Bradykinin-induced translocation of protein kinases C in neuroblastoma NCB-20 cell: dependence on 1,2-diacylglycerol content and free calcium

Bradykinin (BK)-induced production of 1,2-diacylglycerol (1,2-DG) and translocation of protein kinases C (PKCs) were examined in neuroblastoma-derived hybrid NCB-20 cells. Mass analysis of 1,2-DG exhibited a biphasic increase by 1 microM BK stimulation: the first transient phase and the second broad sustained phase. Among three subspecies of PKC expressed in these cells, types II and III were observed to translocate from cytosol to membrane in response to BK as well as PBt2 by Western blotting analysis. Type II translocated more rapidly and distinctly than type III. However, after treatment with quin 2/AM, the second phase of 1,2-DG formation completely disappeared and PKCs translocation by BK or PBt2 was completely abolished. BK-induced IP3 (1,4,5) formation was temporally consistent with the first transient phase of 1,2-DG formation. These findings suggest that PKCs translocation by BK stimulation is caused by 1,2-DG produced not only via phosphoinositide metabolism, but via other phospholipid breakdown which is Ca2+-dependent.     

ADAMTS9 activation by interleukin 1β via NFATc1 in OUMS-27 chondrosarcoma cells and in human chondrocytes

ADAMTS9 is a member of the disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) genes, with aggrecan-degrading activity. It has also been characterized to be reactive and highly activated ADAMTS by IL-1β in both chondrosarcoma cells and human chondrocytes (Demircan et al. Arthritis Rheum 52:1451–1460, 2005). In order to understand the regulation of ADAMTS9 gene expression a functional 3.0 kb human ADAMTS9 promoter has been cloned and characterized. A sequence analysis of the promoter revealed the presence of putative binding sites for Nuclear Factor of Activated T cells (NFAT), which is commonly found in the ADAMTS4 and ADAMTS5 promoters. NFATc1 was up-regulated in an activated form by IL-1β in human chondrocytes. The IL-1β inducible ADAMTS9 expression was inhibited by NFAT inhibitors, FK506 and 11Arg (11R)-VIVIT. Furthermore, direct binding of NFATc1 on distal and proximal promoters of ADAMTS9 was demonstrated by a chromatin immunoprecipitation assay. Promoter-reporter assays supported those results. These findings may provide a better understanding of the regulation of ADAMTS9 expression induced by inflammatory cytokines.     

Monoclonal antibody to galactosylceramide: discrimination of structural difference in the ceramide moiety

A mouse monoclonal antibody (mAb) was developed against monohexaosylceramide. This mAb differentially reacted on thin-layer chromatograms with 3 types of galactosylceramide (GalCer) obtained from bovine brain. Structural analysis of the 3 glycolipids revealed that they consisted of the same galactose and sphingosine but of apparently different fatty acids. Among the GalCers, the mAb reacted with teh two GalCers which contained alpha-hydroxy fatty acids, but not with GalCer composed of nonhydroxy fatty acids. These findings suggest that not only that the mAb discriminated the fatty acid composition in the ceramide moiety of GalCer, but also that the ceramide structure defines the immunological epitope as it is known to do for the carbohydrate moiety of glycosphingolipid.