Figure 1.
ATP-induced BDNF expression in SF.
OASF (n = 11) and RASF (n = 6) were stimulated with ATP (50, 100, 200, 500 µM) for 10 minutes, 2 h, 5 h and 24 h. A. BDNF mRNA levels were induced 2 h after ATP stimulation. B. Influence of different ATP concentrations on the induction levels of BDNF mRNA after 2 h ATP stimulation. C. BDNF protein levels in cell culture supernatants of unstimulated OASF (n = 8) and RASF (n = 6) were measured by ELISA. D. BDNF protein levels in cell culture supernatants of OASF and RASF were elevated 5 h after ATP stimulation. Data are shown as means ± standard deviations. N.s., not significant; *, p<0.05, ***, p<0.001.
Figure 2.
Purinergic receptor P2X, but not P2Y agonists induce BDNF expression.
OASF (n = 8) and RASF (n = 3) were stimulated for 2 h with 100 µM ATP, ADP and UTP, respectively. BDNF mRNA expression was increased by ATP and ADP, but not by UTP. Data are shown as means ± standard deviations. N.s., not significant; *, p<0.05, ***, p<0.001.
Figure 3.
Representative photomicrographs showing immunohistochemical double staining of OA and RA synovial tissues with P2X4 (brown) and fibroblast marker (green). A. original magnification 400×, box shows the Isotype control, magnification 200×. B. original magnification 630×.
Figure 4.
Purinergic receptor P2X4 siRNA transfection in OASF (n = 8).
Endogenous P2X4 expression in OASF was reduced by siRNA transfection before ATP (100 µM) stimulation for 2 h. A. P2X4 mRNA expression was reduced compared to scrambled controls. The ATP-induced BDNF mRNA expression in P2X4 siRNA transfected cells was reduced compared to scrambled controls. B. P2X4 protein levels were reduced by P2X4 siRNA transfection compared to scrambled controls. α-tubulin levels in P2X4 siRNA transfected cells were slightly decreased compared to controls due to experimental conditions. C. Densitometric analysis of Western blot results. Data are shown as means ± standard deviations. N.s., not significant; *, p<0.05, ***, p<0.001.
Figure 5.
Activation of MAPK pathways in OASF after ATP stimulation.
A. OASF were stimulated with ATP (100 µM) for 10 minutes, 2 h, 5 h and 24 h. Western blot analysis of p38 and p44/42 MAPK, as well as phosphorylated p-p38 and p-p44/42 MAPK showed kinase activation after 10 minutes ATP stimulation. B. OASF (n = 10) and RASF (n = 3) were treated with the kinase inhibitors FR180204, SB203580 and PD98059, respectively 15 min before ATP (100 µM) stimulation for 2 h. Data are shown as means ± standard deviations. *, p<0.05.
Figure 6.
Inhibition of p38 and p44/42 MAPK signaling by siRNA transfection.
OASF (n = 6) were transfected with siRNA targeting p38 MAPK, p44/42 MAPK and scrambled siRNA (control) before ATP (100 µM) stimulation for 2 h. A. Decreased p38 and p44/42 MAPK expression after siRNA transfection was verified by Western blot analysis. B. Densitometric analysis of Western blot results. C. The ATP-induced BDNF mRNA expression was reduced in p38 MAPK siRNA transfected OASF but not in p44/42 MAPK siRNA transfected cells. Data are shown as means ± standard deviations. *, p<0.05.