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.

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Chemokines are the inflammatory mediators that modulate liver fibrosis, a common feature of chronic inflammatory liver diseases. CX3CL1/fractalkine is a membrane-associated chemokine that requires step processing for chemotactic activity and has been recently implicated in liver disease. Here, we investigated the potential shedding activities involved in the release of the soluble chemotactic peptides from CX3CL1 in the injured liver. We showed an increased expression of the sheddases ADAM10 and ADAM17 in patients with chronic liver diseases that was associated with the severity of liver fibrosis. We demonstrated that hepatic stellate cells (HSC) were an important source of ADAM10 and ADAM17 and that treatment with the inflammatory cytokine inter-feron-γ induced the expression of CX3CL1 and release of soluble peptides. This release was inhibited by the metalloproteinase inhibitor batimastat; however, ADAM10/ADAM17 inhibitor GW280264X only partially affected shedding activity. By using selective tissue metalloprotease inhibitors and overexpression analyses, we showed that CX3CL1 was mainly processed by matrix metalloproteinase (MMP)-2, a metalloprotease highly expressed by HSC. We further demonstrated that the CX3CL1 soluble peptides released from stimulated HSC induced the activation of the CX3CR1-dependent signalling pathway and promoted chemoattraction of monocytes in vitro. We conclude that ADAM10, ADAM17 and MMP-2 synthesized by activated HSC mediate CX3CL1 shedding and release of chemotactic peptides, thereby facilitating recruitment of inflammatory cells and paracrine stimulation of HSC in chronic liver diseases.

During chronic liver disease, tissue remodeling leads to dramatic changes and accumulation of matrix components. Matrix metalloproteases and their inhibitors have been involved in the regulation of matrix degradation. However, the role of other proteases remains incompletely defined. We undertook a gene‐expression screen of human liver fibrosis samples using a dedicated gene array selected for relevance to protease activities, identifying the ADAMTS1 (A Disintegrin And Metalloproteinase [ADAM] with thrombospondin type 1 motif, 1) gene as an important node of the protease network. Up‐regulation of ADAMTS1 in fibrosis was found to be associated with hepatic stellate cell (HSC) activation. ADAMTS1 is synthesized as 110‐kDa latent forms and is processed by HSCs to accumulate as 87‐kDa mature forms in fibrotic tissues. Structural evidence has suggested that the thrombospondin motif‐containing domain from ADAMTS1 may be involved in interactions with, and activation of, the major fibrogenic cytokine, transforming growth factor beta (TGF‐β). Indeed, we observed direct interactions between ADAMTS1 and latency‐associated peptide‐TGF‐β (LAP‐TGF‐β). ADAMTS1 induces TGF‐β activation through the interaction of the ADAMTS1 KTFR peptide with the LAP‐TGF‐β LKSL peptide. Down‐regulation of ADAMTS1 in HSCs decreases the release of TGF‐β competent for transcriptional activation, and KTFR competitor peptides directed against ADAMTS1 block the HSC‐mediated release of active TGF‐β. Using a mouse liver fibrosis model, we show that carbon tetrachloride treatment induces ADAMTS1 expression in parallel to that of type I collagen. Importantly, concurrent injection of the KTFR peptide prevents liver damage. Conclusion: Our results indicate that up‐regulation of ADAMTS1 in HSCs constitutes a new mechanism for control of TGF‐β activation in chronic liver disease. (HEPATOLOGY 2011)

In the rat model, we used the continuously growing incisor to study the expression pattern of matrix metalloproteinase-20 (MMP-20) during the formation of mineralized dental tissues. Casein zymography analysis of extracts of the forming part of the incisor revealed lysis bands corresponding to both the latent form at 57 kD and the active 46- and 41-kD forms, whereas omission of proteinase inhibitors during protein extraction resulted in a single band at 21 kD. A higher molecular weight form of 78 kD was also stained with MMP-20 and TIMP-2 antibodies in Western blotting, and was therefore believed to correspond to an MMP-20/TIMP-2 complex. Immunohistochemical and immunogold electron microscopic results demonstrated strong MMP-20 staining in the forming outer enamel, which diminished near the dentino-enamel junction, but dentin and predentin were unstained. A strong concentration of MMP-20 was seen in the stratum intermedium (SI), particularly at the earlier stages of enamel development. Our results confirm the presence of MMP-20 protein in ameloblasts and odontoblasts of rat incisor and show it to be localized in the same sites of the forming enamel as amelogenin. Their expression is transient in odontoblasts but persists in ameloblasts, and in both cases the expression of amelogenin preceded that of MMP-20 suggesting a developmentally controlled regulation.

ADAM12 belongs to a disintegrin-like and metalloproteinase-containing protein family that possesses multidomain structures composed of a pro-domain, a metalloprotease, disintegrin-like, cysteine-rich, epidermal growth factor-like, and transmembrane domains, and a cytoplasmic tail. Overexpression of several ADAMs has been reported in human cancer, and we recently described the involvement of ADAM12 in liver injury (Le Pabic, H., Bonnier, D., Wewer, U. M., Coutand, A., Musso, O., Baffet, G., Clement, B., and Theret, N. (2003 ADAMs 2 (a disintegrin and metalloproteinase domain) are a family of cell surface multidomain proteins involved in ectodomain shedding, cell adhesion, and cell signaling (2-5). Common domains to ADAM proteins include propeptide, metalloprotease, disintegrin, cysteine-rich, EGF-like, transmembrane and cytoplasmic domains. More than 30 members have been identified in the ADAM family with broad tissue distribution and have been implicated in highly diverse biological processes, including spermatogenesis/fertilization, neurogenesis, and inflammatory response. In recent years, increased expression of several ADAMs has been reported in human cancers (6 -11) and biological properties of ADAMs suggest an important role in cancer processes including cell adhesion, migration, survival, and proliferation (12, 13). Thus, we have recently described the up-regulation of ADAM12 mRNA levels in patients with hepatocellular carcinoma and the association of ADAM12 expression with liver tumor aggressiveness (1).ADAM12 is expressed as two spliced forms, the secreted form (ADAM12S) has been described as an active metalloprotease that cleaves IGFBP-3 and -5 (14 -16). In addition, by shedding the membrane-bound HB-EGF, ADAM12 was shown to promote cardiac hypertrophy (17) and by cleaving gelatin, fibronectin, and gelatinase IV, ADAM12 was suggested to facilitate tumor invasion in breast cancer (8). The role of ADAM12 in cell-cell and cell-matrix interactions has been supported by the interaction with ␤1-integrin (18, 19) and syndecans (20). The membrane-anchored long form of ADAM12 has a cytoplasmic tail that interacts with several SH3 domain containing proteins including the Src-kinase SRC and YES1 (21), the adapter proteins Grb2 (21) and Fish (22), the regulatory subunit of phosphatidylinositol 3-kinase, p85␣ (17), and PACSIN3, a cytoplasmic phosphoprotein that plays a role in vesicle trafficking (23). In addition, the ADAM12 tail interacts with eve-1, a EEN binding protein that acts as an adaptor module to inhibit the Ras stimulating activity of Sos2 (24) and ␣-actinin-2, an actin cross-linking protein highly expressed in skeletal and cardiac muscle (25). More recently, the role of ADAM12 in modulating the transforming growth factor-␤ signaling pathway has been suggested by the interaction of ADAM12 with both FLRG (follistatin-related gene), binding proteins to transforming growth factor-␤ superfamily members (26), and the transforming growth factor type II receptor (T␤RII) leading to an increase in transforming gr...

ILK is identified as a new partner for ADAM12L cell signaling functions. ADAM12L colocalizes with ILK at focal adhesions and induces the Akt-dependent survival pathway via stimulation of β1 integrins and activation of PI3K. This effect is independent of ADAM12L proteolytic activity and involves its cytoplasmic domain.

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