Physiological regulation of epithelial tight junctions is associated with myosin light-chain phosphorylation

Abstract

Tight junctions serve as the rate-limiting barrier to passive movement of hydrophilic solutes across intestinal epithelia. After activation of Na+-glucose cotransport, the permeability of intestinal tight junctions is increased. Because previous analyses of this physiological tight junction regulation have been restricted to intact mucosae, dissection of the mechanisms underlying this process has been limited. To characterize this process, we have developed a reductionist model consisting of Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, SGLT1. Monolayers of SGLT1 transfectants demonstrate physiological Na+-glucose cotransport. Activation of SGLT1 results in a 22 ± 5% fall in transepithelial resistance (TER) ( P< 0.001). Similarly, inactivation of SGLT1 by addition of phloridzin increases TER by 24 ± 2% ( P < 0.001). The increased tight junction permeability is size selective, with increased flux of small nutrient-sized molecules, e.g., mannitol, but not of larger molecules, e.g., inulin. SGLT1-dependent increases in tight junction permeability are inhibited by myosin light-chain kinase inhibitors (20 μM ML-7 or 40 μM ML-9), suggesting that myosin regulatory light-chain (MLC) phosphorylation is involved in tight junction regulation. Analysis of MLC phosphorylation showed a 2.08-fold increase after activation of SGLT1 ( P< 0.01), which was inhibited by ML-9 ( P < 0.01). Thus monolayers incubated with glucose and myosin light-chain kinase inhibitors are comparable to monolayers incubated with phloridzin. ML-9 also inhibits SGLT1-mediated tight junction regulation in small intestinal mucosa ( P < 0.01). These data demonstrate that epithelial cells are the mediators of physiological tight junction regulation subsequent to SGLT1 activation. The intimate relationship between tight junction regulation and MLC phosphorylation suggests that a critical step in regulation of epithelial tight junction permeability may be myosin ATPase-mediated contraction of the perijunctional actomyosin ring and subsequent physical tension on the tight junction.

Alcohol, intestinal bacterial growth, intestinal permeability to endotoxin, and medical consequences: Summary of a symposium

“…The role of Na+glucose transport in the regulation of epithelial tight junction permeability was further confirmed using Caco-2 intestinal epithelial cells transfected with the intestinal Na+-glucose cotransporter, wherein the activation of the co-transporter increased and inactivation decreased the permeability of the epithelial cells (Turner et al, 1997). Further research showed that the phosphporylation of myosin light-chain (MLC) catalyzed by myosin light-chain kinase (MLCK) was necessary for Na+-glucose co-transport-induced tight junction permeability regulation since pharmacological inhibition of MLCK prevented both MLC phosphorylation and regulation of tight junction barrier function in cultured intestinal epithelial cells (Turner et al, 1997).…”

Enteropathogenic Escherichia coli dephosphorylates and dissociates occludin from intestinal epithelial tight junctions

“…We are far from understanding the regulation of each of these molecules and the interactions that very likely exist between them. It is becoming increasingly clear that tight junctions are altered by both physiological (Madara and Pappenheimer, 1987;Turner et al, 1997) and pathophysiological processes (Spitz et al, 1995;Yuhan et al, 1997;Philpott et al, 1998;Sonoda et al, 1999). Here, we examine the impact of an important and interesting enteric bacterial pathogen, enteropathogenic Escherichia coli, on the tight junction-associated, transmembrane protein occludin.…”

“…Contraction of this ring, which inserts into the cell membrane immediately beneath the tight junction, has been shown to be one mechanism by which paracellular permeability is increased. Both physiological (activation of Na 1 /glucose co-transporter; Madara and Pappenheimer, 1987;Turner et al, 1997) and pathophysiological (EPEC infection; Yuhan et al, 1997) stimuli have been demonstrated to activate this pathway. However, the effects of such stimuli on specific tight junction proteins have not been examined.…”

“…The predominant protein that is phosphorylated in response to EPEC infection is the 20 kDa light chain of myosin (MLC 20 ;Manjarrez-Hernandez et al, 1992;Yuhan et al, 1997). In addition to controlling smooth muscle contraction, MLC 20 phosphorylation also regulates various non-muscle events including tight junction (TJ) permeability (Madara and Pappenheimer, 1987;Hecht et al, 1996;Turner et al, 1997). This event has been demonstrated in intestinal epithelia, in which contraction of the perijunctional actomyosin ring induces an increase in paracellular permeability.…”

How Stress Induces Intestinal Hypersensitivity

“…9 In addition, phosphorylation of myosin light chain by myosin light chain kinase (MLCK) or by Rho kinases may also physiologically regulate paracellular permeability by placing tension on the tight junctional complexes. 10 An ever-increasing number of reports suggest that gastrointestinal pathogens may cause disease, at least in part, by increasing transepithelial permeability. For example, Helicobacter pylori has the ability to increase the passage of food antigens across the gastric epithelium, 11 and infection with this gastric pathogen may be associated with the development of food allergies.…”