YC-1 increases cyclo-oxygenase-2 expression through protein kinase G- and p44/42 mitogen-activated protein kinase-dependent pathways in A549 cells

Abstract

YC-1, an activator of soluble guanylate cyclase (sGC), has been shown to increase the intracellular cGMP concentration. This study was designed to investigate the signaling pathway involved in the YC-1-induced COX-2 expression in A549 cells. YC-1 caused a concentration- and time-dependent increase in COX activity and COX-2 expression in A549 cells. Pretreatment of the cells with the sGC inhibitor (ODQ), the protein kinase G (PKG) inhibitor (KT-5823), and the PKC inhibitors (Go 6976 and GF10923X), attenuated the YC-1-induced increase in COX activity and COX-2 expression. Exposure of A549 cells to YC-1 caused an increase in PKC activity; this effect was inhibited by ODQ, KT-5823 or Go 6976. Western blot analyses showed that PKC-alpha, -iota, -lambda, -zeta and -mu isoforms were detected in A549 cells. Treatment of A549 cells with YC-1 or PMA caused a translocation of PKC-alpha, but not other isoforms, from the cytosol to the membrane fraction. Long-term (24 h) treatment of A549 cells with PMA down-regulated the PKC-alpha. The MEK inhibitor, PD 98059 (10 - 50 microM), concentration-dependently attenuated the YC-1-induced increases in COX activity and COX-2 expression. Treatment of A549 cells with YC-1 caused an activation of p44/42 MAPK; this effect was inhibited by KT-5823, Go 6976, long-term (24 h) PMA treatment or PD98059, but not the p38 MAPK inhibitor, SB 203580. These results indicate that in human pulmonary epithelial cells, YC-1 might activate PKG through an upstream sGC/cGMP pathway to elicit PKC-alpha activation, which in turn, initiates p44/42 MAPK activation, and finally induces COX-2 expression.

Figure 1

Effects of YC-1 on PGE₂ release, COX activity and COX-2 expression in A549 cells. Treatment of the cells with various concentrations of YC-1 for 12 h (a) or YC-1 (50 μM) for the indicated time intervals (b) did not change the PGE₂ release. YC-1 induced a concentration-dependent increase in COX activity (c) and COX-2 expression (e), and a time-dependent increase in COX activity (d) and COX-2 expression (f). The COX activity was measured by examining the PGE₂ formation in the presence of 30 μM exogenous arachidonic acid for 30 min. Results are expressed as means±s.e.mean of four independent experiments performed in duplicate. ^*P<0.05 as compared with the basal level. In (e) and (f), cells were incubated with the indicated concentrations of YC-1 for 12 h (e) or YC-1 (50 μM) for various time intervals (f), and the extracted proteins were then immunodetected with COX-2 or α-tubulin specific antibody as described in Methods. The equal loading in each lane was demonstrated by the similar intensities of α-tubulin. Whole cell lysate of mouse macrophages (RAW 264.7) stimulated by LPS (1 μg ml⁻¹) and INFγ (10 ng ml⁻¹) for 12 h was used as a positive control (P).

Figure 2

Effects of actinomycin D and cycloheximide on the YC-1-induced increase of COX activity and COX-2 expression in A549 cells. In (a), the cells were pretreated with 0.1 μM ActD or 3 μM CHX for 30 min followed by a 12 h YC-1 (50 μM) incubation. The media were then removed, and the COX activity was measured by examining the PGE₂ formation in the presence of 30 μM exogenous arachidonic acid for 30 min. Results are expressed as means±s.e.mean of four independent experiments performed in duplicate. ^*P<0.05 as compared with treatment with YC-1 alone. In (b), the cells were pretreated with 0.1 μM ActD or 3 μM CHX for 30 min before incubation with YC-1 (50 μM) for 12 h. Immunodetection with COX-2 or α-tubulin specific antibody was performed as described in Methods. The equal loading in each lane was demonstrated by the similar intensities of α-tubulin. Data are representative of three independent experiments, which gave essentially identical results. ActD, actinomycin D; CHX, cycloheximide.

Figure 3

Involvement of sGC/cGMP pathway in the YC-1-induced increase of COX activity and COX-2 expression in A549 cells. In (a), the cells were pretreated with various concentrations of ODQ (sGC inhibitor) or KT (PKG inhibitor) for 30 min followed by a 12 h YC-1 (50 μM) incubation. The media were then removed, and the COX activity was measured by examining the PGE₂ formation in the presence of 30 μM exogenous arachidonic acid for 30 min. Results are expressed as means±s.e.mean of four independent experiments performed in duplicate. ^*P<0.05 as compared with treatment with YC-1 alone. In (b), the cells were pretreated with 30 μM ODQ or 5 μM KT for 30 min before incubation with YC-1 (50 μM) for 12 h. Immunodetection with COX-2 or α-tubulin specific antibody was performed as described in Methods. The equal loading in each lane was demonstrated by the similar intensities of α-tubulin. Data are representative of three independent experiments, which gave essentially identical results. KT, KT-5823.

Figure 4

Dibutyryl cGMP and PMA induce a time-dependent increase in COX-2 expression in A549 cells. The cells were incubated with 0.3 μM dibutyryl cGMP (a) or 10 nM PMA (b) for various time intervals, and the extracted proteins were then immunodetected with COX-2 or α-tubulin specific antibody as described in Methods. The extents of COX-2 and α-tubulin protein expression were quantitated using a densitometer with Image-Pro plus software. The relative level was calculated as the ratio of COX-2 to α-tubulin protein level. Results were expressed as mean±s.e.mean (n=3). ^*P<0.05 as compared with the basal level.

Figure 5

Involvement of PKC in the YC-1-induced increase of COX activity and COX-2 expression in A549 cells. In (a), the cells were pretreated with various concentrations of PKC inhibitor (Go or GF) for 30 min followed by a 12 h YC-1 (50 μM) incubation. The media were then removed, and the COX activity was measured by examining the PGE₂ formation in the presence of 30 μM exogenous arachidonic acid for 30 min. Results are expressed as means±s.e.mean of four independent experiments performed in duplicate. ^*P<0.05 as compared with treatment with YC-1 alone. In (b), the cells were pretreated with 1 μM Go or 10 μM GF for 30 min before incubation with YC-1 (50 μM) for 12 h. Immunodetection with COX-2 or α-tubulin specific antibody was performed as described in Methods. The equal loading in each lane was demonstrated by the similar intensities of α-tubulin. Data are representative of three independent experiments, which gave essentially identical results. Go, Go 6976; GF, GF10923X.

Figure 6

The PKC activity caused by YC-1 in the cytosol and membrane and effects of ODQ, KT-5823, and Go 6976 on the YC-1-induced increase in PKC activity in membrane fraction of A549 cells. Cells were treated with 50 μM YC-1 for various time intervals, or 10 nM PMA for 30 min (a), or pretreated with 10 μM ODQ, 3 μM KT, or 10 μM Go for 30 min before incubation with YC-1 (50 μM) for 60 min (b), then subcellular (cytosol and membrane) fractions were isolated. The PKC activity in the cytosol and membrane was measured as described in Methods. Results are expressed as means±s.e. mean of three independent experiments performed in duplicate. ^*P<0.05 as compared with basal level (a) or YC-1 alone (b). KT, KT-5823; Go, Go 6976.

Figure 7

Expression of PKC isoforms in the rat brain and A549 cells. Western blot analysis was conducted to examine the expression of PKC isoforms in A549 cells (right lane) and in the rat brain as a positive control (left lane).

Figure 8

YC-1 and PMA induce translocation of PKC isoforms from cytosol to membrane in A549 cells. The cells were treated with 50 μM YC-1 (a), or 1 μM PMA (b) for various time intervals. The subcellular (cytosol and membrane) fractions were then isolated, the protein levels of PKC isoforms in cytosolic and membrane fractions were determined by Western blotting analysis performed as described in Methods. Data are representative of three independent experiments, which gave essentially identical results.

Figure 9

Involvement of MEK in the YC-1-induced increase of COX activity and COX-2 expression in A549 cells. In (a), cells were pretreated with various concentrations of PD (MEK inhibitor) for 30 min, and then incubated with YC-1 (50 μM) for 12 h. The media were then removed, and the COX activity was measured by examining the PGE₂ formation in the presence of 30 μM exogenous arachidonic acid for 30 min. Results are expressed as means±s.e.mean of three independent experiments performed in duplicate. ^*P<0.05 as compared with treatment with YC-1 alone. In (b), the cells were pretreated with PD (10 and 30 μM) for 30 min and then incubated with YC-1 (50 μM) for 12 h. Immunodetection using anti-COX-2 or α-tubulin specific antibody was performed as described in Methods. The equal loading in each lane was demonstrated by the similar intensities of α-tubulin. Data are representative of three independent experiments, which gave essentially identical results. PD, PD98059.

Figure 10

The activation of p44/42 MAPK induced by YC-1 and PMA and effects of KT-5823, Go 6976, long-term PMA treatment, PD98059 and SB203580 on the YC-1-induced p44/42 MAPK activation in A549 cells. The cells were treated with 50 μM YC-1 (a) or 10 nM PMA (b) for the indicated time intervals. In (c), the cells were pretreated with 3 μM KT, 10 μM Go, 50 μM PD or 10 μM SB for 30 min, or 1 μM PMA for 24 h before incubation with YC-1 (50 μM) for 30 min. The extracted proteins were immunodetected with antibodies specific for phosphorylated p44/42 MAPK (p-p44/42) and nonphosphorylated p44/42 MAPK (p44/42) as described in Methods. The equal loading in each lane was demonstrated by the similar intensities of p44/42. In (d), the extent of p44/42 MAPK activation were quantitated using a densitometer with Image-Pro plus software. Results were expressed as mean±s.e.mean (n=3). ^*P<0.05 as compared with treatment with YC-1 alone. KT, KT-5823; Go, Go 6976; PD, PD98059; SB, SB203580.