3187
Journal of Cell Science 113, 3187-3196 (2000) Printed in Great Britain © The Company of Biologists Limited 2000 JCS1534
Protein kinase C activation leads to dephosphorylation of occludin and tight junction permeability increase in LLC-PK1 epithelial cell sheets Hilary Clarke*, Alejandro Peralta Soler and James M. Mullin Lankenau Medical Research Center, 100 Lancaster Avenue, Wynnewood, PA 19096, USA Author for correspondence (e-mail:
[email protected])
Accepted 6 July; published on WWW 22 August 2000
SUMMARY Activation of protein kinase C by exposure of LLC-PK1 renal epithelial cells to 10−7 M TPA, a tumor promoting phorbol ester, results in a rapid and sustained increase in paracellular permeability as evidenced by a decrease in transepithelial electrical resistance. Occludin, the first identified transmembrane protein to be localized to the tight junction of both epithelial and endothelial cells is thought play an important role in tight junction barriers. Although transepithelial electrical resistance fell to less than 20% of initial values within 1 hour of TPA exposure, transmission electron microscopy showed no change in the gross morphology of the tight junction of cells treated with 10−7 M TPA for up to 2 hours. Immunofluorescence microscopy revealed a more rapid change in the membrane distribution of ZO-1 compared to occludin in the TPAtreated cells. Immunoblot analysis indicated that occludin levels in total cell lysates as well as cytosolic, membrane (Triton-X soluble) and cytoskeletal (Triton-X insoluble) fractions remained unchanged for at least 2 hours in cells treated with 10−7 M TPA compared to their corresponding
control cells. As the phosphorylation state of occludin is thought to be important in both tight junction assembly and regulation, the effect of phorbol ester treatment on the phosphorylation of occludin was investigated. Surprisingly, activation of protein kinase C with 10−7 M TPA resulted in a time-dependent decrease in threonine phosphorylation of occludin which correlated closely with the rapid decrease in transepithelial electrical resistance. This dephosphorylation of occludin, occuring after activation of a serine/threonine kinase by TPA, suggested that protein kinase C was not acting directly on this tight junction target protein. If occludin dephosphorylation is involved in increasing tight junction premeability, then protein kinase C is apparently further upstream in the signaling pathway regulating epithelial barrier function, with a downstream serine/threonine phosphatase acting upon occludin.
INTRODUCTION
function and our increasing knowledge of their molecular composition, relatively little is known about the molecular mechanisms underlying tight junctional physiology. Paracellular permeability is regulated by intracellular signaling and as a pathological consequence of disease (Balda et al., 1991; Bentzel et al., 1980; Madara, 1988; Schneeberger and Lynch, 1992). Protein kinases are thought to play a role in the regulation of tight junction assembly and in paracellular permeability changes. The atypical PKC (protein kinase C) isoforms λ and ζ localize to the cell junctional complex in the renal epithelial MDCK cells (Izumi et al., 1998) and LLC-PK1 cells (Dodane and Kachar, 1996), whereas PKC-ε has been observed in the region of the tight junction in intestinal cells (Saxon et al., 1994). The nonreceptor tyrosine kinases c-src and c-yes are concentrated in the apical junction plaque (Tsukita et al., 1993). Small GTP-binding proteins, rab13 (Zahraoui et al., 1994) and rab3B (Weber et al., 1994), also are concentrated in the tight junction region. In addition, numerous other second messenger pathways have been implicated in influencing perijunctional cytoskeletal organization in concert with changes in junctional permeability (Balda et al., 1991;
The tight junction constitutes the principle barrier in epithelia to passive movement of fluid electrolytes, macromolecules and cells through the paracellular pathway. This route is defined as the space between the cells and includes the tight junction and lateral intercellular space. The tight junction contributes to regulating epithelial transport by maintaining the distinct apical and basolateral surface compositions necessary for vectorial transport across epithelia (Gumbiner, 1990). The tight junction demonstrates ion selectivity, varies significantly in permeability among epithelia found in different tissues, is subject to physiological regulation and undergoes dynamic modulation by agents as diverse as phorbol esters (Ojakian, 1981; Mullin and O’Brien, 1986; Hecht et al., 1994; Stenson et al., 1993), cytokines (Marano et al., 1998; Mullin and Snock, 1990; Madara and Stafford, 1989), calcium (Cereijido et al., 1978; Martinez-Palomo et al., 1980), adenosine triphosphate (Canfield et al., 1991) and bacterial toxins (Moore et al., 1990; Fasano et al., 1995). Despite the great importance of tight junctions to epithelial
Key words: Occludin, Tight junction, Transepithelial, Protein kinase C, LLC-PK1, Phorbol ester, TPA, Phosphorylation, Phosphatase
3188 H. Clarke, A. P. Soler and J. M. Mullin Madara et al., 1992). Despite these studies a comprehensive molecular understanding of the different signaling pathways that mediate control of junctional permeability and their mechanism(s) of action is lacking. Occludin, was the first transmembrane component of the tight junction to be identified. It is a 65 kDa protein predicted to span the membrane four times, having two extracellular loops and cytosolic NH2 and COOH termini (Furuse et al., 1993). Recently an alternatively spliced form of occludin designated occludin 1B had been identified in MDCK cells (Muresan et al., 2000). This occludin 1B transcript contained a 193-base pair insertion encoding a longer form of occludin with a unique N-terminal sequence of 56 amino acids. Occludin 1B seems to have a wide epithelial distribution and a conservation across species which suggests a potentially crucial role for this protein in the structure and function of the tight junction. Additional transmembrane proteins termed claudins, were shown to also constitute tight junction strands (Furuse et al., 1998a,b). Claudins form a new gene family which includes at least 16 members (Morita et al., 1999; Tsukita and Furuse, 1999). Several intracellular tight junctionassociated proteins have been identified, including ZO-1 (Stevenson et al., 1986), ZO-2 (Gumbiner et al., 1991; Jesaitis and Goodenough, 1994), ZO-3 (Haskins et al., 1998), cingulin (Citi et al., 1988), 7H6 (Zhong et al., 1993), symplekin (Keon et al., 1996), and rab3B (Weber et al., 1994). Actin filaments are also known to be associated with the cytoplasmic surface of the tight junction membrane contact sites (Madara, 1987; Madara et al., 1988). The COOH terminal domain of occludin was reported to directly bind to several cytoplasmic proteins, including ZO-1, ZO-2 (Furuse et al., 1994; Itoh et al., 1999) and ZO-3 (Haskins et al., 1998). ZO-1, ZO-2 and ZO-3 also bind to the COOH terminal of claudin-1 to -8 (Itoh et al., 1999). In turn, ZO-1 and ZO-2 bind directly to actin filaments through their COOHterminal regions, suggesting that these molecules function as cross-linkers between tight junction strands and actin filaments (Itoh et al., 1997, 1999; Fanning et al., 1998). ZO-1 has been reported to associate with ZO-2 and ZO-3 (Fanning et al., 1998; Itoh et al., 1999; Wittchen et al., 1999), whereas ZO-2 interacts only with ZO-1 (Haskins et al., 1998). Both tyrosine (Stuart and Nigam, 1995; Tsukamoto and Nigam, 1999) and serine/threonine (Citi and Denisenko, 1995; Sakabibara et al., 1997) protein phosphorylation changes are known to correlate with permeability changes in existing tight junctions, as well as the assembly of tight junctions. Tyrosine kinase agonists and tyrosine phosphatase inhibitors were shown to affect phosphotyrosine levels of ZO-1 and ZO-2, which correlated with altered permeability and redistribution of tight junction components in epithelial and endothelial cells (Staddon et al., 1995; Takeda and Tsukita, 1995; Anderson and Van Itallie, 1995). Like most tight junction proteins, occludin is also a phosphoprotein and in vivo appears to be phosphorylated primarily on serine and threonine residues (Sakakibara et al., 1997; Cordenonsi et al., 1997). Increased occludin serine/threonine phosphorylation shows a strong correlation with tight junction formation in MDCK cells undergoing the calcium switch assay (Sakakibara et al., 1997), whereas in the developing Xenopus embryo, induction of tight junction formation is paralleled by a dephosphorylation of occludin (Cordenonsi et al., 1997). Recent evidence shows that
tyrosine phosphorylation of occludin is necessary for tight junction reassembly during ATP repletion in MDCK cells (Tsukamoto and Nigam, 1999), and is also thought to play some role in tight junction formation in Ras transformed MDCK cells (Chen et al., 2000). Although the exact kinase(s) and phosphatase(s) that alter occludin phosphorylation in vivo have not been identified, the C-terminal domain of chicken occludin can be phosphorylated in vitro by both protein kinase CK2 and the p34cdc2/cyclinB complex (Cordenonsi et al., 1997). Activation of protein kinase C by phorbol esters such as TPA (12-0-tetradecanoylphorbol-13-acetate) results in the immediate translocation of PKC-α from the cytosolic to the membrane-associated compartment in LLC-PK1 cells (Mullin et al., 1997a). The dramatic and sustained increase in tight junction permeability occurs with a similar time-course to PKC-α translocation, and allows the increased paracellular passage of salts and sugars as well as larger macromolecules across the cell sheet (Mullin and O’Brien, 1986; Mullin and McGinn, 1988; Mullin et al., 1992, 1997b). Thus, an increase in membrane-associated PKC-α correlated with increase in tight junction permeability, suggesting a role for this kinase in tight junction regulation. This observation led to the important question of whether the permability changes observed with PKC activation result from direct action of this serine/ threonine kinase on tight junctional proteins. Here, we investigated the subcellular localization and the phosphorylation state of occludin after activation of PKC in LLC-PK1 cells with the phorbol ester, TPA. Treatment of the epithelial cell sheet with TPA did not change the total amount or subcellular distribution of occludin but it did correlate with threonine dephosphorylation of occludin at a rate which correlated closely with the rapid increase in paracellular permeability. The PKC-dependent dephosphorylation of occludin suggested that PKC does not directly target this tight junction protein as predicted. Rather it appears that in LLCPK1 epithelia, PKC is stimulating the activity of a serine/threonine phosphatase which in turn acts on occludin. MATERIALS AND METHODS Cell culture LLC-PK1 cells were a gift from Dr Robert Hull (Hull et al., 1976) and were used between passages 184 and 202. Cells were cultured in α-minimum essential medium without nucleosides (JRH Biosciences) supplemented with 10% fetal bovine serum (Hyclone) and were passaged as described previously (Mullin et al., 1996). Transepithelial electrical measurements Transepithelial electrical resistance was recorded as described previously (Mullin et al., 1996). Cells were seeded onto Millicell PCF filter rings (Millipore) and refed with fresh culture medium 1 hour before the initial transepithelial resistance measurements were recorded. Cell sheets were then refed fresh medium plus the vehicle (100% ethanol) or 10−7 M TPA. Rt was monitored at regular intervals. Rt values of blank filters (without cell sheets) had transepithelial resistances that were always
