Skip to main content
NCBI home page
Gut logoLink to Gut
. 2001 Oct;49(4):577–583. doi: 10.1136/gut.49.4.577

Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo

E Williams 1, R Benyon 1, N Trim 1, R Hadwin 1, B Grove 1, M Arthur 1, E Unemori 1, J Iredale 1
PMCID: PMC1728476  PMID: 11559657

Abstract

BACKGROUND—Following liver injury, hepatic stellate cells (HSC) transform into myofibroblast-like cells (activation) and are the major source of type I collagen and the potent collagenase inhibitors tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) in the fibrotic liver. The reproductive hormone relaxin has been reported to reduce collagen and TIMP-1 expression by dermal and lung fibroblasts and thus has potential antifibrotic activity in liver fibrosis.
AIMS—To determine the effects of relaxin on activated HSC.
METHODS—Following isolation, HSC were activated by culture on plastic and exposed to relaxin (1-100 ng/ml). Collagen deposition was determined by Sirius red dye binding and radiolabelled proline incorporation. Matrix metalloproteinase (MMP) and TIMP expression were assessed by zymography and northern analysis. Transforming growth factor β1 (TGF-β1) mRNA and protein levels were quantified by northern analysis and ELISA, respectively.
RESULTS—Exposure of activated HSC to relaxin resulted in a concentration dependent decrease in both collagen synthesis and deposition. There was a parallel decrease in TIMP-1 and TIMP-2 secretion into the HSC conditioned media but no change in gelatinase expression was observed. Northern analysis demonstrated that primary HSC, continuously exposed to relaxin, had decreased TIMP-1 mRNA expression but unaltered type I collagen, collagenase (MMP-13), alpha smooth muscle actin, and TGF-β1 mRNA expression.
CONCLUSION—These data demonstrate that relaxin modulates effective collagen deposition by HSC, at least in part, due to changes in the pattern of matrix degradation.


Keywords: relaxin; hepatic stellate cell; hepatic fibrosis; type I collagen

Full Text

The Full Text of this article is available as a PDF (188.2 KB).

Figure 1  .

Figure 1  

Open in a new tab

Effect of relaxin on hepatic stellate cell (HSC) collagen deposition. Primary HSC were cultured in the presence of relaxin at three concentrations. (A) Collagen deposition on tissue culture plates quantified by Sirius red binding, as described in the methods. Relaxin treated HSC are shown in comparison with untreated controls (defined as 100%). The results are mean percentage change observed in seven independent experiments (*p<0.05, paired t test). (B) Effect of relaxin on collagen synthesis determined by 3H proline incorporation. The results are mean percentage observed in five independent experiments (*p<0.05 by paired t test).

Figure 2  .

Figure 2  

Open in a new tab

Identification of gelatinase (Gel) A and B by zymography and tissue inhibitor of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) by reverse zymography in conditioned media from cultured hepatic stellate cells (HSC). Five day cultures of primary HSC were incubated in serum free conditions with varying relaxin concentrations for 24 hours. The supernatants were subjected to zymography and reverse zymography, as described in the methods. After staining with Coomassie blue, bands corresponding to the expected molecular weight of gelatinase B (92 kDa) and gelatinase A (72 kDa) were observed. No evidence of matrix metalloproteinase 13 activity was detected. Addition of activated metalloproteinases to the incubation buffer revealed dark bands on a light background corresponding to the expected molecular weights of TIMP-1 (29 kDa) and TIMP-2 (22 kDa). Densitometric analysis of this reverse zymogram indicates that TIMP-1 was reduced by 48% and TIMP-2 by 83% in response to 100 ng/ml relaxin. The results are representative of two independent experiments.

Figure 3  .

Figure 3  

Open in a new tab

Northern blot analysis of hepatic stellate cells (HSC) cultured with a series of concentrations of relaxin. Total RNA was extracted from primary HSC cultured for seven days in the presence or absence of relaxin. Northern analysis was performed and the membranes probed sequentially with [α-32P] labelled random primed cDNA probes, as described in the methods. The results are representative of two independent experiments. TIMP-1, tissue inhibitor of metalloproteinase 1; MMP-13, matrix metalloproteinase 13; α-SMA, alpha smooth muscle actin.

Figure 4  .

Figure 4  

Open in a new tab

Northern blot analysis for transforming growth factor β1 (TGF-β1) mRNA in hepatic stellate cells (HSC) cultured in the presence or absence of relaxin (100 ng/ml for 24 hours). Total RNA was extracted from day 5 primary HSC (A) or passaged P1 HSC (B) and 10 µg aliquots were subjected to northern blotting and probed for TGF-β1. The bottom panel shows the northern blots and the top panel ethidium bromide staining of the 28 S and 18 S ribosomal bands as a control.

Figure 5  .

Figure 5  

Open in a new tab

Transforming growth factor β1 (TGF-β1) levels in the conditioned media of cultured HSC. Primary (day 5) hepatic stellate cells cultured in serum free conditions were incubated in the presence (+) or absence (−) of relaxin 100 ng/ml for 24-72 hours and the total amount of active and latent TGF-β1 determined as described. Levels of active TGF-β1 are represented in (A) and latent (total−active) TGF-β1 in (B). Results shown are representative of one of three independent experiments.

Selected References

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

  1. Aimes R. T., Quigley J. P. Matrix metalloproteinase-2 is an interstitial collagenase. Inhibitor-free enzyme catalyzes the cleavage of collagen fibrils and soluble native type I collagen generating the specific 3/4- and 1/4-length fragments. J Biol Chem. 1995 Mar 17;270(11):5872–5876. doi: 10.1074/jbc.270.11.5872. [DOI] [PubMed] [Google Scholar]
  2. Alcolado R., Arthur M. J., Iredale J. P. Pathogenesis of liver fibrosis. Clin Sci (Lond) 1997 Feb;92(2):103–112. doi: 10.1042/cs0920103. [DOI] [PubMed] [Google Scholar]
  3. Arthur M. J., Friedman S. L., Roll F. J., Bissell D. M. Lipocytes from normal rat liver release a neutral metalloproteinase that degrades basement membrane (type IV) collagen. J Clin Invest. 1989 Oct;84(4):1076–1085. doi: 10.1172/JCI114270. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bani D. Relaxin: a pleiotropic hormone. Gen Pharmacol. 1997 Jan;28(1):13–22. doi: 10.1016/s0306-3623(96)00171-1. [DOI] [PubMed] [Google Scholar]
  5. Bani D. Relaxin: a pleiotropic hormone. Gen Pharmacol. 1997 Jan;28(1):13–22. doi: 10.1016/s0306-3623(96)00171-1. [DOI] [PubMed] [Google Scholar]
  6. Benyon R. C., Iredale J. P., Goddard S., Winwood P. J., Arthur M. J. Expression of tissue inhibitor of metalloproteinases 1 and 2 is increased in fibrotic human liver. Gastroenterology. 1996 Mar;110(3):821–831. doi: 10.1053/gast.1996.v110.pm8608892. [DOI] [PubMed] [Google Scholar]
  7. Bryant-Greenwood G. D., Schwabe C. Human relaxins: chemistry and biology. Endocr Rev. 1994 Feb;15(1):5–26. doi: 10.1210/edrv-15-1-5. [DOI] [PubMed] [Google Scholar]
  8. Cairns J. A., Walls A. F. Mast cell tryptase stimulates the synthesis of type I collagen in human lung fibroblasts. J Clin Invest. 1997 Mar 15;99(6):1313–1321. doi: 10.1172/JCI119290. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Chihal H. J., Espey L. L. Utilization of the releaxed symphysis pubis of guinea pigs for clues to the mechanism of ovulation. Endocrinology. 1973 Dec;93(6):1441–1445. doi: 10.1210/endo-93-6-1441. [DOI] [PubMed] [Google Scholar]
  10. 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]
  11. Edwards D. R., Murphy G., Reynolds J. J., Whitham S. E., Docherty A. J., Angel P., Heath J. K. Transforming growth factor beta modulates the expression of collagenase and metalloproteinase inhibitor. EMBO J. 1987 Jul;6(7):1899–1904. doi: 10.1002/j.1460-2075.1987.tb02449.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Friedman S. L., Roll F. J., Boyles J., Bissell D. M. Hepatic lipocytes: the principal collagen-producing cells of normal rat liver. Proc Natl Acad Sci U S A. 1985 Dec;82(24):8681–8685. doi: 10.1073/pnas.82.24.8681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Iredale J. P., Benyon R. C., Arthur M. J., Ferris W. F., Alcolado R., Winwood P. J., Clark N., Murphy G. Tissue inhibitor of metalloproteinase-1 messenger RNA expression is enhanced relative to interstitial collagenase messenger RNA in experimental liver injury and fibrosis. Hepatology. 1996 Jul;24(1):176–184. doi: 10.1002/hep.510240129. [DOI] [PubMed] [Google Scholar]
  15. Iredale J. P., Goddard S., Murphy G., Benyon R. C., Arthur M. J. Tissue inhibitor of metalloproteinase-I and interstitial collagenase expression in autoimmune chronic active hepatitis and activated human hepatic lipocytes. Clin Sci (Lond) 1995 Jul;89(1):75–81. doi: 10.1042/cs0890075. [DOI] [PubMed] [Google Scholar]
  16. Iredale J. P., Murphy G., Hembry R. M., Friedman S. L., Arthur M. J. Human hepatic lipocytes synthesize tissue inhibitor of metalloproteinases-1. Implications for regulation of matrix degradation in liver. J Clin Invest. 1992 Jul;90(1):282–287. doi: 10.1172/JCI115850. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Labarca C., Paigen K. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem. 1980 Mar 1;102(2):344–352. doi: 10.1016/0003-2697(80)90165-7. [DOI] [PubMed] [Google Scholar]
  18. Lenhart J. A., Ohleth K. M., Ryan P. L., Palmer S. S., Bagnell C. A. Effect of relaxin on tissue inhibitor of metalloproteinase-1 and -2 in the porcine uterus and cervix. Ann N Y Acad Sci. 1999 Jun 30;878:565–566. doi: 10.1111/j.1749-6632.1999.tb07728.x. [DOI] [PubMed] [Google Scholar]
  19. Leyland H., Gentry J., Arthur M. J., Benyon R. C. The plasminogen-activating system in hepatic stellate cells. Hepatology. 1996 Nov;24(5):1172–1178. doi: 10.1002/hep.510240532. [DOI] [PubMed] [Google Scholar]
  20. Maher J. J., McGuire R. F. Extracellular matrix gene expression increases preferentially in rat lipocytes and sinusoidal endothelial cells during hepatic fibrosis in vivo. J Clin Invest. 1990 Nov;86(5):1641–1648. doi: 10.1172/JCI114886. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Musat A. I., Sattler C. A., Sattler G. L., Pitot H. C. Reestablishment of cell polarity of rat hepatocytes in primary culture. Hepatology. 1993 Jul;18(1):198–205. [PubMed] [Google Scholar]
  22. Overall C. M., Wrana J. L., Sodek J. Transcriptional and post-transcriptional regulation of 72-kDa gelatinase/type IV collagenase by transforming growth factor-beta 1 in human fibroblasts. Comparisons with collagenase and tissue inhibitor of matrix metalloproteinase gene expression. J Biol Chem. 1991 Jul 25;266(21):14064–14071. [PubMed] [Google Scholar]
  23. Postlethwaite A. E., Smith G. N., Mainardi C. L., Seyer J. M., Kang A. H. Lymphocyte modulation of fibroblast function in vitro: stimulation and inhibition of collagen production by different effector molecules. J Immunol. 1984 May;132(5):2470–2477. [PubMed] [Google Scholar]
  24. Qin X., Chua P. K., Ohira R. H., Bryant-Greenwood G. D. An autocrine/paracrine role of human decidual relaxin. II. Stromelysin-1 (MMP-3) and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1). Biol Reprod. 1997 Apr;56(4):812–820. doi: 10.1095/biolreprod56.4.812. [DOI] [PubMed] [Google Scholar]
  25. SWEAT F., PUCHTLER H., ROSENTHAL S. I. SIRIUS RED F3BA AS A STAIN FOR CONNECTIVE TISSUE. Arch Pathol. 1964 Jul;78:69–72. [PubMed] [Google Scholar]
  26. Stefanovic B., Hellerbrand C., Holcik M., Briendl M., Aliebhaber S., Brenner D. A. Posttranscriptional regulation of collagen alpha1(I) mRNA in hepatic stellate cells. Mol Cell Biol. 1997 Sep;17(9):5201–5209. doi: 10.1128/mcb.17.9.5201. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Taylor M. J., Clark C. L. Transforming growth factor-beta is a potent inhibitor of basal and stimulated relaxin release by porcine luteal cells maintained in monolayer culture. J Endocrinol. 1992 Dec;135(3):543–550. doi: 10.1677/joe.0.1350543. [DOI] [PubMed] [Google Scholar]
  28. Too C. K., Kong J. K., Greenwood F. C., Bryant-Greenwood G. D. The effect of oestrogen and relaxin on uterine and cervical enzymes: collagenase, proteoglycanase and beta-glycuronidase. Acta Endocrinol (Copenh) 1986 Mar;111(3):394–403. doi: 10.1530/acta.0.1110394. [DOI] [PubMed] [Google Scholar]
  29. Unemori E. N., Amento E. P. Relaxin modulates synthesis and secretion of procollagenase and collagen by human dermal fibroblasts. J Biol Chem. 1990 Jun 25;265(18):10681–10685. [PubMed] [Google Scholar]
  30. Unemori E. N., Bauer E. A., Amento E. P. Relaxin alone and in conjunction with interferon-gamma decreases collagen synthesis by cultured human scleroderma fibroblasts. J Invest Dermatol. 1992 Sep;99(3):337–342. doi: 10.1111/1523-1747.ep12616665. [DOI] [PubMed] [Google Scholar]
  31. Unemori E. N., Beck L. S., Lee W. P., Xu Y., Siegel M., Keller G., Liggitt H. D., Bauer E. A., Amento E. P. Human relaxin decreases collagen accumulation in vivo in two rodent models of fibrosis. J Invest Dermatol. 1993 Sep;101(3):280–285. doi: 10.1111/1523-1747.ep12365206. [DOI] [PubMed] [Google Scholar]
  32. Unemori E. N., Pickford L. B., Salles A. L., Piercy C. E., Grove B. H., Erikson M. E., Amento E. P. Relaxin induces an extracellular matrix-degrading phenotype in human lung fibroblasts in vitro and inhibits lung fibrosis in a murine model in vivo. J Clin Invest. 1996 Dec 15;98(12):2739–2745. doi: 10.1172/JCI119099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Vyas S. K., Leyland H., Gentry J., Arthur M. J. Rat hepatic lipocytes synthesize and secrete transin (stromelysin) in early primary culture. Gastroenterology. 1995 Sep;109(3):889–898. doi: 10.1016/0016-5085(95)90399-2. [DOI] [PubMed] [Google Scholar]
  34. Wiqvist I., Norström A., O'Byrne E., Wiqvist N. Regulatory influence of relaxin on human cervical and uterine connective tissue. Acta Endocrinol (Copenh) 1984 May;106(1):127–132. doi: 10.1530/acta.0.1060127. [DOI] [PubMed] [Google Scholar]

Articles from Gut are provided here courtesy of BMJ Publishing Group

RESOURCES