HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Zheng, S. Articles by Voynow, J. A. Search for Related Content PubMed Articles by Zheng, S. Articles by Voynow, J. A.

Neutrophil Elastase Increases MUC5AC Mucin mRNA Expression via Post-Transcriptional Regulation

Departments of Pediatrics and Pathology, Duke University Medical Center, Durham, North Carolina

Correspondence and requests for reprints should be addressed to Judith A. Voynow, M.D., Duke University Medical Center, Erwin Road, 302 E Bell Building, Durham, NC 27710. E-mail: voyno001{at}mc.duke.edu

Neutrophil elastase (NE) increases the mRNA expression of a major respiratory tract mucin, MUC5AC, by enhancing mRNA stability. We hypothesized that the cis stability domain of MUC5AC was localized to the 3' untranslated region (3'UTR). To test this hypothesis, we performed reporter-3'UTR chimera functional assays and RNA mobility shift assays. A549 cells were transfected with the parent reporter plasmid containing the human growth hormone (hGH) coding sequence linked to the hGH 3'UTR (pTKGH) or a chimera plasmid with the reporter hGH coding sequence linked to the MUC5AC 3'UTR (pTKGH-MUC5AC 3'UTR). After transfection, both pTKGH-transfected cells and pTKGH-MUC5AC 3'UTR–transfected cells were treated with control vehicle or NE (50 nM, 17 h), and then evaluated for hGH mRNA expression. Only the reporter containing the MUC5AC 3'UTR increased hGH expression after NE treatment, suggesting that the MUC5AC 3'UTR contains a domain that regulates NE-induced expression of the reporter. For the RNA mobility shift assays, in vitro radiolabeled sense cRNA was prepared for the full-length MUC5AC-3'UTR and the 5' and 3' half-domains of the MUC5AC-3'UTR. A549 cells were treated with control vehicle or NE (50 nM, 4 h), lysed, and cytosolic protein was isolated and quantitated. Radiolabeled cRNA was incubated with cytosolic protein and analyzed for cRNA–protein binding by nondenaturing polyacrylamide gel electrophoresis and autoradiography. Specificity of the protein–cRNA interaction was tested by competition studies using excess nonradiolabeled cRNA. NE treatment resulted in increased and specific protein binding to the 3' half-domain of the MUC5AC 3'UTR cRNA. These experiments suggest that an NE-responsive RNA stability sequence is located in the 3' half-domain of the MUC5AC 3'UTR.

FOOTNOTES

Supported by the National Institutes of Health. Grants R01 HL65611 and R01 HL082504 both were awarded to J.A.V.

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

(Received in original form March 16, 2006; accepted in final form March 16, 2006)

This Article Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Zheng, S. Articles by Voynow, J. A. Search for Related Content PubMed Articles by Zheng, S. Articles by Voynow, J. A.