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Syncoilin is an intermediate filament protein in activated hepatic stellate cells

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Abstract

Hepatic stellate cells (HSCs) play an important role in several (patho)physiologic conditions in the liver. In response to chronic injury, HSCs are activated and change from quiescent to myofibroblast-like cells with contractile properties. This shift in phenotype is accompanied by a change in expression of intermediate filament (IF) proteins. HSCs express a broad, but variable spectrum of IF proteins. In muscle, syncoilin was identified as an alpha-dystrobrevin binding protein with sequence homology to IF proteins. We investigated the expression of syncoilin in mouse and human HSCs. Syncoilin expression in isolated and cultured HSCs was studied by qPCR, Western blotting, and fluorescence immunocytochemistry. Syncoilin expression was also evaluated in other primary liver cell types and in in vivo-activated HSCs as well as total liver samples from fibrotic mice and cirrhotic patients. Syncoilin mRNA was present in human and mouse HSCs and was highly expressed in in vitro- and in vivo-activated HSCs. Syncoilin protein was strongly upregulated during in vitro activation of HSCs and undetectable in hepatocytes and liver sinusoidal endothelial cells. Syncoilin mRNA levels were elevated in both CCl4- and common bile duct ligation-treated mice. Syncoilin immunocytochemistry revealed filamentous staining in activated mouse HSCs that partially colocalized with α-smooth muscle actin, β-actin, desmin, and α-tubulin. We show that in the liver, syncoilin is predominantly expressed by activated HSCs and displays very low-expression levels in other liver cell types, making it a good marker of activated HSCs. During in vitro activation of mouse HSCs, syncoilin is able to form filamentous structures or at least to closely interact with existing cellular filaments.

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Abbreviations

HSCs:

Hepatic stellate cells

IF:

Intermediate filament

PBS:

Phosphate-buffered saline

(Q)RT-PCR:

(Quantitative) reverse transcriptase polymerase chain reaction

SDS:

Sodium dodecyl sulfate

DAPC:

Dystrophin-associated protein complex

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

LSEC:

Liver sinusoidal endothelial cells

PBS-Tx:

Phosphate-buffered saline with Triton-X

GFAP:

Glial fibrillary acidic protein

CBDL:

Common bile duct ligation

PDGF-bb:

Platelet-derived growth factor-bb

Asgr1:

Asialoglycoprotein receptor 1

CD32b:

Cluster of differentiation

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Acknowledgments

The authors express their warmest thanks to Bill Roskams, Mehiri Chéras, and Eddy Himpe for the acquisition of confocal microscopy images. We thank Dr. P. Sancho-Bru (IDIBAPS, Barcelona, Spain) for providing cDNAs of human liver tissue. We would also like to thank Danielle Blijweert for her technical assistance and Marina Pauwels for critical reading of the manuscript. Grant support: This work was supported by the Vrije Universiteit Brussel, the UZ Brussels, and The Fonds voor wetenschappelijk onderzoek (FWO: G.0652.06 and G.0260.09N) granted to H. R. and L. A. v. G. A. E. T. received a Ph.D. grant from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT-Vlaanderen).

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Authors and Affiliations

  1. Liver Cell Biology Lab, Department of Cell Biology, Faculty of Medicine and Pharmacy, Vrije Universiteit Brussel (VUB), Laarbeeklaan 103, 1090, Brussels, Belgium

    E. Van Rossen, Z. Liu, D. Blijweert, N. Eysackers, I. Mannaerts, B. Schroyen, A. El Taghdouini, H. Reynaert & L. A. van Grunsven

  2. MRC Functional Genomics Unit, Department of Physiology Anatomy and Genetics, University of Oxford, Oxford, UK

    B. Edwards & K. E. Davies

  3. Laboratory of Pediatric Hepatology and Cell Therapy, Institut de Recherche Expérimentale et Clinique (IREC), Université Catholique de Louvain, 1200, Brussels, Belgium

    E. Sokal & M. Najimi

  4. Cliniques Saint Luc, Centre de Thérapie Tissulaire et Cellulaire, 1200, Brussels, Belgium

    E. Sokal

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  1. E. Van Rossen
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  2. Z. Liu
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  3. D. Blijweert
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  5. I. Mannaerts
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  7. A. El Taghdouini
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  11. M. Najimi
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  12. H. Reynaert
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  13. L. A. van Grunsven
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Correspondence to E. Van Rossen.

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418_2013_1142_MOESM1_ESM.tif

ESM_1: mRNA expression patterns during in vitro activation of FACS sorted UV + mouse HSCs and in skin fibroblasts. a) Freshly isolated non-parenchymal fraction was FACS sorted for isolation of pure HSCs. The autofluorescence at ~ 328 nm of vitamin A (as retinyl esters) containing lipid droplets present in quiescent HSCs was used for detection of HSCs. The FACS profile indicates the typical gate for HSCs obtained by flow cytometry when UV excitation was used. HSCs were seeded, and samples were collected at indicated time points during culture. mRNA expression levels of syncoilin isoforms and the activation marker Acta2 were determined by qPCR. b) Primary hHSCs were isolated from liver segments (n = 3). After Nycodenz purification, cells were immediately lyzed (Nyco+) or put in culture for in vitro activation. Activated hHSCs were collected after 4 trypsinization steps (P4). Human skin fibroblasts (FB) were grown until confluency before collection. mRNA levels of syncoilin isoforms, activation markers Acta2 and Lox and extracellular matrix proteins Col1a1, were analyzed by qPCR. (TIFF 879 kb)

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Van Rossen, E., Liu, Z., Blijweert, D. et al. Syncoilin is an intermediate filament protein in activated hepatic stellate cells. Histochem Cell Biol 141, 85–99 (2014). https://doi.org/10.1007/s00418-013-1142-5

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