Abstract-We have recently demonstrated that a developmentally regulated zinc finger protein, basic transcription regulatory element binding protein 2 (BTEB2), is induced in neointimal smooth muscle in response to vascular injury. In this study, we investigated the molecular mechanisms regulating BTEB2 expression in vascular smooth muscle cells (SMCs) in vitro. BTEB2 mRNA expression is rapidly and persistently induced in SMCs by phorbol 12-myristate 13-acetate (PMA) and basic fibroblast growth factor. We have isolated and characterized the promoter region of the human BTEB2 gene to determine the regulatory network controlling expression of this gene in vascular SMCs. Functional studies on the BTEB2 promoter coupled to a luciferase reporter gene demonstrated activation of the promoter by PMA and basic fibroblast growth factor. Both characterization of DNA-protein complexes in vitro and site-specific mutation analysis of the BTEB2 promoter have defined a 9-bp sequence, 5Ј-CGCCCGCGC-3Ј, located at Ϫ25, as the Egr-1 binding site mediating an induction of the BTEB2 promoter activity by PMA. In addition, we show that this site mediates inducible expression through the mitogen-activated protein kinase pathways. These results indicate that BTEB2 is a target of the early-response gene Egr-1, and mitogen-activated protein kinase pathways directly or indirectly activate BTEB2 expression. Given a rapid induction of Egr-1 on stimulation with growth factors or injury, these findings may represent at least one of the molecular mechanisms underlying phenotypic modulation of smooth muscles after vascular injury. (Circ Res. 1999;85:787-795.)

Stem cells in normal tissues and cancer-initiating cells (CICs) are known to be enriched in side population (SP) cells. However, the factors responsible for the regulation of expression of ABCG2, involved in efflux of dyes, in SP cells have not been fully investigated. Here, we characterized the SP cells within diffuse-type gastric carcinoma, and examined the effects of transforming growth factor-b (TGF-b) on SP cells. Diffuse-type gastric carcinoma cells established from four independent patients universally contained SP cells between 1 and 4% of total cells, which displayed greater tumorigenicity than non-SP cells did. TGF-b repressed the transcription of ABCG2 through direct binding of Smad2/3 to its promoter/enhancer, and the number of SP cells and the tumor-forming ability of cancer cells were decreased by TGF-b, although ABCG2 is not directly involved in the tumor-forming ability of SP cells. Cancer cells from metastatic site expressed much higher levels of ABCG2 and included a greater percentage of SP cells than parental cancer cells did. SP cells are thus responsible for the progression of diffuse-type gastric carcinoma, and TGF-b negatively contributes to maintain the CICs within the cancer.

During the process of tumor progression and clinical treatments, tumor cells are exposed to oxidative stress. Tumor cells are frequently resistant to such stress by producing antiapoptotic signaling, including activation of Src family kinases (SFKs), although the molecular mechanism is not clear. In an attempt to identify the SFK-binding proteins selectively phosphorylated in gastric scirrhous carcinoma, we identified an uncharacterized protein, C9orf10. Here we report that C9orf10 (designated Ossa for oxidative stressassociated Src activator) is a novel RNA-binding protein that guards cancer cells from oxidative stressinduced apoptosis by activation of SFKs. Exposure to oxidative stress such as UV irradiation induces the association of Ossa/C9orf10 with regulatory domains of SFKs, which activates these kinases and causes marked tyrosine phosphorylation of C9orf10 in turn. Tyrosine-phosphorylated Ossa recruits p85 subunits of phosphatidylinositol 3-kinase (PI3-kinase) and behaves as a scaffolding protein for PI3-kinase and SFKs, which activates the Akt-mediated antiapoptotic pathway. On the other hand, the carboxyl terminus of Ossa has a distinct function that directly binds RNAs such as insulin-like growth factor II (IGF-II) mRNA and promotes the extracellular secretion of IGF-II. Our findings indicate that Ossa is a dualfunctional protein and might be a novel therapeutic target which modulates the sensitivity of tumors to oxidative stress.

These findings suggest that recovery of muscle fibres atrophied by disuse involves not only growth of atrophied muscle fibres, but also regeneration of muscle fibres. Cross-sectional areas recovery of atrophied muscle fibres thus continues after increases of muscle mass.

Cancer cells undergo multi-step processes in obtaining the ability to metastasize, and are constantly exposed to signals that induce apoptosis. Acquisition of anti-apoptotic properties by cancer cells is important for metastasis, and recent studies suggest that transforming growth factor (TGF)-β promotes the survival of certain types of cancer cells. Here, we found that in highly metastatic breast cancer cells, JygMC(A), JygMC(B) and 4T1, TGF-β ligands were produced in autocrine fashion. Pharmacological inhibition of endogenous TGF-β signalling by a TGF-β type I receptor kinase inhibitor in serum-free conditions increased the expression of BH3-only protein, Bim (also known as Bcl2-like 11) in JygMC(A) and JygMC(B) cells, and caused apoptotic cell death. We also found that induction of Bim by TGF-β was not observed in Foxc1 knocked-down cancer cells. These findings suggest that TGF-β plays a crucial role in the regulation of survival of certain types of cancer cells through the TGF-β-Foxc1-Bim pathway, and that specific inhibitors of TGF-β signalling might be useful as apoptosis inducers in breast cancer cells.