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. 1994 Oct 15;303(Pt 2):475–479. doi: 10.1042/bj3030475

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

C S Hill 1, S Y Oh 1, S A Schmidt 1, K J Clark 1, A W Murray 1
PMCID: PMC1137352  PMID: 7980407

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.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Anderson N. G., Kilgour E., Sturgill T. W. Activation of mitogen-activated protein kinase in BC3H1 myocytes by fluoroaluminate. J Biol Chem. 1991 Jun 5;266(16):10131–10135. [PubMed] [Google Scholar]
  2. Davis R. J. The mitogen-activated protein kinase signal transduction pathway. J Biol Chem. 1993 Jul 15;268(20):14553–14556. [PubMed] [Google Scholar]
  3. Eichholtz T., Jalink K., Fahrenfort I., Moolenaar W. H. The bioactive phospholipid lysophosphatidic acid is released from activated platelets. Biochem J. 1993 May 1;291(Pt 3):677–680. doi: 10.1042/bj2910677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Harrington C. R. Lowry protein assay containing sodium dodecyl sulfate in microtiter plates for protein determinations on fractions from brain tissue. Anal Biochem. 1990 May 1;186(2):285–287. doi: 10.1016/0003-2697(90)90081-j. [DOI] [PubMed] [Google Scholar]
  5. Jaiswal R. K., Murphy M. B., Landreth G. E. Identification and characterization of a nerve growth factor-stimulated mitogen-activated protein kinase activator in PC12 cells. J Biol Chem. 1993 Apr 5;268(10):7055–7063. [PubMed] [Google Scholar]
  6. Kanemitsu M. Y., Lau A. F. Epidermal growth factor stimulates the disruption of gap junctional communication and connexin43 phosphorylation independent of 12-0-tetradecanoylphorbol 13-acetate-sensitive protein kinase C: the possible involvement of mitogen-activated protein kinase. Mol Biol Cell. 1993 Aug;4(8):837–848. doi: 10.1091/mbc.4.8.837. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Krabak M. J., Hui S. W. The mitogenic activities of phosphatidate are acyl-chain-length dependent and calcium independent in C3H/10T1/2 cells. Cell Regul. 1991 Jan;2(1):57–64. doi: 10.1091/mbc.2.1.57. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Kumagai N., Morii N., Fujisawa K., Yoshimasa T., Nakao K., Narumiya S. Lysophosphatidic acid induces tyrosine phosphorylation and activation of MAP-kinase and focal adhesion kinase in cultured Swiss 3T3 cells. FEBS Lett. 1993 Aug 30;329(3):273–276. doi: 10.1016/0014-5793(93)80236-n. [DOI] [PubMed] [Google Scholar]
  9. Lau A. F., Kanemitsu M. Y., Kurata W. E., Danesh S., Boynton A. L. Epidermal growth factor disrupts gap-junctional communication and induces phosphorylation of connexin43 on serine. Mol Biol Cell. 1992 Aug;3(8):865–874. doi: 10.1091/mbc.3.8.865. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Moolenaar W. H., Kruijer W., Tilly B. C., Verlaan I., Bierman A. J., de Laat S. W. Growth factor-like action of phosphatidic acid. Nature. 1986 Sep 11;323(6084):171–173. doi: 10.1038/323171a0. [DOI] [PubMed] [Google Scholar]
  11. Nishida E., Gotoh Y. The MAP kinase cascade is essential for diverse signal transduction pathways. Trends Biochem Sci. 1993 Apr;18(4):128–131. doi: 10.1016/0968-0004(93)90019-j. [DOI] [PubMed] [Google Scholar]
  12. Oh S. Y., Grupen C. G., Murray A. W. Phorbol ester induces phosphorylation and down-regulation of connexin 43 in WB cells. Biochim Biophys Acta. 1991 Sep 3;1094(2):243–245. doi: 10.1016/0167-4889(91)90016-q. [DOI] [PubMed] [Google Scholar]
  13. Oh S. Y., Madhukar B. V., Trosko J. E. Inhibition of gap junctional blockage by palmitoyl carnitine and TMB-8 in a rat liver epithelial cell line. Carcinogenesis. 1988 Jan;9(1):135–139. doi: 10.1093/carcin/9.1.135. [DOI] [PubMed] [Google Scholar]
  14. Oh S. Y., Schmidt S. A., Murray A. W. Epidermal growth factor inhibits gap junctional communication and stimulates serine-phosphorylation of connexin43 in WB cells by a protein kinase C-independent mechanism. Cell Adhes Commun. 1993 Sep;1(2):143–149. doi: 10.3109/15419069309095690. [DOI] [PubMed] [Google Scholar]
  15. Pelech S. L., Sanghera J. S. MAP kinases: charting the regulatory pathways. Science. 1992 Sep 4;257(5075):1355–1356. doi: 10.1126/science.1382311. [DOI] [PubMed] [Google Scholar]
  16. Ray L. B., Sturgill T. W. Characterization of insulin-stimulated microtubule-associated protein kinase. Rapid isolation and stabilization of a novel serine/threonine kinase from 3T3-L1 cells. J Biol Chem. 1988 Sep 5;263(25):12721–12727. [PubMed] [Google Scholar]
  17. Ridley A. J., Paterson H. F., Johnston C. L., Diekmann D., Hall A. The small GTP-binding protein rac regulates growth factor-induced membrane ruffling. Cell. 1992 Aug 7;70(3):401–410. doi: 10.1016/0092-8674(92)90164-8. [DOI] [PubMed] [Google Scholar]
  18. Samiei M., Sanghera J. S., Pelech S. L. Activation of myelin basic protein and S6 peptide kinases in phorbol ester- and PAF-treated sheep platelets. Biochim Biophys Acta. 1993 Apr 16;1176(3):287–298. doi: 10.1016/0167-4889(93)90057-v. [DOI] [PubMed] [Google Scholar]
  19. Sáez J. C., Nairn A. C., Czernik A. J., Spray D. C., Hertzberg E. L., Greengard P., Bennett M. V. Phosphorylation of connexin 32, a hepatocyte gap-junction protein, by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. Eur J Biochem. 1990 Sep 11;192(2):263–273. doi: 10.1111/j.1432-1033.1990.tb19223.x. [DOI] [PubMed] [Google Scholar]
  20. Tsao M. S., Smith J. D., Nelson K. G., Grisham J. W. A diploid epithelial cell line from normal adult rat liver with phenotypic properties of 'oval' cells. Exp Cell Res. 1984 Sep;154(1):38–52. doi: 10.1016/0014-4827(84)90666-9. [DOI] [PubMed] [Google Scholar]
  21. Yu C. L., Tsai M. H., Stacey D. W. Cellular ras activity and phospholipid metabolism. Cell. 1988 Jan 15;52(1):63–71. doi: 10.1016/0092-8674(88)90531-4. [DOI] [PubMed] [Google Scholar]
  22. el Aoumari A., Fromaget C., Dupont E., Reggio H., Durbec P., Briand J. P., Böller K., Kreitman B., Gros D. Conservation of a cytoplasmic carboxy-terminal domain of connexin 43, a gap junctional protein, in mammal heart and brain. J Membr Biol. 1990 May;115(3):229–240. doi: 10.1007/BF01868638. [DOI] [PubMed] [Google Scholar]
  23. van Corven E. J., Groenink A., Jalink K., Eichholtz T., Moolenaar W. H. Lysophosphatidate-induced cell proliferation: identification and dissection of signaling pathways mediated by G proteins. Cell. 1989 Oct 6;59(1):45–54. doi: 10.1016/0092-8674(89)90868-4. [DOI] [PubMed] [Google Scholar]
  24. van Corven E. J., Hordijk P. L., Medema R. H., Bos J. L., Moolenaar W. H. Pertussis toxin-sensitive activation of p21ras by G protein-coupled receptor agonists in fibroblasts. Proc Natl Acad Sci U S A. 1993 Feb 15;90(4):1257–1261. doi: 10.1073/pnas.90.4.1257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. van Corven E. J., van Rijswijk A., Jalink K., van der Bend R. L., van Blitterswijk W. J., Moolenaar W. H. Mitogenic action of lysophosphatidic acid and phosphatidic acid on fibroblasts. Dependence on acyl-chain length and inhibition by suramin. Biochem J. 1992 Jan 1;281(Pt 1):163–169. doi: 10.1042/bj2810163. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. van der Bend R. L., Brunner J., Jalink K., van Corven E. J., Moolenaar W. H., van Blitterswijk W. J. Identification of a putative membrane receptor for the bioactive phospholipid, lysophosphatidic acid. EMBO J. 1992 Jul;11(7):2495–2501. doi: 10.1002/j.1460-2075.1992.tb05314.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. van der Bend R. L., de Widt J., van Corven E. J., Moolenaar W. H., van Blitterswijk W. J. The biologically active phospholipid, lysophosphatidic acid, induces phosphatidylcholine breakdown in fibroblasts via activation of phospholipase D. Comparison with the response to endothelin. Biochem J. 1992 Jul 1;285(Pt 1):235–240. doi: 10.1042/bj2850235. [DOI] [PMC free article] [PubMed] [Google Scholar]

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