Fig 1.
Hypoxia selectively inhibits TNF-α-induced TSLP expression.
HaCaT cells were pretreated under a hypoxic condition (1% O2) for 8 h or indicated time and then stimulated with TNF-α (100 ng/ml) for 2 h. The mRNA levels for TSLP (A, B and D), VEGF-A (C), TNF-α (E), IL-6 (F), MCP-1 (G) and IL-8 (H) were determined by real time PCR. Data are indicated as means ± SEM from 3 samples. Statistical significance: * p < 0.05, ** p < 0.01 vs. corresponding TNF-α group in normoxia.
Fig 2.
NiCl2 and CoCl2 inhibit TNF-α-induced TSLP expression.
(A and B) HaCaT cells were pretreated with NiCl2, CoCl2 and MgCl2 (1 mM) for 24 h and stimulated with TNF-α (100 ng/ml) for 2 h. The expression of mRNAs for TSLP (A) and IL-8 (B) were determined. (C, D) HaCaT cells were pretreated for 8 h with NiCl2 and CoCl2 (1 mM) for 0, 1, 2, 4, 8, 24 h and then stimulated with TNF-α (100 ng/ml) for 2 h. The expression of TSLP mRNA was determined (left panels). Cytotoxic effects by NiCl2 and CoCl2 were evaluated by MTT assay (right panels). (E and F) HaCaT cells were pretreated by NiCl2 (E) or CoCl2 (F) at the concentrations of 0.1, 0.3 and 1 mM for 8 h and stimulated by TNF-α (100 ng/ml) for 2 h. (G) PAM212 cells were pretreated by NiCl2 and CoCl2 (1 mM) for 8 h and stimulated with mouse TNF-α (100 ng/ml) for 2 h. The levels of mRNA for TSLP (left panel) and VEGF (right panel) were determined. Data are indicated as means ± SEM from 3 samples. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. TNF-α only. ## p < 0.01, ### p < 0.001 vs. control.
Fig 3.
DMOG inhibits TNF-α-induced TSLP expression.
(A to C) HaCaT cells were pretreated with NiCl2 (1 mM) and DMOG (1 mM) for 8 h and then stimulated with TNF-α (100 ng/ml) for 2 h. The expressions of mRNA for TSLP (A) and filaggrin (C) were determined. Cytotoxic effects of NiCl2 and DMOG were evaluated by MTT assay (B). (D) HaCaT cells were pretreated with DMOG at the indicated concentrations for 8 h and then stimulated by TNF-α (100 ng/ml) for 2 h. Data are indicated as means ± SEM from 3 samples. Statistical significance: * p < 0.05, ** p < 0.01, *** p < 0.001 vs. corresponding TNF-α only. # p < 0.05, ### p < 0.001 vs. control.
Fig 4.
Hypoxia, NiCl2 and CoCl2 increased levels of HIF-1α and HIF-2α proteins.
HaCaT cells were treated with hypoxia (1% O2), NiCl2 and CoCl2 (1 mM) for 8 h. The expression of HIF-α and actin proteins were determined by western blot.
Fig 5.
Hypoxia and hypoxia-mimicking conditions inhibit TNF-α-induced TSLP expression via HIF-2α but not HIF-1α expression.
(A to E) HaCaT cells were pretreated by HIF-1α inhibitor (10 μM) and HIF-2α antagonist (100 μM) in normoxic and hypoxic condition (1% O2) (A) or with 1 mM NiCl2 (B) and 1 mM CoCl2 (C) for 8 h and stimulated by TNF-α (100 ng/ml) for 2 h. TSLP mRNA levels were determined. (D and E) HaCaT cells treated by NiCl2 (1 mM) and HIF-1α inhibitor (10 μM) (D) or HIF-2α antagonist (100 μM) (E) for 10 h. The levels of mRNA for TSLP (A to C), VEGF-A (D) and filaggrin (E) were determined. (F and G) HaCaT cells were transiently transfected with HRE luciferase reporter constructs, and 24 h later, incubated in normoxia and hypoxia (1% O2) for 8 h (F) or treated with 1 mM NiCl2 and 1 mM CoCl2 for 8 h (G) in the presence or absence of HIF-1α inhibitor (10 μM) and HIF-2α antagonist (100 μM). Firefly luciferase activity was normalized to Renilla luciferase activity. Data are indicated as means ± SEM from 3 or 4 samples. Statistical significance: * p < 0.05, ** p < 0.01 between the indicated groups.
Fig 6.
The region -71 and +185 bp of TSLP promoter is involved in DMOG-induced repression of TSLP expression.
(A) Schematic diagram of TSLP reporter plasmid used in this study. HRE: putative hypoxia responsive element, N1 to N4: NF-κB binding sites, Luc: Luciferase. (B) HaCaT cells were transiently transfected with HRE luciferase reporter constructs, and 24 h later, stimulated with NiCl2 and DMOG (1 mM) for 8 h. Firefly luciferase activity was normalized to Renilla luciferase activity. (C-G) HaCaT cells were transiently transfected with TSLP promoter luciferase reporter constructs indicated in A. Twenty four hours later, the cells were treated for 8 h with DMOG (1 mM) and then stimulated with TNF-α (100 ng/ml) for 8 h. Firefly luciferase activity was normalized to Renilla luciferase activity. Data are indicated as means ± SEM from 3 or 4 samples. Statistical significance: * p < 0.05 vs. corresponding TNF-α control, N.S.: Not significant.