Although the causal relationship between chronic inflammation and carcinogenesis has long been discussed, the molecular basis of the relation is poorly understood. In the present study, we focused on reactive oxygen species (ROS) and their signals under inflammatory conditions leading to the carcinogenesis of epithelial cells and found that repeated treatment with a low dose of H 2 O 2 (0.2 mmol/L) for periods of 2 to 4 days caused a phenotypic conversion of mouse NMuMG mammary epithelial cells from epithelial to fibroblast-like as in malignant transformation. The phenotypic conversion included the dissolution of cell-cell contacts, redistribution of E-cadherin in the cytoplasm, and up-regulation of a set of integrin family members (integrin ␣2, ␣6, and ␤3) and matrix metalloproteinases (MMPs; MMP-3, -10, and -13), as analyzed using Northern blot analysis and quantitative reverse transcription-PCR. Gelatin zymography indicated post-transcriptional activation of gelatinases, including MMP-2 and -9. In parallel, p38 mitogen-activated protein kinase and extracellular signal-regulated kinase 1/2 were activated, which contributed to the induction of MMP-13, and a glutathione S-transferase pull-down assay showed the activation of a small GTPase, Rac1. Surprisingly, the prolonged oxidative treatment was sufficient to induce all of the aforementioned events. Most importantly, depending on the MMP activities, the epithelial cells exposed to oxidative conditions eventually acquired invasiveness in a reconstituted model system with a Matrigel invasion chamber containing normal fibroblasts at the bottom, providing the first substantial evidence supporting the direct role of ROS signals in the malignant transformation of epithelial cells.

hic-5 was originally isolated as an H2O2-inducible cDNA clone whose product was normally found at focal adhesions. In this study, we found that Hic-5 accumulated in the nucleus in response to oxidants such as H2O2. Other focal adhesion proteins including paxillin, the most homologous to Hic-5, remained in the cytoplasm. Mutation analyses revealed that the C- and N-terminal halves of Hic-5 contributed to its nuclear localization in a positive and negative manner, respectively. After the finding that leptomycin B (LMB), an inhibitor of nuclear export signal (NES), caused Hic-5 to be retained in the nucleus, Hic-5 was demonstrated to harbor NES in the N-terminal, which was sensitive to oxidants, thereby regulating the nuclear accumulation of Hic-5. NES consisted of a leucine-rich stretch and two cysteines with a limited similarity to Yap/Pap-type NES. In the nucleus, Hic-5 was suggested to participate in the gene expression of c-fos. Using dominant negative mutants, we found that Hic-5 was actually involved in endogenous c-fos gene expression upon H2O2 treatment. Hic-5 was thus proposed as a focal adhesion protein with the novel aspect of shuttling between focal adhesions and the nucleus through an oxidant-sensitive NES, mediating the redox signaling directly to the nucleus.

ABSTRACT. Changes in the activities of serum cytokines and in acute phase response were observed in dairy cows with naturally occurring coliform mastitis. Seven cows with severe mastitis showed systemic and mammary inflammatory response throughout the observation period, and 11 cows with mild mastitis recovered and were able to be milked within 3 days of onset of mastitis. Serum interleukin (IL)-1 and tumor necrosis factor (TNF) activities were higher in the severe group than in the mild group at the first appearance of symptoms. Elevated IL-1 activity was evident in the severe group throughout the observation period. Serum α-1-acidglycoprotein (α1AG) concentration began to rise with the beginning of mastitis in the severe group, and peaked at 9 days. Serum haptoglobin (Hp) concentrations peaked at 3 days, and decreased gradually after 3 days in the severe group. These results showed that there are dynamic changes in serum IL-1 activity and in serum α1AG and Hp concentrations in cows with severe coliform mastitis. KEY WORDS: acute phase protein, coliform mastitis, cytokine.

Matrix metalloproteinases (MMPs) are tissue‐remodeling enzymes involved in the processing of various biological molecules. MMPs also play important roles in cancer metastasis, contributing to angiogenesis, intravasation of tumor cells, and cell migration and invasion. Accordingly, unraveling the signaling pathways controlling MMP activities could shed additional light on cancer biology. Here, we report a molecular axis, comprising the molecular adaptor hydrogen peroxide‐inducible clone‐5 (HIC‐5), NADPH oxidase 4 (NOX4), and mitochondria‐associated reactive oxygen species (mtROS), that regulates MMP9 expression and may be a target to suppress cancer metastasis. We found that this axis primarily downregulates mtROS levels which stabilize MMP9 mRNA. Specifically, HIC‐5 suppressed the expression of NOX4, the source of the mtROS, thereby decreasing mtROS levels and, consequently, destabilizing MMP9 mRNA. Interestingly, among six cancer cell lines, only EJ‐1 and MDA‐MB‐231 cells exhibited upregulation of NOX4 and MMP9 expression after shRNA‐mediated HIC‐5 knockdown. In these two cell lines, activating RAS mutations commonly occur, suggesting that the HIC‐5–mediated suppression of NOX4 depends on RAS signaling, a hypothesis that was supported experimentally by the introduction of activated RAS into mammary epithelial cells. Notably, HIC‐5 knockdown promoted lung metastasis of MDA‐MB‐231 cancer cells in mice. The tumor growth of HIC‐5–silenced MDA‐MB‐231 cells at the primary sites was comparable to that of control cells. Consistently, the invasive properties of the cells, but not their proliferation, were enhanced by the HIC‐5 knockdown in vitro. We conclude that NOX4‐mediated mtROS signaling increases MMP9 mRNA stability and affects cancer invasiveness but not tumor growth.

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