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doi:10.1016/j.bbamcr.2004.12.009 
Copyright © 2005 Elsevier B.V. All rights reserved.
The plasticity of p19ARF null hepatic stellate cells and the dynamics of activation
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Verena Proell, Mario Mikula, Eva Fuchs and Wolfgang Mikulits
, 
Received 6 October 2004;
Abstract
In the healthy adult liver, quiescent hepatic stellate cells (HSCs) present the major site for vitamin A storage in cytoplasmic lipid droplets. During liver injury due to viral infection or alcohol intoxication, HSCs get activated and produce high amounts of extracellular matrix components for tissue repair and fibrogenesis. Employing p19ARF deficiency, we established a non-transformed murine HSC model to investigate their plasticity and the dynamics of HSC activation. Primary HSCs isolated from livers of adult p19ARF null mice underwent spontaneous activation through long-term passaging without an obvious replicative limit. The immortalized cell line, referred to as M1-4HSC, showed stellate cell characteristics including the expression of desmin, glial fibrillary acidic protein, α-smooth muscle actin and pro-collagen I. Treatment of these non-tumorigenic M1-4HSC with pro-fibrogenic TGF-β1 provoked a morphological transition to a myofibroblastoid cell type which was accompanied by enhanced cellular turnover and impaired migration. In addition, M1-4HSCs expressed constituents of cell adhesion complexes such as p120ctn and β-catenin at cell borders, which dislocalized in the cytoplasm during stimulation to myofibroblasts, pointing to the epitheloid characteristics of HSCs. By virtue of its non-transformed phenotype and unlimited availability of cells, the p19ARF deficient model of activated HSCs and corresponding myofibroblasts render this system a highly valuable tool for studying the cellular and molecular basis of hepatic fibrogenesis.
Keywords: Hepatic stellate cell; TGF-β; Fibrogenesis; Myofibroblast
Article Outline
- 1. Introduction
- 2. Materials and methods
- 2.1. Isolation and immortalization of p19ARF null hepatic stellate cells
- 2.2. Limiting dilution
- 2.3. Tumor formation in vivo
- 2.4. Proliferation kinetics
- 2.5. Reverse transcription polymerase chain reaction (RT-PCR)
- 2.6. Western blot analysis
- 2.7. Transwell assay
- 2.8. Analysis of caspase-3 activity
- 2.9. Analysis of cell viability
- 2.10. Thymidine incorporation
- 2.11. Confocal immunofluorescence microscopy
- 3. Results
- 3.1. Establishment of immortalized p19ARF null hepatic stellate cells (HSCs)
- 3.2. Single cell clones of M1-4HSCs display again a heterogeneous phenotype
- 3.3. The fibrogenic factor TGF-β provokes further activation of M1-4HSCs
- 3.4. Reprogramming of ECM components during activation of HSCs to M-HTs
- 3.5. The balance between cell division rate and apoptosis is shifted by TGF-β
- 3.6. The migration of HSCs is reduced by TGF-β
- 3.7. Cell–cell contact formation of activated mHSCs is affected by TGF-β
- 4. Discussion
- Acknowledgements
- References
