Regulation of epithelial transport and barrier function by distinct protein kinase C isoforms

Abstract

The phorbol ester phorbol 12-myristate 13-acetate (PMA) inhibits Cl(-) secretion (short-circuit current, I(sc)) and decreases barrier function (transepithelial resistance, TER) in T84 epithelia. To elucidate the role of specific protein kinase C (PKC) isoenzymes in this response, we compared PMA with two non-phorbol activators of PKC (bryostatin-1 and carbachol) and utilized three PKC inhibitors (Gö-6850, Gö-6976, and rottlerin) with different isozyme selectivity profiles. PMA sequentially inhibited cAMP-stimulated I(sc) and decreased TER, as measured by voltage-current clamp. By subcellular fractionation and Western blot, PMA (100 nM) induced sequential membrane translocation of the novel PKC epsilon followed by the conventional PKC alpha and activated both isozymes by in vitro kinase assay. PKC delta was activated by PMA but did not translocate. By immunofluorescence, PKC epsilon redistributed to the basolateral domain in response to PMA, whereas PKC alpha moved apically. Inhibition of I(sc) by PMA was prevented by the conventional and novel PKC inhibitor Gö-6850 (5 microM) but not the conventional isoform inhibitor Gö-6976 (5 microM) or the PKC delta inhibitor rottlerin (10 microM), implicating PKC epsilon in inhibition of Cl(-) secretion. In contrast, both Gö-6976 and Gö-6850 prevented the decline of TER, suggesting involvement of PKC alpha. Bryostatin-1 (100 nM) translocated PKC epsilon and PKC alpha and inhibited cAMP-elicited I(sc). However, unlike PMA, bryostatin-1 downregulated PKC alpha protein, and the decrease in TER was only transient. Carbachol (100 microM) translocated only PKC epsilon and inhibited I(sc) with no effect on TER. Gö-6850 but not Gö-6976 or rottlerin blocked bryostatin-1 and carbachol inhibition of I(sc). We conclude that basolateral translocation of PKC epsilon inhibits Cl(-) secretion, while apical translocation of PKC alpha decreases TER. These data suggest that epithelial transport and barrier function can be modulated by distinct PKC isoforms.

Phosphorylation of claudin-4 by PKCε regulates tight junction barrier function in ovarian cancer cells

“…Significantly, the relocalization of PKCε to the membrane led to a colocalization with claudin-4, a finding consistent with claudin-4 being a target of this kinase. Translocation of activated PKCε to the membrane upon TPA treatment has been shown in other studies [42,55] and alteration of TJs by translocation of PKCε has been shown previously in epithelial cells [42]. Various isoforms of PKC have been implicated in ovarian tumorigenesis and drug resistance [56][57][58][59][60][61][62].…”

“…Interestingly, several studies have demonstrated the involvement of various kinases in the phosphorylation and regulation of claudin proteins [29][30][31][32][33][34][35][36][37], and we have recently shown that phosphorylation of claudin-3 by PKA can affect TJ properties in ovarian cancer cells [38]. Protein kinase C (PKC) isoforms are present in ovarian cancer and are known to modulate TJ function by phosphorylation of the proteins in the complex [24,34,[39][40][41][42][43], but it is unclear whether PKC can directly phosphorylate and regulate claudin proteins. We have previously shown that claudin-4 can be phosphorylated in ovarian cancer cells upon 12-OTetradecanoylophorbol-13-acetate (TPA) stimulation [38], but the exact PKC isoforms involved, the phosphorylation sites on claudin-4, and the consequences of this phosphorylation have remained unknown.…”

Phosphorylation of Claudin-3 at Threonine 192 by cAMP-dependent Protein Kinase Regulates Tight Junction Barrier Function in Ovarian Cancer Cells

“…These data are consistent with our finding that claudin-3 can be phosphorylated in vivo and the observation that this phosphorylation interferes with TJ function. Studies have implicated protein kinase C in the regulation of TJs through phorbol ester stimulation (17,34), but PKA-dependent regulation of TJs has been poorly documented and is more controversial (20). PKA activation has been suggested to have a role in TJ disruption (32), and claudin-3 phosphorylation may represent one of the targets to achieve this physiological outcome.…”

Polymorphonuclear Cell Transmigration Induced by Pseudomonas aeruginosa Requires the Eicosanoid Hepoxilin A3

“…A cell fractionation protocol was used to obtain membrane-soluble proteins, as PKC accumulates in the membrane upon activation (24,25). A549 monolayers seeded on 1.…”

“…Lysates were normalized for protein concentration and run on an 8 -16% gradient polyacrylamide gel followed by transference to nitrocellulose. Blots were probed with an anti-phospho-pan PKC Ab (Cell Signaling) followed by incubation with HRP-conjugated goat anti-rabbit Ab and detection with ECL reagent (Pierce/Endogen) (24,25).…”