Serum factors potentiate hypoxia-induced hepatotoxicity in vitro through increasing transforming growth factor-beta1 activation and release.

Abstract

BACKGROUND:

Although transforming growth factor-beta (TGF-beta), a growth regulator of hepatocytes, induces cell death under pathological conditions, responsiveness of hepatocytes to hypoxic stimulus has not been fully defined. This study aimed at investigating the role of TGF-beta1 in hypoxia-induced hepatotoxicity using cultured clone-9 hepatocytes with or without serum supplementation.

METHODS/RESULTS:

Presence of serum significantly potentiated hypoxia-induced hepatotoxicity after 72h of exposure, as evidenced by fluorescent viability stain and LDH cytotoxicity assay. Quantitative PCR showed that TGF-beta1 gene expression decreased, while ELISA revealed that latent TGF-beta1 in conditioned media prominently increased in serum-treated groups under hypoxia. Western blotting indicated that both type I and II receptors of TGF-beta were up-regulated in serum-free groups, but down-regulated in serum-treated groups under hypoxia. Smad2 phosphorylation was only detectable in cells supplemented with serum, and hypoxia potentiated the extent of Smad2 phosphorylation, implicating that the activated TGF-beta1 induces hepatotoxicity in an autocrine manner. Addition of exogenous TGF-beta1 deteriorated, while TGF-beta1 blockade by neutralizing antibody ameliorated hypoxia-induced hepatotoxicity with serum supplementation. Gelatine zymography and immunofluorescent stain evidenced that elevated MMP-2 and MMP-9 activity and serum-dependent CD44 expression and its membranous localization may contribute to TGF-beta1 activation.

CONCLUSION:

The results suggest that the mechanism governing TGF-beta activation plays a crucial role in hypoxia-induced hepatotoxicity. Thus, interventions on TGF-beta1 bioavailability and/or its cognate signaling may be of benefit in preventing hypoxia-related liver injuries.

• Authors: Ying-Hsien Kao, Bruno Jawan, Shigeru Goto, Mei-Chun Pan, Yu-Chun Lin, Cheuk-Kwan Sun, Li-Wen Hsu, Ming-Hong Tai, Yu-Fan Cheng, Toshiaki Nakano, Chih-Shien Wang, Chia-Jung Huang, Chao-Long Chen
• Journal: Cytokine (Cytokine) Vol. 47 Issue 1 Pg. 11-22 (Jul 2009) ISSN: 1096-0023 [Electronic] England
• PMID: 19457680 (Publication Type: Journal Article, Research Support, Non-U.S. Gov't)

Chemical References

• Antibodies, Monoclonal
• Cd44 protein, mouse
• Hyaluronan Receptors
• Nitroimidazoles
• Proliferating Cell Nuclear Antigen
• Receptors, Transforming Growth Factor beta
• Smad2 Protein
• Smad2 protein, mouse
• Transforming Growth Factor beta1
• pimonidazole
• L-Lactate Dehydrogenase
• Protein Serine-Threonine Kinases
• Receptor, Transforming Growth Factor-beta Type I
• Receptor, Transforming Growth Factor-beta Type II
• Tgfbr1 protein, rat
• Matrix Metalloproteinase 9

Topics

• Animals
• Antibodies, Monoclonal (immunology, pharmacology)
• Apoptosis (drug effects, physiology)
• Cell Hypoxia (drug effects, physiology)
• Cell Line
• Cell Survival (drug effects, physiology)
• Gene Expression (physiology)
• Hepatocytes (cytology, drug effects, physiology)
• Hyaluronan Receptors (metabolism)
• L-Lactate Dehydrogenase (metabolism)
• Matrix Metalloproteinase 9 (metabolism)
• Nitroimidazoles (metabolism)
• Phosphorylation (drug effects)
• Proliferating Cell Nuclear Antigen (metabolism)
• Protein Serine-Threonine Kinases (metabolism)
• Rats
• Rats, Sprague-Dawley
• Receptor, Transforming Growth Factor-beta Type I
• Receptor, Transforming Growth Factor-beta Type II
• Receptors, Transforming Growth Factor beta (metabolism)
• Serum (physiology)
• Smad2 Protein (metabolism)
• Transforming Growth Factor beta1 (genetics, immunology, metabolism, pharmacology)