Figure 1.
Anoxia induces the expression of mRNA and production of MUC5AC in NHNE cells.
(A) MTT assay to determine cytotoxic effects of anoxia. Cell viability was not significantly affected by less than 6 h of anoxia (n = 3, *p<0.05, †p<0.0001). (B) The expression of the MUC5AC gene according to anoxia stimulation (95% N2/5% CO2 atmosphere in the hypoxic chamber) for 0–24 h in NHNE cells. MUC5AC gene expression was increased under anoxic conditions, measured by RT-PCR. (C) The expression of MUC5AC mRNA was maximally induced at 12 h during anoxia (measured by real-time PCR, n = 3, *p<0.05, **p<0.001). d) Mucus secretion was sampled at 2, 6, 12, and 24 h after incubating of NHNE cells under anoxic conditions and measured in dot blots with anti-MUC5AC antibody. Secreted MUC5AC was highest at 12 h under anoxic conditions (n = 3, **P<0.001). Data is shown as mean ± standard deviation.
Figure 2.
Anoxia induces HIF-1α protein levels in NHNE cells.
The expression of HIF-1α mRNA and protein in NHNE cells under anoxia stimulation for 0–24 h. (A) The transcription of HIF-1α was not altered by anoxia, measured by RT-PCR. (B) Expression of HIF-1α protein was increased under anoxic conditions compared to the normoxic control (western blot).
Figure 3.
HIF-1α is involved in the regulation of MUC5AC expression.
When HIF-1α expression is increased, MUC5AC expression is induced; conversely, when HIF-1α expression is decreased, MUC5AC expression is reduced accordingly. (A) Gain-of-function study with mammalian HIF-1α expression vector in NCI-H292 cells. DNA of the pCMV vector encoding HIF-1α, or empty pCMV vector without HIF-1α, was transiently transfected. The expression of both HIF-1α and MUC5AC gene was increased when transfected with pCMV-HIF-1α vector, compared to transfection of empty control vectors under normoxic conditions (n = 3, *p<0.05). (B) Loss-of-function study with transfected HIF-1α siRNA in NCI-H292 cells. The expression of both HIF-1α and MUC5AC gene was suppressed when transfected with siRNA of HIF-1α, compared to transfection of siRNA negative control under hypoxic conditions for 6 h (n = 3, *p<0.05). Data is shown as mean ± standard deviation. (N; normoxia, C; control).
Figure 4.
Luciferase reporter assay dependent on MUC5AC promoter sequences.
(A) NCI-H292 cells were transiently transfected with the various deletion mutants and treated under normoxic or anoxic conditions for 6 h. Hypoxia selectively increased luciferase activity driven by sequences corresponding to the −1400/+4 and −776/+4 regions of the MUC5AC promoter, indicating that the −776/+4 region of MUC5AC promoter has a role in the cellular response to anoxia (n = 3, *P<0.01). (B) NCI-H292 cells were transfected with pGL3-basic vectors, pGL3 vectors containing the putative MUC5AC promoter, or pGL3 vectors containing the HRE-mutated MUC5AC promoter. After cells were incubated in the anoxic chamber for 6 h, luciferase activities were measured. Under anoxia conditions, the luciferase reporter activity of the wild-type MUC5AC promoter was increased by 8.3-fold compared to the HRE-mutated MUC5AC promoter, and by 11.2-fold compared to the pGL3-basic vectors (n = 3, **p<0.001). Data is shown as mean ± standard deviation.
Figure 5.
The HRE is required for anoxia-induced MUC5AC transcription.
(A) Chromatin immunoprecipitation (ChIP) assay. NCI-H292 cells were transfected with vectors encoding HIF-1α-binding flanking region of the MUC5AC promoter (HRE site), or a region that does not contain an HRE region (non-HRE site; negative control). The cells were treated under normoxic or anoxic conditions for 6 h and subjected to immunoprecipitation with HIF-1α antibody or control IgG. (B) The MUC5AC mRNA transcript was analyzed by real-time PCR. The binding to HRE under anoxic conditions significantly increased the expression of MUC5AC expression compared to non-HRE region, or HRE region under anoxic conditions (n = 3, *p<0.05). (C) EMSA to determine HIF-1α binding to the HRE region of the MUC5AC promoter in response to anoxia. The activity with HIF-1α-specific HRE-containing oligonucleotides is increased remarkably in response to anoxia. The EMSA band of interest was found to be selectively inhibited by specific HRE-containing competitor oligonucleotide, and it was super-shifted by anti-HIF-1α antibody. Data is shown as mean ± standard deviation. (N; normoxia, A; anoxia).
Figure 6.
Sinus mucosa from chronic sinusitis patients shows high levels of HIF-1α and MUC5AC expression.
(A) The expression of HIF-1α in the control and sinus mucosa from four patients with sinusitis (western blot analysis). The increase of HIF-1α expression in sinusitis was 4.4-fold greater than in control tissue (n = 4; *p<0.05). (B) Immunohistochemistry with anti-HIF-1α antibody in normal (left) and sinusitis (right) tissues. High HIF-1α protein expression is indicated by the strong antibody reactivity in the epithelium of sinus mucosa from a sinusitis patient. (C) Immunohistochemistry with anti-MUC5AC antibody in normal (upper) and sinusitis (lower) tissues. Pronounced MUC5AC expression is seen in the epithelium from the sinusitis patient. Data is shown as mean ± standard deviation. (N; normal, S; sinusitis).
Table 1.
Primers used for PCR.