Membrane fusion is an essential step in the encounter of two nuclei from sex cells-sperm and egg-in fertilization. However, aside from the involvement of two molecules, CD9 and Izumo, the mechanism of fusion remains unclear. Here, we show that spermegg fusion is mediated by vesicles containing CD9 that are released from the egg and interact with sperm. We demonstrate that the CD9 ؊/؊ eggs, which have a defective sperm-fusing ability, have impaired release of CD9-containing vesicles. We investigate the fusion-facilitating activity of CD9-containing vesicles by examining the fusion of sperm to CD9 ؊/؊ eggs with the aid of exogenous CD9-containing vesicles. Moreover, we show, by examining the fusion of sperm to CD9 ؊/؊ eggs, that hamster eggs have a similar fusing ability as mouse eggs. The CD9-containing vesicle release from unfertilized eggs provides insight into the mechanism required for fusion with sperm.

Chondrocytes are critical components for the precise patterning of a developing skeletal framework and articular joint formation. Sox9 is a key transcription factor that is essential for chondrocyte differentiation and chondrocyte-specific gene expressions; however, the precise transcriptional activation mechanism of Sox9 is not fully understood. Here we demonstrate that Sox9 utilizes a cAMP-response element-binding protein (CREB)-binding protein (CBP)/p300 to exert its effects. Sox9 associates with CBP/p300 in the chondrosarcoma cell line SW1353 via its carboxyl termini activation domain in a cell type-specific manner. In promoter assays, CBP/p300 enhances Col2a1, which encodes cartilagespecific type II collagen gene promoter activity via Sox9. Chromatin immunoprecipitation shows that p300 is bound to the Col2a1 promoter region. Furthermore, the CBP/Sox9 complex disrupter peptide suppresses Col2a1 gene expression and chondrogenesis from mesenchymal stem cells. These data demonstrate that CBP and p300 function as co-activators of Sox9 for cartilage tissue-specific gene expression and chondrocyte differentiation.

Pancreatic cancer is the most lethal of all solid tumors partially because of its chemoresistance. Although gemcitabine is widely used as a first selected agent for the treatment of this disease despite low response rate, molecular mechanisms of gemcitabine resistance in pancreatic cancer still remain obscure. The aim of this study is to elucidate the mechanisms of gemcitabine resistance. The 81-fold gemcitabine resistant variant MiaPaCa2-RG was selected from pancreatic cancer cell line MiaPaCa2. By microarray analysis between MiaPaCa2 and MiaPaCa2-RG, 43 genes (0.04%) were altered expression of more than 2-fold. The most upregulated gene in MiaPaCa2-RG was ribonucleotide reductase M1 subunit (RRM1) with 4.5-fold up-regulation. Transfection with RRM1-specific RNAi suppressed more than 90% of RRM1 mRNA and protein expression. After RRM1-specific RNAi transfection, gemcitabine chemoresistance of MiaPaCa2-RG was reduced to the same level of MiaPaCa2. The 18 recurrent pancreatic cancer patients treated by gemcitabine were divided into 2 groups by RRM1 levels. There was a significant association between gemcitabine response and RRM1 expression (p 5 0.018). Patients with high RRM1 levels had poor survival after gemcitabine treatment than those with low RRM1 levels (p 5 0.016). RRM1 should be a key molecule in gemcitabine resistance in human pancreatic cancer through both in vitro and clinical models. RRM1 may have the potential as predictor and modulator of gemcitabine treatment. ' 2006 Wiley-Liss, Inc.Key words: gemcitabine; pancreatic cancer; drug resistance; RRM1; microarray Pancreatic cancer remains one of the most malignant cancers. Although surgery is the only curative treatment currently available, over 80% of patients have advanced regional disease or distant metastasis at the time of diagnosis and less than 20% of the patients are candidates for resection.1 Therefore, chemotherapy, radiation or a combination of these therapies most commonly plays an important role in pancreatic cancer treatment. They have not had a significant impact on survival rates in recent decades, however, despite many clinical trials.

CD9 is a tetraspan protein that associates with several ␤1 integrins, including ␣6␤1. Because ␣6␤1 is present on murine eggs and interacts with the sperm-surface glycoprotein ADAM 2 (fertilin ␤), we first asked whether CD9 is present on murine eggs and whether it functions in sperm-egg binding and fusion. CD9 is present on the plasma membrane of oocytes in the ovary as well as on eggs isolated from the oviduct. The anti-CD9 mAb, JF9, potently inhibits sperm-egg binding and fusion in vitro in a dose-dependent manner. JF9 also disrupts binding of fluorescent beads coated with native fertilin or a recombinant fertilin ␤ disintegrin domain. (Both ligands bind to the egg via ␣6␤1.) Immunohistochemistry showed that CD9 is undetectable in the uterine epithelium, appears basolaterally and as prominent apical patches on the epithelium in the region between the uterus and the oviduct, and then persists apically in the oviduct. The integrin ␣6A subunit is found in similar apical patches in the region between the uterus and oviduct, but is confined to the basal aspect of the epithelium in the uterus and oviduct. Hence, ␣6A and CD9 both are expressed on the apical epithelial surface at the uterine-oviduct junction. These findings correlate with the observation that fertilin ␤ ''knockout'' sperm traverse the uterus but do not progress into the oviduct, contributing to the infertility of fertilin ␤ ؊͞؊ male mice. Our results suggest that high-avidity binding between fertilin ␤ (ADAM 2) and ␣6␤1 requires cooperation between ␣6␤1 and CD9. Such cooperation may assist sperm passage into the oviduct as well as spermegg interactions.

Activating transcription factor (ATF) 5 is a transcription factor belonging to the ATF/cAMP-response element-binding protein gene family. We previously reported that ATF5 mRNA expression increased in response to amino acid limitation. The ATF5 gene allows transcription of mRNAs with at least two alternative 5-untranslated regions (5-UTRs), 5-UTR␣ and 5-UTR␤, derived from exon1␣ and exon1␤. 5-UTR␣ contains highly conserved sequences, in which the upstream open reading frames (uORFs) uORF1 and uORF2 are found in many species. This study was designed to investigate the potential role of 5-UTRs in translational control. These 5-UTRs differentially determined translation efficiency from mRNA. The presence of 5-UTR␣ or 5-UTR␤ represses translation from the downstream ATF5 ORF. Moreover, 5-UTR␣-regulated translational repression is released by amino acid limitation or NaAsO 2 exposure. This release was not seen for 5-UTR␤. Mutation of uAUG2 in the uORF2 of 5-UTR␣ restored the basal expression and abolished the positive regulation by amino acid limitation or arsenite exposure. We demonstrated that phosphorylation of eukaryotic initiation factor 2␣ was required for amino acid limitation-induced translational regulation of ATF5. Furthermore, arsenite exposure activated the exogenously expressed hemeregulated inhibitor kinase and induced the phosphorylation of eukaryotic initiation factor 2␣ in nonerythroid cells. These results suggest that translation of ATF5 is regulated by the alternative 5-UTR region of its mRNA, and ATF5 may play a role in protecting cells from amino acid limitation or arsenite-induced oxidative stress.Activating transcription factor (ATF) 2 -5 (formerly designated ATFx) is a transcription factor of the cAMP-response element-binding protein (CREB)/ATF family that was first identified as a protein that binds to the lipopolysaccharide-response element (GPE-1) on the granulocyte colony-stimulating factor (CSF3) gene along with C/EBP␥ (1). It contains a DNAbinding and dimerization domain (bZIP domain) and regulates processes that are involved in cellular differentiation (2, 3), the cell cycle (4), and apoptosis (5, 6). ATF5 represses cAMP-induced transcription in cultured cells (4) and is shown to inhibit apoptosis (6). Angelastro et al. (2) demonstrated that ATF5 inhibits CRE-mediated expression of neural genes and neural differentiation. Cdc34 is the G 2 checkpoint gene, and ATF5 is a target of Cdc34-dependent ubiquitin-mediated proteolysis (4), expression of which is affected by the cell cycle. Recently, Monaco et al. (7) showed that ATF5 is widely expressed in carcinomas, and interference with its function caused apoptotic cell death of neoplastic breast cell lines. This suggests that ATF5 may be a target for cancer therapy and that studies of the mechanism by which ATF5 expression is regulated might be important in the investigation of treatments for cancer.Mammalian cells have the ability to alter their gene expression to adapt to a variety of environmental stresses, including nutrient limitation, ...

Japan. SinarySerum interleukin 6 (IL-6) levels were measured in 75 patients with lung cancer and in 20 patients with benign lung diseases. IL-6 was detectable in 29 patients with lung cancer (39%), but was not detectable in any of the patients with benign lung diseases. Serum C-reactive protein levels and plasma fibrinogen levels were significantly higher and serum albumin concentration was significantly lower in lung cancer patients with detectable serum IL-6 levels than in those without detectable serum IL-6 levels and in patients with benign lung diseases. On the other hand, no significant difference was observed in blood platelet counts in these three groups. Moreover, serum IL-6 levels were not significantly different in lung cancer patients with or without clinically demonstrated distant metastasis. These results suggest that IL-6 may be a mediator of various reactions including an inflammatory response in lung cancer patients.Keywords interleukin 6; lung cancer; C-reactive protein; fibrinogen; cachexia; inflammatory response Interleukin 6 (IL-6) is known as a multifunctional cytokine which plays a central role in the host defence mechanism by regulating immune responses, haematopoiesis and acutephase reactions (Kishimoto, 1989). Recently, much attention has been focused on its role in the pathogenesis and progression of various malignancies. It has been reported that IL-6 is an autocrine growth factor for renal cell carcinoma cells (Miki et al., 1989;Koo et al., 1992) and that IL-6 is produced by other non-haematopoietic tumour cells, including bladder carcinoma (Rawle et al., 1989), ovarian carcinoma (Watson et al., 1990) and glioblastoma (Meir et al., 1990). In animal systems, IL-6 appears to have an important role in mediating cancer cachexia (Strassmann et al., 1992). Moreover, elevation of the serum IL-6 level is an adverse prognositic factor in patients with metastatic renal cell carcinoma (Blay et al., 1992). However, there is still little information about the role of IL-6 in vivo in patients with various malignancies.In patients with lung cancer, we have already reported that malignant pleural effusions contain IL-6 (Yanagawa et al., 1992), and we (Mizuno et al., 1994) and others (Matsuguchi et al., 1991) have reported that several lung cancer cell lines constitutively produce IL-6 in vitro. To clarify further the role of IL-6, we examined serum levels of IL-6 in patients with lung cancer. The relationship between serum IL-6 levels and the characteristics of the patients was also analysed. Enzyme immunoassay of IL-6 Serum levels of IL-6 were assayed essentially as described previously (Yanagawa et al., 1992). In brief, microtitre plates (Nunc, Naperville, IL, USA) were coated with anti-IL-6 monoclonal antibody in 100 per well phosphate-buffered saline (PBS). After overnight incubation at 4°C, the wells were blocked with 0.1% bovine serum albumin (BSA) in PBS and washed three times. Volumes of 200 MI of test samples were added to the duplicate wells. The plates were incubated at 37C for 24 h. Aft...

Accumulated evidence indicates that hypoxia activates collagen synthesis in tissues. To explore the molecular mechanism of activation, we screened genes that are up-regulated or down-regulated by hypoxia. Fibroblasts isolated from fetal rat lung were cultured under hypoxia. Differential display technique showed that the mRNA level of prolyl 4-hydroxylase (PH) ␣(I), an active subunit that catalyzes the oxygen-dependent hydroxylation of proline residue in procollagen, increased 2-3-fold after an 8-h exposure to hypoxia. This elevated level was maintained over 40 h and returned to the basal level after reoxygenation. The transcription rate, protein level, and hydroxyproline content (an indicator of the prolyl hydroxylation) were all elevated by hypoxic culture. Analysis of the promotor region of PH␣(I) gene indicated that a motif similar to hypoxia-responsive element (HRE) of hypoxia-inducible genes such as erythropoietin, was identified within a 120-base pair sequence upstream of the transcription start site. Luciferase reporter assay and mutational analysis showed that a site similar to the HRE in this motif is functionally essential to hypoxic response. Electrophoretic mobility shift assay revealed that hypoxia-inducible factor-1 was stimulated and bound to the PH␣(I) HRE upon hypoxic challenge. Our results indicate that PH␣(I), an essential enzyme for collagen synthesis, is a target gene for hypoxia-inducible factor-1.Restricted oxygen availability is a feature of many physiologic and pathologic conditions, including high altitude residence, fetal development in the uterus, pulmonary fibrosis, wounded tissue, and neoplasm (1). Systemic and cellular responses to reduced oxygen tension (hypoxia) are initiated by activation and/or inactivation of gene expression. Hypoxia-inducible factor-1 (HIF-1), 1 which was originally found to be a critical mediator for the inducible expression of the erythropoietin (Epo) gene by hypoxia (2), is a heterodimer composed of HIF-1␣ and arylhydrocarbon receptor nuclear translocator (ARNT). HIF-1␣ and ARNT retain a basic helix-loop-helix domain and a Per-ARNT/aryl hydrocarbon receptor Sim domain in their N termini (2). Hypoxia induces stabilization of HIF-1␣ (3), heterodimerization of HIF-1␣ and ARNT (4), and the binding of the heterodimer to the hypoxia-responsive element (HRE) in the regulatory region of the target genes with the transcriptional coactivator p300/CREB-binding protein (5). Although posttranscriptional mechanisms may contribute to the induction of hypoxia-sensitive genes, activation of the HIF-1 complex is an important step leading to hypoxia-mediated induction of glycolytic enzymes (6 -9), Epo (2), vascular endothelial growth factor (10), and tyrosine hydroxylase (11).In the remodeling of the small muscular pulmonary artery observed in hypoxia-induced pulmonary hypertension, type I collagen is actively synthesized and accumulated in the media and the adventitia of the artery (12). Recent studies have revealed that in vivo exposure of rats to hypoxia increases prolyl ...

Little is yet known about the biological and biochemical properties of the disintegrin-like domains of ADAM (a disintegrin and metalloprotease) proteins. Mouse ADAM 2 (mADAM 2; fertilin ␤) is a sperm surface protein involved in murine fertilization. We produced recombinant proteins containing the disintegrin-like domain of mADAM 2 in both insect cells and in bacteria. The protein produced in insect cells (baculo D؉C) contained a signal sequence followed by the disintegrin-like and cysteine-rich domains; it was purified from the medium of recombinant baculovirus-infected cells. A bacterial construct containing the disintegrin-like domain was produced in Escherichia coli as a glutathione S-transferase chimera. Baculo D؉C, as well as the D domain of the bacterial construct (released with thrombin), bound to the microvillar surface of murine eggs. Using concentrations in the range of 1 to 5 M, both recombinant proteins strongly inhibited sperm-egg binding and fusion; the baculovirus-produced protein exhibited a somewhat greater extent of inhibition (ϳ75 versus ϳ55% maximal inhibition). Substitution of alanine for each of the five charged residues within the disintegrin loop of mADAM 2 revealed a critical importance for the aspartic acid at position nine. Binding of both recombinant proteins to the egg was inhibited by the function blocking anti-␣ 6 monoclonal antibody, GoH3, but not by a nonfunctionblocking anti-␣ 6 monoclonal antibody. Binding was also inhibited by a peptide analogue of, and with an antibody against, the disintegrin loop of mADAM 2. ADAMs1 are a large group of type I integral membrane glycoproteins that contain a disintegrin and a metalloprotease domain (1, 2). Following their metalloprotease and disintegrinlike domains, they contain a cysteine-rich domain, an EGF repeat, a transmembrane domain, and a cytoplasmic tail. Their closest relatives are the P-III snake venom metalloproteases (SVMPs), which are secreted proteins that contain a disintegrin-like and a metalloprotease domain as well as a cysteinerich domain. P-II SVMPs contain metalloprotease and disintegrin domains but lack the cysteine-rich (and other) domains.The disintegrin domains of the P-II SVMPs have a 13-amino acid loop containing, at their tips, sequences such as RGD (kistrin and echistatin), KGD (barbourin), and MVD (atrolysin E). Several P-II snake disintegrins have been shown to bind to the platelet integrin, ␣ IIb ␤ 3, thereby preventing binding of fibrinogen and inhibiting platelet aggregation. Inhibition of platelet aggregation accounts, in part, for the severe hemorrhagic response in snakebite victims. The disintegrin-like domains of P-III SVMPs differ from their P-II counterparts in having two additional cysteine residues and a 14-residue predicted loop that aligns with the 13-amino acid "RGD" loop found in the P-II disintegrins. One of the additional cysteine residues is near the center of the disintegrin loop. The disintegrin-like domain of the P-III SVMP atrolysin A has been characterized. Atrolysin A purified from snake ...