Activation of TGF-beta within cultured hepatocytes and in liver injury leads to intracrine signaling with expression of connective tissue growth factor.
Recently, synthesis and secretion of connective tissue growth factor (CTGF)/CYR61/CTGF/NOV-family member 2 (CCN2) in cultures of hepatocytes were shown, which are sensitively up-regulated by exogenous TGF-beta. In this study TGF-beta-dependent CTGF/CCN2 expression in hepatocytes cultured under completely TGF-beta-free conditions was analysed by Western-blots, metabolic labelling, and CTGF-reporter gene assays. In alkaline phosphatase monoclonal anti-alkaline phosphatase complex (APAAP)-staining of cultured hepatocytes it was demonstrated that latent TGF-beta within the hepatocytes becomes rapidly detectable during culture indicating an intracellular demasking of the mature TGF-beta antigen. Subsequent signaling to theCTGF/CCN2 promoter occurs via p-Smad2, whereas p-Smad3 does not seem to be involved. Cycloheximide did not abolish the rapid immunocytochemical appearance of mature TGF-beta, but calpain inhibitors partially suppressed intracellular TGF-beta activation and subsequently CTGF up-regulation. Calpain treatment had the reverse effect. None of the inhibitors of extracellular TGF-beta signalling was effective in the reduction of spontaneous CTGF synthesis, but intracellularly acting Alk 4-/Alk 5-specific inhibitor SB-431542 was able to diminish CTGF expression. The assumption that latent intracellular TGF-beta is activated by calpains during culture-induced stress or injurious conditions in the liver in vivo was further validated by a direct effect of calpains on the activation of recombinant latent TGF-beta. In conclusion, these data are the first to suggest the possibility of intracrine TGF-beta signalling due to calpain-dependent intracellular proteolytic activation leading to transcriptional activation of CTGF/CCN2 as a TGF-beta-sensitive reporter gene. This mechanism might be deleterious for keeping long-term hepatocyte cultures due to TGF-beta-induced apoptosis and, further, might be of relevance for induction of apoptosis or epithelial-mesenchymal transition of hepatocytes in injured liver.
Gressner
Olav A
OA
Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital, Aachen, Germany. ogressner@ukaachen.de
Lahme
Birgit
B
Siluschek
Monika
M
Rehbein
Katharina
K
Herrmann
Jens
J
Weiskirchen
Ralf
R
Gressner
Axel M
AM
D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002154:Calpain / Q000378:metabolism; D002478:Cells, Cultured; D055513:Connective Tissue Growth Factor / Q000235:genetics / Q000378:metabolism*; D004791:Enzyme Inhibitors / Q000494:pharmacology; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D042541:Intracellular Space / Q000187:drug effects / Q000378:metabolism*; D008107:Liver Diseases / Q000378:metabolism* / Q000473:pathology; D008297:Male; D008954:Models, Biological; D010766:Phosphorylation / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects; D051785:Smad Proteins / Q000378:metabolism; D013997:Time Factors; D015533:Transcriptional Activation / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000378:metabolism* / Q000494:pharmacology
D004791:Enzyme Inhibitors; D051785:Smad Proteins; D016212:Transforming Growth Factor beta; D055513:Connective Tissue Growth Factor; D000469:Alkaline Phosphatase; D002154:Calpain