Transdifferentiation-dependent expression of distinct C5a-receptors C5R1 and C5L2 in cultured hepatic stellate cells and their response to C5a-ligand

Background: Recently we reported that the complement factor 5 (C5) is a susceptibility gene for hepatic fibrogenesis in mice and humans (Hillebrandt S et al. Nat Genet 2005). However, the underlying fibrogenic mechanisms of C5 have yet to be defined. Therefore, we initiated the present study with primary cultures of rat hepatic stellate cells (HSC) to analyze the expression of the bona fide receptor C5R1 for the small chemotactic C5a fragment during HSC transdifferentiation in vitro. We also investigated the role of C5L2 during transdifferentiation, in particular since recent studies identified C5L2 as receptor for both C5a and the acylation-stimulating protein (C3adesArg), which is involved in the stimulation of triglyceride synthesis (Kalant D et al. JBC 2005).

Methods: HSC were isolated by pronase-collagenase digestion from rat liver and cultured up to 15 days under standard conditions on plastic in the presence and absence of C5a-ligand (BA–152, BD). At culture days 1, 3, 5, 9, and 15, HSC were analyzed for specific mRNA expression of the receptors C5R1 and C5L2 and collagen α2(I) (Col1a2) using quantitative RT-PCR (TaqMan). Cell surface detection of C5R1 was performed using immunohistochemistry.

Results: Immunohistochemistry revealed a time-dependent increase of C5R1 cell surface expression on HSC. Of note, the expression of both the C5R1 and the C5L2 receptor increases significantly during transdifferentiation of HSC to myofibroblasts, reaching maximum gene expression of both receptors at day 9 of culture. Interestingly, C5R1 is more strongly expressed than C5L2 in the later course of HSC transdifferentiation (days 4–16), which is reflected by an mRNA ratio of C5R1/C5L2 ranging from 1.2 to 2.1, whereas C5L2 expression dominates at earlier points in time (day 2; ratio 0.78). The functional relevance of C5a is shown by a time-dependent stimulation of Col1a2 mRNA expression in response to C5a ligand (2.4-fold on day 2, 3.2-fold on day 4; relative to control).

Conclusions: HSC transdifferentiating spontaneously in culture to myofibroblasts express two relevant C5a-receptors, which might sensitize these cells to specific C5a-dependent effects. Our findings suggest that C5a plays a role in the modulation of both HSC lipid metabolism and extracellular matrix deposition. We speculate that the differential expression of C5L2 and C5R1 contributes to the phenotypic switch of HSC from lipid-storage cells to fibrogenic myofibroblasts.

Key words

C5 - C5a-receptors - hepatic stellate cells - liver fibrogenesis