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J. Biol. Chem., Vol. 281, Issue 36, 26205-26215, September 8, 2006

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Tumor Necrosis Factor-induced Long Myosin Light Chain Kinase Transcription Is Regulated by Differentiation-dependent Signaling Events

CHARACTERIZATION OF THE HUMAN LONG MYOSIN LIGHT CHAIN KINASE PROMOTER^*

W. Vallen Graham, Fengjun Wang, Daniel R. Clayburgh¹, Jason X. Cheng, Bora Yoon, Yingmin Wang, Anning Lin^¶, and Jerrold R. Turner²

From the Department of Pathology and ^¶Ben May Institute for Cancer Research, University of Chicago, Chicago, Illinois 60637 and the State Key Laboratory of Trauma, Burns and Combined Injury, Institute of Burn Research, Southwest Hospital, Third Military Medical University, Chongqing 400038, China

Myosin light chain kinase (MLCK) is expressed as long and short isoforms from unique transcriptional start sites within a single gene. Tumor necrosis factor (TNF) augments intestinal epithelial long MLCK expression, which is critical to cytoskeletal regulation. We found that TNF increases long MLCK mRNA transcription, both in human enterocytes in vitro and murine enterocytes in vivo.5'-RACE identified two novel exons, 1A and 1B, which encode alternative long MLCK transcriptional start sites. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis identified two essential Sp1 sites upstream of the exon 1A long MLCK transcriptional start site. Analysis of deletion and truncation mutants showed that a 102-bp region including these Sp1 sites was necessary for basal transcription. A promoter construct including 4-kb upstream of exon 1A was responsive to TNF, AP-1, or NFB, but all except NFB responses were absent in a shorter 2-kb construct, and all responses were absent in a 1-kb construct. Electrophoretic mobility shift assays, ChIP, and site-directed mutagenesis explained these data by identifying three functional AP-1 sites between 2- and 4-kb upstream of exon 1A and two NFB sites between 1- and 2-kb upstream of exon 1A. Analysis of differentiating epithelia showed that only well differentiated enterocytes activated the 4-kb long MLCK promoter in response to TNF, and consensus promoter reporters demonstrated that TNF-induced NFB activation decreased during differentiation while TNF-induced AP-1 activation increased. Thus either AP-1 or NFB can up-regulate long MLCK transcription, but the mechanisms by which TNF up-regulates intestinal epithelial long MLCK transcription from exon 1A are differentiation-dependent.

Received for publication, March 7, 2006 , and in revised form, May 19, 2006.

^* This work was supported in part by Grants DK61931 and DK68271 from the National Institutes of Health, the Crohn's Colitis Foundation of America, The University of Chicago Digestive Disease Center (DK42086), and The University of Chicago Cancer Center (CA14599). 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 A Predoctoral Fellow of the National Institutes of Health, The University of Chicago Medical Scientist Training Program Grant (T32 GM07281).

² To whom correspondence should be addressed: Dept. of Pathology, The University of Chicago, 5841 South Maryland Ave., MC 1089, Chicago, IL 60637. Tel.: 773-702-2433; E-mail: jturner{at}bsd.uchicago.edu.

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