Molecular details of BMP-7 signaling in hepatic stellate cells and myofibroblasts

Aims: TGF-β1 causes excessive extracellular matrix deposition in the setting of fibrogenic lesions. In the liver, BMP-7 is an antifibrotic ligand abrogating profibrogenic TGF-β1 responses [1]. However, the exact molecular details regulating this antagonism are unknown. We employed a model system (L 6 E 9 myoblasts) which shows a myofibroblastic phenotype upon treatment with TGF-β1 [2] resembling that of the transition of hepatic stellate cell (HSC) into myofibroblast-like cells (MFB). Previously, we have already shown that TGF-β1 responses are counteracted by BMP-7 in (L 6 E 9 -) myoblasts [3]. Methods and Results: Myoblasts express the BMP-receptors ALK2 and ALK3 but not full-length ALK6 as shown by RT-PCR. The strongest enhancement of BMP-7 signaling as measured by activation of a BRE 2 -MLP-Luc reporter was achieved by overexpression of rat ALK2 in (L 6 E 9 -) myoblasts and HSC indicating similar signaling mechanisms in both cell types. To confirm these findings we established siRNAs which effectively knock down ALK2 and ALK3. As the main downstream target of BMP-7, we identified Smad5 using different phospho-Smad-specific antibodies. Since TGF-β1 activates primarily ALK1 and Smad1– in addition to ALK5/Smad2/Smad3–in HSC and putatively in myoblasts [4, 5], BMP-7 and TGF-β1 use different receptors and mediators. Nevertheless, both ligands regulate Id proteins indicated by the finding that heterologous expression of Smad1 and Smad5 activates the Id1-derived promoter-construct (BRE 2 -MLP-Luc) in HSC and myoblasts. Conclusions: BMP-7 specifically activates the ALK2/Smad5 signaling pathway in myoblasts and HSC implicating that TGF-β1 and BMP-7 use completely different signaling molecules. There might be a direct mechanism at the receptor-level causing a reduction in Smad3 phosphorylation [3] or an indirect mechanism via the induction of Id proteins [6]. However, since Id proteins are induced by both ligands these proteins are unlikely key regulators in that process. Literatur: [1] Weiskirchen R, Meurer SK, Gressner OA, Herrmann J, Borkham-Kamphorst E, Gressner AM. Front Biosci. (2009) 14, 4992-5012 [2] Ignotz RA, Endo T and Massague J. J Biol Chem. (1987) 262, 6443-6446 [3] Scherner O, Meurer SK, Tihaa L, Gressner AM, and Weiskirchen R. J Biol Chem. (2007) 282, 13934-13943 [4] Wiercinska E, Wickert L, Denecke B, Said HM, Hamzavi J, Gressner AM, Thorikay M, ten Dijke P, Mertens PR, Breitkopf K, Dooley S. Hepatology (2006) 43, 1032-1041 [5] Velasco S, Alvarez-Muñoz P, Pericacho M, Dijke PT, Bernabéu C, López-Novoa JM, Rodríguez-Barbero A. J Cell Sci. (2008) 121, 913-919 [6] Kinoshita K, Iimuro Y, Otogawa K, Saika S, Inagaki Y, Nakajima Y, Kawada N, Fujimoto J, Friedman SL, Ikeda K. GUT (2007) 56, 706-714 ALK - BMP-7 - TGF-beta - hepatic stellate cells. myofibroblasts - signaling