A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability

Clayburgh, Daniel; Rosen, Shari; Witkowski, Edwina D.; Wang, Fengjun; Blair, Stephanie A; Dudek, Steven M.; Garcia, Joe G.N.; Alverdy, John C.; Turner, Jerrold R.

doi:10.1074/jbc.m408822200

Cited by 159 publications

(152 citation statements)

References 34 publications

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“…In control tissues the subcellular localization of MLCK expression was similar to that previously reported in human jejunal enterocytes 18 and was concentrated within the apical actomyosin ring and a less intense juxtanuclear pool ( Figure 2). This distribution was qualitatively indistinguishable from that seen in ileal specimens with inactive CD.…”

Section: Ileal Epithelial Mlck Expression Is Increased In CDsupporting

confidence: 64%

“…18 This isoform is localized specifically to the apical actomyosin ring where it regulates epithelial barrier function by modifying tight junction permeability. 18 We therefore asked if this MLCK isoform was increased in ileal epithelium of CD patients. MLCK1 expression was analyzed in 18 CD specimens and three controls.…”

Section: Ileal Epithelial Mlck1 Isoform Expression Is Upregulated In mentioning

confidence: 99%

“…Primary antisera and concentrations used were: monoclonal mouse anti-pan MLCK, clone K36 (Sigma), 7.4 mg/ml; affinity-purified anti-MLCK1-specific rabbit antisera, 18 4 mg/ml; affinity-purified rabbit anti-serine-19-phosphorylated myosin light chain antisera, 19 and chicken antitotal myosin light chain antisera, 12 5 mg/ml. Secondary antisera were all used at 8 mg/ml and were Alexa 488-or Alexa 594-conjugated goat anti-mouse immunoglobulin (Invitrogen, Carlsbad, CA, USA), Alexa 488-conjugated goat anti-chicken immunoglobulin (Invitrogen), and Alexa 594-conjugated goat anti-rabbit immunoglobulin (Invitrogen).…”

Section: Tissue Stainingmentioning

confidence: 99%

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Epithelial myosin light chain kinase expression and activity are upregulated in inflammatory bowel disease

Blair

Kane

Clayburgh

et al. 2006

Laboratory Investigation

266

198

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The intestinal epithelial barrier is frequently disrupted in inflammatory bowel disease (IBD) and this has been proposed to play a role in disease pathogenesis and reactivation. In vitro studies show that cytokine-induced epithelial barrier dysfunction can be mediated by increased myosin light chain kinase (MLCK) expression and subsequent myosin II regulatory light chain (MLC) phosphorylation. However, this has never been examined in human disease. The aim of these studies, therefore, was to determine whether MLCK is upregulated in the intestinal epithelium of IBD patients. MLCK expression and MLC phosphorylation in human intestinal resection and biopsy specimens were determined by quantitative immunofluorescence microscopy and correlated with clinical and histopathological data. The data show that ileal epithelial MLCK expression was mildly upregulated in inactive IBD. Expression increased further in active disease, with progressive increases in MLCK expression correlating positively with histological disease activity. This correlation between activity and MLCK expression was also seen in individual patients where areas of differing disease activity were analyzed. Colonic epithelial MLCK expression was similarly increased in active IBD and these increases also correlated positively with disease activity, both in individual patients and the overall study group. To evaluate MLCK enzymatic activity, MLC phosphorylation was assessed in snap-frozen colon biopsies. MLC phosphorylation was significantly increased in biopsies with active, but not inactive, IBD. Therefore, these data show that MLCK expression and enzymatic activity are increased in IBD. Moreover, the correlation with disease activity suggests that MLCK upregulation may contribute to barrier dysfunction and IBD pathogenesis.

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Section: Ileal Epithelial Mlck Expression Is Increased In CDsupporting

confidence: 64%

Section: Ileal Epithelial Mlck1 Isoform Expression Is Upregulated In mentioning

confidence: 99%

Section: Tissue Stainingmentioning

confidence: 99%

See 1 more Smart Citation

Epithelial myosin light chain kinase expression and activity are upregulated in inflammatory bowel disease

Blair

Kane

Clayburgh

et al. 2006

Laboratory Investigation

266

198

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“…To assess this, mRFP1-ZO-1 transgenic mice were bred with knockout mice that lack long MLCK, the isoform expressed in intestinal epithelia (19,20). The ZO-1 mobile fraction and t 1/2 were not significantly different from those measured in wild-type mice (Fig.…”

Section: Mlck-dependent Barrier Regulationmentioning

confidence: 99%

MLCK-dependent exchange and actin binding region-dependent anchoring of ZO-1 regulate tight junction barrier function

Marchiando

Weber

et al. 2010

Proc. Natl. Acad. Sci. U.S.A.

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The perijunctional actomyosin ring contributes to myosin light chain kinase (MLCK)-dependent tight junction regulation. However, the specific protein interactions involved in this process are unknown. To test the hypothesis that molecular remodeling contributes to barrier regulation, tight junction protein dynamic behavior was assessed by fluorescence recovery after photobleaching (FRAP). MLCK inhibition increased barrier function and stabilized ZO-1 at the tight junction but did not affect claudin-1, occludin, or actin exchange in vitro. Pharmacologic MLCK inhibition also blocked in vivo ZO-1 exchange in wild-type, but not long MLCK −/− , mice. Conversely, ZO-1 exchange was accelerated in transgenic mice expressing constitutively active MLCK. In vitro, ZO-1 lacking the actin binding region (ABR) was not stabilized by MLCK inhibition, either in the presence or absence of endogenous ZO-1. Moreover, the free ABR interfered with full-length ZO-1 exchange and reduced basal barrier function. The free ABR also prevented increases in barrier function following MLCK inhibition in a manner that required endogenous ZO-1 expression. In silico modeling of the FRAP data suggests that tight junction-associated ZO-1 exists in three pools, two of which exchange with cytosolic ZO-1. Transport of the ABR-anchored exchangeable pool is regulated by MLCK. These data demonstrate a critical role for the ZO-1 ABR in barrier function and suggest that MLCK-dependent ZO-1 exchange is essential to this mechanism of barrier regulation.fluorescence recovery after photobleaching | mathematical models | myosin light chain kinase | paracellular permeability | intestinal epithelium G reat progress has been made toward identifying components of the tight junction. These include transmembrane, peripheral membrane, and regulatory proteins, many of which contain one or more domains that mediate interactions with other tight junction and cytoskeletal proteins (1). These and other observations drove development of models that depicted the tight junction as a static, heavily cross-linked protein complex (2). However, recent data showing rapid and continuous remodeling of the tight junction refuted the previous models and led to the hypothesis that modulation of protein remodeling behavior could be a mechanism of tight junction barrier regulation (3, 4).The perijunctional ring of F-actin and myosin II that supports the tight junction is essential to physiological and pathophysiological barrier regulation (5). For example, activation of perijunctional myosin light chain kinase (MLCK) is sufficient to enhance paracellular permeability (6, 7) and is required for tight junction barrier regulation in response to Na + -nutrient cotransport, inflammatory cytokines, or pathogenic bacteria (8). Thus, modulation of MLCK activity represents a point of convergence for multiple signaling pathways that regulate tight junction barrier function.To assess the role of molecular remodeling in barrier regulation, dynamic behaviors of tight junction proteins were assessed b...

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“…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.…”

Section: Tnf Up-regulates Mlck Expression In Vitro and In Vivomentioning

confidence: 99%

Molecular Basis of Epithelial Barrier Regulation

Turner

2006

The American Journal of Pathology

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166

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The intestinal epithelium is faced with the complex task of providing a barrier while also allowing nutrient and water absorption. The frequency with which these processes are disrupted in disease can be taken as evidence of their importance. It is therefore of interest to define the mechanisms of altered intestinal barrier and transport function and develop means to correct diseaseassociated defects. Over the past 10 years, some of the molecular events underlying physiological epithelial barrier regulation have been described. Remarkably, recent advances have shown that activation of the same mechanisms is central to barrier dysfunction in both in vitro and in vivo models of disease. Although the contribution of barrier dysfunction to pathogenesis of chronic disease remains incompletely understood, it is now clear that cytoskeletal regulation of barrier function is both an important pathogenic process and that targeted inhibition of myosin light chain kinase, which affects this cytoskeleton-dependent tight junction dysfunction, is an attractive candidate for therapeutic intervention.

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A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability

Cited by 159 publications

References 34 publications

Epithelial myosin light chain kinase expression and activity are upregulated in inflammatory bowel disease

Epithelial myosin light chain kinase expression and activity are upregulated in inflammatory bowel disease

MLCK-dependent exchange and actin binding region-dependent anchoring of ZO-1 regulate tight junction barrier function

Molecular Basis of Epithelial Barrier Regulation

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