[Show abstract][Hide abstract] ABSTRACT: Diacylglycerol kinase (DGK) is involved in the regulation of lipid-mediated signal transduction through the metabolism of a second messenger diacylglycerol. Of the DGK family, DGKζ, which contains a nuclear localization signal, localizes mainly to the nucleus but translocates to the cytoplasm under pathological conditions. However, the detailed mechanism of translocation and its functional significance remain unclear. To elucidate these issues, we used a proteomic approach to search for protein targets that interact with DGKζ. Results show that nucleosome assembly protein (NAP) 1-like 1 (NAP1L1) and NAP1-like 4 (NAP1L4) are identified as novel DGKζ binding partners. NAP1Ls constitutively shuttle between the nucleus and the cytoplasm in transfected HEK293 cells. The molecular interaction of DGKζ and NAP1Ls prohibits nuclear import of DGKζ because binding of NAP1Ls to DGKζ blocks import carrier proteins, Qip1 and NPI1, to interact with DGKζ, leading to cytoplasmic tethering of DGKζ. In addition, overexpression of NAP1Ls exerts a protective effect against doxorubicin-induced cytotoxicity. These findings suggest that NAP1Ls are involved in a novel molecular basis for the regulation of nucleocytoplasmic shuttling of DGKζ and provide a clue to examine functional significance of its translocation under pathological conditions.
Experimental Cell Research 12/2011; 317(20):2853-63. DOI:10.1016/j.yexcr.2011.09.014 · 3.37 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Diacylglycerol kinase (DGK) converts diacylglycerol (DG) to phosphatidic acid, both of which act as second messengers to mediate a variety of cellular mechanisms. Therefore, DGK contributes to the regulation of these messengers in cellular signal transduction. Of DGK isozymes cloned, DGKzeta is characterized by a nuclear localization signal that overlaps with a sequence similar to the myristoylated alanine-rich C-kinase substrate. Previous studies showed that nuclear DG is differentially regulated from plasma membrane DG and that the nuclear DG levels fluctuate in correlation with cell cycle progression, suggesting the importance of nuclear DG in cell cycle control. In this connection, DGKzeta has been shown to localize to the nucleus in fully differentiated cells, such as neurons and lung cells, although it remains elusive how DGK behaves during the cell cycle in proliferating cells. Here we demonstrate that DGKzeta localizes to the nucleus during interphase including G1, S, and G2 phases and is associated with chromatin although it dissociates from condensed chromatin during mitotic phase in NIH3T3 cells. Furthermore, this localization pattern is also observed in proliferating spermatogonia in the testis. These results suggest a reversible association of DGKzeta with histone or its related proteins in cell cycle, plausibly dependent on their post-translational modifications.
[Show abstract][Hide abstract] ABSTRACT: Total hip replacement can be complicated by periprosthetic osteolysis. Monocytes/macrophages play a major role in the formation of the foreign body granulomas induced by wear debris. We hypothesized that periprosthetic monocytes/macrophages do not only accelerate inflammatory and osteoclast-mediated osteolytic processes, but also resorb periprosthetic bone directly by themselves. This study was designed to evaluate the osteolytic potential in vitro of monocytes/macrophages derived from bone marrow.
Monocytes/macrophages were produced by filtration of rat bone marrow cells, followed by culture in the presence of macrophage-colony stimulating factor (M-CSF). Monocyte/macrophage properties were ascertained using immunocytochemistry and phagocytic activity. Osteolytic cytokines and extracellular matrix degrading proteinases were quantified at the mRNA level.
Adherent cell fraction was immunoreactive for the monocyte/macrophage specific marker CD68 and active in the phagocytosis of carbon particles up to 72 h. They also showed immunoreactivity to cathepsin K, IL-1beta, IL-6, and M-CSF, but mostly did not react to TRAP. mRNA levels of osteolytic cytokines and extracellular matrix degrading proteinases were enhanced, but that of RANKL were not. Monocytes/macrophages resorbed dentine discs and carbonated calcium phosphate was very actively resorbed after stimulation with titanium particles.
Harvested bone marrow cells expressed monocyte/macrophage phenotype, but not osteoclastic markers. The capacity of these cathepsin-K-positive phagocytic cells to resorb dentine discs and carbonated calcium phosphate in vitro suggests a direct role of monocytes/macrophages in bone resorption and periprosthetic osteolysis. The finding supports our hypothesis and previous histomorphometric observations on the presence of such osteolytic macrophages in vivo around loosening prosthesis.
Journal of Biomedical Materials Research Part B Applied Biomaterials 01/2008; 84(1):191-204. DOI:10.1002/jbm.b.30861 · 2.33 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Toll-like receptors (TLRs) have been known to act as sensors of innate immunity and respond to ligands of microbial and endogenous components. Tissues and cells typical for interface membrane of foreign body reaction were analyzed to evaluate potential role of TLRs in the pathogenesis of the so called "aseptic loosening of total hip replacement." Fourteen cases of interface membrane around aseptic loose total hip replacement implants were stained by single and double immunohistochemical methods to examine cellular localization of toll-like receptor (TLR)-4 and TLR-9. Osteoarthritic synovium was used as control tissues. Cultured macrophages were used to study TLR-4 and TLR-9 mRNA levels by quantitative reverse transcriptase-polymerase chain reaction. The effect of titanium particle stimulation on macrophages was also examined in the culture. Extensive immunolocalization of TLR-4 and TLR-9 positive cells was observed in the synovial membrane-like interface membrane of foreign body granulomas compared with control synovial membranes. TLR and CD68 double staining demonstrated that the TLR positive cells in aseptic loosening were mostly monocyte/macrophages and foreign body giant cells. TLR-4 and TLR-9 mRNA expression was also found in macrophage-colony stimulating factor treated rat macrophages, but this expression decreased (p < 0.05 or less) upon stimulation with titanium particles although matrix metalloproteinase (MMP)-9 mRNA levels used as macrophage activation marker were increased (p = 0.01). The interface membrane around loosening total hip replacement implants is apparently well equipped with TLRs and, thus, probably very sensitive to various structural components of microbes and to endogenous TLR ligands. This seems to be due to recruitment of monocyte/macrophages as particles per se seemed to down-regulate some of the key TLRs. This suppression after particle phagocytosis might prevent excessive and harmful host responses, and injury to innocent bystander cells/tissues.
Journal of Biomedical Materials Research Part A 06/2007; 81(4):1017-26. DOI:10.1002/jbm.a.31235 · 2.84 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: The dorsal root ganglion (DRG) and dorsal horn of the spinal cord are areas through which primary afferent information passes enroute to the brain. Previous studies have reported that, during normal neuronal activity, the regional distribution of a second messenger, diacylglycerol (DG), which is derived from phosphoinositide turnover, is diverse in these areas. However, the way that DG is regulated in these organs remains unknown. The present study was performed to investigate mRNA expression and protein localization of DG kinase (DGK) isozymes, which play a central role in DG metabolism. Gene expression for DGK isozymes was detected with variable regional distributions and intensities in the spinal cord. Among the isozymes, most intense signals were found for DGKzeta and DGKiota in the DRG. By immunohistochemical analysis, DGKzeta immunoreactivity was detected heterogeneously in the nucleus and cytoplasm of small DRG neurons with variable levels of distribution, whereas it was detected exclusively in the cytoplasm of large neurons. On the other hand, DGKiota immunoreactivity was distributed solely in the cytoplasm of most of the DRG neurons. Double-immunofluorescent imaging of these isozymes showed that they coexisted in a large population of DRG neurons at distinct subcellular sites, i.e., DGKzeta in the nucleus and DGKiota in the cytoplasm. Thus, DGK isozymes may have different functional roles at distinct subcellular sites. Furthermore, the heterogeneous subcellular localization of DGKzeta between the nucleus and cytoplasm implies the possible translocation of this isozyme in small DRG neurons under various conditions.
Cell and Tissue Research 11/2006; 326(1):35-42. DOI:10.1007/s00441-006-0219-z · 3.33 Impact Factor