Hypoxia-Mediated Mechanism of MUC5AC Production in Human Nasal Epithelia and Its Implication in Rhinosinusitis

<div><p>Background</p><p>Excessive mucus production is typical in various upper airway diseases. In sinusitis, the expression of MUC5AC, a major respiratory mucin gene, increases. However, the mechanisms leading to mucus hypersecretion in sinusitis have not been characterized. Hypoxia due to occlusion of the sinus ostium is one of the major pathologic mechanisms of sinusitis, but there have been no reports regarding the mechanism of hypoxia-induced mucus hypersecretion.</p><p>Methods and Findings</p><p>This study aims to identify whether hypoxia may induce mucus hypersecretion and elucidate its mechanism. Normal human nasal epithelial (NHNE) cells and human lung mucoepidermoid carcinoma cell line (NCI-H292) were used. Sinus mucosa from patients was also tested. Anoxic condition was in an anaerobic chamber with a 95% N₂/5% CO₂ atmosphere. The regulatory mechanism of MUC5AC by anoxia was investigated using RT-PCR, real-time PCR, western blot, ChIP, electrophoretic mobility shift, and luciferase assay. We show that levels of MUC5AC mRNA and the corresponding secreted protein increase in anoxic cultured NHNE cells. The major transcription factor for hypoxia-related signaling, HIF-1α, is induced during hypoxia, and transfection of a mammalian expression vector encoding HIF-1α results in increased MUC5AC mRNA levels under normoxic conditions. Moreover, hypoxia-induced expression of MUC5AC mRNA is down-regulated by transfected HIF-1α siRNA. We found increased MUC5AC promoter activity under anoxic conditions, as indicated by a luciferase reporter assay, and mutation of the putative hypoxia-response element in MUC5AC promoter attenuated this activity. Binding of over-expressed HIF-1α to the hypoxia-response element in the MUC5AC promoter was confirmed. In human sinusitis mucosa, which is supposed to be hypoxic, expression of MUC5AC and HIF-1α is higher than in control mucosa.</p><p>Conclusion</p><p>The results indicate that anoxia up-regulates MUC5AC by the HIF-1α signaling pathway in human nasal epithelia and suggest that hypoxia might be a pathogenic mechanism of mucus hypersecretion in sinusitis.</p></div

Anoxia induces HIF-1α protein levels in NHNE cells.

<p>The expression of HIF-1α mRNA and protein in NHNE cells under anoxia stimulation for 0–24 h. (<b>A</b>) The transcription of HIF-1α was not altered by anoxia, measured by RT-PCR. (<b>B</b>) Expression of HIF-1α protein was increased under anoxic conditions compared to the normoxic control (western blot).</p

Luciferase reporter assay dependent on MUC5AC promoter sequences.

<p>(<b>A</b>) 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>B</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.</p

Primers used for PCR.

<p>RT-PCR; reverse transcriptase polymerase chain reaction.</p

Sinus mucosa from chronic sinusitis patients shows high levels of HIF-1α and MUC5AC expression.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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. <i>(N; normal, S; sinusitis)</i>.</p

Anoxia induces the expression of mRNA and production of MUC5AC in NHNE cells.

<p>(<b>A</b>) 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>B</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. (<b>C</b>) 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.</p

HIF-1α is involved in the regulation of MUC5AC expression.

<p>When HIF-1α expression is increased, MUC5AC expression is induced; conversely, when HIF-1α expression is decreased, MUC5AC expression is reduced accordingly. (<b>A</b>) 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>B</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. <i>(N; normoxia, C; control)</i>.</p

A Far-Red-Emitting Fluorescence Probe for Sensitive and Selective Detection of Peroxynitrite in Live Cells and Tissues

In this study, the far-red-emitting fluorescence probe <b>1</b>, containing a rhodamine derivative and a hydrazide reactive group, was developed for peroxynitrite detection and imaging. This probe, which is cell permeable and shows high sensitivity and selectivity in fluorometric detection of peroxynitrite over other ROS/RNS, was successfully utilized to detect exogenous and endogenous peroxynitrite in HeLa and RAW 264.7 cells, respectively. More importantly, <b>1</b> can also be used to detect endogenous peroxynitrite generated in <i>Pseudomonas aeruginosa</i> (PAO1)-infected mouse bone marrow-derived neutrophils. We anticipate that the new probe will serve as a powerful molecular imaging tool in investigations of the role(s) played by peroxynitrite in a variety of physiological and pathological contexts

Effects of PAR activators in pig airway gland.

<p>(A) Approximate dose-response relationship for mucus secretion in pig submucosal glands. Each point represents the average of 10–14 glands from 3–4 different pig trachea. <b>(</b>B) Secretion rates from airway gland in response to PAR activators. Data represent mean ± S.E.M. secretion rates from 11 to 41 glands from two to four pigs. (C) Typical gland response to repeated PAR2-AP application. Similar secretory responses were noted with repeated PAR2-AP treatment. (D) Trypsin (10 µM) induced vigorous mucus secretion, which desensitized the PAR2-AP (10 µM) response.</p

Ca²⁺ and anion dependency of PAR2-AP-induced mucus secretion in pig airway glands.

<p>(A) Images of mucus bubbles with and without BAPTA-AM (50 µM). Scale bar: 0.5 mm. (B) Summary data from 21 to 33 glands from three pigs. Data represent mean ± S.E.M. mucus secretion rates. * and ** indicate significant differences from the response seen with PAR2-AP alone, <i>P</i><0.05 and 0.005, respectively.</p