Objective: To explore the mechanism of signal transduction in respiratory syncytial virus (RSV)-induced expression of thymic stromal lymphopoietin (TSLP) in bronchial epithelial cells.
Methods: The eukaryotic expression vector of RSV F protein, pcDNA3-F, was constructed and transfected into in vitro cultured human bronchial epithelial cell line CRL-9483, which was also transfected with Smad7 expression vector pcDNA3/Smad7 or exposed to p38, ERK1/2, JNK, and JAK/STAT1 inhibitors. The mRNA levels of TSLP and the housekeeping GAPDH gene were analyzed 24 h later with semi-quantitative RT-PCR. In cells with downregulated TSLP mRNA expression due to the addition of the signal inhibitors, cytoplasm TSLP or GAPDH protein levels were further analyzed using Western blotting.
Results: Virtually no TSLP mRNA expression was detected by RT-PCR in cultured CRL-9483 cells transfected with pcDNA3 exclusively (TSLP/GAPDH relative total gray scale of 0.10-/+0.03), while cell transfection with pcDNA3-F resulted in significantly increased TSLP mRNA level (0.42-/+0.20, P=0.024). In the presence of F protein expression, both p38 and JNK inhibitors could downregulate TSLP mRNA levels (0.14-/+0.04, P=0.036; 0.23-/+0.07, P=0.048, respectively), while TGF-beta-Smad inhibiting protein Smad7 (0.60-/+0.25), ERK 1/2 inhibitor (0.45-/+0.23), and JAK/STAT1inhibitor (0.44-/+0.25) failed to block TSLP expression (P>0.05). Western blotting showed that p38 inhibitor (TSLP/GAPDH relative total gray scale=3.67-/+1.18, P=0.018) and JNK inhibitor (1.48-/+0.77, P=0.004) also downregulated the protein levels of TSLP as compared with pcDNA3-F transfection group (8.13-/+2.20).
Conclusion: RSV F protein can stimulate TSLP expression in human bronchial epithelial cells mediated partially by p38 and JNK signal pathways.