To understand the molecular events coupling between cell proliferation and differentiation by elucidating genes essential for the process, we conducted a large scale gene expression analysis of an in vitro osteoclastogenesis system consisting of recombinant RANKL and mouse RAW264 cells. The entire process leading to the formation of tartrate resistant acid phosphatase-positive multinucleated cells takes 3 days and plates become fully covered with multinucleated cells at 4 days. Microarray probing at eight time points revealed 635 genes that showed greater than 2-fold differential expression for at least one time point and they could be classified into six groups by the "k-means" clustering analysis. Among a group of 106 early inducible genes (within 2-5 h after RANKL stimulation), four genes including NFAT2 were identified as genes whose enhanced expressions were fairly correlated with an efficient induction of matured osteoclasts. Moreover, cyclosporin A significantly suppressed the multinucleated cell formation accompanying the reduction of the nuclear localization of NFAT2. When the expression of NFAT2 was suppressed by introducing antisense NFAT2, multinucleated cell formation was severely hampered. Functional analysis thus combined with gene analysis by microarray technology elucidated a key role of NFAT2 in osteoclastogenesis in vitro.Specific factors/regulatory genes playing essential roles for cellular differentiation have been identified in various systems, and they have been shown to exert their effects eventually through the induction or repression of certain groups of genes (1-4). Therefore, gene expression profiling based on fine statistical analysis in addition to an elucidation of key factors/ genes might be essential to understand the molecular mechanisms underlying the differentiation process of a certain cell type. Fortunately, recent advances in the technology for assaying RNA in a highly parallel fashion (5-7), coupled with the completion/progress of several mammalian genome projects, make the approach feasible if a refined system is available. Here, we describe the broad outlines of gene expression during osteoclastogenesis in vitro, in particular during the initial stage, and explain the identification and characterization of genes essential for osteoclastogenesis on the basis of profiling characteristics. A similar approach using a different cell system was reported recently (8).Osteoclasts are multinucleated (MN) 1 giant cells and present only in bone with the capacity to resorb mineralized tissues (9). They were reported to be formed by fusion of mononuclear precursor cells derived from colony-forming unit granulocyte macrophages (CFU-GM) and branch from the monocyte-macrophage lineage during the early stage of the differentiation process (9, 10). Recently, a key factor responsible for initiating this differentiation process was identified and named receptor activator of NFB ligand (RANKL) (or osteoclast differentiation factor (ODF)/TNF-related activation-induced cytokine TRANCE) (11-13)...

The gap junctional intercellular communication mediated by Cx43 plays indispensable roles in both germ line development and postnatal folliculogenesis. In this study, we focused on the e¡ect of follicle-stimulating hormone (FSH) on the Cx43 protein in rat primary granulosa cells and found that FSH stimulation elevated the phosphorylation in addition to the protein level of Cx43. Serine residues in the carboxyl-terminal region were exclusively phosphorylated in this system and we identi¢ed Ser365, Ser368, Ser369 and Ser373 as major phosphorylation sites by FSH stimulation. A Cx43 variant containing mutations at all these serine residues was found to severely reduce dye transfer activity when assayed in HeLa cells. The present study revealed a novel regulatory mechanism of Cx43-mediated gap junctional intercellular communication through phosphorylation in the carboxyl-terminus.

Abstract.Changes in activities and levels of superoxide dismutase (SOD, EC 1.15.1.1) isoenzymes were studied during air adaptation of submerged rice (Oryza sativa L.) seedlings. Seeds were germinated for 6 d in the dark under water (submerged), and then for another day in air (airadapted). For a control, seeds were germinated for 6 d throughout in air (aerobic). Staining for activity of SOD of shoot extracts showed a total of five major distinct forms of SOD: one mitochondrial Mn enzyme (mtSOD) and four CuZn enzymes, one of which was plastidic (plSOD) and the other three cytosolic (cytSOD). Activity of plSOD was much lower in submerged seedlings than in aerobic controls and increased after exposure to air. In contrast, mtSOD activity in submerged seedlings was as high as that found in aerobic controls, and did not increase upon exposure to air. One of the cytSODs showed responses similar to those of plSOD. The activities of another two cytSODs were slightly lower in submerged seedlings than those in aerobic controls, but decreased after 24 h of air adaptation. Western blot analysis revealed that these changes in activities of SODs are due to changes in the levels of their enzyme proteins. We also followed changes in the levels of cytochrome c and ferredoxin-NADP + reductase (EC 1.6.99.4) as indices of the development of mitochondria and plastids, respectively. Organelle SODs were always present at higher levels than would be expected in view of the development of the electron-transport systems of the corresponding organelles during submergence and the subsequent air-adaptation period.

No abstract

No abstract