Cyclooxygenase-2 enhances alpha2beta1 integrin expression and cell migration via EP1 dependent signaling pathway in human chondrosarcoma cells

Abstract

Background: Cyclooxygenase (COX)-2, the inducible isoform of prostaglandin (PG) synthase, has been implicated in tumor metastasis. Interaction of COX-2 with its specific EP receptors on the surface of cancer cells has been reported to induce cancer invasion. However, the effects of COX-2 on migration activity in human chondrosarcoma cells are mostly unknown. In this study, we examined whether COX-2 and EP interaction are involved in metastasis of human chondrosarcoma.

Results: We found that over-expression of COX-2 or exogenous PGE2 increased the migration of human chondrosarcoma cells. We also found that human chondrosarcoma tissues and chondrosarcoma cell lines had significant expression of the COX-2 which was higher than that in normal cartilage. By using pharmacological inhibitors or activators or genetic inhibition by the EP receptors, we discovered that the EP1 receptor but not other PGE receptors is involved in PGE2-mediated cell migration and alpha2beta1 integrin expression. Furthermore, we found that human chondrosarcoma tissues expressed a higher level of EP1 receptor than normal cartilage. PGE2-mediated migration and integrin up-regulation were attenuated by phospholipase C (PLC), protein kinase C (PKC) and c-Src inhibitor. Activation of the PLCbeta, PKCalpha, c-Src and NF-kappaB signaling pathway after PGE2 treatment was demonstrated, and PGE2-induced expression of integrin and migration activity were inhibited by the specific inhibitor, siRNA and mutants of PLC, PKC, c-Src and NF-kappaB cascades.

Conclusions: Our results indicated that PGE2 enhances the migration of chondrosarcoma cells by increasing alpha2beta1 integrin expression through the EP1/PLC/PKCalpha/c-Src/NF-kappaB signal transduction pathway.

Figure 1

COX-2-directed migration of human chondrosarcoma cells. JJ012 cells were transfected with IPTG/COX-2 expression plasmid or control vector for 24 hr followed by stimulation with IPTG (5 mM) for 24 hr, the COX-2 expression, PGE₂ production and migration activity were determined by Western blot analysis (A), ELISA assay (B) and Transwell (C). JJ012 cells were transfected with IPTG/COX-2 expression plasmid or control vector for 24 hr, and pretreated with valeryl salicylate (20 μM), celebrex (10 μM) or NS-398 (20 μM) for 30 min followed by stimulation with IPTG (5 mM), and in vitro migration was measured with the Transwell after 24 hr (D). JJ012 cells were incubated with various concentrations of PGE₂, and in vitro migration activity measured with the Transwell after 24 hr (E). Total RNA were extracted from normal cartilage (lines 1-4), chondrosarcoma patients (lines 5-8) or from chondrosarcoma cell lines (SW1353 and JJ012), and subjected to qPCR analysis for COX-2 (F). The migration activity of each cells measured in vitro with the Transwell chamber after 24 h showed a significantly higher migration activity in primary chondrosarcoma and chondrosarcoma cell lines as compared with primary chondrocyte (G). Results are expressed as the mean ± S.E. *, p < 0.05 compared with control; #, p < 0.05 compared with PGE₂-treated group.

Figure 2

EP1 receptor is involved in PGE₂-mediated migration of human chondrosarcoma cells. (A) JJ012 cells were transfected with IPTG/COX-2 expression plasmid or control vector for 24 hr followed by stimulation with IPTG (5 mM) for 24 hr, the mRNA expression of EP receptors was determined by qPCR. (B) JJ012 cells were incubated with PGE₂ for 24 hr, and the mRNA expression of EP receptors was determined by qPCR. (C) Total RNA were extracted from normal cartilage (lines 1-4), chondrosarcoma patients (lines 5-8) or from chondrosarcoma cell lines (SW1353 and JJ012), and subjected to qPCR analysis for EP1 receptor. (D) JJ012 cells were 17-phenyl trinor PGE₂ (3 μM), butaprost (10 μM), sulprostone (10 μM), 11-deoxy-PGE₁ (10 μM) and PGE2 plus SC19220 (10 μM), and in vitro migration activity measured with the Transwell after 24 hr. (E) Cells were transfected with EP receptors siRNA for 24 hr followed by stimulation with PGE₂, and in vitro migration measured with the Transwell after 24 hr. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control; #, p < 0.05 compared with PGE₂-treated group.

Figure 3

COX-2-directed migration of human chondrosarcoma cells involves up-regulation of α2β1 integrin. (A) JJ012 cells were incubated with PGE₂ for 24 hr, and the cells surface α5, α2, β3, α5β1, αvβ3 and α2β1 integrin was determined using flow cytometry. (B) Cells were incubated with PGE₂ for 24 hr, and the mRNA levels of α2 and β1 integrin was determined using qPCR. (C) JJ012 cells were transfected with IPTG/COX-2 expression plasmid or control vector for 24 hr followed by stimulation with IPTG (5 mM) for 24 hr, the mRNA expression of α2 and β1 integrin was determined by qPCR. JJ012 cells were incubated with PGE₂ for indicated time intervals, and mRNA and cell surface α2β1 integrin were examined by qPCR (D) and flow cytometry (E). (F) Cells were pretreated with α2β1 monoclonal antibody (3 μg/ml) for 30 min followed by stimulation with PGE₂. The in vitro migration activity measured after 24 hr. (E) JJ012 cells were treated with 17-phenyl trinor PGE₂ (3 μM), 11-deoxy-PGE₁ (10 μM), PGE₂, and PGE₂ plus SC19220 (10 μM), and cells surface α2β1 integrin was determined using flow cytometry. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control; #, p < 0.05 compared with PGE₂-treated group.

Figure 4

PLC/PKC/c-Src signaling pathway is involved in PGE₂-mediated migration and integrin upregulation in human chondrosarcoma cells. (A) JJ012 cells were incubated with PGE₂ for indicated time intervals, and p-PLCβ3, p-PKCα and p-c-Src expression was determined by Western blot analysis. (B&D) JJ012 cells were pretreated for 30 min with U73122, GF109203X or PP2. Then they were followed by stimulation with PGE₂, and in vitro migration and cell surface α2β1 integrin were measured with the Transwell and flow cytometry after 24 hr. (C) Cells were transfected with PKCα or c-Src mutant and PLCβ siRNA for 24 hr followed by stimulation with PGE₂, and in vitro migration measured with the Transwell after 24 hr. JJ012 cells were incubated with PGE₂ for indicated time intervals, and PKCα activity was determined by the PKCα kinase assay kit. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control; #, p < 0.05 compared with PGE₂-treated group

Figure 5

PGE₂ induces α2β1 integrin upregulation and cell migration through NF-κB activation. Cells were pretreated with PDTC or TPCK for 30 min, then they were followed by stimulation with PGE₂, and in vitro migration (A) and α2β1 integrin expression (B) were measured with the Transwell and flow cytometry after 24 hr. (C) JJ012 cells were incubated with PGE₂ for indicated time intervals, p-IKK, p-IκBα and p-p65 expression was determined by Western blot analysis. (D) Cells were transfected with IKKα or IKKβ mutant for 24 hr followed by stimulation with PGE₂, and in vitro migration measured with the Transwell after 24 hr. (E) Cells were pretreated with U73122, GF109203X, PP2, PDTC, TPCK, PDTC for 30 min or co-transfected with PLCβ siRNA, PKCα mutant, IKKα mutant, IKKβ mutant for 24 hr before incubation with PGE₂ for 24 hr. The NF-κB activity was measured. (F) Cells were pretreated with U73122, GF109203X or PP2 for 30 min followed stimulation with PGE₂ for 60 min, and p-p65 expression was examined by Western blot analysis. Results are expressed as the mean ± S.E. *, p < 0.05 compared with control; #, p < 0.05 compared with PGE₂-treated group