A mitochondrial thioredoxin-sensitive mechanism regulates TGF-ß-mediated gene expression associated with epithelial-mesenchymal transition.

Transforming growth factor (TGF)-ß is a pro-oncogenic cytokine that induces the epithelial-mesenchymal transition (EMT), a crucial event in tumor progression. During TGF-ß-mediated EMT in NMuMG mouse mammary epithelial cells, we observed sustained increases in reactive oxygen species (ROS) levels in the cytoplasm and mitochondria with a concomitant decrease in mitochondrial membrane potential and intracellular glutathione levels. In pseudo ρ0 cells, whose respiratory chain function was impaired, the increase in intracellular ROS levels was abrogated, suggesting an important role of mitochondrial activity as a trigger for TGF-ß-stimulated ROS generation. In line with this, TGF-ß-mediated expression of the EMT marker fibronectin was inhibited not only by chemicals that interfere with ROS signaling but also by exogenously expressed mitochondrial thioredoxin (TXN2) independent of Smad signaling. Of note, TGF-ß-mediated induction of HMGA2, a central mediator of EMT and metastatic progression, was similarly impaired by TXN2 expression, revealing a novel mechanism involving a thiol oxidation reaction in mitochondria, which regulates TGF-ß-mediated gene expression associated with EMT.

A HIC-5- and KLF4-dependent mechanism transactivates p21(Cip1) in response to anchorage loss.

Anchorage loss elicits a set of responses in cells, such as transcriptional changes, in order to prevent inappropriate cell growth in ectopic environments. However, the mechanisms underlying these responses are poorly understood. In this study, we investigated the transcriptional up-regulation of cyclin-dependent kinase inhibitor p21(Cip1) during anchorage loss, which is important for cell cycle arrest of nonadherent cells in the G1 phase. Up-regulation was mediated by an upstream element, designated as the detachment-responsive element (DRE), that contained Kruppel-like factor 4 (KLF4) and runt-related transcription factor 1 (RUNX1) recognition sites; both of these together were necessary for transactivation, as individually they were insufficient. RNAi experiments revealed that KLF4 and a multidomain adaptor protein, hydrogen peroxide-inducible clone 5 (HIC-5), were critically involved in DRE transactivation. The role of HIC-5 in this mechanism was to tether KLF4 to DNA sites in response to cellular detachment. In addition, further analysis suggested that oligomerization and subsequent nuclear matrix localization of HIC-5, which was accelerated spontaneously in cells during anchorage loss, was assumed to potentiate the scaffolding function of HIC-5 in the nucleus and consequently regulate p21(Cip1) transcription in a manner responding to anchorage loss. At the RUNX1 site, a LIM-only protein, CRP2, imposed negative regulation on transcription, which appeared to be removed by anchorage loss and contributed to increased transcriptional activity of DRE together with regulation at the KLF4 sites. In conclusion, this study revealed a novel transcriptional mechanism that regulated gene expression in a detachment-dependent manner, thereby contributing to anchorage-dependent cell growth.

Critical roles of the cAMP-responsive element-binding protein-mediated pathway in disorganized epithelial phenotypes caused by mitochondrial dysfunction.

In most human cancers, somatic mutations have been identified in the mtDNA; however, their significance remains unclear. We recently discovered that NMuMG mouse mammary epithelial cells, when deprived of mitochondria or following inhibition of respiratory activity, undergo epithelial morphological disruption accompanied with irregular edging of E-cadherin, the appearance of actin stress fibers, and an altered gene expression profile. In this study, using the mtDNA-less pseudo ρ0 cells obtained from NMuMG mouse mammary epithelial cells, we examined the roles of two mitochondrial stress-associated transcription factors, cAMP-responsive element-binding protein (CREB) and C/EBP homologous protein-10 (CHOP), in the disorganization of epithelial phenotypes. We found that the expression of matrix metalloproteinase-13 and that of GADD45A, SNAIL and integrin α1 in the ρ0 cells were regulated by CHOP and CREB, respectively. Of note, knockdown and pharmacological inhibition of CREB ameliorated the disrupted epithelial morphology. It is interesting to note that the expression of high mobility group AT-hook 2 (HMGA2), a non-histone chromatin protein implicated in malignant neoplasms, was increased at the protein level through the CREB pathway. Here, we reveal how the activation of the CREB/HMGA2 pathway is implicated in the repression of integrin α1 expression in HepG2 human cancer cells, highlighting the importance of the CREB/HMGA2 pathway in malignant transformation associated with mitochondrial dysfunction, thereby raising the possibility that the pathway indirectly interferes with the cell-cell adhesion structure by influencing the cell-extracellular matrix adhesion status. Overall, the data suggest that mitochondrial dysfunction potentially contributes to neoplastic transformation of epithelial cells through the activation of these transcriptional pathways.

HIC-5: A Mobile Molecular Scaffold Regulating the Anchorage Dependence of Cell Growth.

HIC-5 is a multidomain LIM protein homologous to paxillin that serves as a molecular scaffold at focal adhesions and in the nucleus. It forms mobile molecular units with LIM-only proteins, PINCH, and CRP2 and translocates in and out of the nucleus via a nuclear export signal (NES). Of note, NES of HIC-5 is distinctive in its sensitivity to the cellular redox state. Recently, the mobile units of HIC-5 have been suggested to be involved in the regulation of the anchorage dependence of cell growth. On loss of adhesion, an increase in reactive oxygen species in the cells modifies NES and stops shuttling, which leads to cell-cycle control. More specifically, the system circumvents nuclear localization of cyclin D1 and transactivates p21(Cip1) in detached cells, thereby avoiding anchorage-independent cell growth. Thus, the HIC-5-LIM only protein complex has emerged as a fail-safe system for regulating the anchorage dependence of cell growth.

Importance of mitochondrial dysfunction in oxidative stress response: A comparative study of gene expression profiles.

Mitochondria are considered to play an important role in oxidative stress response since they are a source of reactive oxygen species and are also targeted by these species. This study examined the mitochondrial conditions in cells of epithelial origin that were exposed to H(2)O(2) and found a decline in the membrane potential along with a specific loss of UQCRC1, a sub-unit of complex III, suggesting that mitochondrial dysfunction occurs upon exposure to oxidative stress. This observation led to the hypothesis that certain cellular responses to oxidative stress occurred because of mitochondrial dysfunction. When mitochondria-less (pseudo ρ0) cells were examined as a model of mitochondrial dysfunction, striking similarities were found in their cellular responses compared with those found in cells exposed to oxidative stress, including changes in gene expression and gelatinolytic enzyme activities, thus suggesting that cellular responses to oxidative stress were partly mediated by mitochondrial dysfunction. This possibility was further validated by microarray analysis, which suggested that almost one-fourth of the cellular responses to oxidative stress were mediated by mitochondrial dysfunction that accompanies oxidative stress, thereby warranting a therapeutic strategy that targets mitochondria for the treatment of oxidative stress-associated diseases.

Hic-5 deficiency enhances mechanosensitive apoptosis and modulates vascular remodeling.

Forces associated with blood flow are crucial not only for blood vessel development but also for regulation of vascular pathology. Although there have been many studies characterizing the responses to mechanical stimuli, molecular mechanisms linking biological responses to mechanical forces remain unclear. Hic-5 (hydrogen peroxide-inducible clone-5) is a focal adhesion adaptor protein proposed as a candidate for a mediator of mechanotransduction. In the present study, we generated Hic-5-deficient mice by targeted mutation. Mice lacking Hic-5 are viable and fertile, and show no obvious histological abnormalities including vasculature. However, after wire injury of the femoral artery in Hic-5 deficient mice, histological recovery of arterial media was delayed due to enhanced apoptosis of vascular wall cells, whereas neointima formation was enhanced. Stretch-induced apoptosis was enhanced in cultured vascular smooth muscle cells (vascular SMCs) from Hic-5 deficient mice. Mechanical stress also induced the alteration of intracellular distribution of vinculin from focal adhesions to the whole cytoplasm in SMCs. Immunoelectron microscopic study of vascular SMCs from a wire-injured artery demonstrated that vinculin was dispersed in the nucleus and the cytoplasm in Hic-5-deficient mice whereas vinculin was localized mainly in the sub-plasma membrane region in wild type mice. Our findings indicate that Hic-5 may serve as a key regulator in mechanosensitive vascular remodeling.

Gene expression profiling identifies a role for CHOP during inhibition of the mitochondrial respiratory chain.

Mitochondrial dysfunction, in particular, interference in the respiratory chain, is often responsible for the toxicogenic effects of xenobiotics. In this study, changes in gene expression resulting from pharmacological inhibition of the respiratory chain were studied by DNA microarray analysis using cells treated with rotenone or antimycin A, which inhibit complexes I and III of the electron transport system, respectively. Forty-eight genes were either up- or down-regulated more than 3-fold. These included stress- and/or metabolic-related effector genes and several transcriptional regulators represented by CHOP-10. Further study using siRNA showed that among the four genes studied, up-regulation of three was dependent on CHOP-10. C/EBPbeta, a dimerizing partner of CHOP-10, was also involved in two of the three genes including Trib3, implying that CHOP-10, heterodimerizing with C/EBPbeta or another partner played a key role in the expression of a set of genes under stress. Although CHOP-10 and Trib3 were both ER-stress response genes, signal inducing Trib3 during mitochondrial stress was distinct from that during ER stress. Cytotoxicity caused by inhibition of the respiratory chain was attenuated by treatment with siRNA for CHOP-10. This study demonstrated the importance of CHOP-10 in coordinating individual gene expression in response to the mitochondrial stress.

Competitive nuclear export of cyclin D1 and Hic-5 regulates anchorage dependence of cell growth and survival.

Anchorage dependence of cell growth and survival is a critical trait that distinguishes nontransformed cells from transformed cells. We demonstrate that anchorage dependence is determined by anchorage-dependent nuclear retention of cyclin D1, which is regulated by the focal adhesion protein, Hic-5, whose CRM1-dependent nuclear export counteracts that of cyclin D1. An adaptor protein, PINCH, interacts with cyclin D1 and Hic-5 and potentially serves as an interface for the competition between cyclin D1 and Hic-5 for CRM1. In nonadherent cells, the nuclear export of Hic-5, which is redox-sensitive, was interrupted due to elevated production of reactive oxygen species, and cyclin D1 was exported from the nucleus. When an Hic-5 mutant that was continuously exported in a reactive oxygen species-insensitive manner was introduced into the cells, cyclin D1 was retained in the nucleus under nonadherent conditions, and a significant population of cells escaped from growth arrest or apoptosis. Interestingly, activated ras achieved predominant cyclin D1 nuclear localization and thus, growth in nonadherent cells. We report a failsafe system for anchorage dependence of cell growth and survival.

Hic-5, an adaptor protein expressed in vascular smooth muscle cells, modulates the arterial response to injury in vivo.

Focal adhesion components are targets for biochemical and mechanical stimuli that evoke crucial injury. Hic-5 (hydrogen peroxide-inducible clone 5) is a multidomain adaptor protein which is implicated in the regulation of integrin signaling in focal adhesion. The aim of this research was to test the hypothesis that Hic-5, a focal adhesion LIM protein expressed in smooth muscle cells, is involved in dynamic processes by pathological stimuli in the vessel wall. Here, we describe the analysis of the function of Hic-5 using a mouse model of vascular injury that may mimic balloon angioplasty. At 4 days after vascular injury, marked down-regulation of the Hic-5 expression was observed in the smooth muscle layer, and local delivery of the Hic-5 using adenovirus vectors repressed injury-induced neointimal expansion. In addition, Hic-5 reduced cells migration into three-dimensional collagen gels, and the forced expression of Hic-5 in cells embedded in the collagen gel matrix repressed the expression of uPA that participates in smooth muscle cell migration. These results suggest that Hic-5 modulates cellular responses to pathological stimuli in the vessel wall.

Downregulation of hepatocyte nuclear factor-4alpha and its role in regulation of gene expression by TGF-beta in mammary epithelial cells.

We found that a specific isoform of hepatocyte nuclear factor 4alpha (HNF-4alpha), HNF-4alpha8, was expressed in mouse mammary epithelial NMuMG cells, and that its expression was repressed by TGF-beta. The repression was interfered by dominant negative forms of activin receptor-like kinase 5 (ALK5) and Smad3, and sensitive to cycloheximide, suggesting the involvement of additional protein(s) as well as ALK5 and Smad3 in the repression. Further study showed that high mobility group A2 (HMGA2), which is reported to be directly upregulated by Smads, repressed HNF-4alpha8 expression. Therefore, it is likely that HMGA2 mediates the downregulation of HNF-4alpha8 downstream of ALK5 and Smads To determine the significance of the downregulation of HNF-4alpha8 in TGF-beta signaling, we performed DNA microarray analysis and extracted a subgroup of TGF-beta1-regulated genes, including tenascin C and tissue inhibitor of metalloproteinase 3 (TIMP-3), whose regulation by TGF-beta1 was attenuated by forced expression of HNF-4alpha8. HMGA2 has recently emerged as a transcriptional organizer of TGF-beta signaling, regulating several key factors involved in epithelial-mesenchymal transition (EMT). In this study, we identified an isoform of HNF-4alpha as a new target downstream of HMGA2 and assigned a new role to HNF-4alpha in the TGF-beta signaling/transcriptional cascade driven by ALK5/Smad/HMGA2 and associated with the malignant transformation of cells.

Impact of salusin-alpha and -beta on human macrophage foam cell formation and coronary atherosclerosis.

BACKGROUND: Human salusins, related bioactive polypeptides with mitogenic effects on vascular smooth muscle cells and fibroblasts and roles in hemodynamic homeostasis, may be involved in the origin of coronary atherosclerosis. Macrophage foam cell formation, characterized by cholesterol ester accumulation, is modulated by scavenger receptor (cholesterol influx), acyl-coenzyme A:cholesterol acyltransferase-1 (ACAT-1; storage cholesterol ester converted from free cholesterol), and ATP-binding cassette transporter A1 (cholesterol efflux). METHODS AND RESULTS: Serum salusin-alpha levels were decreased in 173 patients with angiographically proven coronary artery disease compared with 40 patients with mild hypertension and 55 healthy volunteers (4.9+/-0.6 versus 15.4+/-1.1 and 20.7+/-1.5 pmol/L, respectively; P<0.0001). Immunoreactive salusin-alpha and -beta were detected in human coronary atherosclerotic plaques, with dominance of salusin-beta in vascular smooth muscle cells and fibroblasts. After 7 days in primary culture, acetylated low-density lipoprotein-induced cholesterol ester accumulation in human monocyte-derived macrophages was significantly decreased by salusin-alpha and increased by salusin-beta. Salusin-alpha significantly reduced ACAT-1 expression in a concentration-dependent manner. In contrast, salusin-beta significantly increased ACAT-1 expression by 2.1-fold, with a maximal effect at 0.6 nmol/L. These effects of salusins were abolished by G-protein, c-Src tyrosine kinase, protein kinase C, and mitogen-activated protein kinase kinase inhibitors. ACAT activity and ACAT-1 mRNA levels were also significantly decreased by salusin-alpha and increased by salusin-beta; however, neither salusin-alpha nor salusin-beta affected scavenger receptor A function assessed by [125I]acetylated low-density lipoprotein endocytosis or scavenger receptor class A and ATP-binding cassette transporter A1 expression. CONCLUSIONS: Our results indicate that the 2 salusin isoforms have opposite effects on foam cell formation in human monocyte-derived macrophages. Development of atherosclerosis may be accelerated by salusin-beta and suppressed by salusin-alpha via ACAT-1 regulation.

Circulating progenitor cells contribute to neointimal formation in nonirradiated chimeric mice.

Recent evidence suggests that bone marrow-derived cells may contribute to repair and lesion formation following vascular injury. In most studies, bone marrow-derived cells were tracked by transplanting exogenous cells into bone marrow that had been compromised by irradiation. It remains to be determined whether endogenous circulating progenitors actually contribute to arterial remodeling under physiological conditions. Here, we established a parabiotic model in which two mice were conjoined subcutaneously without any vascular anastomosis. When wild-type mice were joined with transgenic mice that expressed green fluorescent protein (GFP) in all tissues, GFP-positive cells were detected not only in the peripheral blood but also in the bone marrow of the wild-type mice. The femoral arteries of the wild-type mice were mechanically injured by insertion of a large wire. At 4 wk, there was neointima hyperplasia that mainly consisted of alpha-smooth muscle actin-positive cells. GFP-positive cells were readily detected in the neointima (14.8+/-4.5%) and media (31.1+/-8.8%) of the injured artery. Some GFP-positive cells expressed alpha-smooth muscle actin or an endothelial cell marker. These results indicate that circulating progenitors contribute to re-endothelialization and neointimal formation after mechanical vascular injury even in nonirradiated mice.

Induction of matrix metalloproteinase gene expression in an endothelial cell line by direct interaction with malignant cells.

Mouse endothelial TKD2 cells in monolayers were cocultured with various human cell lines for 24 h, and the expression of several secreted matrix metalloproteinases (MMP) and cell adhesion molecules was examined by real-time reverse transcription-polymerase chain reaction using mouse-specific primers. Coculture with normal fibroblasts did not elicit the expression of these molecules, but coculture with cancer cells induced the expression of MMP-3, MMP-9 and MMP-10 mRNA in endothelial cells, and in normal mouse embryonic fibroblasts. The induction of MMP mRNA was dependent on direct cell adhesion, as separate culture of A549 cells in Boyden chambers did not induce MMP mRNA, and neutralizing antibody against VLA-4 abolished the induction. An inhibitor of phosphatidylinositol-3-phosphate kinase strongly suppressed the induction of MMP-3, MMP-9 and MMP-10 mRNA, and expression of the dominant-negative mutant of phosphatidylinositol-3-phosphate kinase also decreased the induction. It was suggested that intracellular reactive oxygen species (ROS) levels were increased in TKD2 cells following adhesion to cancer cells. ROS scavengers decreased the levels of MMP induction, and roterone, an inhibitor of mitochondrial complex I, strongly suppressed the induction of MMP-3, MMP-9 and MMP-10. The depletion of mitochondria in TKD2 cells decreased the induction of MMP-9, but the induction of MMP-3 and MMP-10 was not affected. These results indicate that the adhesion of cancer cells to endothelial cells activates several distinct signaling pathways to induce MMP gene expression, and the pathways for MMP-3, MMP-9 and MMP-10 are partly different. For the induction of MMP-9, mitochondria participate in induction, possibly through the production of ROS.

Anthracyclines, small-molecule inhibitors of hypoxia-inducible factor-1 alpha activation.

Hypoxia-inducible factor-1 (HIF-1) is a central mediator of cellular responses to low oxygen and has recently become an important therapeutic target for solid tumor therapy. To identify small molecule inhibitors of the HIF-1 transcriptional activation, we have established a high through-put assay system using a stable transformant of mammalian cells that express the luciferase reporter gene construct containing a HIF-1 binding site. Using this system, we screened 5000 cultured broths of microorganisms, and we found that cinerubin (1-hydroxy aclacinomycin B) showed a significant inhibition of the reporter activity induced by hypoxic conditions. In addition, we demonstrated that aclarubicin also inhibited the HIF-1 transcriptional activity under hypoxic conditions, but neither doxorubicin nor daunorubicin inhibited it. Consistent with these results, cinerubin and aclarubicin inhibited the hypoxic induction of the vascular endothelial growth factor (VEGF) protein in HepG2 cells, but neither doxorubicin nor daunorubicin affected it. Thus, our results suggested that some anthracyclines are also acting as angiogenesis inhibitors.

Inhibition of angiogenesis and HIF-1alpha activity by antimycin A1.

We identified antimycin A1 as an inhibitor of the hypoxia-response element (HRE) from screening using a reporter under the control of HRE under hypoxic conditions. Antimycin A1 was effective at 20 pg/ml in inhibiting the reporter activity. The expression of vascular endothelial growth factor (VEGF) mRNA during hypoxia was also inhibited by antimycin A1. Angiogenesis induced by implantation of mouse sarcoma-180 cells was significantly inhibited by non-toxic doses of antimycin A1. Hypoxia inducible factor (HIF)-1alpha protein levels were significantly decreased by antimycin A1, but its mRNA level was not affected. Antimycin A1 is known to be an inhibitor of mitochondrial electron transport system, and depletion of mitochondria abolished antimycin A1-effect, at least in part. Inhibitors of proteasome or protein synthesis did not affect the decrease in HIF-1alpha level induced by antimycin A1. These results indicate that antimycin A1 inhibited angiogenesis through decrease in VEGF production caused by inhibition of HIF-1alpha activation.

Oligomerizing potential of a focal adhesion LIM protein Hic-5 organizing a nuclear-cytoplasmic shuttling complex.

Hic-5 is a focal adhesion LIM protein serving as a scaffold in integrin signaling. The protein comprises four LD domains in its N-terminal half and four LIM domains in its C-terminal half with a nuclear export signal in LD3 and is shuttled between the cytoplasmic and nuclear compartments. In this study, immunoprecipitation and in vitro cross-linking experiments showed that Hic-5 homo-oligomerized through its most C-terminal LIM domain, LIM4. Strikingly, paxillin, the protein most homologous to Hic-5, did not show this capability. Gel filtration analysis also revealed that Hic-5 differs from paxillin in that it has multiple forms in the cellular environment, and Hic-5 but not paxillin was capable of hetero-oligomerization with a LIM-only protein, PINCH, another molecular scaffold at focal adhesions. The fourth LIM domain of Hic-5 and the fifth LIM domain region of PINCH constituted the interface for the interaction. The complex included integrin-linked kinase, a binding partner of PINCH, which also interacted with Hic-5 through the region encompassing the pleckstrin homology-like domain and LIM domains of Hic-5. Of note, Hic-5 marginally affected the subcellular distribution of PINCH but directed its shuttling between the cytoplasmic and nuclear compartments in the presence of integrin-linked kinase. Uncoupling of the two signaling platforms of Hic-5 and PINCH through interference with the hetero-oligomerization resulted in impairment of cellular growth. Hic-5 is, thus, a molecular scaffold with the potential to dock with another scaffold through the LIM domain, organizing a mobile supramolecular unit and coordinating the adhesion signal with cellular activities in the two compartments.

Involvement of FAK and PTP-PEST in the regulation of redox-sensitive nuclear-cytoplasmic shuttling of a LIM protein, Hic-5.

The LIM protein Hic-5 is a focal adhesion protein shuttling in and out of the nucleus through the redox-sensitive nuclear export signal, and unlike other focal adhesion proteins including paxillin, the protein most homologous to Hic-5, it accumulates in the nucleus under oxidative conditions and participates in the transcription of c-fos and p21(Cip1) genes. Here, we examined the roles of the interacting partners of Hic-5, focal adhesion kinase (FAK) and protein tyrosine phosphatase PEST (PTP-PEST), in the nuclear translocation of Hic-5 and found that they were inhibitory. Interestingly, the interaction of Hic-5 with FAK was regulated by specific cysteines near the binding site and decreased in cells under oxidative conditions. Its interaction with PTP-PEST was also sensitive to the oxidant. These results suggest that the nuclear-cytoplasmic shuttling of Hic-5 is regulated by its interacting partners at focal adhesions or in the cytoplasm in a redox-sensitive manner, coordinating its role at focal adhesions with that in the nucleus, depending on the redox state of cells. Cytochalasin D or a phorbol ester also induced nuclear accumulation of Hic-5, which was inhibited by scavengers of reactive oxygen species (ROS), suggesting that besides oxidants, endogenously produced ROS induced the nuclear accumulation of Hic-5.

Uni-axial stretching regulates intracellular localization of Hic-5 expressed in smooth-muscle cells in vivo.

Hic-5 is a focal adhesion protein belonging to the paxillin LIM family that shuttles in and out of the nucleus. In the present study, we examined the expression of Hic-5 among mouse tissues by immunohistochemistry and found its expression only in smooth-muscle cells in several tissues. This result is consistent with a previous report on adult human tissues and contradicts the relatively ubiquitous expression of paxillin, the protein most homologous to Hic-5. One factor characterizing smooth-muscle cells in vivo is a continuous exposure to mechanical stretching in the organs. To study the involvement of Hic-5 in cellular responses to mechanical stress, we exposed mouse embryo fibroblasts to a uni-axial cyclic stretching and found that Hic-5 was relocalized from focal adhesions to stress fibers through its C-terminal LIM domains during the stress. In sharp contrast to this, paxillin did not change its focal-adhesion-based localization. Of the factors tested, which included interacting partners of Hic-5, only CRP2 (an only-LIM protein expressed in vascular smooth-muscle cells) and GIT1 were, like Hic-5, localized to stress fibers during the cyclic stretching. Interestingly, Hic-5 showed a suppressive effect on the contractile capability of cells embedded in three-dimensional collagen gels, and the effect was further augmented when CRP2 co-localized with Hic-5 to fiber structures of those cells. These results suggested that Hic-5 was a mediator of tensional force, translocating directly from focal adhesions to actin stress fibers upon mechanical stress and regulating the contractile capability of cells in the stress fibers.

Invasive potential induced under long-term oxidative stress in mammary epithelial cells.

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 alpha2, alpha6, and beta3) 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.