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