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CCN3/NOV small interfering RNA enhances fibrogenic gene expression in primary hepatic stellate cells and cirrhotic fat storing cell line CFSC

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Journal of Cell Communication and Signaling Aims and scope

Abstract

Nephroblastoma overexpressed gene encodes a matricellular protein (CCN3/NOV) of the CCN family, comprising CCN1 (CYR61), CCN2 (CTGF), CCN4 (WISP-1), CCN5 (WISP-2), and CCN6 (WISP-3). CCN proteins are involved in the regulation of mitosis, adhesion, apoptosis, extracellular matrix production, growth arrest and migration in multiple cell types. Compared to CCN2/CTGF, known as a profibrotic protein, the biological role of CCN3/NOV in liver fibrosis remains obscure. In this study we showed ccn3/nov mRNA to increase dramatically following hepatic stellate cell activation, reaching peak levels in fully transdifferentiated myofibroblasts. In models of experimental hepatic fibrosis, CCN3/NOV increased significantly at the mRNA and protein levels. CCN3/NOV was found mainly in non-parenchymal cells along the areas of tissue damage and repair. In the bile-duct ligation model, CCN3/NOV was localized mainly along portal tracts, while the repeated application of carbon tetrachloride resulted in CCN3/NOV expression mainly in the centrilobular areas. In contrast to CCN2/CTGF, the profibrotic cytokines platelet-derived growth factor-B and -D as well as transforming growth factor-β suppressed CCN3/NOV expression. In vitro, CCN3/NOV siRNA attenuated migration in the cirrhotic fat storing cell line CFSC well in line with in vivo findings that various types of cells expressing CCN3/NOV migrate into the area of tissue damage and regeneration. The suppression of CCN3/NOV enhanced expression of profibrotic marker proteins, such as α-smooth muscle actin, collagen type I, fibronectin, CCN2/CTGF and TIMP-1 in primary rat hepatic stellate cells and in CFSC. We further found that adenoviral overexpression of CCN2/CTGF suppressed CCN3/NOV expression, while the overexpression of CCN3/NOV as well as the suppression of CCN3/NOV by targeting siRNAs both resulted in enhanced CCN2/CTGF expression. These results indicate the complexity of CCN actions that are far beyond the classic Yin/Yang interplay.

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Abbreviations

Ad5:

adenovirus type 5

α-SMA:

α-smooth muscle actin

BDL:

bile duct ligation

CCN2/CTGF:

connective tissue growth factor

CCN3/NOV:

Nephroblastoma overexpressed gene

CFSC:

cirrhotic fat storing cells

EMT:

epithelial-to-mesenchymal transition

HSC:

hepatic stellate cells

KC:

Kupffer cell(s)

LSEC:

liver sinusoidal endothelial cell(s)

MFB:

myofibroblast(s)

MMP:

matrix metalloproteinase

PDGF:

platelet-derived growth factor

PDGFRβ:

platelet-derived growth factor receptor type β

pMF:

periportal myofibroblasts

TGF-β:

transforming growth factor-β

TIMP-1:

tissue inhibitor of metalloproteinases-1.

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Acknowledgements

This work was supported by the Deutsche Forschungsgemeinschaft (SFB/TRR57) to RW. We wish to extend our special thanks to U. Haas and G. Dietzel for their kind assistance.

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Authors and Affiliations

  1. Institute of Clinical Chemistry and Pathobiochemistry, RWTH-University Hospital, 52074, Aachen, Germany

    Erawan Borkham-Kamphorst, Eddy Van de Leur & Ralf Weiskirchen

  2. Department of Nephrology, RWTH-University Hospital Aachen, Aachen, Germany

    Claudia R. van Roeyen & Jürgen Floege

Authors
  1. Erawan Borkham-Kamphorst
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  2. Claudia R. van Roeyen
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  3. Eddy Van de Leur
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  4. Jürgen Floege
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  5. Ralf Weiskirchen
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Corresponding authors

Correspondence to Erawan Borkham-Kamphorst or Ralf Weiskirchen.

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Suppl. Fig. 1

Expression of CCN3/NOV in liver cell subpopulation and during transdifferentiation. (A) Quantitative RT-PCR analysis (TaqMan) from culture-activated HSC showing significant upregulation of CCN3/NOV, CCN2/CTGF and α-SMA that reach peak levels in fully transdifferentiated MFB. (B) No CCN3/NOV expression was found in naive hepatocytes that were cultured for indicated time intervals. (C) A standard RT-PCR for analysis of CCN3/NOV expression in different primary liver cells that were cultured for indicated time intervals was performed showing minimal expression in Kupffer cells (KC) and sinusoidal endothelial cells (SLEC) compared to culture-activated HSC and periportal miofibroblasts (pMF) in secondary culture (D) Double staining immunohistochemistry of CCN3/NOV (green) and ED2 (red). ED2, a specific Kupffer cell marker, showed no co-localization with CCN3/NOV (PPT 11.2 MB)

Suppl. Fig. 2

CCN3/NOV and PDGF signalling. (A) CFSC were transfected with control siRNA and CCN3/NOV siRNAs and starved for 24 h followed by stimulation with PDGF-B (20 ng/ml) for 15 min. Western blot analysis of respective cell lysates showed no effects of CCN3/NOV siRNA in PDGF-induced phosphorylation of PDGF receptors, AKT, ERK1/2, and p38. (B) The efficacy of CCN3/NOV knock down was confirmed in CCN3/NOV ELISA using cell supernatants taken from the same set of experiments (PPT 145 kb)

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Borkham-Kamphorst, E., van Roeyen, C.R., Van de Leur, E. et al. CCN3/NOV small interfering RNA enhances fibrogenic gene expression in primary hepatic stellate cells and cirrhotic fat storing cell line CFSC. J. Cell Commun. Signal. 6, 11–25 (2012). https://doi.org/10.1007/s12079-011-0141-3

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  • DOI: https://doi.org/10.1007/s12079-011-0141-3

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