Serum factors potentiate hypoxia-induced hepatotoxicity in vitro through increasing transforming growth factor-b1 activation and release Ying-Hsien Kao a,1 , Bruno Jawan a,1 , Shigeru Goto b,c , Mei-Chun Pan a , Yu-Chun Lin b , Cheuk-Kwan Sun b , Li-Wen Hsu b , Ming-Hong Tai d , Yu-Fan Cheng b , Toshiaki Nakano b , Chih-Shien Wang a , Chia-Jung Huang a , Chao-Long Chen b, * a Liver Transplantation Program and Department of Anesthesiology, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung 833, Taiwan b Liver Transplantation Program and Department of Surgery, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Chang Gung University College of Medicine, 123, Ta-Pei Rd., Niao-Sung, Kaohsiung 833, Taiwan c Department of Surgery, Iwao Hospital, Yufuin, Oita 879-5102, Japan d Institute of Biomedical Sciences, National Sun Yat-Sen University, Kaohsiung 804, Taiwan article info Article history: Received 9 September 2008 Received in revised form 2 February 2009 Accepted 10 March 2009 Keywords: TGF-b receptors Smad2 phosphorylation Hepatotoxicity Matrix metalloproteinases CD44 abstract Background: Although transforming growth factor-b (TGF-b), 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-b1 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 72 h of exposure, as evidenced by fluorescent viability stain and LDH cytotoxicity assay. Quantitative PCR showed that TGF-b1 gene expression decreased, while ELISA revealed that latent TGF-b1 in conditioned media prominently increased in serum-treated groups under hypoxia. Western blotting indicated that both type I and II receptors of TGF-b 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-b1 induces hepatotoxicity in an autocrine manner. Addition of exogenous TGF-b1 deteriorated, while TGF-b1 block- ade 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-b1 acti- vation. Conclusion: The results suggest that the mechanism governing TGF-b activation plays a crucial role in hypoxia-induced hepatotoxicity. Thus, interventions on TGF-b1 bioavailability and/or its cognate sig- naling may be of benefit in preventing hypoxia-related liver injuries. Ó 2009 Elsevier Ltd. All rights reserved. 1. Introduction Transforming growth factor-b (TGF-b) is a pleiotropic cytokine that is ubiquitously expressed by all cells and tissues in body. TGF-b is synthesized as an inactive, latent dimeric precursor, and the biologically active TGF-b is formed upon cleavage and dissoci- ation of its amino terminal portion [1]. Through its effects on cell growth, differentiation, motility, adhesion, and apoptosis, TGF-b is an influential regulator in determining cell fate and in modulat- ing tissue morphogenesis [2]. TGF-b signals through a heteromeric receptor complex assembled in response to ligand-binding and composed of type I (TbRI) and type II (TbRII) serine/threonine ki- nase receptors. TbRII exists as a homodimer on cell surface even in the absence of ligand. Binding of TGF-b1 to the extracellular do- main of TbRII causes the formation of tetraheteromeric complex, followed by transphosphorylation of TbRI by TbRII and subsequent activation of downstream substrate Smad proteins [3,4]. Mutations or deletions in TbRII can result in a loss of sensitivity to TGF-b1, a process that frequently accompanies the progression of malig- nancy [5–7]. Thus, genetic alteration and expression levels of TbRI and TbRII may relate to and reflect the responsiveness of cells to external TGF-b1 stimulation. TGF-b1 is known to be an inducer of hepatocyte growth arrest and apoptosis in primary culture [8,9] and a growth inhibitor in regenerating liver [10], hence playing significant roles in both pathological and physiological hepatic conditions. Quiescent liver contains only modest amounts of TGF-b, whereas injury to liver re- sults in the production of TGF-b1. TGF-b1 overexpression is known 1043-4666/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.cyto.2009.03.004 * Corresponding author. Fax: +886 7 7324855. E-mail address: clchen@adm.cmgh.org.tw (C.-L. Chen). 1 These authors contributed equally to this work. Cytokine 47 (2009) 11–22 Contents lists available at ScienceDirect Cytokine journal homepage: www.elsevier.com/locate/issn/10434666