Modulation of intracellular TGF-beta signalling by different PDGF isoforms and MEK1/2 in a murine hepatic stellate cell line

Background: TGF-β signalling plays an important, pro-fibrogenic role in liver fibrogenesis [1]. In addition it is known that the biological sequestering of platelet-derived growth factor (PDGF)-BB and PDGF-DD abrogates fibrogenic attributes in culture-activated hepatic stellate cells and in models of experimental hepatic fibrogenesis [2, 3]. Several previous reports further suggested that members of the PDGF family are prominent modulators of pro-fibrogenic TGF-β signalling in many cell systems. Methods: In the current study, we have tested the impact of different PDGF isoforms (PDGF-AA, PDGF-BB, PDGF-CC, PDGF-DD) on TGF-β signalling in a murine immortalized hepatic stellate cell line, i.e. the GRX cell line [4]. Therefore, we stimulated respective cells with a combination of the individual PDGFs and TGF-β in the CAGA-Smad3-dependent luciferase reporter gene assay. In addition, we examined the expression and secretion of collagen type I in various co-stimulation assays. We further investigated the influence of MEK1/2 on TGF-β signalling using the aforementioned reporter gene assay and the MEK1/2-specific inhibitor PD098059. Results: All tested PDGF isoforms showed no significant impact on TGF-β signalling in the TGF-β sensitive reporter gene assay in GRX cells. However, PDGF-BB was able to down-regulate the secretion of collagen type I and the inhibition of MEK1/2 caused a strong enhancement of TGF-β signalling. Conclusions: We conclude that the MEK1/2 signalling has an antagonistic role on classical TGF-β/Smad3-dependent pathway in GRX cells. Moreover, the demonstration that none of the tested PDGF isoforms was able to enhance the effects of TGF-β further suggests that the pro-fibrogenic activities of these cytokines are mediated in an independent manner in GRX cells (induction of extracellular matrix protein expression vs. stimulation of proliferation).

Literatur:

[1] Gressner AM, Weiskirchen R. (2006) J Cell Mol Med. 10, 76-99. [2] Borkham-Kamphorst E, Herrmann J, Stoll D, Treptau J, Gressner AM, Weiskirchen R. (2004) Lab Invest. 84, 766-777. [3] Borkham-Kamphorst E, van Roeyen CR, Ostendorf T, Floege J, Gressner AM, Weiskirchen R. (2007) J Hepatol. 46, 1064-1074. [4] Borojevic R, Monteiro AN, Vinhas SA, Domont GB, Mourão PA, Emonard H, Grimaldi G, Grimaud JA. (1985) In Vitro Cell Dev Biol. 21, 382-390.

GRX - PDGF - TGF-beta - collagen type I - costimulation - reporter assay