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Double-Stranded RNA and TGF- Promote MUC5AC Induction in Respiratory Cells

Hiromi Tadaki^*,, Hirokazu Arakawa^1,*, Takahisa Mizuno^*, Tomoko Suzuki^*, Kiyoshi Takeyama, Hiroyuki Mochizuki^*, Kenichi Tokuyama, Shumpei Yokota and Akihiro Morikawa^*

^* Department of Pediatrics and Developmental Medicine, Gunma University Graduate School of Medicine, Gunma, Japan; Department of Pediatrics, Yokohama City University School of Medicine, Kanagawa, Japan; First Department of Medicine, Tokyo Women’s Medical University, Tokyo, Japan; and Department of Pharmacy, Takasaki University of Health and Welfare, Gunma, Japan

Viral infection is a major trigger for exacerbation of asthma and induces overproduction of mucins. We investigated whether dsRNA could amplify the induction of mucin by TGF- in human bronchial epithelial cells, as well as the molecular mechanisms regulating MUC5AC expression. Human pulmonary mucoepidermoid carcinoma (NCI-H292) cells and normal human bronchial epithelial cells were exposed to polyinosinic-cytidyric acid (poly(I:C)) and TGF-. Then, MUC5AC protein production, mRNA expression, and promoter activity were evaluated. Cells were pretreated with a selective inhibitor of ERK, and phosphorylation of ERK was examined by Western blotting. Furthermore, the expression of MAPK phosphatase 3 (MKP3) mRNA was evaluated and the effect of MKP3 overexpression was assessed. Poly(I:C) synergistically increased MUC5AC induction by TGF- in both NCI-H292 and normal human bronchial epithelial cells. This increase was dependent on MUC5AC gene transcription. A MEK1/2 inhibitor (U0126) significantly inhibited MUC5AC production. Phosphorylation of ERK was enhanced by poly(I:C). TGF- stimulation up-regulated MKP3 mRNA expression, while costimulation with poly(I:C) inhibited this up-regulation dose-dependently. Enhanced expression of MUC5AC mRNA by poly(I:C) in wild-type cells was completely suppressed in cells transfected with the MKP3 expression vector. dsRNA can synergistically amplify the induction of MUC5AC mucin by TGF-. This synergistic effect on MUC5AC production may be due to enhanced activation of ERK through inhibition of MKP3 by poly(I:C).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Address correspondence and reprint requests to Dr. Hirokazu Arakawa, Department of Pediatrics and Developmental Medicine, Gunma University Graduate School of Medicine, 3-39-15 Showa-machi, Maebashi, Gunma 371-8511, Japan. E-mail address: harakawa{at}showa.gunma-u.ac.jp

² Abbreviations used in this paper: poly(I:C), polyinosinic-cytidyric acid; AB-PAS, Alcian blue/periodic acid-Schiff; C_T, threshold cycle; EGFR, epidermal growth factor receptor; MKP, MAPK phosphatase; NHBE, normal human bronchial epithelial; RT, room temperature.