Motoko Shibanuma

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 H2O2 (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 α2, α6, and β3) 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.

Induction of senescence-like phenotypes by forced expression of hic-5, which encodes a novel LIM motif protein, in immortalized human fibroblasts.

The hic-5 gene encodes a novel protein with Zn finger-like (LIM) motifs, the expression of which increases during cellular senescence. The ectopic expression of hic-5 in nontumorigenic immortalized human fibroblasts, whose expression levels of hic-5 were significantly reduced in comparison with those of mortal cells, decreased colony-forming efficiency. Stable clones expressing high levels of hic-5 mRNA showed higher levels of mRNAs for several extracellular matrix-related proteins, along with the alteration of an alternative splicing as seen in senescent cells and decreased c-fos inducibility. Furthermore, these clones acquired a senescence-like phenotype, such as growth retardation; senescence-like morphology; and increased expression of Cip1/WAF1/sdi1 after 20 to 40 population doublings. On the other hand, antisense RNA expression of hic-5 in human normal diploid fibroblasts delayed the senescence process. HIC-5 was localized in nuclei and had affinity for DNA. Based on these observations, we speculated that HIC-5 affected the expression of senescence-related genes through interacting with DNA and thereby induced the senescence-like phenotypes. To our knowledge, hic-5 is the first single gene that could induce senescence-like phenotypes in a certain type of immortalized human cell and mediate the normal process of senescence.

Hic-5-Reduced Cell Spreading on Fibronectin: Competitive Effects between Paxillin and Hic-5 through Interaction with Focal Adhesion Kinase

ABSTRACT Hic-5 is a paxillin homologue that is localized to focal adhesion complexes. Hic-5 and paxillin share structural homology and interacting factors such as focal adhesion kinase (FAK), Pyk2/CAKβ/RAFTK, and PTP-PEST. Here, we showed that Hic-5 inhibits integrin-mediated cell spreading on fibronectin in a competitive manner with paxillin in NIH 3T3 cells. The overexpression of Hic-5 sequestered FAK from paxillin, reduced tyrosine phosphorylation of paxillin and FAK, and prevented paxillin-Crk complex formation. In addition, Hic-5-mediated inhibition of spreading was not observed in mouse embryo fibroblasts (MEFs) derived from FAK−/− mice. The activity of c-Src following fibronectin stimulation was decreased by about 30% in Hic-5-expressing cells, and the effect of Hic-5 was restored by the overexpression of FAK and the constitutively active forms of Rho-family GTPases, Rac1 V12 and Cdc42 V12, but not RhoA V14. These observations suggested that Hic-5 inhibits cell spreading through competition with paxillin for FAK and subsequent prevention of downstream signal transduction. Moreover, expression of antisense Hic-5 increased spreading in primary MEFs. These results suggested that the counterbalance of paxillin and Hic-5 expression may be a novel mechanism regulating integrin-mediated signal transduction.

Significance of nuclear relocalization of ERK1/2 in reactivation of c-fos transcription and DNA synthesis in senescent fibroblasts.

Two of mitogen-activated protein kinases (MAPK), p44mapk/p42mapk extracellular signal-regulated kinases (ERK1/2), translocate into nuclei following activation and play critical roles in connecting the signal to gene expression and allowing cell-cycle entry. Here we found that the nuclear translocation of ERK1/2 in response to growth stimuli was significantly inhibited in senescent cells that were irreversibly growth arrested, compared with presenescent cells. The activation step of these enzymes was not impaired, since ERK1/2 were phosphorylated and activated in senescent cells as efficiently as in presenescent cells. By elaborately localizing ERK2 in the nuclei of senescent cells, we could restore c-fos transcriptional activity upon growth stimuli, which was repressed in senescent cells. Furthermore, the nuclear localization of ERK1/2 has been suggested to potentiate the proliferative activity of the senescent cells in collaboration with adenovirus E1A protein. More importantly, SV40 large T antigen, the strong inducer of DNA synthesis, had the inherent ability to restore nuclear relocalization of active ERK1/2 in senescent cells, which was essentially required for the reinitiation of DNA synthesis. Thus, manipulating the relocalization of ERK1/2 into nuclei was expected to open the way to overcome some of the senescent phenotypes.

A LIM protein, Hic-5, functions as a potential coactivator for Sp1

Hic‐5 is a LIM protein with striking similarity to paxillin, and shuttles between focal adhesions and the nucleus. Our previous study suggested that Hic‐5 participates in the transcriptional control of several genes such as the c‐fos and p21 genes. In the present study, we examined the function of Hic‐5 in the nucleus using the transcriptional promoter region of the p21 gene. When localized to the nucleus, Hic‐5 was found to transactivate the p21 promoter through two of five Sp1 sites in the region proximal to the TATA box. The Hic‐5 effect was mediated by a transactivation domain of Sp1 and functional interaction with p300 through the LIM4 domain. Hic‐5 was also shown to interact functionally and physically with Smad3 through the LIM domains and to potentiate p21 promoter activity together with Smad3 and Sp1. These properties were confirmed in an artificial system using GAL4‐fusion protein. Thus, Hic‐5 was suggested to have a potential function as a cofactor in the transcriptional complex that contains Sp1, playing a role in gene transcription in the nucleus as well as in integrin signaling at focal adhesion sites.

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 α‐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 α‐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. — Tanaka, K., Sata, M., Natori, T., Kim‐Kaneyama, J., Nose, K., Shibanuma, M., Hirata, Y., and Nagai, R. Circulating progenitor cells contribute to neointimal formation in nonirradiated chimeric mice. FASEB J. 22, 428–436 (2008)

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.

Inhibition by N‐acetyl‐l‐cysteine of interleukin‐6 mRNA induction and activation of NFκB by tumor necrosis factor α in a mouse fibroblastic cell line, Balb/3T3

Redox‐based modulation plays a role in transcriptional control of gene expression. In the present study, we investigated the possible role of reactive oxygen species in the induction of interleukin‐6 (IL‐6) mRNA and in increases in NFκB binding activity by tumor necrosis factor (TNF) α using a mouse fibroblastic cell line, Balb/3T3. Expression of IL‐6 mRNA is known to be dependent upon NFκB that binds to the 5′‐flanking region of the IL‐6 gene. We found that: (i) TNFα increased IL‐6 mRNA levels and this increase was inhibited by N‐acetyl‐l‐cysteine (NAC), a scavenger of reactive oxygen species. (ii) NFκB binding activity in this cell line was also increased by TNFα, and the increase was inhibited in the presence of NAC. (iii) The treatment of cells with low doses of hydrogen peroxide increased the NFκB binding activity. (iv) Expression of a reporter gene in which the chloramphenicol acetyltransferase (CAT) gene was under the control of NFκB binding sites was induced by hydrogen peroxide. These results suggest that the induction of IL‐6 mRNA is regulated by a mechanism involving reactive oxygen species and that NFκB, whose activity is sensitive to the cellular redox state, plays an important role in this induction in a fibroblastic cell line, Balb/3T3, stimulated with TNFα.

Specific decrease in the level of Hic-5, a focal adhesion protein, during immortalization of mouse embryonic fibroblasts, and its association with focal adhesion kinase

Hic‐5 is a paxillin homologue with four LIM domains in its C‐terminal region, localized mainly in focal adhesions in normal fibroblasts. Hic‐5 is also known to associate with focal adhesion kinase (FAK) or the related CAKβ, and with vinculin. In the present study, we examined changes in Hic‐5 and paxillin protein levels in primary mouse embryo fibroblasts (MEF) during mortal and immortal stages. The Hic‐5 level was markedly decreased when cells became immortalized, whereas that of paxillin was increased. The vinculin level was not changed significantly. Hic‐5 was mainly localized in focal adhesion plaques of mortal MEF but was localized in the nuclear periphery in the immortalized MEF; the number of focal adhesion plaques was decreased in these cells. Mouse Hic‐5 contains three LD domains in its N‐terminal half, and the first LD domain (LD1) appears to be involved in interaction with FAK. However, this interaction was not essential for recruitment of Hic‐5 to focal adhesions, since its subcellular localization was similar in FAK−/− cells. Forced expression of Hic‐5 decreased colony forming ability of MEF from FAK+/+ mice, but not of FAK−/− cells. These observations suggested the involvement of Hic‐5 in determination of cellular proliferative capacity in collaboration with other cytoskeletal components. J. Cell. Biochem. 76:411–419, 2000.

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.