Acrolein decreases SUMO modification of glucocorticoid receptor and reduces its sensitivity to corticosteroids in airway hypersecretion

Abstract

To explore the variation of small ubiquitin-related modification (SUMO) level of glucocorticoid receptors (GRs) exposed to acrolein stimulation as well as the influence of the variation on mucus hypersecretion. The recombinant plasmid was constructed by inserting small ubiquitin-like modifier 1 (SUMO1) cDNA into eukaryotic expression plasmid pcDNA3.1-EGFP with a flag sequence marker. The recombinant plasmid pcDNA3.1-EGFP-flag-SUMO1 was identified by enzyme digestion analysis. The 16HBE cells were cultured and randomized divided into 9 following groups: A. control; B. acrolein stimulated; C. dexamethasone incubated; D. acrolein stimulated and dexamethasone incubated; E. SUMO1 transfected; F. pcDNA3.1-EGFP vector transfected; G. SUMO1 transfected and acrolein stimulated; H. SUMO1 transfected, acrolein stimulated and dexamethasone incubated; I. pcDNA3.1-EGFP vector transfected, acrolein stimulated and dexamethasone incubated. Each group consisted of 5 parallel wells and experiments were repeated for 4 times for statistical analysis. The transfection efficiency was evaluated by the expression of enhanced green fluorescent protein (EGFP) via fluorescence microscope. The SUMO modification level of GRs was measured with co-immunoprecipitation and Western blot. The transcription level of mucin (MUC) 5AC was evaluated with reverse transcription-polymerase chain reaction (RT-PCR). The MUC5AC secreted in supernatant was measured by enzyme-linked immunosorbent assay (ELISA). The SUMO modification level was much lower in group B (0.079 ± 0.023) than that in group A (0.446 ± 0.068) (t = -23.13, P = 0.000) and in group D (0.574 ± 0.018) than that in group C (0.843 ± 0.028) (t = -36.34, P = 0.000). The transcription level of MUC5AC was significantly lower in group H (0.330 ± 0.063) than group D (0.617 ± 0.190) (q = -7.80, P = 0.000). Through ELISA, there was no significance between groups G and B in the term of secretion level of MUC5AC. While the secretion level of MUC5AC was much lower in group H ((0.416 ± 0.092) µg/L) than group D ((0.663 ± 0.104) µg/L) and group G ((0.740 ± 0.343) µg/L) (q = -7.31, -9.59; P = 0.001, 0.000). Acrolein decreases the SUMO modification of GRs and reduces the inhibitory effect of dexamethasone on the transcription of MUC5AC.