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


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.


CONCLUSIONS
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.
Kao
Ying-Hsien
YH
Liver Transplantation Program and Department of Anesthesiology, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung 833, Taiwan.
Jawan
Bruno
B
Goto
Shigeru
S
Pan
Mei-Chun
MC
Lin
Yu-Chun
YC
Sun
Cheuk-Kwan
CK
Hsu
Li-Wen
LW
Tai
Ming-Hong
MH
Cheng
Yu-Fan
YF
Nakano
Toshiaki
T
Wang
Chih-Shien
CS
Huang
Chia-Jung
CJ
Chen
Chao-Long
CL
D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology / Q000494:pharmacology; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D015687:Cell Hypoxia / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D015870:Gene Expression / Q000502:physiology; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D018960:Hyaluronan Receptors / Q000378:metabolism; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D009593:Nitroimidazoles / Q000378:metabolism; D010766:Phosphorylation / Q000187:drug effects; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; D018125:Receptors, Transforming Growth Factor beta / Q000378:metabolism; D044967:Serum / Q000502:physiology*; D051899:Smad2 Protein / Q000378:metabolism; D053773:Transforming Growth Factor beta1 / Q000235:genetics / Q000276:immunology / Q000378:metabolism* / Q000494:pharmacology
D000911:Antibodies, Monoclonal; C476399:Cd44 protein, mouse; D018960:Hyaluronan Receptors; D009593:Nitroimidazoles; D018809:Proliferating Cell Nuclear Antigen; D018125:Receptors, Transforming Growth Factor beta; D051899:Smad2 Protein; C497783:Smad2 protein, mouse; D053773:Transforming Growth Factor beta1; C033815:pimonidazole; D007770:L-Lactate Dehydrogenase; D017346:Protein Serine-Threonine Kinases; D000077293:Receptor, Transforming Growth Factor-beta Type I; D000077294:Receptor, Transforming Growth Factor-beta Type II; C000625843:Tgfbr1 protein, rat; D020780:Matrix Metalloproteinase 9