[Regulation of sphingosine kinase 1 in the TNF-α-induced expression of MUC5AC in airway epithelial cells].

OBJECTIVE

To explore the effects of sphingosine kinase 1 (SphK1) on mucin (MUC)5AC overexpression under the induction of tumor necrosis factor-α (TNF-α).

METHODS

The HBE16 airway epithelial cells were cultured, pretreated with SphK1-specific inhibitor DMS or transfected with SphK1 siRNA. Then each group was stimulated by a certain concentration of TNF-α. The relative contents of SphK1, COX-2, p-P38, p-ERK and IκBα were detected by Western blot while the levels of MUC5AC, PGE2 and cAMP analyzed by enzyme linked immunoassay. The transcription activities of cAMP-response-element-binding protein and nuclear factor κB (NF-κB) were detected by luciferase reporter gene detection system. The mRNA expressions of SphK1 and MUC5AC were detected by RT-PCR (reverse transcription-polymerase chain reaction). And the intracellular MUC5AC protein was detected by immunofluorescence.

RESULTS

The levels of protein and mRNA expression of SphK1 and MUC5AC were obviously elevated [(0.69 ± 0.12, 0.86 ± 0.07), (0.60 ± 0.09, 0.79 ± 0.05)]. They were all significantly higher than those in the control group (0.26 ± 0.03, 0.33 ± 0.04, 0.18 ± 0.06, 0.22 ± 0.10). There was an elevation of COX-2, PGE2, cAMP, p-P38, p-ERK, CREB and NF-κB activity (all P < 0.01); the production of IκBα protein was lower than that in the control group (P = 0.003). DMS decreased the levels of MUC5AC mRNA expression (0.37 ± 0.06) and protein production (0.27 ± 0.04), lowered the production of COX-2, PGE2, cAMP, p-P38 and p-ERK, inhibited the activity of CREB and NF-κB and increased the IκBα production (all P < 0.01). Transfection of SphK1 siRNA showed the similar effects as DMS (all P < 0.01).

CONCLUSION

SphK1 plays an important role in regulating the MUC5AC expression under the induction of TNF-α. And the activation of critical signal factors in that process is dependent on SphK1.