TGF-beta1 increases proliferation of airway smooth muscle cells by phosphorylation of map kinases

Abstract

Background: Airway remodeling in asthma is the result of increased expression of connective tissue proteins, airway smooth muscle cell (ASMC) hyperplasia and hypertrophy. TGF-beta1 has been found to increase ASMC proliferation. The activation of mitogen-activated protein kinases (MAPKs), p38, ERK, and JNK, is critical to the signal transduction associated with cell proliferation. In the present study, we determined the role of phosphorylated MAPKs in TGF-beta1 induced ASMC proliferation.

Methods: Confluent and growth-arrested bovine ASMCs were treated with TGF-beta1. Proliferation was measured by [3H]-thymidine incorporation and cell counting. Expressions of phosphorylated p38, ERK1/2, and JNK were determined by Western analysis.

Results: In a concentration-dependent manner, TGF-beta1 increased [3H]-thymidine incorporation and cell number of ASMCs. TGF-beta1 also enhanced serum-induced ASMC proliferation. Although ASMCs cultured with TGF-beta1 had a significant increase in phosphorylated p38, ERK1/2, and JNK, the maximal phosphorylation of each MAPK had a varied onset after incubation with TGF-beta1. TGF-beta1 induced DNA synthesis was inhibited by SB 203580 or PD 98059, selective inhibitors of p38 and MAP kinase kinase (MEK), respectively. Antibodies against EGF, FGF-2, IGF-I, and PDGF did not inhibit the TGF-beta1 induced DNA synthesis.

Conclusion: Our data indicate that ASMCs proliferate in response to TGF-beta1, which is mediated by phosphorylation of p38 and ERK1/2. These findings suggest that TGF-beta1 which is expressed in airways of asthmatics may contribute to irreversible airway remodeling by enhancing ASMC proliferation.

Figure 1

ASMC responses to TGF-β1 and serum in different culture conditions. ASMCs were cultured with DMEM/10% FBS to confluence and then changed to DMEM/0.2% BSA, DMEM/0.5% FBS, or DMEM/10% FBS for 72 hours, followed by treatment with 5 ng/ml of TGF-β1 or 10% FBS for 24 hours prior to [³H]-thymidine incorporation assay. * p < 0.05, *** p < 0.001 compared to control of the same condition. n = 4–6.

Figure 2

TGF-β1 concentration-dependently increased proliferation of ASMCs. Confluent and growth-arrested ASMCs were incubated with various concentrations of TGF-β1 for 24 or 48 hours prior to [³H]-thymidine incorporation assay (A) or cell counting (B). Significant differences were detected at all concentrations of TGF-β1 treatment compared to the untreated control, p < 0.05 to p < 0.0001, n = 4–18.

Figure 3

TGF-β1 enhanced serum-induced proliferation of ASMCs. Confluent and growth-arrested ASMCs were treated with 10% FBS in the absence or presence of TGF-β1 (1 ng/ml) for 48 hours and the changes of [³H]-thymidine incorporation (n = 9) and cell number (n = 6) were determined. All values are % of untreated control cultured in 0.2% BSA/DMEM. p values indicated were compared to control (10% FBS only).

Figure 4

TGF-β1 increased expression of phosphorylated MAPKs in ASMCs. Confluent and growth-arrested ASMCs were incubated with 1 ng/ml of TGF-β1 for 1, 5, 30 minutes, 24 or 48 hours prior to protein extraction and Western analysis for phosphorylated or total p38 (Panel A), ERK1/2 (Panel B), and JNK (Panel C). * p < 0.05, ** p < 0.01, ** p < 0.001 compared to control, n = 4–10, C = control.

Figure 5

Effects of MAPKs inhibitors on TGF-β1 induced increase of proliferation in ASMCs. Confluent and growth-arrested ASMCs were pretreated for 1 hour with SB 203580, PD 98059, or SP 600125, prior to 24-hour treatment with 1 ng/ml of TGF-β1 (T). DNA synthesis was measured by [³H]-thymidine incorporation assay. Inhibition of phosphorylated p38 and ERK1/2 reduced TGF-β1 induced DNA synthesis. ## p < 0.01 compared to untreated control (C), ** p < 0.01, *** p < 0.001 compared to T, n = 7–8.

Figure 6

Effects of MAPKs inhibitors on TGF-β1 induced activation of MAPKs. Confluent and growth-arrested ASMCs were pretreated for 1 hour with SB 203580, PD 98059, or SP 600125, prior to 24-hour treatment with 1 ng/ml of TGF-β1 (T), followed by protein extraction and Western analysis for phosphorylated or total p38 (Panel A), ERK1/2 (Panel B), and JNK (Panel C). The blots are representatives of 3 independent experiments. C = control. ** p < 0.01 *** p < 0.001 compared to T.

Figure 7

Role of EGF, FGF-2, PDGF and IGF-I in TGF-β1 induced proliferation of ASMCs. Confluent and growth-arrested ASMCs were treated with TGF-β1 (1 ng/ml) in the absence or presence of neutralizing antibodies to EGF, FGF-2, PDGF and IGF-I for 48 hours prior to [³H]-thymidine incorporation assay. # p < 0.01, compared to untreated control (C). There were no significant differences (p > 0.05) in the DNA synthesis between TGF-β1 treated cells with and without pretreatment with these antibodies.