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. Clayburgh, Jason X. Cheng, Bora Yoon, Yingmin Wang, Anning Lin, Jerrold R. Turner

Research output: Contribution to journalArticle

94 Scopus citations

Abstract

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, 1Aand 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 NFκB, but all except NFκB 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 NFκB 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 NFκB activation decreased during differentiation while TNF-induced AP-1 activation increased. Thus either AP-1 or NFκB can up-regulate long MLCK transcription, but the mechanisms by which TNF upregulates intestinal epithelial long MLCK transcription from exon 1A are differentiation-dependent.

Original languageEnglish (US)
Pages (from-to)26205-26215
Number of pages11
JournalJournal of Biological Chemistry
Volume281
Issue number36
DOIs
StatePublished - Sep 8 2006

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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