Elsevier

Journal of Hepatology

Volume 36, Issue 3, March 2002, Pages 362-369
Journal of Hepatology

Regulation of hepatic stellate cell proliferation and collagen synthesis by proteinase-activated receptors

https://doi.org/10.1016/S0168-8278(01)00285-9Get rights and content

Abstract

Background/Aims: Thrombin and MC tryptase, which are agonists for proteinase-activated receptors-1 and -2, respectively, are both increased in injured liver. We have examined if rat stellate cells express these receptors and if receptor agonists influence stellate cell activation.

Methods: Expression of mRNA for proteinase activated receptors-1 and -2 were examined by RT-PCR and Northern blotting in lysates of cultured stellate cells and receptor protein examined by Western blotting. The effects of receptor agonists on cell proliferation and collagen synthesis were examined by 3H-thymidine and 3H-proline incorporation assays, respectively.

Results: Rat stellate cells activated by culture on plastic showed a progressive increase in expression of proteinase-activated receptor-1 and -2 mRNA and proteinase-activated receptor-2 protein as they transformed to a myofibroblastic phenotype. Proteinase-activated receptor-1 agonists thrombin and the peptide SFFLRN, and proteinase-activated receptor-2 agonists tryptase and the peptide SLIGRL induced stellate cell proliferation and the rapid phosphorylation of 44 and 42 kDa mitogen-activated protein kinases. PD98059, an inhibitor of these kinases, inhibited this proliferative response. Both tryptase and SLIGRL increased collagen secretion by stellate cells.

Conclusions: This study indicates that the natural proteinase-activated receptor agonists thrombin and MC tryptase might sustain liver fibrosis by promoting stellate cell proliferation and collagen synthesis.

Introduction

Hepatic stellate cells (HSC) have a central role in the pathogenesis of liver fibrosis. In normal liver, HSC store vitamin A and show minimal proliferation and collagen synthesis. However, in injured liver, HSC lose vitamin A and transform to myofibroblastic cells, termed activated HSC, which express α-smooth muscle actin (α-SMA) and synthesise various extracellular matrix protein components [1], [2], [3], [4], [5].

Proteases produced by inflammatory cells might contribute to HSC activation. Mast cells (MC) accumulate in the portal tracts, sinusoids and the fibrotic septae during human and rat liver injury and fibrosis [6], [7], [8], [9], [10], [11]. Activated HSC might recruit MC as they synthesise stem cell factor [12], a potent MC chemoattractant and maturation factor [13], [14]. MC in turn may support fibrogenesis by releasing fibrogenic mediators [15]. In particular, the major constituent of MC granules, the serine protease tryptase, is a mitogen for epithelial cells and fibroblasts and increases collagen synthesis by the latter [16], [17], [18], [19], [20].

Tryptase exerts its proliferative effects through proteinase activated receptor (PAR)-2 [20]. Tryptase (and trypsin) cleaves the N-terminus of the receptor and the new N-terminus becomes a tethered ligand which binds an extracellular domain of the receptor [21], [22], [23] initiating signalling pathways including activation of mitogen-activated protein (MAP) kinases [24], [25]. Tryptase does not activate PAR-1 which has thrombin as its major agonist [21]. Synthetic hexa-peptides corresponding to the N-terminus of the tethered ligand (SFFLRN for rat PAR-1, SLIGRL for rat PAR-2) can also activate the receptor non-proteolytically by binding directly to the extracellular activation domain [23]. PAR activation may represent a novel mechanism contributing to HSC activation during liver injury and fibrosis. In support of this, Marra and coworkers have shown that expression of PAR-1 is increased in HSC in injured liver and PAR-1 agonists increase proliferation of HSC in vitro [26], [27]. These findings suggest thrombin generated following tissue injury might initiate or perpetuate fibrogenesis. However, it is uncertain if PAR-2 plays a role in liver fibrogenesis. PAR-2 has a wide tissue and cell distribution [28], [29], [30], [31], [32], [33] but its expression by HSC and possible modulation of cellular function remain uninvestigated.

In this study, we report that HSC express both PAR-1 and PAR-2 following activation in vitro and agonists to these receptors increase the proliferation of HSC. We show that PAR-2 agonists induce HSC proliferation by a MAP kinase dependent pathway and that PAR-2 activation increases collagen synthesis by HSC. Therefore, endogenous PAR-1 and -2 agonists such as thrombin and tryptase might positively contribute to fibrogenesis in the liver.

Section snippets

Materials

Chemicals were obtained from the following sources: Moloney Murine Leukemia Virus reverse transcriptase from Immunogen International Limited (Sunderland, UK), DyNAzyme™ II DNA polymerase recombinant enzyme from Flowgen (Lichfield, UK) All cell culture media and supplements were from Life Technologies (Renfrewshire, UK). Radionuclides, nylon membranes, Megaprime DNA labelling system and Enhanced Chemiluminscence detection kits were from Amersham International (Amersham, UK). Purified human lung

Cultured HSC express PAR-1 and PAR-2 mRNA

Freshly isolated rat HSC were induced to transform to a myofibroblastic phenotype by culture on plastic in serum-containing media. Progressive activation of HSC following plating on plastic was demonstrated by their expression of the mRNA for the myofibroblastic marker α-SMA (Fig. 1A). Northern blotting showed that mRNA for PAR-1 was increasingly expressed following HSC activation, with maximum expression at 14 days of culture. A 3.5 kilobase (kb) product was detected in the HSC-derived mRNA

Discussion

Our studies demonstrate that HSC activation might be promoted by serine proteases which activate proteinase-activated receptors. We have shown that activated HSC express both PAR-1 and -2 and respond to receptor agonists with proliferation and collagen synthesis. Our findings with PAR-1 confirm previous findings by Marra et al. [26], [27] who identified PAR-1 expression in injured liver and activated HSC and demonstrated PAR-1 mediated proliferation in these cells. Whilst one suggested agonist

Acknowledgements

Dr M. Gaca was funded by an unrestricted grant from Bayer, A.G., Wuppertal, Germany whilst these studies were being performed.

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    Present address: Section of Digestive Diseases, Department of Internal Medicine, Yale Univeristy, New Haven, CT 06510, USA.

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