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DNA methylation at CpG sequences is involved in tissue-specific and developmentally regulated gene expression. The Sry (sex-determining region on the Y chromosome) gene encodes a master protein for initiating testis differentiation in mammals, and its expression is restricted to gonadal somatic cells at 10.5-12.5 days postcoitum (dpc) in the mouse. We found that in vitro methylation of the 5'-flanking region of the Sry gene caused suppression of reporter activity, implying that Sry gene expression could be regulated by DNA methylation-mediated gene silencing. Bisulfite restriction mapping and sodium bisulfite sequencing revealed that the 5'-flanking region of the Sry gene was hypermethylated in the 8.5-dpc embryos in which the Sty gene was not expressed. Importantly, this region was specifically hypomethylated in the gonad at 11.5 dpc, while the hypermethylated status was maintained in tissues that do not express the Sry gene. We concluded that expression of the Sry gene is under the control of an epigenetic mechanism mediated by DNA methylation. [ABSTRACT FROM AUTHOR]

Investigates the role of prostaglandins E[sub 2] (PGE) in mouse mastocytoma P-815 cell adhesion to extracellular matrix proteins in vitro. Critical mediators of the PGE; Level and cell adhesion of cAMP IN PGE[sub 2]; Augmentation of EP4-mediated cAMP synthesis.

Accumulating evidence suggests that IgE-mediated activation of mast cells occurs even in the absence of antigen, which is referred to as ‘monomeric IgE’ responses. Although monomeric IgE was found to induce a wide variety of responses, such as up-regulation of the Fc∈RI, survival, cytokine production, histamine synthesis, and adhesion to fibronectin, it remains to be clarified how mast cells are activated in the absence of antigen. It has been controversial whether monomeric IgE responses are mediated by a similar signaling mechanism to antigen stimulation, although recent studies suggest that IgE can induce the Fc∈RI aggregation even in the absence of antigen. In this study, we focused on the role of conventional protein kinase C (cPKC), since this response is suppressed by a specific inhibitor for cPKC. Monomeric IgE-induced Ca2+ influx was not observed in a mouse mastocytoma cell line, which lacks the expression of PKCβII, although Ca2+ influx induced by cross-linking of the Fc∈RI was intact. Transfection of PKCβII cDNA was found to restore the Ca2+ influx induced by monomeric IgE in this cell line. Furthermore, the dominant negative form of PKβII (PKβII/T500V) significantly suppressed the Ca2+ influx, histamine synthesis, and interleukin-6 production in another mouse mast cell line, which is highly sensitive to monomeric IgE. Expression of PKCβII/T500V was found not to affect the antigen-induced responses. These results suggest that PKβII plays a critical rote in monomeric IgE responses, but not in antigen responses. [ABSTRACT FROM AUTHOR]

Methylation of DNA is involved in tissue-specific gene control, and establishment of DNA methylation pattern in the genome is thought to be essential for embryonic development. Three isoforms of Dnmt1 (DNA methyltransferase 1) transcripts, Dnmt1s, Dnmt1o, and Dnmt1p, are produced by alternative usage of multiple first exons. Dnmt1s is expressed in somatic cells. Dnmt1p is found only in pachytene spermatocytes, whereas Dnmt1o is specific to oocytes and preimplantation embryos. Here we determined that there is a tissue-dependent differentially methylated region (T-DMR) in the 5' region of Dnmt1o but not in that of the Dnmt1s/1p. The methylation status of the Dnmt1o T-DMR was distinctively different in the oocyte from that in the sperm and adult somatic tissues and changed at each stage from fertilization to blastocyst stage, suggesting that active methylation and demethylation occur during preimplantation development. The T-DMR was highly methylated in somatic cells and embryonic stem cells. Analysis using Dnmt-deficient embryonic stem cell lines revealed that Dnmt1, Dnmt3a, and Dnmt3b are each partially responsible for maintenance of methylation of Dnmt1o T-DMR. In particular, there are compensatory and cooperative roles between Dnmt3a and Dnmt3b. Thus, the regulatory region of Dnmt1o, but not of Dnmt1s/1p, appeared to be a target of DNA methylation. The present study also suggested that the DNA methylation status of the gene region dynamically changes during embryogenesis independently of the change in the bulk DNA methylation status. [ABSTRACT FROM AUTHOR]

To assess the structural requirements for Gs coupling by prostaglandin E receptors (EPs), the Gs-coupled EP2 and Gi-coupled EP3β receptors were used to generate hybrid receptors. Interchanging of the whole i2 loop and its N-terminal half (i2N) had no effect on the binding of both receptors expressed in HEK293 cells. Agonist-induced cAMP formation was observed in wild type EP2 but not in the i2 loop- or i2N-substituted EP2. Wild type EP3β left cAMP levels unaffected, whereas i2 loop- and i2N-substituted EP3 gained agonist-induced adenylyl cyclase stimulation. In EP2, the ability to stimulate cAMP formation was lost by mutation of Tyr143 into Ala but retained by mutations into Phe, Trp, and Leu. Consistent with this observation, substitution of the equivalent His140 enabled EP3β to stimulate cAMP formation with the rank order of Phe > Tyr > Trp > Leu. The point mutation of His140 into Phe was effective in another EP3 variant in which its C-terminal tail is different or lacking. Simultaneous mutation of the adjacent TFO141 to Ala but not at the following Tyr142 weakened the acquired ability to stimulate cAMP levels in the EP3 mutant. Mutation of EP2 at adjacent Phe144 to Ala but not at Tyr145 reduced the efficiency of agonist-induced cAMP formation. In Chinese hamster ovary cells stably expressing Gs-acquired EP3 mutant, an agonist-dependent cAMP formation was observed, and pertussis toxin markedly augmented cAMP formation. These results suggest that a cluster of hydrophobic aromatic amino acids in the i2 loop plays a key role for Gs coupling. [ABSTRACT FROM AUTHOR]

Accumulating evidence suggests that IgE-mediated activation of mast cells occurs even in the absence of antigen, which is referred to as "monomeric IgE" responses. Although monomeric IgE was found to induce a wide variety of responses, such as up-regulation of the FcepsilonRI, survival, cytokine production, histamine synthesis, and adhesion to fibronectin, it remains to be clarified how mast cells are activated in the absence of antigen. It has been controversial whether monomeric IgE responses are mediated by a similar signaling mechanism to antigen stimulation, although recent studies suggest that IgE can induce the FcepsilonRI aggregation even in the absence of antigen. In this study, we focused on the role of conventional protein kinase C (cPKC), since this response is suppressed by a specific inhibitor for cPKC. Monomeric IgE-induced Ca(2+) influx was not observed in a mouse mastocytoma cell line, which lacks the expression of PKCbetaII, although Ca(2+) influx induced by cross-linking of the FcepsilonRI was intact. Transfection of PKCbetaII cDNA was found to restore the Ca(2+) influx induced by monomeric IgE in this cell line. Furthermore, the dominant negative form of PKCbetaII (PKCbetaII/T500V) significantly suppressed the Ca(2+) influx, histamine synthesis, and interleukin-6 production in another mouse mast cell line, which is highly sensitive to monomeric IgE. Expression of PKCbetaII/T500V was found not to affect the antigen-induced responses. These results suggest that PKCbetaII plays a critical role in monomeric IgE responses, but not in antigen responses.