OPN expression (Fig. 5A–D). We next investigated whether c-Jun binds to the AP-1 element on the OPN promoter after HGF stimulation. The in vivo recruitment of c-Jun to the OPN promoter was assessed via chromatin immunoprecipitation assay. In vivo binding of c-Jun to the AP-1 element of the OPN promoter occurred after HGF stimulation (Fig. 5F). The binding of c-Jun to the AP-1 element by HGF was attenuated by c-Met inhibitor, Ly294002, Akt inhibitor, and PP2 or c-Met and Akt siRNA (Fig. 5F). In addition, pretreatment of cells with c-Met inhibitor, Ly294002, Akt inhibitor, and PP2 also reduced HGF-induced accumulation of c-Jun into the nucleus (Fig. 5G).


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)

AP-1 is involved in the potentiation of OPN production by HGF.Cells were pretreated for 30 min with curcumin or transfected with c-Jun 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 = 5) (A–D). Primary osteoblasts were incubated with HGF for indicated time intervals, and c-Jun phosphorylation was determined by Western blotting (E). Primary osteoblasts were pretreated with c-Met inhibitor, Ly294002, Akt inhibitor, and PP2 or transfected with c-Met and Akt siRNA, and then stimulated with HGF for 120 min. A chromatin immunoprecipitation assay was then performed. The chromatin was immunoprecipitated with anti-c-Jun. One percent of the precipitated chromatin was assayed to verify equal loading (input) (F). MG63 cells were pretreated with c-Met inhibitor, Ly294002, Akt inhibitor, or PP2, and then stimulated with HGF for 120 min, and c-Jun immunofluorescence staining was examined (G). *: p<0.05 as compared with HGF-treated group.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3366938&req=5

pone-0038378-g005: AP-1 is involved in the potentiation of OPN production by HGF.Cells were pretreated for 30 min with curcumin or transfected with c-Jun 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 = 5) (A–D). Primary osteoblasts were incubated with HGF for indicated time intervals, and c-Jun phosphorylation was determined by Western blotting (E). Primary osteoblasts were pretreated with c-Met inhibitor, Ly294002, Akt inhibitor, and PP2 or transfected with c-Met and Akt siRNA, and then stimulated with HGF for 120 min. A chromatin immunoprecipitation assay was then performed. The chromatin was immunoprecipitated with anti-c-Jun. One percent of the precipitated chromatin was assayed to verify equal loading (input) (F). MG63 cells were pretreated with c-Met inhibitor, Ly294002, Akt inhibitor, or PP2, and then stimulated with HGF for 120 min, and c-Jun immunofluorescence staining was examined (G). *: p<0.05 as compared with HGF-treated group.
Mentions: The promoter region of human OPN contains AP-1 binding site [20]. To examine the role of the AP-1 binding site in HGF-mediated OPN expression, the AP-1 inhibitor curcumin was used. Pretreatment of cells with curcumin reduced HGF-enhanced OPN expression (Fig. 5A–D). AP-1 activation was further evaluated by analyzing the phosphorylation of c-Jun as well as by a chromatin immunoprecipitation assay. Treatment of primary osteoblasts with HGF increased c-Jun phosphorylation (Fig. 5E). In addition, transfection of cells with c-Jun siRNA suppressed HGF-induced OPN expression (Fig. 5A–D). We next investigated whether c-Jun binds to the AP-1 element on the OPN promoter after HGF stimulation. The in vivo recruitment of c-Jun to the OPN promoter was assessed via chromatin immunoprecipitation assay. In vivo binding of c-Jun to the AP-1 element of the OPN promoter occurred after HGF stimulation (Fig. 5F). The binding of c-Jun to the AP-1 element by HGF was attenuated by c-Met inhibitor, Ly294002, Akt inhibitor, and PP2 or c-Met and Akt siRNA (Fig. 5F). In addition, pretreatment of cells with c-Met inhibitor, Ly294002, Akt inhibitor, and PP2 also reduced HGF-induced accumulation of c-Jun into the nucleus (Fig. 5G).

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