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author:("Wang, fengyun")
1.  Amelioration of IFN-γ and TNF-α-Induced Intestinal Epithelial Barrier Dysfunction by Berberine via Suppression of MLCK-MLC Phosphorylation Signaling Pathway 
PLoS ONE  2013;8(5):e61944.
Intestinal barrier dysfunction occurs in many intestinal diseases, in which proinflammatory cytokines play critical roles. However, researchers are still on the way to defining the underlying mechanisms and to evaluate therapeutic strategies for restoring intestinal barrier function. Berberine, a drug that has clinically been used to treat gastroenteritis and diarrhea for thousands of years, has been shown to protect barrier function in both endothelial and epithelial cells, but the mechanisms are completely unknown. In this study, we investigate the protective actions of berberine on barrier function and the underlying mechanisms in Caco-2 monolayers challenged with IFN-γ and TNF-α. Caco-2 monolayers were treated without or with simultaneous IFN-γ and TNF-α in the absence or presence of berberine. Both transepithelial electrical resistance (TER) and paracellular permeability were measured to evaluate barrier function. The expression and distribution of tight junction proteins ZO-1, occluding, and claudin-1 were respectively analyzed by immunoblot or immunofluorescence. The expressions of phosphorylated myosin light chain (pMLC), MLC kinase (MLCK) and hypoxia-inducible factor-1α (HIF-1α) were determined by immunoblot. The translocation of NF-κB p65 to nuclei was analyzed by immunofluorescence and immunoblot, respectively. The results showed that berberine significantly attenuated TER decrease and paracellular permeability increase in Caco-2 monolayers treated with IFN-γ and TNF-α. Berberine also dramatically alleviated IFN-γ and TNF-α-induced morphological alteration of tight junction proteins ZO-1, occluding, and claudin-1. The increase of both MLC phosphorylation and MLCK protein expression induced by IFN-γ and TNF-α was significantly inhibited by berberine treatment. Additionally, berberine suppressed the activation of HIF-1α, but not NF-κB. Taken together, it is suggested that berberine attenuates IFN-γ and TNF-α-induced intestinal epithelial barrier dysfunction by inhibiting the signaling pathway of MLCK-dependent MLC phosphorylation mediated by HIF-1α.
doi:10.1371/journal.pone.0061944
PMCID: PMC3643960  PMID: 23671580
2.  Myosin Light Chain Kinase Mediates Intestinal Barrier Disruption following Burn Injury 
PLoS ONE  2012;7(4):e34946.
Background
Severe burn injury results in the loss of intestinal barrier function, however, the underlying mechanism remains unclear. Myosin light chain (MLC) phosphorylation mediated by MLC kinase (MLCK) is critical to the pathophysiological regulation of intestinal barrier function. We hypothesized that the MLCK-dependent MLC phosphorylation mediates the regulation of intestinal barrier function following burn injury, and that MLCK inhibition attenuates the burn-induced intestinal barrier disfunction.
Methodology/Principal Findings
Male balb/c mice were assigned randomly to either sham burn (control) or 30% total body surface area (TBSA) full thickness burn without or with intraperitoneal injection of ML-9 (2 mg/kg), an MLCK inhibitor. In vivo intestinal permeability to fluorescein isothiocyanate (FITC)-dextran was measured. Intestinal mucosa injury was assessed histologically. Tight junction proteins ZO-1, occludin and claudin-1 was analyzed by immunofluorescent assay. Expression of MLCK and phosphorylated MLC in ileal mucosa was assessed by Western blot. Intestinal permeability was increased significantly after burn injury, which was accompanied by mucosa injury, tight junction protein alterations, and increase of both MLCK and MLC phosphorylation. Treatment with ML-9 attenuated the burn-caused increase of intestinal permeability, mucosa injury, tight junction protein alterations, and decreased MLC phosphorylation, but not MLCK expression.
Conclusions/Significance
The MLCK-dependent MLC phosphorylation mediates intestinal epithelial barrier dysfunction after severe burn injury. It is suggested that MLCK-dependent MLC phosphorylation may be a critical target for the therapeutic treatment of intestinal epithelial barrier disruption after severe burn injury.
doi:10.1371/journal.pone.0034946
PMCID: PMC3329538  PMID: 22529961
3.  LIGHT signals directly to intestinal epithelia to cause barrier dysfunction via cytoskeletal and endocytic mechanisms 
Gastroenterology  2007;132(7):2383-2394.
BACKGROUND & AIMS
LIGHT (lymphotoxin-like inducible protein that competes with glycoprotein D for herpes virus entry on T cells) is a TNF core family member that regulates T cell activation and causes experimental inflammatory bowel disease. Additional data suggest that LIGHT may be involved in the pathogenesis of human inflammatory bowel disease. The aim of this study was to determine if LIGHT was capable of signaling directly to intestinal epithelia and to define the mechanisms and consequences of such signaling.
METHODS
The effects of LIGHT and interferon-γ (IFN-γ) on barrier function, cytoskeletal regulation, and tight junction structure were assessed in mice and intestinal epithelial monolayers.
RESULTS
LIGHT induced barrier loss in cultured epithelia via myosin II regulatory light chain (MLC) phosphorylation; both barrier loss and MLC phosphorylation were reversed by MLC kinase (MLCK) inhibition. IFN-γ pretreatment, which induced lymphotoxin β receptor (LTβR) expression, was required for these effects and neither barrier dysfunction nor intestinal epithelial MLC phosphorylation occurred in LTβR-knockout mice. In cultured monolayers, endocytosis of the tight junction protein occludin correlated with barrier loss. Internalized occludin co-localized with caveolin-1. LIGHT-induced occludin endocytosis and barrier loss were both prevented by inhibition of caveolar endocytosis.
CONCLUSIONS
T cell-derived LIGHT activates intestinal epithelial LTβR to disrupt barrier function. This requires MLCK activation and caveolar endocytosis. These data suggest a novel role for LIGHT in disease pathogenesis and suggest that inhibition of MLCK-dependent caveolar endocytosis may represent an approach to restoring barrier function in inflammatory bowel disease.
doi:10.1053/j.gastro.2007.02.052
PMCID: PMC2709832  PMID: 17570213
tight junction; interferon-γ; tumor necrosis factor; LIGHT; lymphotoxin; endocytosis; cytoskeleton; myosin; inflammatory bowel disease
4.  IFN-γ-induced TNFR2 Upregulation is Required for TNF-dependent Intestinal Epithelial Barrier Dysfunction 
Gastroenterology  2006;131(4):1153-1163.
Background & Aims:
Tumor necrosis factor (TNF) plays critical roles in intestinal disease. In intestinal epithelia, TNF causes tight junction disruption and epithelial barrier loss by upregulating myosin light chain kinase (MLCK) activity and expression. The aim of this study was to determine the signaling pathways by which TNF causes intestinal epithelial barrier loss.
Methods:
Caco-2 cells that were either non-transfected or stably-transfected with human TNF receptor 1 (TNFR1) or TNFR2 and mouse colonocytes were used for physiological, morphological, and biochemical analyses.
Results:
Colitis induced in vivo by adoptive transfer of CD4+CD45RBhi T cells was associated with increased epithelial myosin light chain kinase (MLCK) expression and myosin II regulatory light chain (MLC) phosphorylation as well as morphological tight junction disruption. In vitro studies showed that TNF caused similar increases in MLCK expression and MLC phosphorylation, as well as barrier dysfunction, in Caco-2 monolayers only after IFN-γ pretreatment. This reductionist model was therefore used to determine the molecular mechanism by which IFN-γ and TNF synergize to cause intestinal epithelial barrier loss. IFN-γ priming increased TNFR1 and TNFR2 expression and blocking antibody studies showed that TNFR2, but not TNFR1, was required for TNF-induced barrier dysfunction. Transgenic TNFR2, but not TNFR1, expression allowed IFN-γ-independent TNF responses.
Conclusions:
IFN-γ primes intestinal epithelia to respond to TNF by inducing TNFR2 upregulation, which in turn mediates the TNF-induced MLCK-dependent barrier dysfunction. The data further suggest that epithelial TNFR2 blockade may be a novel approach to restore barrier function in intestinal disease.
doi:10.1053/j.gastro.2006.08.022
PMCID: PMC1693969  PMID: 17030185
tight junction; interferon-γ; tumor necrosis factor; cytoskeleton; myosin; inflammatory bowel disease
5.  A Differentiation-dependent Splice Variant of Myosin Light Chain Kinase, MLCK1, Regulates Epithelial Tight Junction Permeability* 
The Journal of biological chemistry  2004;279(53):55506-55513.
Activation of Na+-nutrient cotransport leads to increased tight junction permeability in intestinal absorptive (villus) enterocytes. This regulation requires myosin II regulatory light chain (MLC) phosphorylation mediated by MLC kinase (MLCK). We examined the spatiotemporal segregation of MLCK isoform function and expression along the crypt-villus axis and found that long MLCK, which is expressed as two alternatively spliced isoforms, accounts for 97 ± 4% of MLC kinase activity in interphase intestinal epithelial cells. Expression of the MLCK1 isoform is limited to well differentiated enterocytes, both in vitro and in vivo, and this expression correlates closely with development of Na+-nutrient cotransport-dependent tight junction regulation. Consistent with this role, MLCK1 is localized to the perijunctional actomyosin ring. Furthermore, specific knockdown of MLCK1 using siRNA reduced tight junction permeability in monolayers with active Na+-glucose cotransport, confirming a functional role for MLCK1. These results demonstrate unique physiologically relevant patterns of expression and subcellular localization for long MLCK isoforms and show that MLCK1 is the isoform responsible for tight junction regulation in absorptive enterocytes.
doi:10.1074/jbc.M408822200
PMCID: PMC1237105  PMID: 15507455

Results 1-5 (5)