The importance of ERK activity in the regulation of cyclin D1 levels and DNA synthesis in human cultured airway smooth muscle

Abstract

The relationship between persistent ERK (extracellular signal-regulated kinase) activity, cyclin D1 protein and mRNA levels and cell cycle progression in human cultured airway smooth muscle was examined in response to stimulation by ET-1 (endothelin-1), thrombin and bFGF (basic fibroblast growth factor). Thrombin (0.3 and 3 u ml(-1)) and bFGF (0.3 and 3 nM) increased ERK activity for more than 2 h and increased cell number, whereas ET-1 (100 nM) transiently stimulated ERK activity and was non-mitogenic. The MEK1 (mitogen-activated ERK kinase) inhibitor, PD 98059 (30 microM), inhibited both ERK phosphorylation and activity, and either prevented (thrombin 0.3 and 3 u ml(-1), bFGF 300 pM) or attenuated (bFGF 3 nM) DNA synthesis. Thrombin and bFGF increased both cyclin D1 mRNA and protein levels. PD 98059 decreased cyclin D1 protein levels stimulated by the lower but not higher thrombin concentrations. Moreover, increases in cyclin D1 mRNA levels were unaffected by PD 98059 pretreatment, irrespective of the mitogen or its concentration, suggesting that inhibition of cyclin D1 protein levels occurred by a post-transcriptional mechanism. These findings indicate that the control of cyclin D1 protein levels may occur independently of the MEK1/ERK signalling pathways. The inhibition of S phase entry by PD 98059 at higher thrombin concentrations appears to result from effects on pathways downstream or parallel to those regulating cyclin D1 protein levels. These findings suggest heterogeneity in the signalling of DNA synthesis in human cultured airway smooth muscle.

Figure 1

FACS analysis and cell counts were performed to measure the extent of cell cycle progression. The media was changed and bFGF, thrombin (Thr) or ET-1 were re-added after 24 h in preparation for FACS analysis and cell counts after 48 h. Monomed A was added to all cells (including control cells) at the time of mitogen addition. (a) Representative flow cytometric DNA content profiles. DNA staining by propidium iodide (DNA content) is plotted on the x-axis. The cell number is plotted on the y-axis. As indicated on the histogram profile of the control cells for each treatment, the proportion of cells in G₁ is represented between 50–60 on the x-axis; the proportion of cells in the G₂/M phases are represented between 100–120 on the x-axis; and the events in the area between the two peaks represent the proportion of cells in S phase. (b) FACS data are expressed as a percentage of the number of cells in each phase of the cell cycle. FACS values represent the mean and s.e.m. from at least four different cell lines. ^*P<0.05, ^**P<0.01, Responses are compared to the control response for each phase. Repeated measures ANOVA, followed by Dunnet's post-hoc test for multiple comparisons. (c) Cell number data represents the mean and s.e.m. of results from six different cell lines and are expressed as fold increments over the baseline number of cells. ^*P<0.05, Responses are compared to the number of cells treated with Monomed A (1%) alone (control). Repeated measures ANOVA, followed by Dunnet's post-hoc test was used for multiple comparisons. The mean number of control cells was 1.8±0.2 × 10⁵ cells.

Figure 2

Increases in ERK phosphorylation measured by Western blotting after 20 h stimulation by thrombin or bFGF and Monomed A (1%) in the presence and absence of PD 98059 (30 μM) (0.3% DMSO control). Proteins from cell lysates were separated by SDS–PAGE and transferred to a nitrocellulose membrane. Proteins were labelled by Western blotting and detected by enhanced chemiluminescence (ECL). (a) Representative blots and grouped data from four experiments of identical design showing levels of ERK phosphorylation following (b) stimulation with high mitogen concentrations and (c) stimulation with low mitogen concentrations. The immunostained panels showing levels detected in response to low and high mitogen concentrations in (a) are from separate experiments. Each histogram represents the mean and s.e.m. of results from four different cell lines. ^*P<0.05, ^**P<0.01, Mitogen responses are compared to those of cells incubated with Monomed A (1%) alone. †P<0.05, Responses in cells treated with PD 98059 are compared to those of control cells treated with PD 98059. Repeated measures ANOVA, followed by Dunnet's post-hoc test was used for multiple comparisons.

Figure 3

ERK activity levels in ASM incubated with bFGF (3 nM), thrombin (3 u ml⁻¹), or ET-1 (100 nM) after 5 min, 2 h and 20 h. Monomed A (1%) was added to all cells. Increases in ERK activity in cell lysates were detected by a specific kinase assay (Amersham). Total ERK activity is expressed as nmol of ³²P transferred min⁻¹ mg⁻¹ of protein. Each histogram represents the mean and s.e.m. of data from at least three different cell lines. ^*P<0.05 denotes increase in ERK activity over the control level. Repeated measures ANOVA, followed by Dunnet's post-hoc test was used to determine whether ERK activity levels in mitogen-stimulated cells were greater than those in control cells incubated with Monomed A (1%) alone at each respective time point (5 min, 2 h and 20 h). Basal ERK activities were 183±82, 704±45 and 210±58 pmol min⁻¹ mg⁻¹ at 5 min, 2 h and 20 h, respectively.

Figure 4

ERK activity as measured by Biotrak ERK enzyme activity assay kit (Amersham, U.K.) after 2 h stimulation by thrombin (3 u ml⁻¹) and bFGF (3 nM). Total ERK activity is expressed as nmol of ³²P transferred min⁻¹ mg⁻¹ protein per lysate. Each histogram represents the mean and s.e.m. of results from three different cell lines. ^*P<0.05, ^**P<0.01, mitogen-stimulated ERK activity is compared to the activity in control cells treated with Monomed A (1%) alone. †P<0.05 Mitogen-stimulated ERK activity in the presence of PD 98059 is compared to the ERK activity levels in control cells incubated with PD 98059. Repeated measures ANOVA, followed by Dunnet's post-hoc test was used for multiple comparisons. Basal ERK activity was 516±194 pmol min⁻¹ mg⁻¹.

Figure 5

DNA synthesis stimulated by ET-1 (100 nM), thrombin (0.3 and 3 u ml⁻¹) and bFGF (3 nM and 300 pM) for 24 h in the absence or presence of PD 98059 (30 μM), as measured by [³H]-thymidine incorporation assay. Monomed A (1%) was added to all cells. Each histogram represents the mean and s.e.m. of data from separate experiments in at least three different cell lines. ^*P<0.05, ^**P<0.01, Responses are compared to the DNA synthesis in control cells stimulated by Monomed A (1%) alone. †P<0.05, Responses are compared to the DNA synthesis in cells incubated with Monomed A and PD 98059. Repeated measures ANOVA, followed by Dunnet's post-hoc test was used for multiple comparisons.

Figure 6

Cyclin D1 protein levels determined by Western blot in response to 20 h treatment with thrombin (0.3 and 3 u ml⁻¹), bFGF (300 pM and 3 nM) and ET-1 (100 nM) in the absence and presence of PD 98059 (30 μM). Monomed A (1%) was added to all cells. (a) Representative blots (left-hand side and right-hand side panels are from two separate experiments) and (b) pooled data. Cyclin D1 protein levels are expressed as fold increments over the cyclin D1 levels in control cells. Each histogram is representative of the mean and s.e.m. of five different cell lines. ^*P<0.05, ^**P<0.01 Mitogen responses are compared to the cyclin D1 protein levels in control cells treated with Monomed A (1%) alone. Repeated measures ANOVA, followed by Dunnet's post-hoc test was used for multiple comparisons.

Figure 7

Cyclin D1 mRNA levels measured by Northern blot analysis in response to 16 h treatment with thrombin and bFGF in the absence or presence of PD 98059 (30 μM). Monomed A (1%) was added to all cells. The mRNA from cell extracts was separated on a formaldehyde denaturing gel and transferred to a nylon membrane. Cyclin D1 mRNA was hybridized with a radiolabelled DNA probe and then detected by apposition to X-ray film or phosphorimaging. (a) Representative blot of the responses to the high mitogen concentrations and (b) pooled data, expressed as the mean and s.e.m. of six experiments of identical design from at least three different cell lines. Results are expressed as a fold increment over the mRNA levels in control cells. GAPDH mRNA levels are shown as a control for loading differences (a). ^*P<0.05, ^**P<0.01 Responses are compared to the cyclin D1 mRNA levels in control cells stimulated with Monomed A (1%) alone. Repeated measures ANOVA, followed by Dunnet's post-hoc test was used for multiple comparisons (b).