p38MAPK is involved in CCL2-induced VCAM-1 expression in synovial fibroblasts.

<p>(A) OASFs were pretreated for 30 min with SB203580 (10 µM), PD98059 (30 µM), or SP600125 (10 µM) followed by stimulation with CCL2 (30 ng/ml) for 24 h, and VCAM-1 expression was examined by qPCR (n = 5). OASFs were pretreated for 30 min with SB203580 followed by stimulation with CCL2 for 24 h, and VCAM-1 expression was examined by flow cytometry (B) and Western blotting (C) (n = 5). OASFs were transfected with p38MAPK mutant for 24 h followed by stimulation with CCL2 for 24 h, and VCAM-1 expression was examined by qPCR (D) and flow cytometry (E) (n = 4). OASFs were incubated with CCL2 for indicated time intervals (F) or pretreated with RS102895 or rottlerin for 30 min before incubation with CCL2 for 30 min (G), and p38 phosphorylation was determined by Western blotting (n = 5). Results are expressed as the mean ± S.E. *: p<0.05 as compared with basal level. #: p<0.05 as compared with CCL2-treated group.</p

PKCδ is involved in CCL2-induced VCAM-1 expression in synovial fibroblasts.

<p>OASFs were pretreated for 30 min with GF109203X (3 µM) or rottlerin (10 µM) followed by stimulation with CCL2 (30 ng/ml) for 24 h, and VCAM-1 expression was examined by qPCR (A), flow cytometry (B), and Western blotting (C) (n = 4–6). OASFs were transfected with PKCδ siRNA for 24 h followed by stimulation with CCL2 for 24 h, and VCAM-1 expression was examined by qPCR (D) and flow cytometry (E). OASFs were incubated with CCL2 for indicated time intervals (n = 4) (F) or pretreated with RS102895 for 30 min before incubation with CCL2 for 30 min (n = 4) (G), and PKCδ phosphorylation was determined by Western blotting (n = 4). Results are expressed as the mean ± S.E. *: p<0.05 as compared with basal level. #: p<0.05 as compared with CCL2-treated group.</p

CCL2 increases VCAM-1 expression.

<p>(A) Synovial fluid was obtained from normal (n = 12) or osteoarthritis patients (n = 11) and examined with ELISA for the expression of CCL2. (B) Human synovial fibroblasts were cultured for 48 h, and media were collected to measure CCL2 (n = 6). OASFs were incubated with various concentrations of CCL2 for 24 h. The mRNA (C), cell surface (D), and protein expression (E) of VCAM-1 was examined by qPCR, flow cytometry, and Western blotting (n = 4–6). Results are expressed as the mean ± S.E. *: p<0.05 as compared with basal level. #: p<0.05 as compared with CCL2-treated group.</p

CCL2 increases VCAM-1 expression through CCR2 receptor.

<p>OASFs were pretreated for 30 min with RS102895 (400 nM) or C0214 (400 nM) followed by stimulation with CCL2 (30 ng/ml) for 24 h, and VCAM-1 expression was examined by qPCR (A; β-actin was used as internal control), flow cytometry (B), and Western blotting (C) (n = 5–6). Results are expressed as the mean ± S.E. *: p<0.05 as compared with basal level. #: p<0.05 as compared with CCL2-treated group.</p

AP-1 is involved in the potentiation of VCAM-1 expression by CCL2.

<p>OASFs were pretreated for 30 min with curcumin (3 µM) or tanshinone IIA (5 µM) followed by stimulation with CCL2 (30 ng/ml) for 24 h, and VCAM-1 expression was examined by qPCR (A), flow cytometry (B), and Western blotting (C) (n = 4–6). OASFs were transfected with c-Jun siRNA for 24 h followed by stimulation with CCL2 for 24 h, and VCAM-1 expression was examined by qPCR (D) and flow cytometry (E) (n = 4). (F) OASFs were incubated with CCL2 for indicated time intervals and c-Jun phosphorylation was determined by Western blotting (n = 5). Results are expressed as the mean ± S.E. *: p<0.05 as compared with basal level. #: p<0.05 as compared with CCL2-treated group.</p

The c-Met receptor is involved in HGF-mediated VEGF-A production.

<p>(A) OASFs were transfected with c-Met siRNA for 24 h, and c-Met expression was examined by Western blotting. (B–D) OASFs were pretreated with the c-Met inhibitor (3 µM) for 30 min or transfected with c-Met siRNA for 24 h followed by treatment with HGF for 24 h, the VEGF-A expression was examined by qPCR, Western blotting, and ELISA. Results are expressed as the mean ± S.E. *, <i>p</i><0.05 compared with control; #, <i>p</i><0.05 compared with HGF-treated group.</p

HGF enhances HIF-1α activation.

<p>(A) OASFs were incubated with HGF for indicated time intervals, and HIF-1α expression was examined by Western blotting. (B) OASFs were incubated with HGF for indicated time intervals, and nucleus HIF-1α accumulation was determent by Western blotting. (C) OASFs were incubated with HGF for indicated time intervals, and HIF-1α mRNA expression was examined by qPCR. (D&E) OASFs were pretreated for 30 min with HIF-1α inhibitor followed by treatment with HGF for 24 h, the VEGF-A expression was examined by qPCR and ELISA. (F) OASFs were transfected with HIF-1α mutant followed by stimulation with HGF for 24 h, the VEGF-A expression was examined by ELISA. Results are expressed as the mean ± S.E. *, <i>p</i><0.05 compared with control; #, <i>p</i><0.05 compared with HGF-treated group.</p

HGF stimulates concentration- and time-dependent increases in VEGF-A production.

<p>OASFs were incubated with HGF (3–100 ng/ml) for 24 h (A) or with HGF (30 ng/ml) for 6, 12, or 24 h (B), and VEGF-A mRNA was examined by qPCR. (C–F) OASFs were incubated with HGF (3–100 ng/ml) for 24 h or with HGF (30 ng/ml) for 6, 12, or 24 h, and VEGF-A protein expression was examined by Western blotting (whole cells lysate) and ELISA (medium). Results are expressed as the mean ± S.E. *, <i>p</i><0.05 compared with control; #, <i>p</i><0.05 compared with HGF-treated group.</p

The PI3K/Akt signaling pathway is activated in response to HGF treatment of synovial fibroblasts.

<p>(A) OASFs were incubated with HGF for indicated time intervals, and p85 and Akt phosphorylation was examined by Western blotting. (B–D) OASFs were pretreated for 30 min with Ly294002 (10 µM) or Akt inhibitor (10 µM) followed by treatment with HGF for 24 h, the VEGF-A expression was examined by qPCR, Western blotting, and ELISA. (E) OASFs were transfected with p85 or Akt mutant followed by stimulation with HGF for 24 h, the VEGF-A expression was examined by ELISA. OASFs were pretreated for 30 min with c-Met inhibitor (F) or c-Met inhibitor and Ly294002 for 30 min (G) followed by stimulation with HGF for 15 min, and p85 and Akt phosphorylation was determined by Western blotting. Results are expressed as the mean ± S.E. *, <i>p</i><0.05 compared with control; #, <i>p</i><0.05 compared with HGF-treated group.</p

mTORC1 activation is involved in HGF-mediated VEGF-A production.

<p>(A) OASFs were incubated with HGF for indicated time intervals, mTORC1 and S6K phosphorylation was examined by Western blotting. (B–D) OASFs were pretreated for 30 min with rapamycin (30 nM) followed by treatment with HGF for 24 h, the VEGF-A expression was examined by qPCR, Western blotting, and ELISA. (E) OASFs were transfected with mTORC1 siRNA followed by stimulation with HGF for 24 h, the VEGF-A expression was examined by ELISA. (F) OASFs were pretreated for 30 min with c-Met inhibitor, Ly294002, and Akt inhibitor for 30 min, followed by stimulation with HGF for 30 min, and mTORC1 phosphorylation was determined by Western blotting. Results are expressed as the mean ± S.E. *, <i>p</i><0.05 compared with control; #, <i>p</i><0.05 compared with HGF-treated group.</p