Lysophosphatidic acid inhibits gap-junctional communication and stimulates phosphorylation of connexin-43 in WB cells: possible involvement of the mitogen-activated protein kinase cascade

Abstract

Lysophosphatidic acid (LPA) was shown to be a powerful inhibitor of gap-junctional communication between cultured rat liver WB cells, as determined by the transfer of Lucifer Yellow, with 50% inhibition obtained at about 0.3 microM LPA. Inhibition of communication was rapid (5 min) and was maintained for at least 80 min. After incubation for 3 h with LPA, communication competence was partially restored and dye transfer was refractory to further addition of LPA. Communication in LPA-refractory cells retained sensitivity to inhibition by phorbol ester and by epidermal growth factor (EGF). LPA-induced inhibition was associated with phosphorylation of connexin-43 protein, as detected by slower migration of the protein detected on Western blots, which could be eliminated by incubation of samples with alkaline phosphatase. A close correspondence was observed between the time- and dose-dependency of LPA effects on communication and the induction of mitogen-activated protein kinase (MAP kinase). Activation of both the 42 kDa and 44 kDa subspecies were confirmed by mobility shifts on Western blots using an anti-(MAP kinase R1) (erk 1-III) antibody and by fractionation on Mono Q columns. Cells pretreated with phorbol ester for 24 h were insensitive to phorbol ester inhibition of communication or activation of MAP kinase, but retained their sensitivity to LPA. The results indicate that LPA initiates the activation of protein kinase cascades in WB cells that are probably independent of protein kinase C and identifies connexin-43 as one substrate for the activated kinases.

Inhibition of Endothelial Cell Migration, Intercellular Communication, and Vascular Tube Formation by Thromboxane A2

“…Lysophosphatidic acid, thrombin, and endothelin (ET) all inhibit junctional communication in various cells (40,41). With lysophosphatidic acid, ET, and thrombin, however, maximal inhibition occurs within 5-10 min of ligand binding with the restoration of junctional function at 3 h mediated through receptor desensitization (40,41). In those studies, gap junction closure by lysophosphatidic acid, thrombin, and ET was not accompanied by changes to the morphology of junctional plaques.…”

“…Indeed, endothelial cell migration and angiogenesis are sensitive to inhibition of gap junction-mediated intercellular communication, and antiangiogenic cytokines, such as interleukin-1␤, potently inhibit intercellular communication in endothelium (37)(38)(39). Inhibition of junctional communication alone is not sufficient to explain the loss of EC function, however, since many proangiogenic G-proteinlinked receptor ligands also decrease junctional communication (40,41). The difference between the effects of TXA 2 and other G-protein-linked receptor ligands may be related to the kinetics of the inhibition and the mode of action.…”

“…In those studies, gap junction closure by lysophosphatidic acid, thrombin, and ET was not accompanied by changes to the morphology of junctional plaques. Rather, the loss of intercellular communication was associated with changes in the phosphorylation state of Cx43, mediated by either c-Src or MAP kinase activation (40,41).…”

“…The difference between the effects of TXA 2 and other G-protein-linked receptor ligands may be related to the kinetics of the inhibition and the mode of action. Lysophosphatidic acid, thrombin, and endothelin (ET) all inhibit junctional communication in various cells (40,41). With lysophosphatidic acid, ET, and thrombin, however, maximal inhibition occurs within 5-10 min of ligand binding with the restoration of junctional function at 3 h mediated through receptor desensitization (40,41).…”

Chloral hydrate decreases gap junction communication in rat liver epithelial cells

“…Although the cytoplasmic, carboxyl-terminal domain of Cx 43 contains consensus phosphorylation sequences for protein kinases , neither the kinases nor the phosphorylated amino acid residues responsible for closing or opening the Cx 43 gap junction channel have been fully characterized. However, given that GJC disruption is correlated with phosphorylation of Cx 43 at Ser/Thr (serine/ threonine) residues by TPA, epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and LPA (lysophosphatidic acid; Hossain et al 1998;Oh et al 1991;Kanemitsu and Lau 1993;Lau et al 1992;Hill et al 1994), it appears that phosphorylation at these sites may play a key role in regulating GJC and gap junction turnover.…”