Abstract
The turnover of connexin 43 (Cx43) is very rapid in many cells and involves both the lysosomal and proteasomal protease pathways. Here we show that Ca(2+)-mobilizing agonists such as angiotensin II (Ang II) can up-regulate the expression of Cx43 in WB rat liver epithelial cells. Vasopressin had the same effect in A7R5 smooth-muscle cells. The effect of Ang II was not prevented by pretreatment with proteasomal or lysosomal inhibitors and was associated with an enhanced biosynthesis of Cx43 as measured by metabolic labelling experiments. The accumulation of Cx43 occurred in intracellular compartments and at the cell surface, as determined by confocal immunofluorescence studies and by immunoblotting of fractions soluble and insoluble in Triton X-100. Chronic treatment of WB cells with ethanol inhibited Cx43 expression; this was associated with decreased biosynthesis of Cx43. Neither treatment with Ang II nor treatment with ethanol altered the levels of Cx43 mRNA. Incubation of WB cells with Ang II did not alter gap-junctional communication as judged by a dye-coupling assay. However, treatment with ethanol markedly decreased gap-junctional communication and this effect was diminished in Ang-II-treated cells, demonstrating that gap-junctional communication is linked to the level of Cx43 expression. We conclude that Cx43 biosynthesis is regulated by Ca(2+)-mobilizing agonists and ethanol in WB cells. The changes in Cx43 expression might have a role in modifying the conduction of metabolites and second messengers between cells.
Full Text
The Full Text of this article is available as a PDF (306.4 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Abou Hashieh I., Mathieu S., Besson F., Gerolami A. Inhibition of gap junction intercellular communications of cultured rat hepatocytes by ethanol: role of ethanol metabolism. J Hepatol. 1996 Mar;24(3):360–367. doi: 10.1016/s0168-8278(96)80017-1. [DOI] [PubMed] [Google Scholar]
- Banoub R. W., Fernstrom M., Malkinson A. M., Ruch R. J. Enhancement of gap junctional intercellular communication by dibutyryl cyclic AMP in lung epithelial cells. Anticancer Res. 1996 Nov-Dec;16(6B):3715–3719. [PubMed] [Google Scholar]
- Bex V., Mercier T., Chaumontet C., Gaillard-Sanchez I., Flechon B., Mazet F., Traub O., Martel P. Retinoic acid enhances connexin43 expression at the post-transcriptional level in rat liver epithelial cells. Cell Biochem Funct. 1995 Mar;13(1):69–77. doi: 10.1002/cbf.290130112. [DOI] [PubMed] [Google Scholar]
- Beyer E. C., Kistler J., Paul D. L., Goodenough D. A. Antisera directed against connexin43 peptides react with a 43-kD protein localized to gap junctions in myocardium and other tissues. J Cell Biol. 1989 Feb;108(2):595–605. doi: 10.1083/jcb.108.2.595. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Bokkala S., Joseph S. K. Angiotensin II-induced down-regulation of inositol trisphosphate receptors in WB rat liver epithelial cells. Evidence for involvement of the proteasome pathway. J Biol Chem. 1997 May 9;272(19):12454–12461. doi: 10.1074/jbc.272.19.12454. [DOI] [PubMed] [Google Scholar]
- Bokkala S., Rubin E., Joseph S. K. Effect of chronic ethanol exposure on inositol trisphosphate receptors in WB rat liver epithelial cells. Alcohol Clin Exp Res. 1999 Dec;23(12):1875–1883. [PubMed] [Google Scholar]
- Bukauskas F. F., Jordan K., Bukauskiene A., Bennett M. V., Lampe P. D., Laird D. W., Verselis V. K. Clustering of connexin 43-enhanced green fluorescent protein gap junction channels and functional coupling in living cells. Proc Natl Acad Sci U S A. 2000 Mar 14;97(6):2556–2561. doi: 10.1073/pnas.050588497. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chanson M., Bruzzone R., Bosco D., Meda P. Effects of n-alcohols on junctional coupling and amylase secretion of pancreatic acinar cells. J Cell Physiol. 1989 Apr;139(1):147–156. doi: 10.1002/jcp.1041390121. [DOI] [PubMed] [Google Scholar]
- Christ G. J., Spray D. C., el-Sabban M., Moore L. K., Brink P. R. Gap junctions in vascular tissues. Evaluating the role of intercellular communication in the modulation of vasomotor tone. Circ Res. 1996 Oct;79(4):631–646. doi: 10.1161/01.res.79.4.631. [DOI] [PubMed] [Google Scholar]
- Civitelli R., Ziambaras K., Warlow P. M., Lecanda F., Nelson T., Harley J., Atal N., Beyer E. C., Steinberg T. H. Regulation of connexin43 expression and function by prostaglandin E2 (PGE2) and parathyroid hormone (PTH) in osteoblastic cells. J Cell Biochem. 1998 Jan 1;68(1):8–21. doi: 10.1002/(sici)1097-4644(19980101)68:1<8::aid-jcb2>3.0.co;2-#. [DOI] [PubMed] [Google Scholar]
- Darrow B. J., Fast V. G., Kléber A. G., Beyer E. C., Saffitz J. E. Functional and structural assessment of intercellular communication. Increased conduction velocity and enhanced connexin expression in dibutyryl cAMP-treated cultured cardiac myocytes. Circ Res. 1996 Aug;79(2):174–183. doi: 10.1161/01.res.79.2.174. [DOI] [PubMed] [Google Scholar]
- Dodge S. M., Beardslee M. A., Darrow B. J., Green K. G., Beyer E. C., Saffitz J. E. Effects of angiotensin II on expression of the gap junction channel protein connexin43 in neonatal rat ventricular myocytes. J Am Coll Cardiol. 1998 Sep;32(3):800–807. doi: 10.1016/s0735-1097(98)00317-9. [DOI] [PubMed] [Google Scholar]
- Dupont G., Tordjmann T., Clair C., Swillens S., Claret M., Combettes L. Mechanism of receptor-oriented intercellular calcium wave propagation in hepatocytes. FASEB J. 2000 Feb;14(2):279–289. doi: 10.1096/fasebj.14.2.279. [DOI] [PubMed] [Google Scholar]
- Fernandez-Cobo M., Gingalewski C., Drujan D., De Maio A. Downregulation of connexin 43 gene expression in rat heart during inflammation. The role of tumour necrosis factor. Cytokine. 1999 Mar;11(3):216–224. doi: 10.1006/cyto.1998.0422. [DOI] [PubMed] [Google Scholar]
- Guan X., Ruch R. J. Gap junction endocytosis and lysosomal degradation of connexin43-P2 in WB-F344 rat liver epithelial cells treated with DDT and lindane. Carcinogenesis. 1996 Sep;17(9):1791–1798. doi: 10.1093/carcin/17.9.1791. [DOI] [PubMed] [Google Scholar]
- Hashieh I. A., Mathieu S., Gerolami A. Effects of ethanol on intercellular communications and polarization of hepatocytes in short-term culture. Hepatology. 1992 May;15(5):751–756. doi: 10.1002/hep.1840150502. [DOI] [PubMed] [Google Scholar]
- Hess B. Periodic patterns in biology. Naturwissenschaften. 2000 May;87(5):199–211. doi: 10.1007/s001140050704. [DOI] [PubMed] [Google Scholar]
- Hill C. S., Oh S. Y., Schmidt S. A., Clark K. J., Murray A. W. Lysophosphatidic acid inhibits gap-junctional communication and stimulates phosphorylation of connexin-43 in WB cells: possible involvement of the mitogen-activated protein kinase cascade. Biochem J. 1994 Oct 15;303(Pt 2):475–479. doi: 10.1042/bj3030475. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Höfer T. Model of intercellular calcium oscillations in hepatocytes: synchronization of heterogeneous cells. Biophys J. 1999 Sep;77(3):1244–1256. doi: 10.1016/S0006-3495(99)76976-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Johnston M. F., Simon S. A., Ramón F. Interaction of anaesthetics with electrical synapses. Nature. 1980 Jul 31;286(5772):498–500. doi: 10.1038/286498a0. [DOI] [PubMed] [Google Scholar]
- Kumar N. M., Gilula N. B. The gap junction communication channel. Cell. 1996 Feb 9;84(3):381–388. doi: 10.1016/s0092-8674(00)81282-9. [DOI] [PubMed] [Google Scholar]
- Laing J. G., Beyer E. C. The gap junction protein connexin43 is degraded via the ubiquitin proteasome pathway. J Biol Chem. 1995 Nov 3;270(44):26399–26403. doi: 10.1074/jbc.270.44.26399. [DOI] [PubMed] [Google Scholar]
- Laing J. G., Tadros P. N., Westphale E. M., Beyer E. C. Degradation of connexin43 gap junctions involves both the proteasome and the lysosome. Exp Cell Res. 1997 Nov 1;236(2):482–492. doi: 10.1006/excr.1997.3747. [DOI] [PubMed] [Google Scholar]
- Laird D. W. The life cycle of a connexin: gap junction formation, removal, and degradation. J Bioenerg Biomembr. 1996 Aug;28(4):311–318. doi: 10.1007/BF02110107. [DOI] [PubMed] [Google Scholar]
- Leybaert L., Paemeleire K., Strahonja A., Sanderson M. J. Inositol-trisphosphate-dependent intercellular calcium signaling in and between astrocytes and endothelial cells. Glia. 1998 Dec;24(4):398–407. [PubMed] [Google Scholar]
- Madhukar B. V., de Feijter-Rupp H. L., Trosko J. E. Pulse treatment with the tumor promoter TPA delays the onset of desensitization response and prolongs the inhibitory effect on gap junctional intercellular communication of a rat liver epithelial cell line WB F-344. Cancer Lett. 1996 Aug 23;106(1):117–123. doi: 10.1016/0304-3835(96)04315-7. [DOI] [PubMed] [Google Scholar]
- Musil L. S., Goodenough D. A. Biochemical analysis of connexin43 intracellular transport, phosphorylation, and assembly into gap junctional plaques. J Cell Biol. 1991 Dec;115(5):1357–1374. doi: 10.1083/jcb.115.5.1357. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Niessen H., Harz H., Bedner P., Krämer K., Willecke K. Selective permeability of different connexin channels to the second messenger inositol 1,4,5-trisphosphate. J Cell Sci. 2000 Apr;113(Pt 8):1365–1372. doi: 10.1242/jcs.113.8.1365. [DOI] [PubMed] [Google Scholar]
- Oh S. Y., Dupont E., Madhukar B. V., Briand J. P., Chang C. C., Beyer E., Trosko J. E. Characterization of gap junctional communication-deficient mutants of a rat liver epithelial cell line. Eur J Cell Biol. 1993 Apr;60(2):250–255. [PubMed] [Google Scholar]
- 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]
- 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]
- 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]
- Paemeleire K., Martin P. E., Coleman S. L., Fogarty K. E., Carrington W. A., Leybaert L., Tuft R. A., Evans W. H., Sanderson M. J. Intercellular calcium waves in HeLa cells expressing GFP-labeled connexin 43, 32, or 26. Mol Biol Cell. 2000 May;11(5):1815–1827. doi: 10.1091/mbc.11.5.1815. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Peracchia C., Sotkis A., Wang X. G., Peracchia L. L., Persechini A. Calmodulin directly gates gap junction channels. J Biol Chem. 2000 Aug 25;275(34):26220–26224. doi: 10.1074/jbc.M004007200. [DOI] [PubMed] [Google Scholar]
- Rivedal E., Mollerup S., Haugen A., Vikhamar G. Modulation of gap junctional intercellular communication by EGF in human kidney epithelial cells. Carcinogenesis. 1996 Nov;17(11):2321–2328. doi: 10.1093/carcin/17.11.2321. [DOI] [PubMed] [Google Scholar]
- Rose B., Loewenstein W. R. Permeability of cell junction depends on local cytoplasmic calcium activity. Nature. 1975 Mar 20;254(5497):250–252. doi: 10.1038/254250a0. [DOI] [PubMed] [Google Scholar]
- Saffitz J. E., Laing J. G., Yamada K. A. Connexin expression and turnover : implications for cardiac excitability. Circ Res. 2000 Apr 14;86(7):723–728. doi: 10.1161/01.res.86.7.723. [DOI] [PubMed] [Google Scholar]
- Schiavi A., Hudder A., Werner R. Connexin43 mRNA contains a functional internal ribosome entry site. FEBS Lett. 1999 Dec 31;464(3):118–122. doi: 10.1016/s0014-5793(99)01699-3. [DOI] [PubMed] [Google Scholar]
- Sipma H., Deelman L., Smedt H. D., Missiaen L., Parys J. B., Vanlingen S., Henning R. H., Casteels R. Agonist-induced down-regulation of type 1 and type 3 inositol 1,4,5-trisphosphate receptors in A7r5 and DDT1 MF-2 smooth muscle cells. Cell Calcium. 1998 Jan;23(1):11–21. doi: 10.1016/s0143-4160(98)90070-7. [DOI] [PubMed] [Google Scholar]
- Sneyd J., Keizer J., Sanderson M. J. Mechanisms of calcium oscillations and waves: a quantitative analysis. FASEB J. 1995 Nov;9(14):1463–1472. doi: 10.1096/fasebj.9.14.7589988. [DOI] [PubMed] [Google Scholar]
- Spray D. C., Chanson M., Moreno A. P., Dermietzel R., Meda P. Distinctive gap junction channel types connect WB cells, a clonal cell line derived from rat liver. Am J Physiol. 1991 Mar;260(3 Pt 1):C513–C527. doi: 10.1152/ajpcell.1991.260.3.C513. [DOI] [PubMed] [Google Scholar]
- Spray D. C. Gap junction proteins: where they live and how they die. Circ Res. 1998 Sep 21;83(6):679–681. doi: 10.1161/01.res.83.6.679. [DOI] [PubMed] [Google Scholar]
- Spray D. C., Ginzberg R. D., Morales E. A., Gatmaitan Z., Arias I. M. Electrophysiological properties of gap junctions between dissociated pairs of rat hepatocytes. J Cell Biol. 1986 Jul;103(1):135–144. doi: 10.1083/jcb.103.1.135. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Stock A., Sies H., Stahl W. Enhancement of gap junctional communication and connexin43 expression by thyroid hormones. Biochem Pharmacol. 1998 Feb 15;55(4):475–479. doi: 10.1016/s0006-2952(97)00473-5. [DOI] [PubMed] [Google Scholar]
- Thomas A. P., Bird G. S., Hajnóczky G., Robb-Gaspers L. D., Putney J. W., Jr Spatial and temporal aspects of cellular calcium signaling. FASEB J. 1996 Nov;10(13):1505–1517. [PubMed] [Google Scholar]
- Wojcikiewicz R. J., Ernst S. A., Yule D. I. Secretagogues cause ubiquitination and down-regulation of inositol 1, 4,5-trisphosphate receptors in rat pancreatic acinar cells. Gastroenterology. 1999 May;116(5):1194–1201. doi: 10.1016/s0016-5085(99)70023-5. [DOI] [PubMed] [Google Scholar]
- Ziambaras K., Lecanda F., Steinberg T. H., Civitelli R. Cyclic stretch enhances gap junctional communication between osteoblastic cells. J Bone Miner Res. 1998 Feb;13(2):218–228. doi: 10.1359/jbmr.1998.13.2.218. [DOI] [PubMed] [Google Scholar]