Contribution of the p38MAPK signalling pathway to proliferation in human cultured airway smooth muscle cells is mitogen-specific

Abstract

We have investigated the role of p38MAPK in human airway smooth muscle (HASM) proliferation in response to thrombin and bFGF. The regulation of cyclin D1 mRNA, cyclin D1, cyclin E and p21Cip1 protein levels, and the extent of retinoblastoma protein (pRb) phosphorylation in response to activation of p38MAPK have also been examined. Two distinct inhibitors of p38MAPK, SB 203580 (10 microm) and SB 202190 (10 microm), prevented bFGF (0.3-3 nm)-stimulated cell proliferation, but had no effect on the response to thrombin (0.3-3 U ml(-1)). In cells incubated with thrombin or bFGF for 20 h, there was an increase in p38MAPK phosphorylation in response to bFGF, but not to thrombin. Thrombin and bFGF-stimulated increases in ERK phosphorylation and cyclin D1 mRNA and protein levels were not influenced by SB 203580 pre-treatment. Similarly, cyclin E and p21Cip1 protein levels, measured after 20 h incubation with mitogen, did not appear to be regulated by SB 203580 (10 microm). Although both thrombin and bFGF significantly increased levels of pRb phosphorylation, SB 203580 (10 microm) inhibited only bFGF-stimulated pRb phosphorylation. In addition, SB 203580 (10 microm) selectively inhibited bFGF-stimulated DNA synthesis, suggesting that the antimitogenic actions of SB 203580 on pRb phosphorylation cause cell cycle arrest at late G1 phase. In conclusion, these results indicate that p38MAPK is involved in bFGF-, but not in thrombin-stimulated HASM proliferation. The activation of the p38MAPK pathway by bFGF, but not by thrombin, regulates the phosphorylation of pRb without influencing cyclin D1 expression.

Figure 1

Phosphorylation levels of p38^MAPK measured by Western blot analysis in response to 20 h incubation with thrombin (0.3 and 3 U ml⁻¹) and bFGF (0.3 and 3 nM). Each histogram represents the mean and s.e.m. of results from 10 different cell lines. The mitogen-stimulated phosphorylation levels are compared to the phosphorylation levels detected in control cells. ^*P<0.05; Differences were identified by repeated-measures ANOVA, followed by Dunnett's post-hoc test.

Figure 2

Increases in ERK phosphorylation measured by Western blot analysis in response to 20 h incubation with thrombin (0.3 and 3 U ml⁻¹) or bFGF (0.3 and 3 nM) in the absence and presence of SB 203580 (10 μM). (a) Representative blots and pooled data of ERK phosphorylation levels measured in response to (b) thrombin and (c) bFGF. Each histogram represents the mean and s.e.m. of results from six different cell lines. Responses are compared to ERK phosphorylation levels detected in control cells. Differences were identified using a one-way ANOVA with repeated measures, followed by Dunnett's post-hoc test. ^*P<0.05 cf. control, ^**P<0.05 cf. SB 203580 alone.

Figure 3

Cyclin D1 protein levels determined by Western blot analysis in response to 20 h treatment with thrombin (0.3 and 3 U ml⁻¹) and bFGF (0.3 and 3 nM) in the absence and presence of SB 203580 (10 μM). (a) Representative blot and pooled data of cyclin D1 levels measured in response to thrombin (b) and bFGF (c). Cyclin D1 protein levels are expressed as fold increments over the levels measured in control cells. Each histogram is representative of the mean and s.e.m. of nine different cell lines. ^**P<0.01, ^*P<0.05, differences were identified using a paired t-test. Cyclin D1 levels following mitogen stimulation are compared to those of control cells.

Figure 4

Increases in pRb phosphorylation detected by Western blot analysis in response to 20 h incubation with thrombin (0.3 and 3 U ml⁻¹) or bFGF (0.3 and 3 nM) in the absence and presence of SB 203580 (10 μM). (a) Representative blots and pooled data showing pRb phosphorylation levels in response to stimulation by (b) thrombin or (c) bFGF. Levels of pRb phosphorylation are expressed as fold increments over the levels detected in control cells. Each histogram is representative of the mean and s.e.m. of eight different cell lines. Differences were identified using a one-way ANOVA with repeated measures, followed by Dunnett's post hoc test. ^*P<0.05 cf. control, ^**P<0.05 cf. SB 203580 alone. ^†P<0.05, SB 203580-treated cells compared with the corresponding level in non-pretreated cells under the same incubation conditions.