Biomedical subjects

Le Shen

Publications and source records attributed to Le Shen.

7 recordsLinked to original sources

PubMed2006

Myosin light chain phosphorylation regulates barrier function by remodeling tight junction structure.

Epithelial tight junctions form a barrier against passive paracellular flux. This barrier is regulated by complex physiologic and pathophysiologic signals that acutely fine-tune tight junction permeability. Although actomyosin contraction and myosin light chain phosphorylation are clearly involved in some forms of tight junction regulation, the contributions of other signaling events and the role of myosin light chain phosphorylation in this response are poorly understood. Here we ask if activation of myosin light chain kinase alone is sufficient to induce downstream tight junction regulation. We use a confluent polarized intestinal epithelial cell model system in which constitutively active myosin light chain kinase, tMLCK, is expressed using an inducible promoter. tMLCK expression increases myosin light chain phosphorylation, reorganizes perijunctional F-actin, and increases tight junction permeability. TJ proteins ZO-1 and occludin are markedly redistributed, morphologically and biochemically, but effects on claudin-1 and claudin-2 are limited. tMLCK inhibition prevents changes in barrier function and tight junction organization induced by tMLCK expression, suggesting that these events both require myosin light chain phosphorylation. We conclude that myosin light chain phosphorylation alone is sufficient to induce tight junction regulation and provide new insights into the molecular mechanisms that mediate this regulation.

Actins↗

PubMed2006

Role of epithelial cells in initiation and propagation of intestinal inflammation. Eliminating the static: tight junction dynamics exposed.

Like all mucosal surfaces, the intestine forms a barrier that separates the external environment, i.e., the gut lumen, from the protected internal milieu. The intestinal barrier is formed by the epithelial cells that line the luminal surface. Plasma membranes of these cells prevent free passage of hydrophilic molecules across this barrier but do not seal the space between cells. This function is provided by the tight junction. Each cell is encircled at the apicolateral boundary by the tight junction, which seals the paracellular space. The tight junction does not form a completely impermeant seal, however, because that would prevent paracellular absorption of essential nutrients and ions; intestinal tight junctions are "leaky" and allow solutes to be transported paracellularly according to size and charge. Abundant data are available to demonstrate that barrier properties of tight junctions can be modulated in response to physiological, pharmacological, and pathophysiological stimuli, but the structural modifications responsible for these responses are poorly defined. Recent advances in understanding the role of tight junction dynamics in response to such stimuli are the focus of this review.

Animals↗

PubMed2005

Recognition of host immune activation by Pseudomonas aeruginosa.

It is generally reasoned that lethal infections caused by opportunistic pathogens develop permissively by invading a host that is both physiologically stressed and immunologically compromised. However, an alternative hypothesis might be that opportunistic pathogens actively sense alterations in host immune function and respond by enhancing their virulence phenotype. We demonstrate that interferon-gamma binds to an outer membrane protein in Pseudomonas aeruginosa, OprF, resulting in the expression of a quorum-sensing dependent virulence determinant, the PA-I lectin. These observations provide details of the mechanisms by which prokaryotic organisms are directly signaled by immune activation in their eukaryotic host.

Adhesins, Bacterial↗

PubMed2005

Actin depolymerization disrupts tight junctions via caveolae-mediated endocytosis.

The tight junction (TJ) determines epithelial barrier function. Actin depolymerization disrupts TJ structure and barrier function, but the mechanisms of this effect remain poorly understood. The goal of this study was to define these mechanisms. Madin-Darby canine kidney (MDCK) cells expressing enhanced green fluorescent protein-, enhanced yellow fluorescent protein-, or monomeric red fluorescent protein 1-fusion proteins of beta-actin, occludin, claudin-1, ZO-1, clathrin light chain A1, and caveolin-1 were imaged by time-lapse multidimensional fluorescence microscopy with simultaneous measurement of transepithelial electrical resistance (TER). Actin depolymerization was induced with latrunculin A (LatA). Within minutes of LatA addition TER began to fall. This coincided with occludin redistribution and internalization. In contrast, ZO-1 and claudin-1 redistribution occurred well after maximal TER loss. Occludin internalization and TER loss, but not actin depolymerization, were blocked at 14 degrees C, suggesting that membrane traffic is required for both events. Inhibition of membrane traffic with 0.4 M sucrose also blocked occludin internalization and TER loss. Internalized occludin colocalized with caveolin-1 and dynamin II, but not with clathrin, and internalization was blocked by dominant negative dynamin II (K44A), but not by Eps15Delta95-295 expression. Inhibition of caveolae-mediated endocytosis by cholesterol extraction prevented both LatA-induced TER loss and occludin internalization. Thus, LatA-induced actin depolymerization causes TJ structural and functional disruption by mechanisms that include caveolae-mediated endocytosis of TJ components.

Actins↗

PubMed2005

Distinct temporal-spatial roles for rho kinase and myosin light chain kinase in epithelial purse-string wound closure.

BACKGROUND & AIMS: Small epithelial wounds heal by purse-string contraction of an actomyosin ring that is regulated by myosin light chain (MLC) kinase (MLCK) and rho kinase (ROCK). These studies aimed to define the roles of these kinases in purse-string wound closure. METHODS: Oligocellular and single-cell wounds were created in intestinal epithelial monolayers. Fluorescence imaging and electrophysiologic data were collected during wound closure. Human biopsies were studied immunohistochemically. RESULTS: Live-cell imaging of enhanced green fluorescent protein-beta-actin defined rapid actin ring assembly within 2 minutes after wounding. This progressed to a circumferential ring within 8 minutes that subsequently contracted and closed the wound. We therefore divided this process into 2 phases: ring assembly and wound contraction. Activated rho and ROCK localized to the wound edge during ring assembly. Consistent with a primary role in the assembly phase, ROCK inhibition prevented actin ring assembly and wound closure. ROCK inhibition after ring assembly was complete had no effect. Recruitment and activation of MLCK occurred after ring assembly was complete and coincided with ring contraction. MLCK inhibition slowed and then stopped contraction but did not prevent ring assembly. MLCK inhibition also delayed barrier function recovery. Studies of human colonic biopsy specimens suggest that purse-string wound closure also occurs in vivo, because MLC phosphorylation was enhanced surrounding oligocellular wounds. CONCLUSIONS: These results suggest complementary roles for these kinases in purse-string closure of experimental and in vivo oligocellular epithelial wounds; rho and ROCK are critical for actin ring assembly, while the activity of MLCK drives contraction.

Actin Cytoskeleton↗

PubMed2004

A porous defense: the leaky epithelial barrier in intestinal disease.

A critical function of the intestinal mucosa is to form a barrier that separates luminal contents from the interstitium. This intestinal barrier is compromised in a number of intestinal diseases, most notably inflammatory bowel disease. In vitro studies have demonstrated that cytokines elaborated by immune cells can cause the mucosal barrier to become leaky; these cytokines are known to be increased in intestinal mucosa involved in inflammatory bowel disease. Detailed information describing the mechanisms by which altered cytokine signaling occurs is not available, but recent data implicate the cytoskeleton within epithelial cells as a critical regulator of the mucosal barrier under physiological and pathophysiological conditions. Using available data, we describe a model of intestinal disease where an initial insult to the epithelial barrier may trigger a self-amplifying cycle of immune activation, cytokine release, and further barrier dysfunction. This model is supported by the observation that pharmacological abrogation of cytokine signaling corrects both barrier defects and clinical disease in animal models and human patients, although such therapy clearly has multiple mechanisms. Other therapeutic targets that represent strategies to prevent or reverse disease processes are also considered. The overarching hypothesis is that modulation of the mucosal epithelial barrier plays a critical role in the initiation and propogation of inflammatory intestinal diseases.

Animals↗

PubMed2002

Molecular ordering of the initial signaling events of CD95.

Binding of either ligand or agonistic antibodies to the death receptor CD95 (APO-1/Fas) induces the formation of the death-inducing signaling complex (DISC). We now show that signal initiation of CD95 in type I cells can be further separated into at least four distinct steps. (i) The first step is ligand-induced formation of CD95 microaggregates at the cell surface. (ii) The second step is recruitment of FADD to form a DISC. This step is dependent on actin filaments. (iii) The third step involves formation of large CD95 surface clusters. This event is positively regulated by DISC-generated caspase 8. (iv) The fourth step is internalization of activated CD95 through an endosomal pathway. The latter step is again dependent on the presence of actin filaments. The data indicate that the signal initiation by CD95 is a complex process actively regulated at various levels, providing a number of new drug targets to specifically modulate CD95 signaling.

Actin Cytoskeleton↗