Biochem. J. (2001) 359
(485–496) (Printed in Great Britain)
LIM-domain protein cysteine- and glycine-rich protein 2 (CRP2) is a novel marker of hepatic stellate cells and binding partner of the protein inhibitor of activated STAT1
Ralf WEISKIRCHEN*1, Markus MOSER†, Sabine WEISKIRCHEN*, Martin ERDEL‡, Sandra DAHMEN†, Reinhard BUETTNER§ and Axel M. GRESSNER*
*Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital, Pauwelsstrasse 30, D-52074 Aachen, Germany, †Institute of Pathology, RWTH-University Hospital, Pauwelsstrasse 30, D-52074 Aachen, Germany, ‡Institute of Medical Biology and Human Genetics, University of Innsbruck, A-6020 Innsbruck, Austria, and §Institute of Pathology, University Hospital Bonn, D-53127 Bonn, Germany
Activation of hepatic stellate cells is considered to be the main step in the development of liver fibrosis, which is characterized by the transition of quiescent vitamin-A-rich cells to proliferative, fibrogenic and contractile myofibroblasts. The identification of regulatory genes during early cell activation and transdifferentiation is essential to extend our knowledge of hepatic fibrogenesis. In liver, the gene CSRP2 is exclusively expressed by stellate cells, whereas no transcripts are detectable in hepatocytes, sinusoidal endothelial cells or Kupffer cells. The early activation of stellate cells induced by platelet-derived growth factor is accompanied by an enhanced expression of CSRP2. During later stages of transdifferentiation, the expression of CSRP2 in these cells is suppressed in vitro and in vivo. The CSRP2-encoded cysteine- and glycine-rich double-LIM-domain protein (CRP)2 is proposed to function as a molecular adapter, arranging two or more as yet unidentified protein constituents into a macromolecular complex. To identify these proteins and assign a cellular function to CRP2, a human cDNA library was screened with full-length CRP2 as bait in a yeast two-hybrid screen. The protein inhibitor of activated STAT1 ('PIAS1') was shown to associate selectively with the C-terminal LIM domain of CRP2. Physical interaction of both proteins in the cellular environment was confirmed by co-localization experiments with confocal laser scanning microscopy and co-immunoprecipitation analysis. These results establish CRP2 as a potential new factor in the JAK/STAT-signalling pathway and suggest that the suppression of CSRP2 might be a prerequisite for the myofibroblastic transition of hepatic stellate cells.
Key words: bile duct ligation, JAK/STAT pathway, LIM-domain proteins, liver fibrosis, platelet-derived growth factor.
Abbreviations used: a-SMA, a-smooth-muscle actin; CRP, cysteine- and glycine-rich protein; DAPI, 4,6-diamidino-2-phenylindole; DMEM, Dulbecco's modified Eagle's medium; FCS, fetal calf serum; FISH, fluorescence in situ hybridization; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HBSS, Hanks buffered standard saline; HSCs, hepatic stellate cells; LIM1, N-terminal LIM domain of CRP; LIM2, C-terminal LIM domain of CRP protein; MFBs, myofibroblastic cells; MLP, muscle LIM-domain protein, PDGF, platelet-derived growth factor; PIAS1, protein inhibitor of activated STAT1, TGF-b1, transforming growth factor b1; X-Gal, 5-bromo-4-chloroindol-3-yl b-D-galactopyranoside.
1To whom correspondence should be addressed (e-mail rweiskirchen@post.klinikum.rwth-aachen.de).
Received 26 June 2001/7 August 2001; accepted 24 August 2001
The Biochemical Society, London ©
2001
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