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. 1996 Dec 15;98(12):2771–2778. doi: 10.1172/JCI119103

Growth inhibitory properties of endothelin-1 in activated human hepatic stellate cells: a cyclic adenosine monophosphate-mediated pathway. Inhibition of both extracellular signal-regulated kinase and c-Jun kinase and upregulation of endothelin B receptors.

A Mallat 1, A M Préaux 1, C Serradeil-Le Gal 1, D Raufaste 1, C Gallois 1, D A Brenner 1, C Bradham 1, J Maclouf 1, V Iourgenko 1, L Fouassier 1, D Dhumeaux 1, P Mavier 1, S Lotersztajn 1
PMCID: PMC507742  PMID: 8981923

Abstract

During chronic liver diseases, hepatic stellate cells (HSC) acquire an activated myofibroblast-like phenotype, proliferate, and synthetize fibrosis components. We have shown that endothelin-1 (ET-1) inhibits the proliferation of activated human HSC via endothelin B (ETB) receptors. We now investigate the transduction pathway involved in the growth inhibitory effect of ET-1 in activated HSC. Endothelin-1 and the ETB receptor agonist, sarafotoxin-S6C, increased synthesis of PGI2 and PGE2, leading to elevation of cAMP. The cyclooxygenase inhibitor ibuprofen and the adenylyl cyclase inhibitor SQ22536 both blunted the growth inhibitory effect of ET-1. Analysis of early steps associated with growth inhibition indicated that: (a) similar to ET-1, forskolin decreased c-jun mRNA induction without affecting c-fos and krox 24 mRNA expression; (b) ET-1, sarafotoxin-S6C, as well as forskolin, reduced activation of both c-Jun kinase and extracellular signal-regulated kinase. Finally, forskolin, PGI2, and PGE2 raised by fivefold the number of ET binding sites after 6 h, and increased the proportion of ETB receptors from 50% in control cells to 80% in treated cells. In conclusion, ET-1 inhibits proliferation of activated HSC via ETB receptors, through a prostaglandin/cAMP pathway that leads to inhibition of both extracellular signal-regulated kinase and c-Jun kinase activities. Upregulation of ETB receptors by prostaglandin/cAMP raises the possibility of a positive feedback loop that would amplify the growth inhibitory response. These results suggest that ET-1 and agents that increase cAMP might be of interest to limit proliferation of activated HSC during chronic liver diseases.

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Selected References

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  1. Battistini B., Chailler P., D'Orléans-Juste P., Brière N., Sirois P. Growth regulatory properties of endothelins. Peptides. 1993 Mar-Apr;14(2):385–399. doi: 10.1016/0196-9781(93)90057-n. [DOI] [PubMed] [Google Scholar]
  2. Beno D. W., Rapp U. R., Davis B. H. Prostaglandin E suppression of platelet-derived-growth-factor-induced Ito cell mitogenesis occurs independent of raf perinuclear translocation and nuclear proto-oncogene expression. Biochim Biophys Acta. 1994 Jun 30;1222(2):292–300. doi: 10.1016/0167-4889(94)90181-3. [DOI] [PubMed] [Google Scholar]
  3. Bird T. A., Kyriakis J. M., Tyshler L., Gayle M., Milne A., Virca G. D. Interleukin-1 activates p54 mitogen-activated protein (MAP) kinase/stress-activated protein kinase by a pathway that is independent of p21ras, Raf-1, and MAP kinase kinase. J Biol Chem. 1994 Dec 16;269(50):31836–31844. [PubMed] [Google Scholar]
  4. Blumer K. J., Johnson G. L. Diversity in function and regulation of MAP kinase pathways. Trends Biochem Sci. 1994 Jun;19(6):236–240. doi: 10.1016/0968-0004(94)90147-3. [DOI] [PubMed] [Google Scholar]
  5. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  6. Burgering B. M., Bos J. L. Regulation of Ras-mediated signalling: more than one way to skin a cat. Trends Biochem Sci. 1995 Jan;20(1):18–22. doi: 10.1016/s0968-0004(00)88944-6. [DOI] [PubMed] [Google Scholar]
  7. Cano E., Mahadevan L. C. Parallel signal processing among mammalian MAPKs. Trends Biochem Sci. 1995 Mar;20(3):117–122. doi: 10.1016/s0968-0004(00)88978-1. [DOI] [PubMed] [Google Scholar]
  8. Chiu R., Angel P., Karin M. Jun-B differs in its biological properties from, and is a negative regulator of, c-Jun. Cell. 1989 Dec 22;59(6):979–986. doi: 10.1016/0092-8674(89)90754-x. [DOI] [PubMed] [Google Scholar]
  9. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  10. Davis R. J. MAPKs: new JNK expands the group. Trends Biochem Sci. 1994 Nov;19(11):470–473. doi: 10.1016/0968-0004(94)90132-5. [DOI] [PubMed] [Google Scholar]
  11. Diehl A. M., Yin M., Fleckenstein J., Yang S. Q., Lin H. Z., Brenner D. A., Westwick J., Bagby G., Nelson S. Tumor necrosis factor-alpha induces c-jun during the regenerative response to liver injury. Am J Physiol. 1994 Oct;267(4 Pt 1):G552–G561. doi: 10.1152/ajpgi.1994.267.4.G552. [DOI] [PubMed] [Google Scholar]
  12. Domae M., Yamada K., Inoue T., Satoh M., Furukawa T. Endothelins stimulate cyclic AMP accumulation in the isolated rat anterior pituitary gland: possible involvement of ETA receptor activation and prostaglandin E2 production. J Pharmacol Exp Ther. 1994 Jul;270(1):55–60. [PubMed] [Google Scholar]
  13. Eguchi S., Hirata Y., Imai T., Marumo F. Endothelin receptor subtypes are coupled to adenylate cyclase via different guanyl nucleotide-binding proteins in vasculature. Endocrinology. 1993 Feb;132(2):524–529. doi: 10.1210/endo.132.2.7678793. [DOI] [PubMed] [Google Scholar]
  14. Friedman S. L. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N Engl J Med. 1993 Jun 24;328(25):1828–1835. doi: 10.1056/NEJM199306243282508. [DOI] [PubMed] [Google Scholar]
  15. Gandhi C. R., Stephenson K., Olson M. S. A comparative study of endothelin- and platelet-activating-factor-mediated signal transduction and prostaglandin synthesis in rat Kupffer cells. Biochem J. 1992 Jan 15;281(Pt 2):485–492. doi: 10.1042/bj2810485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Gandhi C. R., Stephenson K., Olson M. S. Endothelin, a potent peptide agonist in the liver. J Biol Chem. 1990 Oct 15;265(29):17432–17435. [PubMed] [Google Scholar]
  17. Geerts A., Lazou J. M., De Bleser P., Wisse E. Tissue distribution, quantitation and proliferation kinetics of fat-storing cells in carbon tetrachloride-injured rat liver. Hepatology. 1991 Jun;13(6):1193–1202. [PubMed] [Google Scholar]
  18. Haraguchi S., Good R. A., James-Yarish M., Cianciolo G. J., Day N. K. Induction of intracellular cAMP by a synthetic retroviral envelope peptide: a possible mechanism of immunopathogenesis in retroviral infections. Proc Natl Acad Sci U S A. 1995 Jun 6;92(12):5568–5571. doi: 10.1073/pnas.92.12.5568. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Heasley L. E., Zamarripa J., Storey B., Helfrich B., Mitchell F. M., Bunn P. A., Jr, Johnson G. L. Discordant signal transduction and growth inhibition of small cell lung carcinomas induced by expression of GTPase-deficient G alpha 16. J Biol Chem. 1996 Jan 5;271(1):349–354. doi: 10.1074/jbc.271.1.349. [DOI] [PubMed] [Google Scholar]
  20. Hibi M., Lin A., Smeal T., Minden A., Karin M. Identification of an oncoprotein- and UV-responsive protein kinase that binds and potentiates the c-Jun activation domain. Genes Dev. 1993 Nov;7(11):2135–2148. doi: 10.1101/gad.7.11.2135. [DOI] [PubMed] [Google Scholar]
  21. Housset C., Rockey D. C., Bissell D. M. Endothelin receptors in rat liver: lipocytes as a contractile target for endothelin 1. Proc Natl Acad Sci U S A. 1993 Oct 15;90(20):9266–9270. doi: 10.1073/pnas.90.20.9266. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Hsueh Y. P., Lai M. Z. c-Jun N-terminal kinase but not mitogen-activated protein kinase is sensitive to cAMP inhibition in T lymphocytes. J Biol Chem. 1995 Jul 28;270(30):18094–18098. doi: 10.1074/jbc.270.30.18094. [DOI] [PubMed] [Google Scholar]
  23. Jouneaux C., Audigier Y., Goldsmith P., Pecker F., Lotersztajn S. Gs mediates hormonal inhibition of the calcium pump in liver plasma membranes. J Biol Chem. 1993 Feb 5;268(4):2368–2372. [PubMed] [Google Scholar]
  24. Jouneaux C., Mallat A., Serradeil-Le Gal C., Goldsmith P., Hanoune J., Lotersztajn S. Coupling of endothelin B receptors to the calcium pump and phospholipase C via Gs and Gq in rat liver. J Biol Chem. 1994 Jan 21;269(3):1845–1851. [PubMed] [Google Scholar]
  25. Karin M. The regulation of AP-1 activity by mitogen-activated protein kinases. J Biol Chem. 1995 Jul 14;270(28):16483–16486. doi: 10.1074/jbc.270.28.16483. [DOI] [PubMed] [Google Scholar]
  26. Karne S., Jayawickreme C. K., Lerner M. R. Cloning and characterization of an endothelin-3 specific receptor (ETC receptor) from Xenopus laevis dermal melanophores. J Biol Chem. 1993 Sep 5;268(25):19126–19133. [PubMed] [Google Scholar]
  27. Kyriakis J. M., Banerjee P., Nikolakaki E., Dai T., Rubie E. A., Ahmad M. F., Avruch J., Woodgett J. R. The stress-activated protein kinase subfamily of c-Jun kinases. Nature. 1994 May 12;369(6476):156–160. doi: 10.1038/369156a0. [DOI] [PubMed] [Google Scholar]
  28. Malarkey K., Belham C. M., Paul A., Graham A., McLees A., Scott P. H., Plevin R. The regulation of tyrosine kinase signalling pathways by growth factor and G-protein-coupled receptors. Biochem J. 1995 Jul 15;309(Pt 2):361–375. doi: 10.1042/bj3090361. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Mallat A., Fouassier L., Préaux A. M., Gal C. S., Raufaste D., Rosenbaum J., Dhumeaux D., Jouneaux C., Mavier P., Lotersztajn S. Growth inhibitory properties of endothelin-1 in human hepatic myofibroblastic Ito cells. An endothelin B receptor-mediated pathway. J Clin Invest. 1995 Jul;96(1):42–49. doi: 10.1172/JCI118052. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Mallat A., Lotersztajn S. Multiple hepatic functions of endothelin-1: physiopathological relevance. J Hepatol. 1996 Sep;25(3):405–413. doi: 10.1016/s0168-8278(96)80130-9. [DOI] [PubMed] [Google Scholar]
  31. Mallat A., Preaux A. M., Blazejewski S., Dhumeaux D., Rosenbaum J., Mavier P. Effect of simvastatin, an inhibitor of hydroxy-methylglutaryl coenzyme A reductase, on the growth of human Ito cells. Hepatology. 1994 Dec;20(6):1589–1594. doi: 10.1002/hep.1840200631. [DOI] [PubMed] [Google Scholar]
  32. Mallat A., Preaux A. M., Blazejewski S., Rosenbaum J., Dhumeaux D., Mavier P. Interferon alfa and gamma inhibit proliferation and collagen synthesis of human Ito cells in culture. Hepatology. 1995 Apr;21(4):1003–1010. [PubMed] [Google Scholar]
  33. Mancini R., Jezequel A. M., Benedetti A., Paolucci F., Trozzi L., Orlandi F. Quantitative analysis of proliferating sinusoidal cells in dimethylnitrosamine-induced cirrhosis. An immunohistochemical study. J Hepatol. 1992 Jul;15(3):361–366. doi: 10.1016/0168-8278(92)90069-2. [DOI] [PubMed] [Google Scholar]
  34. Markewitz B. A., Kohan D. E., Michael J. R. Endothelin-1 synthesis, receptors, and signal transduction in alveolar epithelium: evidence for an autocrine role. Am J Physiol. 1995 Feb;268(2 Pt 1):L192–L200. doi: 10.1152/ajplung.1995.268.2.L192. [DOI] [PubMed] [Google Scholar]
  35. Masaki T., Yanagisawa M., Goto K. Physiology and pharmacology of endothelins. Med Res Rev. 1992 Jul;12(4):391–421. doi: 10.1002/med.2610120405. [DOI] [PubMed] [Google Scholar]
  36. Minden A., Lin A., McMahon M., Lange-Carter C., Dérijard B., Davis R. J., Johnson G. L., Karin M. Differential activation of ERK and JNK mitogen-activated protein kinases by Raf-1 and MEKK. Science. 1994 Dec 9;266(5191):1719–1723. doi: 10.1126/science.7992057. [DOI] [PubMed] [Google Scholar]
  37. Mitchell F. M., Russell M., Johnson G. L. Differential calcium dependence in the activation of c-Jun kinase and mitogen-activated protein kinase by muscarinic acetylcholine receptors in rat 1a cells. Biochem J. 1995 Jul 15;309(Pt 2):381–384. doi: 10.1042/bj3090381. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Negishi M., Sugimoto Y., Irie A., Narumiya S., Ichikawa A. Two isoforms of prostaglandin E receptor EP3 subtype. Different COOH-terminal domains determine sensitivity to agonist-induced desensitization. J Biol Chem. 1993 May 5;268(13):9517–9521. [PubMed] [Google Scholar]
  39. Nishimura J., Chen X., Jahan H., Shikasho T., Kobayashi S., Kanaide H. cAMP induces up-regulation of ETA receptor mRNA and increases responsiveness to endothelin-1 of rat aortic smooth muscle cells in primary culture. Biochem Biophys Res Commun. 1992 Oct 30;188(2):719–726. doi: 10.1016/0006-291x(92)91115-7. [DOI] [PubMed] [Google Scholar]
  40. Pagès G., Lenormand P., L'Allemain G., Chambard J. C., Meloche S., Pouysségur J. Mitogen-activated protein kinases p42mapk and p44mapk are required for fibroblast proliferation. Proc Natl Acad Sci U S A. 1993 Sep 15;90(18):8319–8323. doi: 10.1073/pnas.90.18.8319. [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Peterson G. L. A simplification of the protein assay method of Lowry et al. which is more generally applicable. Anal Biochem. 1977 Dec;83(2):346–356. doi: 10.1016/0003-2697(77)90043-4. [DOI] [PubMed] [Google Scholar]
  42. Pinzani M., Failli P., Ruocco C., Casini A., Milani S., Baldi E., Giotti A., Gentilini P. Fat-storing cells as liver-specific pericytes. Spatial dynamics of agonist-stimulated intracellular calcium transients. J Clin Invest. 1992 Aug;90(2):642–646. doi: 10.1172/JCI115905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  43. Pinzani M., Milani S., De Franco R., Grappone C., Caligiuri A., Gentilini A., Tosti-Guerra C., Maggi M., Failli P., Ruocco C. Endothelin 1 is overexpressed in human cirrhotic liver and exerts multiple effects on activated hepatic stellate cells. Gastroenterology. 1996 Feb;110(2):534–548. doi: 10.1053/gast.1996.v110.pm8566602. [DOI] [PubMed] [Google Scholar]
  44. Pradelles P., Grassi J., Maclouf J. Enzyme immunoassays of eicosanoids using acetylcholine esterase as label: an alternative to radioimmunoassay. Anal Chem. 1985 Jun;57(7):1170–1173. doi: 10.1021/ac00284a003. [DOI] [PubMed] [Google Scholar]
  45. Prasad M. V., Dermott J. M., Heasley L. E., Johnson G. L., Dhanasekaran N. Activation of Jun kinase/stress-activated protein kinase by GTPase-deficient mutants of G alpha 12 and G alpha 13. J Biol Chem. 1995 Aug 4;270(31):18655–18659. doi: 10.1074/jbc.270.31.18655. [DOI] [PubMed] [Google Scholar]
  46. Raitano A. B., Halpern J. R., Hambuch T. M., Sawyers C. L. The Bcr-Abl leukemia oncogene activates Jun kinase and requires Jun for transformation. Proc Natl Acad Sci U S A. 1995 Dec 5;92(25):11746–11750. doi: 10.1073/pnas.92.25.11746. [DOI] [PMC free article] [PubMed] [Google Scholar]
  47. Rockey D. C., Housset C. N., Friedman S. L. Activation-dependent contractility of rat hepatic lipocytes in culture and in vivo. J Clin Invest. 1993 Oct;92(4):1795–1804. doi: 10.1172/JCI116769. [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Schmitt-Gräff A., Krüger S., Bochard F., Gabbiani G., Denk H. Modulation of alpha smooth muscle actin and desmin expression in perisinusoidal cells of normal and diseased human livers. Am J Pathol. 1991 May;138(5):1233–1242. [PMC free article] [PubMed] [Google Scholar]
  49. Serradeil-Le Gal C., Jouneaux C., Sanchez-Bueno A., Raufaste D., Roche B., Préaux A. M., Maffrand J. P., Cobbold P. H., Hanoune J., Lotersztajn S. Endothelin action in rat liver. Receptors, free Ca2+ oscillations, and activation of glycogenolysis. J Clin Invest. 1991 Jan;87(1):133–138. doi: 10.1172/JCI114962. [DOI] [PMC free article] [PubMed] [Google Scholar]
  50. Sokolovsky M. Endothelin receptor subtypes and their role in transmembrane signaling mechanisms. Pharmacol Ther. 1995;68(3):435–471. doi: 10.1016/0163-7258(95)02015-2. [DOI] [PubMed] [Google Scholar]
  51. Westwick J. K., Cox A. D., Der C. J., Cobb M. H., Hibi M., Karin M., Brenner D. A. Oncogenic Ras activates c-Jun via a separate pathway from the activation of extracellular signal-regulated kinases. Proc Natl Acad Sci U S A. 1994 Jun 21;91(13):6030–6034. doi: 10.1073/pnas.91.13.6030. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Westwick J. K., Weitzel C., Leffert H. L., Brenner D. A. Activation of Jun kinase is an early event in hepatic regeneration. J Clin Invest. 1995 Feb;95(2):803–810. doi: 10.1172/JCI117730. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Westwick J. K., Weitzel C., Minden A., Karin M., Brenner D. A. Tumor necrosis factor alpha stimulates AP-1 activity through prolonged activation of the c-Jun kinase. J Biol Chem. 1994 Oct 21;269(42):26396–26401. [PubMed] [Google Scholar]
  54. Williams D. L., Jr, Jones K. L., Pettibone D. J., Lis E. V., Clineschmidt B. V. Sarafotoxin S6c: an agonist which distinguishes between endothelin receptor subtypes. Biochem Biophys Res Commun. 1991 Mar 15;175(2):556–561. doi: 10.1016/0006-291x(91)91601-8. [DOI] [PubMed] [Google Scholar]
  55. Win K. M., Charlotte F., Mallat A., Cherqui D., Martin N., Mavier P., Preaux A. M., Dhumeaux D., Rosenbaum J. Mitogenic effect of transforming growth factor-beta 1 on human Ito cells in culture: evidence for mediation by endogenous platelet-derived growth factor. Hepatology. 1993 Jul;18(1):137–145. [PubMed] [Google Scholar]
  56. Xia Z., Dickens M., Raingeaud J., Davis R. J., Greenberg M. E. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science. 1995 Nov 24;270(5240):1326–1331. doi: 10.1126/science.270.5240.1326. [DOI] [PubMed] [Google Scholar]
  57. Yanagisawa M., Kurihara H., Kimura S., Tomobe Y., Kobayashi M., Mitsui Y., Yazaki Y., Goto K., Masaki T. A novel potent vasoconstrictor peptide produced by vascular endothelial cells. Nature. 1988 Mar 31;332(6163):411–415. doi: 10.1038/332411a0. [DOI] [PubMed] [Google Scholar]
  58. Zohn I. E., Yu H., Li X., Cox A. D., Earp H. S. Angiotensin II stimulates calcium-dependent activation of c-Jun N-terminal kinase. Mol Cell Biol. 1995 Nov;15(11):6160–6168. doi: 10.1128/mcb.15.11.6160. [DOI] [PMC free article] [PubMed] [Google Scholar]
  59. el-Mowafy A. M., Abdel-Latif A. A. Characterization of iris sphincter smooth muscle endothelin receptor subtypes which are coupled to cyclic AMP formation and polyphosphoinositide hydrolysis. J Pharmacol Exp Ther. 1994 Mar;268(3):1343–1351. [PubMed] [Google Scholar]

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