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. 2001 Apr 24;98(9):5175-80.
doi: 10.1073/pnas.081510898. Epub 2001 Apr 10.

Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma

A Nakanishi  1 , S Morita, H Iwashita, Y Sagiya, Y Ashida, H Shirafuji, Y Fujisawa, O Nishimura, M Fujino
Affiliations

Role of gob-5 in mucus overproduction and airway hyperresponsiveness in asthma

A Nakanishi et al. Proc Natl Acad Sci U S A. .

Abstract

Airway hyperresponsiveness (AHR), goblet cell metaplasia, and mucus overproduction are important features of bronchial asthma. To elucidate the molecular mechanisms behind these pulmonary pathologies, we examined for genes preferentially expressed in the lungs of a murine model of allergic asthma by using suppression subtractive hybridization (SSH). We identified a gene called gob-5 that had a selective expression pattern in the airway epithelium with AHR. Here, we show that gob-5, a member of the calcium-activated chloride channel family, is a key molecule in the induction of murine asthma. Intratracheal administration of adenovirus-expressing antisense gob-5 RNA into AHR-model mice efficiently suppressed the asthma phenotype, including AHR and mucus overproduction. In contrast, overexpression of gob-5 in airway epithelia by using an adenoviral vector exacerbated the asthma phenotype. Introduction of either gob-5 or hCLCA1, the human counterpart of gob-5, into the human mucoepidermoid cell line NCI-H292 induced mucus production as well as MUC5AC expression. Our results indicated that gob-5 may play a critical role in murine asthma, and its human counterpart hCLCA1 is therefore a potential target for asthma therapy.

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Figures

Figure 1
Figure 1
Selective expression of gob-5 mRNA in airway epithelia from AHR-model mice. (A) Tissue distribution of gob-5 was determined by Northern blot analysis in normal and AHR-model mice. (Upper) The results obtained by hybridization with a gob-5 cDNA probe. The murine tissues from which the poly(A)+ RNA was prepared are indicated at the top. The same filter was rehybridized with a β-actin probe to control for equal loading (Lower). (B, C, E, and F) Lung sections of normal (B and C) and AHR-model mice (E and F) were in situ hybridized with DIG-labeled, single-stranded antisense (B and E) and sense (C and F) RNA probes. (D and G) The same lung sections of normal (D) and AHR-model mice (G) were stained with PAS and counterstained with hematoxylin. Original magnification, ×200. Three different experiments showed similar results.
Figure 2
Figure 2
Induction of gob-5 before the onset of AHR and eosinophilic inflammation. (A) Time-related changes in airway responsiveness after OVA inhalation were measured (n = 7–11). **, P < 0.01, compared with day 0 group. (B) Time-related changes in cell recruitment in BAL fluid after OVA inhalation. BAL was performed 24 h after inhalation of aerosolized OVA (n = 6–7). Total cells (●) were classified as macrophages (■), eosinophils (▴), neutrophils (○), or lymphocytes (□). (C) Time-dependent expression of gob-5 was determined by Northern blot analysis in lung tissue after OVA inhalation. Presented figures are representative of three separate experiments. Three different experiments showed similar results.
Figure 3
Figure 3
Functional analysis of gob-5 in the development of AHR and mucus production by in vivo adenoviral gene transfer. (A) Reduction in airway responsiveness by the administration of adenovirus-expressing gob-5 antisense RNA in AHR-model mice (n = 5–10). Recombinant adenovirus-expressing gob-5 antisense RNA transcripts (Ad-gob-5-AS) and control adenovirus (Ad-wt) were intratracheally administered one day before the start of OVA inhalation. Airway responsiveness was measured 24 h after the final inhalation carried out for 6 consecutive days. *, P < 0.05; **, P < 0.01, compared with the OVA-inhaled group by Dunnett type test. (B–E) Suppression of airway mucus secretion by the administration of adenovirus-expressing gob-5 antisense RNA in AHR-model mice. Lung sections of Ad-wt-administered mice (B and D) and Ad-gob-5-AS-administered mice (C and E) were stained with PAS, counterstained with hematoxylin. Original magnifications, ×40 (B and C), ×200 (D and E). (F) Promotion of airway responsiveness by overexpressing gob-5 in OVA-inhaled mice (n = 6–10). Recombinant adenovirus expressing gob-5 RNA transcripts (Ad-gob-5-S) and control adenovirus (Ad-wt) were intratracheally administered 1 day before the start of OVA inhalation. Airway reactivity was measured 24 h after the final inhalation carried out for 3 consecutive days. **, P < 0.01, compared with OVA-inhaled group by Dunnett type test. (G–J) Induction of airway mucus secretion by overexpressing gob-5. Lung sections of Ad-wt-administered mice (G and I) and Ad-gob-5-S-administered mice (H and J) were stained with PAS, counterstained with hematoxylin. Original magnifications, ×40 (G and H), ×200 (I and J).
Figure 4
Figure 4
Mucus production in human mucoepidermoid cells transfected with gob-5 or hCLCA1. (A) PAS staining was performed on NCI-H292 cells after transfection with control vector (pcDNA3.1), gob-5 expression vector (pcDNA-gob-5), or hCLCA1 expression vector (pcDNA-hCLCA1). (B) Induction of MUC5AC gene expression by transfection with gob-5 or hCLCA1 genes in NCI-H292 cells. Total RNA was extracted from transfected cells 3 days after transfection, and reverse transcription (RT)-PCR amplification was performed for hCLCA1, gob-5, and MUC5AC gene detection. Amplification of the GAPDH gene was used as a control for RNA loading. cDNA reactions in which the RT was omitted (− lanes) were used as negative controls for each condition. These data are representative of three independent experiments.
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