ABSTRACT
Little is known about the expression or role of ADAMTS-1, -4 and -5 and their endogenous inhibitor TIMP3 in the liver in physiological and pathological conditions. Their expression was, therefore, investigated in the hepatocellular carcinoma cell lines HepG2 and HuH-7 using qRT-PCR and western blotting, and their cellular localisation by immunocytochemistry. Cytokine treatments were used to assess mRNA and protein modulation. ADAMTS-1, -4, -5 and TIMP3 mRNA and protein were detected in both HepG2 and HuH-7 cells. IL-1ß and IL-6 treatments significantly modulated ADAMTS-1 mRNA expression and IL-1ß treatment ADAMTS-4 mRNA expression in HepG2 cells. Modulations of mRNA by ≥ 5-fold did not translate to increased protein expression. This study showed that ADAMTS-1, -4, -5 and TIMP3 were expressed at differential levels in hepatocellular carcinoma cell lines. The pro-inflammatory cytokines IL-1ß, TNF-α or IL-6 induced changes in mRNA expression, although these did not translate to the protein level.
Subject(s)
ADAM Proteins/biosynthesis , Carcinoma, Hepatocellular/metabolism , Procollagen N-Endopeptidase/biosynthesis , Tissue Inhibitor of Metalloproteinase-3/biosynthesis , ADAMTS1 Protein , ADAMTS4 Protein , ADAMTS5 Protein , Cell Line, Tumor , Hep G2 Cells , Humans , Interleukin-1beta/pharmacology , Interleukin-6/pharmacology , Liver/metabolism , Liver Neoplasms/metabolism , RNA, Messenger/genetics , RNA, Messenger/metabolism , Tumor Necrosis Factor-alpha/pharmacologyABSTRACT
BACKGROUND & AIMS: Soluble fractalkine is increased in the liver during times of injury; however the effect of pro-inflammatory cytokines in this process is currently unknown. The aim of this study was to determine whether pro-inflammatory cytokines elevated in patients with hepatocellular carcinoma influence fractalkine shedding from HepG2 cells and whether ADAM17 was involved in this process. METHODS: In vitro experiments were performed in the human hepatocellular carcinoma cell line HepG2. Soluble fractalkine was detected using an ELISA. ADAM17 expression was investigated using quantitative real time (reverse transcription)-polymerase chain reaction and flow cytometry. Short interfering RNA transfection was used to down-regulate ADAM17 expression. RESULTS: Soluble fractalkine was present in supernatants of HepG2 cells, and was significantly increased by interleukin-1ß (p ≤ 0.005) and tumour necrosis factor-α (p ≤ 0.043), but not by interleukin-6 (p ≥ 0.316). This corresponded to minor increases in ADAM17 protein, but not ADAM17 mRNA, following the same treatments. However, the down-regulation of ADAM17 protein did not affect fractalkine shedding. CONCLUSIONS: This study showed that soluble fractalkine is up-regulated under inflammatory conditions associated with hepatocellular carcinoma development, but ADAM17 does not appear to be responsible for regulating this process.
