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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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c-Src is involved in HGF-mediated OPN production in osteoblasts.Cells were pretreated for 30 min with PP2 or transfected with c-Src 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 = 5) (A–D). Primary osteoblasts were incubated with HGF for indicated time intervals, and c-Src phosphorylation was examined by Western blotting (E). Primary osteoblasts were incubated with HGF for indicated time intervals, and c-Src kinase activity was examined by c-Src kinase assay kit (F). Primary osteoblasts were pretreated with c-Met inhibitor, Ly294002, and Akt inhibitor for 30 min or transfected with c-Met and Akt siRNA for 24 h followed by stimulation with HGF for 30 min, and c-Src kinase activity was examined by c-Src kinase assay kit (G). *: p<0.05 as compared with basal level (F) or HGF-treated group (A-D&G).
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pone-0038378-g004: c-Src is involved in HGF-mediated OPN production in osteoblasts.Cells were pretreated for 30 min with PP2 or transfected with c-Src 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 = 5) (A–D). Primary osteoblasts were incubated with HGF for indicated time intervals, and c-Src phosphorylation was examined by Western blotting (E). Primary osteoblasts were incubated with HGF for indicated time intervals, and c-Src kinase activity was examined by c-Src kinase assay kit (F). Primary osteoblasts were pretreated with c-Met inhibitor, Ly294002, and Akt inhibitor for 30 min or transfected with c-Met and Akt siRNA for 24 h followed by stimulation with HGF for 30 min, and c-Src kinase activity was examined by c-Src kinase assay kit (G). *: p<0.05 as compared with basal level (F) or HGF-treated group (A-D&G).

Mentions: PI3K/Akt-dependent c-Src activation is involved in the regulation of gene expression [24]. Therefore, we investigated the role of Src in mediating HGF-induced OPN expression with the specific Src inhibitor PP2. As shown in Figure 4A–D, HGF-induced OPN expression was markedly attenuated by pretreatment of cells for 30 min with PP2 or transfected of cells for 24 h with c-Src mutant and siRNA. The major phosphorylation site of c-Src at the Tyr416 residue results in activation from c-Src autophosphorylation [25]. To directly confirm the crucial role of c-Src in OPN expression, we measured the level of c-Src phosphorylation at Tyr416 in response to HGF. As shown in Figure 4E, treatment of primary osteoblasts with HGF resulted in a time-dependent phosphorylation of c-Src at Tyr416. In addition, HGF also increased c-Src kinase activity (Fig. 4F). Pretreatment of primary osteoblasts with c-Met inhibitor, Ly294002, and Akt inhibitor or transfection with c-Met and Akt siRNA markedly inhibited the HGF-induced c-Src kinase activity (Fig. 4G). Based on these results, HGF appears to act through a signaling pathway involving c-Met receptor, PI3K, Akt, and c-Src to enhance OPN expression 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)

c-Src is involved in HGF-mediated OPN production in osteoblasts.Cells were pretreated for 30 min with PP2 or transfected with c-Src 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 = 5) (A–D). Primary osteoblasts were incubated with HGF for indicated time intervals, and c-Src phosphorylation was examined by Western blotting (E). Primary osteoblasts were incubated with HGF for indicated time intervals, and c-Src kinase activity was examined by c-Src kinase assay kit (F). Primary osteoblasts were pretreated with c-Met inhibitor, Ly294002, and Akt inhibitor for 30 min or transfected with c-Met and Akt siRNA for 24 h followed by stimulation with HGF for 30 min, and c-Src kinase activity was examined by c-Src kinase assay kit (G). *: p<0.05 as compared with basal level (F) or HGF-treated group (A-D&G).
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Related In: Results  -  Collection

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

pone-0038378-g004: c-Src is involved in HGF-mediated OPN production in osteoblasts.Cells were pretreated for 30 min with PP2 or transfected with c-Src 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 = 5) (A–D). Primary osteoblasts were incubated with HGF for indicated time intervals, and c-Src phosphorylation was examined by Western blotting (E). Primary osteoblasts were incubated with HGF for indicated time intervals, and c-Src kinase activity was examined by c-Src kinase assay kit (F). Primary osteoblasts were pretreated with c-Met inhibitor, Ly294002, and Akt inhibitor for 30 min or transfected with c-Met and Akt siRNA for 24 h followed by stimulation with HGF for 30 min, and c-Src kinase activity was examined by c-Src kinase assay kit (G). *: p<0.05 as compared with basal level (F) or HGF-treated group (A-D&G).
Mentions: PI3K/Akt-dependent c-Src activation is involved in the regulation of gene expression [24]. Therefore, we investigated the role of Src in mediating HGF-induced OPN expression with the specific Src inhibitor PP2. As shown in Figure 4A–D, HGF-induced OPN expression was markedly attenuated by pretreatment of cells for 30 min with PP2 or transfected of cells for 24 h with c-Src mutant and siRNA. The major phosphorylation site of c-Src at the Tyr416 residue results in activation from c-Src autophosphorylation [25]. To directly confirm the crucial role of c-Src in OPN expression, we measured the level of c-Src phosphorylation at Tyr416 in response to HGF. As shown in Figure 4E, treatment of primary osteoblasts with HGF resulted in a time-dependent phosphorylation of c-Src at Tyr416. In addition, HGF also increased c-Src kinase activity (Fig. 4F). Pretreatment of primary osteoblasts with c-Met inhibitor, Ly294002, and Akt inhibitor or transfection with c-Met and Akt siRNA markedly inhibited the HGF-induced c-Src kinase activity (Fig. 4G). Based on these results, HGF appears to act through a signaling pathway involving c-Met receptor, PI3K, Akt, and c-Src to enhance OPN expression 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