Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2022 Sep 25;18(15):5873-5884.
doi: 10.7150/ijbs.73192. eCollection 2022.

C3G down-regulation enhances pro-migratory and stemness properties of oval cells by promoting an epithelial-mesenchymal-like process

Nerea Palao  1   2 , Celia Sequera  1   2   3 , Ángel M Cuesta  1   2 , Cristina Baquero  1   2 , Paloma Bragado  1   2 , Alvaro Gutierrez-Uzquiza  1   2 , Aránzazu Sánchez  1   2 , Carmen Guerrero  4   5   6 , Almudena Porras  1   2
Affiliations

C3G down-regulation enhances pro-migratory and stemness properties of oval cells by promoting an epithelial-mesenchymal-like process

Nerea Palao et al. Int J Biol Sci. .

Abstract

Previous data indicate that C3G (RapGEF1) main isoform is highly expressed in liver progenitor cells (or oval cells) compared to adult mature hepatocytes, suggesting it may play an important role in oval cell biology. Hence, we have explored C3G function in the regulation of oval cell properties by permanent gene silencing using shRNAs. We found that C3G knock-down enhanced migratory and invasive ability of oval cells by promoting a partial epithelial to mesenchymal transition (EMT). This is likely mediated by upregulation of mRNA expression of the EMT-inducing transcription factors, Snail1, Zeb1 and Zeb2, induced in C3G-silenced oval cells. This EMT is associated to a higher expression of the stemness markers, CD133 and CD44. Moreover, C3G down-regulation increased oval cells clonogenic capacity by enhancing cell scattering. However, C3G knock-down did not impair oval cell differentiation into hepatocyte lineage. Mechanistic studies revealed that HGF/MET signaling and its pro-invasive activity was impaired in oval cells with low levels of C3G, while TGF-β signaling was increased. Altogether, these data suggest that C3G might be tightly regulated to ensure liver repair in chronic liver diseases such as non-alcoholic steatohepatitis. Hence, reduced C3G levels could facilitate oval cell expansion, after the proliferation peak, by enhancing migration.

Keywords: C3G; epithelial mesenchymal transition; migration; oval cells; stemness.

PubMed Disclaimer

Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
C3G knock-down enhances migration and invasion in oval cells, while decreases adhesion. Oval cells with permanent C3G knock-down using different shRNAs (shC3G-1 and shC3G-3) and non-silenced controls (-) were used. A) Western-blot analysis of C3G protein levels normalized with β-actin in cells maintained in the presence of serum. Different C3G isoforms are detected as a double band. B) C3G immunofluorescence (red) in cells maintained with either 10% FBS or 0% FBS for 16h as indicated. The histogram shows the mean value ± S.E.M of integrated intensity (ID) per DAPI area (n=4). Images were taken at the same exposure time. C) Wound-healing assay. Top panels, phase contrast microscopy images from cells at 0, 6, 8 and 24h after wound generation. Cells were maintained in the absence of serum. Lower panel, histogram showing the mean value ± S.E.M. of wound closure (n=4). D) Invasion through Matrigel using serum as chemoattractant. Top panel, representative images from invading cells. Lower panel, histogram showing the mean value ± S.E.M. of the number of invading cells (n=3-6). E) Adhesion assay. Top panel, representative images of adhered cells at 15 and 30 min after seeding in a medium supplemented with 10% FBS. Lower panel, histogram showing the mean value ± S.E.M. of adhered cells (percentage) (n=4-11). *p≤0.05, **p≤0.01 and ***p≤0.001, C3G-knock-down versus non-silenced cells.
Figure 2
Figure 2
C3G down-regulation promotes EMT in oval cells. C3G knock-down (shC3G-3) oval cells and its non-silenced control (-) were used. A) Levels of Snail1, Zeb1, Zeb2 and Twist1 mRNAs in cells maintained either with 10%FBS or 0% FBS as indicated. Histograms show RQ mean value ± S.E.M. (n=4). *p≤0.05, and ***p≤0.001, C3G-knock-down versus non-silenced cells. B) Western-blot analysis of N-Cadherin and Vimentin protein levels normalized with β-actin. Cells were maintained with 10% FBS for 24h. Confocal microscopy images of Vimentin (C), E-Cadherin (D), ZO-1 (E) and F-actin (F) staining in cells maintained with 10% FBS. Nuclei were visualized with DAPI (blue). Images were taken at the same exposure time. Scale bars: 20 μm.
Figure 3
Figure 3
C3G knock-down enhances oval cell stemness. Oval cells with permanent C3G knock-down using different shRNAs (shC3G-1 and shC3G-3) and its non-silenced controls (-) maintained either with 10% FBS or 0% FBS. A) Cd133 and Cd44 mRNA levels. Histograms show RQ mean value ± S.E.M. (n=4). Cytometry analysis of CD44 (B) and EpCAM (C). Left panels, histograms showing percentage of positive cells (mean value ± S.E.M.) (n=4). Right panels, fluorescence intensity. D) Clonogenic assay. Left panel, macroscopic view of colonies at day 11 (upper images), microscopic view of an individual colony (lower images). Right panels, histograms showing the mean value ± S.E.M. of the number of colonies or cells per colony (n=3-10). E) Microscopic view of individual colonies at days 4-8. F) Histograms showing mean value ± S.E.M. of scattered colonies (percentage) at day 4 (n=3). *p≤0.05, **p≤0.01 and ***p≤0.001, C3G-knock-down versus non-silenced cells.
Figure 4
Figure 4
Effect of C3G down-regulation on the differentiation of oval cells to hepatocytes. C3G knock-down (shC3G-3) oval cells and its non-silenced control (-) were treated with HGF, dexamethasone and oncostatin M in a medium supplemented with 10% FBS for 3-9 days. N.H. (Neonatal hepatocytes). A) HNF4α mRNA levels. Histogram shows RQ mean value ± S.E.M. (n=2). B) Western-blot analysis of E-Cadherin and CK19 protein levels normalized with β-actin at time 0 (-) and at day 3 and 9 (n=3).
Figure 5
Figure 5
C3G knock-down impairs HGF/MET signaling, while enhances the TGF-β one. C3G knock-down (shC3G-3) oval cells and its non-silenced control (-) were used. A) Time-course analysis of P-MET, P-Akt, P-SHP2, P-p38MAPK, P-ERKs and total Akt, p38 and ERKs levels by western-blot normalized with β-actin (n=3). Cells maintained in the absence of serum for 16h, were treated with HGF for the indicated periods. B) HGF-induced invasion through Matrigel without chemoattractant. Top panel, representative images from invading cells. Lower panel, histogram showing the mean value ± S.E.M. of invading cells (percentage) (n=3). C) Cytometry analysis of MET cell surface levels in untreated or HGF-treated cells (10 min or 2h) maintained in the absence of serum. Histogram shows the mean value ± S.E.M. of the percentage of MET positive cells (n=4). D) Time-course analysis of P-Smad2, total Smad2, P-ERKs and total ERKs levels by western-blot normalized with β-actin, Tubulin or the corresponding non-phosphorylated protein (n=2). Cells maintained in the absence of serum for 16h, were treated with TGF-β for the indicated time periods. *p≤0.05, **p≤0.01 and ***p≤0.001, C3G-knock-down versus non-silenced cells or as indicated.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

References

    1. Bria A, Marda J, Zhou J, Sun X, Cao Q, Petersen BE. et al. Hepatic progenitor cell activation in liver repair. Liver Res. 2017;1:81–7. - PMC - PubMed
    1. Ko S, Russell JO, Molina LM, Monga SP. Liver Progenitors and Adult Cell Plasticity in Hepatic Injury and Repair: Knowns and Unknowns. Annu Rev Pathol. 2020;15:23–50. - PMC - PubMed
    1. Chen J, Chen L, Zern MA, Theise ND, Diehl AM, Liu P. et al. The diversity and plasticity of adult hepatic progenitor cells and their niche. Liver international: official journal of the International Association for the Study of the Liver. 2017;37:1260–71. - PMC - PubMed
    1. del Castillo G, Alvarez-Barrientos A, Carmona-Cuenca I, Fernandez M, Sanchez A, Fabregat I. Isolation and characterization of a putative liver progenitor population after treatment of fetal rat hepatocytes with TGF-beta. Journal of cellular physiology. 2008;215:846–55. - PubMed
    1. del Castillo G, Factor VM, Fernandez M, Alvarez-Barrientos A, Fabregat I, Thorgeirsson SS. et al. Deletion of the Met tyrosine kinase in liver progenitor oval cells increases sensitivity to apoptosis in vitro. Am J Pathol. 2008;172:1238–47. - PMC - PubMed

Publication types

MeSH terms