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

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, 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 upregulates intestinal epithelial long MLCK transcription from exon 1A are differentiation-dependent.

The glial cell-line derived neurotrophic factor: a novel regulator of intestinal barrier function in health and disease

“…Another important regulator of the actin cytoskeleton is the myosin light chain kinase (MLCK). In IBD, TNF-␣ leads to an upregulation of MLCK (21). This leads to a perijunctional contraction of the actin cytoskeleton and, consequently, to barrier destabilization (11).…”

“…Additional barrier-protective effects can be assumed from the immunomodulatory effects by GDNF. Apart from these effects, it is obvious that GDNF targets major regulatory mechanisms, which are consistently reported to be involved in the loss of intestinal barrier functions in IBD: 1) In enterocytes of patients with IBD the typical contraction of the cortical actin-myosin ring is present as a consequence of increased p38 MAPK phosphorylation (13,21). 2) A constant increase of apoptosis and cell shedding is known in patients with IBD (30)…”

Molecular Basis of Epithelial Barrier Regulation

“…However, only the AP-1 sites appeared to be used in well-differentiated intestinal epithelial monolayers. 59 Thus, MLCK transcriptional regulation is likely to be both tissue and differentiation specific.…”

“…61 To understand better the means by which TNF increases MLCK expression, the transcriptional start site of long MLCK in cultured human intestinal epithelia was identified. 59 Two novel exons that encode alternative transcriptional start sites were found, and the long MLCK promoter upstream of one of these sites was cloned. It identified functional response elements for both activator protein-1 (AP-1) and nuclear factor-B.…”

“…56 Although epithelial MLCK is transcribed from the same gene as smooth muscle MLCK, transcription begins at upstream start sites, resulting in a larger MLCK, termed long MLCK, 57 with an amino-terminal extension. 58,59 Alternative splicing of long MLCK transcripts leads to translation of two different MLCK isoforms in intestinal epithelia, 58 of which one can be regulated by Src kinase as well as Ca ϩ2 /calmodulin. 60 Thus, whereas the mechanisms by which TNF activates MLCK activity in intestinal epithelia are not yet described, both Src kinase and Ca ϩ2 / calmodulin are potential signaling intermediates.…”