. 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 ␤ (TGF-␤) in human activated HSCs. The PI3K inhibitor LY294002 and the mitogen-activated protein kinase kinase (MEK) inhibitor UO126 prevented ADAM12 induction by TGF-␤, 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. I ncreased expression and activities of matrix metalloproteinase (MMP) has been shown widely in malignant phenotypes facilitating the breakdown of extracellular matrix component and cell evasion, but also unmasking bioactive cryptic fragments and releasing active growth factors which, in turn, favor tumor growth. 1 The ADAMs (a disintegrin and metalloproteinase-containing proteins) are a family of multidomain glycoproteins highly homologous to the class III snake venom metalloprotease-disintegrins. 2 The common extracellular part of the proteins includes a regulatory prodomain and metalloprotease, disintegrin-like and cystein-rich domains. Further, ADAMs are characterized by an epidermal growth factor (EGF)-like domain, a transmembrane domain, and a cytoplasmic tail. More than 30 members have been identified in the ADAM family with a broad tissue distribution and have been involved in specific cellular processes including sperm-egg interaction, 3 myocyte fusion, 4 neurogenesis, 5 and adipogenesis. 6 Recently, ADAM12 gene therapy was shown to rescue the pathology of mdx-gene deficient dystrophic mice.

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Staphylococcus aureus pathogenesis is directed by regulatory proteins and RNAs. We report the case of an RNA attenuating virulence and host uptake, possibly to sustain commensalism. A S. aureus sRNA, SprC (srn_3610), reduced virulence and bacterial loads in a mouse infection model. S. aureus deleted for sprC became more virulent and increased bacterial dissemination in colonized animals. Conversely, inducing SprC expression lowered virulence and the bacterial load. Without sprC, S. aureus phagocytosis by monocytes and macrophages was higher, whereas bacteria were internalized at lower yields when SprC expression was stimulated. Without sprC, higher internalization led to a greater number of extracellular bacteria, facilitating colonization. SprC expression decreased after phagocytosis, concurring with the facilitated growth of bacteria lacking the sRNA in the presence of an oxidant. The major staphylococcal autolysin facilitates S. aureus uptake by human phagocytes. ATL proved to be negatively regulated by SprC. The SprC domains involved in pairing with atl mRNA were analyzed. The addition of ATL reduced phagocytosis of bacteria lacking sprC with no effects on wild-type bacterial uptake, implying that SprC influences phagocytosis, at least in part, by controlling ATL. Since the control of SprC on ATL was modest, other factors must contribute to atl 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...

Selective RNA extractions are required when studying bacterial gene expression within complex mixtures of pathogens and human cells, during adhesion, internalization and survival within the host. New technologies should be developed and implemented to enrich the amount of bacterial RNAs since the majority of RNAs are from the eukaryotic host cells, requiring high read depth coverage to capture the bacterial transcriptomes in dual-RNAseq studies. This will improve our understanding about bacterial adaptation to the host cell defenses, and about how they will adapt to an intracellular life. Here we present an RNA extraction protocol to selectively enrich the lowest bacterial RNA fraction from a mixture of human and bacterial cells, using zirconium beads, with minimal RNA degradation. Zirconium beads have higher capacity to extract bacterial RNAs than glass beads after pathogen internalization. We optimized the beads size and composition for an optimal bacterial lysis and RNA extraction. The protocol was validated on two human cell lines, differentiated macrophages and osteoblasts, with either Gram-positive (Staphylococcus aureus) or -negative (Salmonella typhimurium) bacteria. Relative to other published protocols, yield of total RNA recovery was significantly improved, while host cell infection was performed with a lower bacterial inoculum. Within the host, bacterial RNA recovery yields were about six-fold lower than an RNA extraction from pure bacteria, but the quality of the RNA recovered was essentially similar. Bacterial RNA recovery was more efficient for S. aureus than for S. typhimurium, probably due to their higher protection by the Gram-positive cell walls during the early step of eukaryotic cell lysis. These purified bacterial RNAs allow subsequent genes expression studies in the course of host cell-bacteria interactions.