Overexpression of Endoglin Modulates TGF-β1-signalling Pathways in a Novel Immortalized Mouse Hepatic Stellate Cell Line

PLoS One. 2013;8(2):e56116. doi: 10.1371/journal.pone.0056116. Epub 2013 Feb 20.

Abstract

Hepatic stellate cells (HSCs) play a major role in the pathogenesis of liver fibrosis. Working on primary HSCs requires difficult isolation procedures; therefore we have generated and here characterize a mouse hepatic stellate cell line expressing GFP under control of the collagen 1(I) promoter/enhancer. These cells are responsive to pro-fibrogenic stimuIi, such as PDGF or TGF-β1, and are able to activate intracellular signalling pathways including Smads and MAP kinases. Nevertheless, due to the basal level of activation, TGF-β1 did not significantly induce GFP expression contrasting the TGF-β1 regulated endogenous collagen I expression. We could demonstrate that the accessory TGF-β-receptor endoglin, which is endogenously expressed at very low levels, has a differential effect on signalling of these cells when transiently overexpressed. In the presence of endoglin activation of Smad1/5/8 was drastically enhanced. Moreover, the phosphorylation of ERK1/2 was increased, and the expression of vimentin, α-smooth muscle actin and connective tissue growth factor was upregulated. Endoglin induced a slight increase in expression of the inhibitor of differentiation-2 while the amount of endogenous collagen type I was reduced. Therefore, this profibrogenic cell line with hepatic stellate cell origin is not only a promising novel experimental tool, which can be used in vivo for cell tracing experiments. Furthermore it allows investigating the impact of various regulatory proteins (e.g. endoglin) on profibrogenic signal transduction, differentiation and hepatic stellate cell biology.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Becaplermin
  • Biomarkers / metabolism
  • Cell Line, Transformed
  • Collagen / metabolism
  • Endoglin
  • Enzyme Activation / drug effects
  • Extracellular Signal-Regulated MAP Kinases / metabolism
  • Fluorescence
  • Green Fluorescent Proteins / metabolism
  • Hepatic Stellate Cells / drug effects
  • Hepatic Stellate Cells / enzymology
  • Hepatic Stellate Cells / metabolism*
  • Hepatic Stellate Cells / pathology
  • Intracellular Signaling Peptides and Proteins / metabolism*
  • Ligands
  • Liver Cirrhosis / enzymology
  • Liver Cirrhosis / pathology
  • Mice
  • Mice, Inbred C57BL
  • Proto-Oncogene Proteins c-sis / pharmacology
  • Rats
  • Rats, Sprague-Dawley
  • Signal Transduction* / drug effects
  • Transforming Growth Factor beta1 / metabolism*

Substances

  • Biomarkers
  • Endoglin
  • Eng protein, mouse
  • Intracellular Signaling Peptides and Proteins
  • Ligands
  • Proto-Oncogene Proteins c-sis
  • Transforming Growth Factor beta1
  • Green Fluorescent Proteins
  • Becaplermin
  • Collagen
  • Extracellular Signal-Regulated MAP Kinases

Grant support

This work was supported by grants from the Deutsche Forschungsgemeinschaft (SFB/TRR 57 P13), the IZKF Aachen, and the START Program of the Medical Faculty of RWTH Aachen. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.