Cyclophilin A up-regulates MMP-9 expression and adhesion of monocytes/macrophages via CD147 signalling pathway in rheumatoid arthritis

Abstract

Objectives: To investigate whether cyclophilin A (CypA) can up-regulate the expression of MMP-2 and MMP-9 in monocytes/macrophages and whether CD147 facilitates this regulation in RA. Methods. Peripheral blood monocytes were isolated from RA patients and differentiated into macrophages by M-CSF (15 ng/ml). Under CypA stimulation (200 ng/ml), the protein release and activation of MMPs were detected by gelatin zymography and invasion assay. Human monocyte cell line THP-1 cells were selected for the advanced searching for potential interaction between CypA and CD147 in production of MMPs and cell adhesion to extracellular matrix (ECM).

Results: CypA significantly increased production and activation of MMP-9, not MMP-2, in the monocytes/macrophages derived from RA SF. CSA and HAb18G/CD147 antagonistic peptide AP-9 against CD147, respectively, dramatically decreased MMP-2 and MMP-9 expression, both in the absence or presence of CypA. Similar effects of CypA on MMP-9 production and cell invasion were observed in THP-1 cells. CypA-induced nuclear factor kappaB (NF-kappaB) activity for MMP-9 transcription were strongly blocked by extracellular signal-regulated kinase (ERK), c-Jun amino terminal kinase (JNK) inhibitors (U0126 and SP600125, respectively), but not by p38 mitogen-activated protein kinase (MAPK) inhibitors (SB203580). CypA also induced calcium mobilization and increased the adhesion of THP-1 cells to ECM.

Conclusions: These findings suggest that in RA, the abundant CypA, by its direct binding to CD147, up-regulates MMP-9 expression and adhesion of monocytes/macrophages to ECM, and the cyclophilin-CD147 interactions might contribute to the destruction of cartilage and bone.

Fig. 1.

Detection of CD147, CD14 and CD68 expressions in monocytes/macrophages by flow cytometry. Flow cytometry showed that CD147, CD14 and CD68 expressions were all increased both in mature macrophages (shaded peaks) differentiated from monocytes of normal human and RA peripheral blood (unshaded peaks). CD147 expression on monocytes/macrophages was highest among these cells. Data are expressed as means ± s.d. (n = 8).

Fig. 2.

Gelatin zymography of culture medium conditioned by monocytes/macrophages with CypA stimulation. The protein expression of pro-MMP-9 (d) and MMP-9 (e) were significantly increased when monocytes were differentiated into marophages. CypA increased the secretion and activation of pro-MMP-9, but not that of pro-MMP-2, in macrophages and monocytes/macrophages from RA SF. Both CSA and AP-9 could inhibit CypA's effect on MMP-9 production. Data are expressed as means ± s.d. (n = 8). *P < 0.05, **P < 0.05 vs negative control of monocytes and macrophages derived from normal human peripheral blood, respectively. P < 0.05, P < 0.05 vs negative control of monocytes and macrophages derived from RA patients’ peripheral blood; ★P < 0.05 vs negative control of monocytes/macrophages RA patients’ SF, respectively.

Fig. 3.

Invasion assay of monocytes/macrophages with CypA stimulation. Monocytes and macrophages from normal human (a) and RA patients’ (b) peripheral blood were pre-treated, respectively with CSA, or AP-9, and then were stimulated by CypA for invasion assay. A higher number of macrophages (cells/filter) were found to have invaded through transwell chambers than monocytes. CypA significantly increased the number of the invading macrophages, while it had little effect on monocytes. CSA or AP-9 could inhibit CypA's function. Data are expressed as means ± s.d. (n = 8). *P < 0.05 and P < 0.05 vs negative control of monocytes and macrophages from normal human peripheral blood, respectively. *P < 0.05 and P < 0.05 vs negative control of monocytes and macrophages from RA patients’ peripheral blood, respectively.

Fig. 4.

Gelatin zymography of culture medium conditioned by THP-1 cells with CypA stimulation. (a) uTHP-1 and dTHP-1 cells were pre-treated, respectively, with siRNA, CSA or AP-9, and then were stimulated with CypA for 24 h. The supernatants were collected for gel zymography. The statistical data showed relative enzyme activity of pro-MMP-9 (b), MMP-9 (c) and pro-MMP-2 (d) with different treatments. CypA increased total MMP-9, but not MMP-2, secretion in the uTHP-1 and dTHP-1 cells. RNAi, CSA or AP-9 decreased the secretion of pro-MMP-2 and pro-MMP-9 in uTHP-1 or dTHP-1 cells, respectively, both in the absence or presence of CypA. Data are expressed as means ± s.d. (n = 3). *P < 0.05 vs the normal negative uTHP-1 cells. P < 0.05 vs the normal negative dTHP-1 cells.

Fig. 5.

qRT–PCR of MMP-2, -9 and CD147 mRNA in THP-1 cells with CypA stimulation. The uTHP-1 and dTHP-1 cells were pre-treated, respectively, with CSA, RNAi or AP-9 against CD147 and then were stimulated by CypA. qRT–PCR was run for MMP-9 (a), MMP-2 (b) and CD147 (c) mRNA. CypA increased MMP-9, not MMP-2, mRNA expression, in both uTHP-1 and dTHP-1 cells (P < 0.05). RNAi, CSA or AP-9 decreased MMP-2 and MMP-9 expression in the uTHP-1 or dTHP-1 cells, respectively (P < 0.05), both in the absence or presence of CypA. (c) CypA had no significant effect on CD147 mRNA expression, while CSA dramatically decreased it. (d) Flow cytometry showed that CSA dramatically suppressed membrane CD147 expression ∼52% and AP-9 blocked membrane CD147 ∼ 63% in THP-1 cells. Data are expressed as means ± s.d. (n = 3). *P < 0.05 vs the normal negative uTHP-1 cells. P < 0.05 vs the normal negative dTHP-1 cells.

Fig. 6.

IF of NF-κB activity induced by CypA in the THP-1 cell. (a) The u and d THP-1 cells were precubated with control (a, b, i, j) and AP-9 (c, d, k, l), then stimulated with CypA (200 ng/mL, b, d, j, l), CypA/CSA (f, n), LPS (1 µg/mL, g, o) or LPS/CSA (h, p) respectively for 1h. The p50 protein of NF-κB activity was analysed by IF with FITC-conjugated anti-p50 antibody (green fluorescence) for the uTHP-1 and Cy3-conjugated anti-p50 antibody (red fluorescence) for the dTHP-1, respectively. CypA-induced nuclear translocation of NF-κB 2 h after treatment (b and h). Nuclear translocation of NF-κB induced by CypA was blockaded by AP-9 (d and l) or CSA (f and n) respectively. (b) Merged images of Cy3 and DAPI in the dTHP-1 were obtained using computer software. After CypA stimulating THP-1, the red color for NF-κB overlayed with the blue color for nucleus in cell nucleus under immunofluorescence microscopy, which showed plenty of p50 transducted into nucleus. The scale bars indicate 25 μm (a) and 10 μm (b). The data shown are representative of similar results from three independent experiments.

Fig. 7.

Effect of MAPK inhibitors on MMP-9 secretion and NF-κB sactivity in the THP-1. (a) The uTHP-1 and dTHP-1 cells were pre-treated with 10 μM U0126, SB203580 and SP600125 for 2 h, respectively and then were stimulated with CypA for 2 h. The cells were collected for detection of NF-κB activity by IF. The scale bars indicate 25 μm. (b) The uTHP-1 and dTHP-1 cells were pre-treated with U0126, SB203580 and SP600125 for 2 h, respectively and then were stimulated with CypA for 48 h in serum-free culture. The supernatants of each sample were collected for gelatin zymography. SB203580 had no effect on the MMP-9 secretion in the uTHP-1 or dTHP-1 cells, while U0126 and SP600125 significantly decreased MMP-9 secretion, both in the absence or presence of CypA. Similar results were observed in NF-κB activity by IF. The data shown are representative of similar results from three independent experiments.

Fig. 8.

CypA-mediated Ca²⁺ increase and adhesion of the THP-1 cells. (a) Ca²⁺ mobilization in the Fluo-3-loaded uTHP-1 (a ∼ d) and dTHP-1 (e ∼ h) cells was measured in the presence of CypA (200 ng/ml) (a, e), CypA/CSA (b, f), HAb18 pre-incubation (d, h) or pre-stimulation (c, g). The arrows indicate the addition of the agonist. The calcium intensity that was detected by intensity of green fluorescence correlated well with cytosolic Ca²⁺ concentration and showed the changing of Ca²⁺ levels in THP-1 cells. CypA-induced Ca²⁺ mobilization in both uTHP-1 (a) and dTHP-1 (e) cells while HAb 18 and CSA inhibited CypA-induced Ca²⁺ mobilization. (b) The uTHP-1 and dTHP-1 cells were pre-treated with HAb18 antibody or CSA and then were stimulated with CypA in Matrigel Matrix-coated 96-well microtitre plates. A significant increase in both uTHP-1 and dTHP-1 cell adhesion was observed with CypA. In contrast, CSA or HAb18 dramatically reduced THP-1 cell adhesion respectively, both in the absence or presence of CypA. Data are expressed as means ± s.d. (n = 3). P < 0.05 vs the normal negative uTHP-1 cells. *P < 0.05 vs the normal negative dTHP-1 cells.

Fig. 8.

CypA-mediated Ca²⁺ increase and adhesion of the THP-1 cells. (a) Ca²⁺ mobilization in the Fluo-3-loaded uTHP-1 (a ∼ d) and dTHP-1 (e ∼ h) cells was measured in the presence of CypA (200 ng/ml) (a, e), CypA/CSA (b, f), HAb18 pre-incubation (d, h) or pre-stimulation (c, g). The arrows indicate the addition of the agonist. The calcium intensity that was detected by intensity of green fluorescence correlated well with cytosolic Ca²⁺ concentration and showed the changing of Ca²⁺ levels in THP-1 cells. CypA-induced Ca²⁺ mobilization in both uTHP-1 (a) and dTHP-1 (e) cells while HAb 18 and CSA inhibited CypA-induced Ca²⁺ mobilization. (b) The uTHP-1 and dTHP-1 cells were pre-treated with HAb18 antibody or CSA and then were stimulated with CypA in Matrigel Matrix-coated 96-well microtitre plates. A significant increase in both uTHP-1 and dTHP-1 cell adhesion was observed with CypA. In contrast, CSA or HAb18 dramatically reduced THP-1 cell adhesion respectively, both in the absence or presence of CypA. Data are expressed as means ± s.d. (n = 3). P < 0.05 vs the normal negative uTHP-1 cells. *P < 0.05 vs the normal negative dTHP-1 cells.