A pertussis toxin sensitive G-protein-independent pathway is involved in serum amyloid A-induced formyl peptide receptor 2-mediated CCL2 production

Abstract

Serum amyloid A (SAA) induced CCL2 production via a pertussis toxin (PTX)-insensitive pathway in human umbilical vein endothelial cells (HUVECs). SAA induced the activation of three MAPKs (ERK, p38 MAPK, and JNK), which were completely inhibited by knock-down of formyl peptide receptor 2 (FPR2). Inhibition of p38 MAPK and JNK by their specific inhibitors (SB203580 and SP600125), or inhibition by a dominant negative mutant of p38 MAPK dramatically decreased SAA-induced CCL2 production. Inactivation of G((i)) protein(s) by PTX inhibited the activation of SAA-induced ERK, but not p38 MAPK or JNK. The results indicate that SAA stimulates FPR2-mediated activation of p38 MAPK and JNK, which are independent of a PTX-sensitive G-protein and are essential for SAA-induced CCL2 production.

Figure 1

SAA-induced CCL2 production from HUVECs is PTX-insensitive. HUVECs were cultured for 24 h in the presence or absence of PTX (100 ng/ml), and then stimulated for 24 h with different concentrations of SAA (0, 0.1, 0.5, 1, and 2 µM) or 50 µM of LPC (A). HUVECs were stimulated for varying times (0, 1, 3, 6, 12, and 24 h) with 2 µM SAA in the absence or presence of PTX (100 ng/ml) (B). HUVECs were stimulated for 24 h with 2 µM SAA in the absence or presence of WRW⁴ (10 µM) (C). The secreted CCL2 levels were determined by ELISA. The results are means ± SEM of 3 independent experiments performed in duplicate (A-C). *P < 0.05 compared to the control (0 µM). #Significantly different (P < 0.05) from treatment with LPC or SAA alone.

Figure 2

SAA stimulates MAPKs activities in HUVECs. (A) HUVECs were stimulated for different lengths of time (0, 2, 5, 10, and 30 min) with 2 µM of SAA. (B) HUVECs were stimulated for 5 min with different concentrations of SAA (0, 0.01, 0.1, 0.5, 1, and 2 µM). (C) HUVECs were cultured for 24 h in the presence or absence of PTX (100 ng/ml), and then stimulated for 5 min with 2 µM of SAA or vehicle. (A-C) Phosphorylated ERK, phosphorylated p38 MAPK, or phosphorylated JNK levels were determined by immunoblot analysis using anti-phospho-ERK antibody, anti-phospho-p38 MAPK antibody, or anti-phospho-JNK antibody. The results shown in (A-C) are representative of at least 3 independent experiments.

Figure 3

p38 MAPK and JNK are essential for SAA-induced CCL2 production by HUVECs. (A) HUVECs were pre-incubated with several concentrations (0, 1, 5, 10, 20, or 50 µM) of PD98059 (60 min), SB203580 (15 min), or SP600125 (15 min) prior to stimulation with 2 µM of SAA for 24 h. (B) The genes for GFP (AdTrack) or dominant negative p38 (AdDNp38) (MOI; 50) were transferred into HUVECs using recombinant adenoviruses prior to SAA (2 µM) stimulation for 24 h (B) or for 2, 5, 10, or 30 min (B insert). The amounts of secreted CCL2 were measured by ELISA. Phosphorylated ATF-2 levels were measured by Western blot analysis (B insert). Results are means ± SEM of 3 independent experiments performed in duplicate (A, B). ^*P < 0.05 compared to the control (SAA only treated).

Figure 4

SAA-induced MAPK activity is mediated by FPR2. HUVECs were transfected with 20 nM of FPR2 siRNA or control siRNA for 48 h. The transfected cells were stimulated with vehicle, 2 µM of SAA, or 100 nM of PMA for 5 min. Phosphorylated ERK, phosphorylated p38 MAPK, or phosphorylated JNK levels were determined by immunoblot analysis using anti-phospho-ERK antibody, anti-phospho-p38 MAPK antibody or anti-phospho-JNK antibody. The results shown are representative of 3 independent experiments.

Figure 5

SAA-induced CCL2 production from HUAECs is PTX-insensitive. HUAECs were stimulated for 24 h with different concentrations of SAA (0, 0.1, 0.5, 1, and 2 µM) or 1 µg/ml of LPS (A). HUAECs were cultured for 24 h in the presence or absence of PTX (100 ng/ml), and then stimulated for 24 h with 2 µM SAA or 50 µM LPC (B). The secreted CCL2 levels were determined by ELISA. Results are the means ± SEM of 3 independent experiments performed in duplicate (A, B). ^*P < 0.05 compared to the control (0 µM). ^#Significantly different (P < 0.05) from treatment with LPC alone.