Interleukin‐33 (IL‐33), the most recently identified member of the IL‐1 family, induces synthesis of T Helper 2 (Th2)‐type cytokines via its heterodimeric ST2/IL‐1RAcP receptor. Th2‐type cytokines play an important role in fibrosis; thus, we investigated the role of IL‐33 in liver fibrosis. IL‐33, ST2 and IL‐1RAcP gene expression was analysed in mouse and human normal (n= 6) and fibrotic livers (n= 28), and in human hepatocellular carcinoma (HCC; n= 22), using real‐time PCR. IL‐33 protein was detected in normal and fibrotic liver sections and in isolated liver cells using Western blotting and immunolocalization approaches. Our results showed that IL‐33 and ST2 mRNA was overproduced in mouse and human fibrotic livers, but not in human HCC. IL‐33 expression correlated with ST2 expression and also with collagen expression in fibrotic livers. The major sources of IL‐33 in normal liver from both mice and human beings are the liver sinusoidal endothelial cells and, in fibrotic liver, the activated hepatic stellate cells (HSC). Moreover, IL‐33 expression was increased in cultured HSC when stimulated by pro‐inflammatory cytokines. In conclusion, IL‐33 is strongly associated with fibrosis in chronic liver injury and activated HSC are a source of IL‐33.

“A disintegrin and metalloproteinases” (ADAMs) form a family of cell–surface glycoproteins with potential protease and cell–adhesion activities. We have investigated ADAM expression in human liver cancers and their regulation by several cytokines involved in liver injury. Using degenerative RT–PCR, cDNA encoding sequences for ADAM9 and ADAM12 were identified in human activated hepatic stellate cells (HSCs). Northern blot analyses showed that HSCs, but not hepatocytes, expressed transcripts for ADAM9 messenger RNA (mRNA) and both the long and short forms of ADAM12. This expression was associated with the transition from quiescent to activated state of rat HSCs and markedly increased in human livers with cirrhosis. ADAM12 but not ADAM9 expression was up–regulated by transforming growth factor β (TGF–β) in human activated HSCs. The PI3K inhibitor LY294002 and the mitogen–activated protein kinase kinase (MEK) inhibitor UO126 prevented ADAM12 induction by TGF–β, suggesting the involvement of PI3K and MEK activities. In vivo, the steady–state of both ADAM9 and ADAM12 mRNA levels was nearly undetectable in both normal livers and benign tumors and increased in hepatocellular carcinomas (up to 3– and 6–fold, respectively) and liver metastases from colonic carcinomas (up to 40– and 60–fold, respectively). The up–regulation of both ADAM9 and ADAM12 was correlated with an increase in matrix metalloproteinase 2 expression and activity. In conclusion, in liver cancers ADAM9 and ADAM12 expression is associated with tumor aggressiveness and progression.

Matrix metalloproteinase–2 (MMP2) is a key enzyme in the process of extracellular matrix remodeling involved in tumor invasion and metastasis. The activation of MMP2 involves interplay with the membrane type–matrix metalloproteinase–1 (MT1–MMP) and the tissue inhibitor of metalloproteinase–2 (TIMP2). In vitro, activated hepatic stellate cells are a main source of MMP2 and collagen I induces MMP2 activation. The steady–state mRNA levels of MMP2, MT1–MMP, TIMP2, collagen I, collagen IV, and laminin γ1 were compared with MMP2 activity in 55 hepatocellular carcinomas, 47 matching nontumor biopsies and 19 histologically normal livers. In hepatocellular carcinomas, increased collagen I mRNA levels were strongly associated with those of MMP2 (Spearman R = .74, P < .001), MT1–MMP (R = .65, P < .001) and TIMP2 (R = 0.61, P < .001). MMP2 activity was correlated with the mRNA expression of collagen I (R = .45 P < .01), collagen IV (R = .40, P < .01) and laminin γ1 (R = .33, P < .05). Unlike collagen IV and laminin γ1 mRNAs, MMP2, MT1–MMP, TIMP2, collagen I mRNA levels were increased in nonencapsulated compared with encapsulated tumors (P < .05). In addition, MMP2 activity was fourfold higher (P < .01) in tumors arising in cirrhotic livers than in those arising in noncirrhotic livers. Moreover, tumor recurrence was associated with 4.6– and 2.8–fold (P < .05) higher collagen I and MMP2 mRNA levels, respectively, in hepatocellular carcinomas arising in cirrhotic livers. Thus, a high extracellular matrix remodeling favors tumor progression in hepatocellular carcinomas.

Kappa class glutathione S-transferase (GST) cDNA sequences have been identified in rat, mouse, and human. In the present study, we determined the structure and chromosomal location of the human GST Kappa 1 (hGSTK1) gene, characterized the protein, and demonstrated its subcellular localization. The human gene spans approximately 5 kb, has 8 exons, and maps onto chromosome 7q34. The 5'-flanking region lacks TATA or CCAAT boxes, but there is an initiator element overlapping the transcription start site. hGSTK1 amino acid sequence showed homology to bacterial 2-hydroxychromene-2-carboxylate isomerase, an enzyme involved in naphthalene degradation pathway. hGSTK1 mRNA was expressed in all of the organs examined. Subcellular fractionation of HepG2 cells showed that the protein was located in peroxisomes and mitochondria and was not detectable in cytoplasm. The peroxisomal localization was confirmed by transfection of HepG2 cells with a plasmid coding a green fluorescent protein fused inframe to the N terminus of hGSTK1. The C terminus of hGSTK1 was essential for localization of the protein to peroxisomes, and the C-terminal sequence Ala-Arg-Leu represents a peroxisome targeting signal. This is the first time that a human GST has been found in peroxisomes, suggesting a new function for this family of enzymes.