PPAR gamma ligands, 15-deoxy-delta12,14-prostaglandin J2 and rosiglitazone regulate human cultured airway smooth muscle proliferation through different mechanisms

Abstract

The influence of two peroxisome proliferator-activated receptor gamma (PPARgamma) ligands, a thiazolidinedione, rosiglitazone (RG) and the prostaglandin D2 metabolite 15-deoxy-Delta(12,14)-prostaglandin J2 (15d-PGJ2) on the proliferation of human cultured airway smooth muscle (HASM) was examined. The increases in HASM cell number in response to basic fibroblast growth factor (bFGF, 300 pm) or thrombin (0.3 U ml-1) were significantly inhibited by either RG (1-10 microM) or 15d-PGJ2 (1-10 microM). The effects of RG, but not 15d-PGJ2, were reversed by the selective PPARgamma antagonist GW9662 (1 microM). Neither RG nor 15d-PGJ2 (10 microM) decreased cell viability, or induced apoptosis, suggesting that the regulation of cell number was due to inhibition of proliferation, rather than increased cell death. Flow-cytometric analysis of HASM cell cycle distribution 24 h after bFGF addition showed that RG prevented the progression of cells from G1 to S phase. In contrast, 15d-PGJ2 caused an increase in the proportion of cells in S phase, and a decrease in G2/M, compared to bFGF alone. Neither RG nor 15d-PGJ2 inhibited ERK phosphorylation measured 6 h post mitogen addition. The bFGF-mediated increase in cyclin D1 protein levels after 8 h was reduced in the presence of 15d-PGJ2, but not RG. Although both RG and 15d-PGJ2 can inhibit proliferation of HASM irrespective of the mitogen used, only the antiproliferative effects of RG appear to be PPARgamma-dependent. The different antimitogenic mechanisms of 15d-PGJ2 and synthetic ligands for PPARgamma may be exploited to optimise the potential for these compounds to inhibit airway remodelling in asthma. British Journal of Pharmacology (2004) 141, 517-525. doi:10.1038/sj.bjp.0705630

Figures

Figure 1

Effects of RG and 15d-PGJ2 on bFGF-induced proliferation of HASM. Serum-deprived cells were incubated in the presence of the PPARγ ligand (1–10 μM) for 30 min prior to the addition of bFGF (300 pM). Cell enumeration data at 48 h are expressed as mean percentage±s.e.m. of the control cell number (no mitogen, 2.34±0.46 × 105, n=9, upper panel, and 2.40±0.54 × 105 cells, n=7, lower panel). *P<0.05, **P<0.01, ***P<0.001 cf. control, †P<0.001 cf. bFGF.

Figure 2

Effect of RG and 15d-PGJ2 on thrombin-induced proliferation of HASM. Serum-deprived cells were incubated in the presence of the PPARγ ligand (1–10 μM) for 30 min before the addition of thrombin (0.3 U ml−1). Cell enumeration data at 48 h are expressed as the mean percentage±s.e.m. of the control cell number (no mitogen) for n=9 (RG) or n=7 (15d-PGJ2). *P<0.05, **P<0.01 cf. control, †P<0.05 cf. thrombin.

Figure 3

Effect of the selective PPARγ antagonist GW9662 on the ability of RG and 15d-PGJ2 to decrease thrombin-mediated proliferation. Serum-deprived cells were incubated with either: vehicle (control); thrombin (0.3 U ml−1); thrombin and PPARγ ligand (RG or 15d-PGJ2, 10 μM, added 30 min prior to mitogen); or thrombin, PPARγ ligand and GW9662 (0.1–1 μM, added 30 min prior to RG or 15d-PGJ2). Cell enumeration data at 48 h are expressed as mean percentage±s.e.m. of control for n=7 (RG) or n=5 (15d-PGJ2). **P<0.01, ***P<0.001 cf. control, †P<0.05 cf. thrombin.

Figure 4

Effect of PPARγ ligands on the distribution of events in the cell cycle of HASM. (a) shows the representative FACS profile of the distribution of cells detected in G0/G1, S and G2/M phases of the cell cycle under control conditions (no PPARγ ligand or mitogen). The histogram shows DNA staining by PI, with an increase in PI fluorescence linearly related to an increase in DNA content. The frequency of events with each level of PI fluorescence is shown. The greatest proportion of events is detected in G0/G1 phase. Cells with approximately twice the PI fluorescence of G0/G1 cells represent those cells in G2/M phase of the cell cycle. Cells with intermediate staining represent cells with varying DNA content, that is, those in S phase. (b, c) show the effects of 10 μM RG or 15d-PGJ2 on control cell cycle distribution, respectively. The distribution profile 24 h after mitogen addition (bFGF, 300 pM) in the absence (d) and presence of 10 μM RG (e) or 10 μM 15d-PGJ2 (f) is also shown. All representative profiles have been obtained from experiments performed on one culture, and are consistent with the results from the five cultures examined.

Figure 5

Effect of PPARγ ligands on the cell cycle distribution of HASM. Cells were incubated for 24 h in the absence or presence of bFGF (300 pM), 30 min after addition of vehicle control, 10 μM RG or 10 μM 15d-PGJ2. The frequency of events detected by fluorescence of PI in (a) G0/G1, (b) S and (c) G2/M phases of the cell cycle are shown as mean±s.e.m. for n=5 cultures. *P<0.05, **P<0.01, ***P<0.001 cf. control, †P<0.05 cf. bFGF.

Figure 6

Effects of (a) 10 μM RG and (b) 10 μM 15d-PGJ2 on cyclin D1 protein levels at 8 h after stimulation with bFGF (300 pM). Western blots are representative of experiments in six different HASM cultures. Representative blots of the effects of 15d-PGJ2 and RG on cyclin D1 levels were obtained using 60 μg of protein. The densitometry results are shown as mean percentage±s.e.m. of bFGF. ***P<0.001 cf. control.