homology 2 domain-containing inositol 5'-phosphatase; RhoGDI, Rho GDP dissociation inhibitor; ROK, Rho-dependent protein kinase; ROS, reactive oxygen species, TNFR, tumor necrosis factor receptor; SAPK, stress activating protein kinase; SH2, Src homology 2; SNARE, soluble NSF attachment protein receptor; TNF-α, tumor necorsis factor-α; VAMP, Vesicle-associated membrane protein; WASP, Wiskott-Aldrich syndrome protein A bstractPhagocytosis by inflam m atory cells is an essential step and a part of innate im m unity for protection against foreign pathogens, m icroorganism or dead cells. Phagocytosis, endocytotic events sequel to binding particle ligands to the specific receptors on phagocyte cell surface such as Fcγ recptor (FcγR ), com plem ent receptor (CR ), β-glucan receptor, and phosphatidylserine (PS) receptor, require actin assem bly, pseudopod extension and phagosom e closure. Rho GTPases (R hoA, C dc42, and R ac1) are critically involved in these processes. A brupt superoxide form ation, called as oxidative burst, occurs through NADPH oxidase complex in leukocytes following phagocytosis. NADPH oxidase com plex is com posed of m em brane proteins, p22 PHOX and gp91 PHOX , and cytosolic proteins, p40 PHOX , p47 PHOX and p67 PHOX . The cytosolic subunits and Rac-GTP are translocated to the m embrane, form ing com plete NADPH oxidase com plex with m em brane part subunits. B inding of im unoglobulin G (IgG)-and com plem ent-opsonized particles to FcγR and C R of leukocytes induces apoptosis of the cells, which m ay be due to oxidative burst and accom panying cytochrom e c release and casapase-3 activation.

Crosstalk of signaling pathways play crucial roles in cell proliferation, cell differentiation, and cell fate determination for development. In the case of ventx1.1 in Xenopus embryos, both BMP-4/Smad-1 and FGF/Xbra signaling induce the expression of neural repressor ventx1.1. However, the details of how these two pathways interact and lead to neural inhibition by ventx1.1 remain largely unknown. In the present study, Xbra directly bound to the ventx1.1 promoter region and inhibited neurogenesis in a Ventx1.1-dependent manner. Furthermore, Smad-1 and Xbra physically interacted and regulated ventx1.1 transcription in a synergistic fashion. Xbra and Smad-1 interaction cooperatively enhanced the binding of an interacting partner within the ventx1.1 promoter and maximum cooperation was achieved in presence of intact DNA binding sites for both Smad-1 and Xbra. Collectively, BMP-4/Smad-1 and FGF/Xbra signal crosstalk cooperate to activate the transcription of neural repressor ventx1.1 in Xenopus embryos. This suggests that the crosstalk between BMP-4 and FGF signaling negatively regulates early neurogenesis by synergistic activation of ventx1.1 in Xenopus embryos.

Background: RhoA GTPase is essential for integrin ␣M␤2-mediated phagocytosis. Results: Activation of Rap1 GTPase can induce phagocytosis even when RhoA is inactivated. Conclusion: Rap1 GTPase can replace the function of RhoA GTPase in phagocytosis. Significance: This might be the first observation that Rap1 and RhoA GTPases collectively regulate phagocytosis in macrophages.Phagocytosis occurs primarily through two main processes in macrophages: the Fc␥ receptor-and the integrin ␣M␤2-mediated processes. Complement C3bi-opsonized particles are known to be engulfed through integrin ␣M␤2-mediated process, which is regulated by RhoA GTPase. C3 toxin fused with Tat-peptide (Tat-C3 toxin), an inhibitor of the Rho GTPases, was shown to markedly inhibit the phagocytosis of serum (C3bi)-opsonized zymosans (SOZs). However, 8CPT-2Me-cAMP, an activator of exchange protein directly activated by cAMP (Epac, Rap1 guanine nucleotide exchange factor), restored the phagocytosis of the SOZs that was previously inhibited by the Tat-C3 toxin. In addition, a constitutively active form of Rap1 GTPase (CA-Rap1) also restored the phagocytosis that was previously reduced by a dominant negative form of RhoA GTPase (DN-RhoA). This suggests that Rap1 can replace the function of RhoA in the phagocytosis. Inversely, CA-RhoA rescued the phagocytosis that was suppressed by DN-Rap1. These findings suggest that both RhoA and Rap1 GTPases collectively regulate the phagocytosis of SOZs. In addition, filamentous actin was reduced by the Tat-C3 toxin, which was again restored by 8CPT-2Me-cAMP. Small interfering profilin suppressed the phagocytosis, suggesting that profilin is essential for the phagocytosis of SOZs. Furthermore, 8CPT-2Me-cAMP increased the co-immunoprecipitation of profilin with Rap1, whereas Tat-C3 toxin decreased that of profilin with RhoA. Co-immunoprecipitations of profilin with actin, Rap1, and RhoA GTPases were augmented in the presence of GTP␥S rather than GDP. Therefore, we propose that both Rap1 and RhoA GTPases regulate the formation of filamentous actin through the interaction between actin and profilin, thereby collectively inducing the phagocytosis of SOZs in macrophages.

B cell-activating factor belonging to the TNF family (BAFF) is primarily expressed by macrophages and dendritic cells and stimulates the proliferation, differentiation, and survival of B cells and their Ig production. In the present study, we examined the pathways by which TGF-beta1 and IFN-gamma induce BAFF expression to see if TGF-beta1 and IFN-gamma regulate B cell differentiation via macrophages. We found that TGF-beta1 stimulated mouse macrophages to express BAFF and that a typical TGF-beta signaling pathway was involved. Thus, Smad3 and Smad4 promoted BAFF promoter activity, and Smad7 inhibited it, and the BAFF promoter was shown to contain three Smad-binding elements. Importantly, TGF-beta1 enhanced the expression of membrane-bound and soluble forms of BAFF. IFN-gamma further augmented TGF-beta1-induced BAFF expression. IFN-gamma caused phosphorylation of CREB, and overexpression of CREB increased IFN-gamma-induced BAFF promoter activity. Furthermore, H89, a protein kinase A (PKA) inhibitor, abrogated the promoter activity. Neither Stat1alpha (a well-known transducing molecule of IFN-gamma) nor AG490 (a JAK inhibitor) affected BAFF expression in response to IFN-gamma. Taken together, these results demonstrate that TGF-beta1 and IFN-gamma up-regulate BAFF expression through independent mechanisms, i.e., mainly Smad3/4 and PKA/CREB, respectively.

Cyclin-dependent kinase inhibitors (CDKI) are negative regulators of cell cycle progression by binding the cyclin-CDK complex and inhibiting the CDK activity. Genetic alteration in the CDKI genes has been implicated for carcinogenesis. To test the genetic alteration in the p27 and p57 genes, KIP family CDKI genes, 30 gastric tumor-normal pairs and 8 gastric cancer cell lines were analyzed for mutations by polymerase chain reaction-single strand conformational polymorphism (PCR-SSCP). No mutation was detected in these genes although length polymorphisms in the proline-alanine repeat of the p57 gene were detected. When the p27 and p57 mRNAs were analyzed in gastric cancer cell lines by RT-PCR, the p27 mRNA was expressed considerably high in tumor cells but expression of the p57 mRNA was much low in gastric cancer cell lines compared to that of normal cells. The result suggests that inactivation of gene expression rather than mutations in the p57 gene accounts possibly for the involvement of this gene in tumorigenesis of gastric cancer. However, expression of the p27 gene seems to be essential for cell survival.