Proteinase 3 (PR3) is a 29-kDa serine proteinase, with homology to human leukocyte elastase (HLE) and cathepsin G (Cat G), secreted from activated neutrophils. It was found that PR3, but not HLE and Cat G, activated the oral epithelial cells to produce interleukin (IL)-8 and monocyte chemoattractant protein (MCP)-1 and to express intracellular adhesion molecule (ICAM)-1. Oral epithelial cells express protease-activated receptor (PAR)-1, -2, and -3 mRNA, and the expression of PAR-2 on the cell surface was promoted by PR3. PR3 cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand. The production of cytokine induced by PR3 was completely abolished by a phospholipase C inhibitor. These findings suggested that PR3 but not HLE and Cat G activated oral epithelial cells through G protein-coupled PAR-2. In human gingival fibroblasts (HGF), HLE and Cat G as well as PR3 were active to produce IL-8 and MCP-1. Human oral epithelial cells but not HGF express mRNA and protein of secretory leukocyte protease inhibitor (SLPI), an inhibitor of HLE and Cat G, and recombinant SLPI clearly inhibited the activation of HGF induced by HLE and Cat G but not by PR3. HLE and Cat G cleaved the peptide corresponding to the N terminus of PAR-2 with exposure of its tethered ligand, as well as PR3. These results suggest that neutrophil serine proteinases have equal ability to activate human nonepithelial cells through PAR-2 to produce inflammatory cytokines.
Recent evidence has been accumulated that microbial lipoprotein (LP) plays pathological roles in bacterial infection. The part of LP responsible for the expression of biological activities has been demonstrated to be the N-terminal lipopeptide moiety. Mycoplasmas, wall-less microorganisms, also possess LP capable of activating macrophages or fibroblasts. We have found that M. salivarium LP activates normal human gingival fibroblasts and macrophages to induce production of inflammatory cytokines, and we have purified a 44-kDa LP (LP44) responsible for the activity. In addition, the structure of the N-terminal lipopeptide moiety of LP44 has been determined to be S-(2,3-bispalmitoyloxypropyl) Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe-Thr-Gly-Trp-Val-Ala-. The lipopeptide S-(2,3-bispalmitoyloxypropyl)Cys-Gly-Asp-Pro-Lys-His-Pro-Lys-Ser-Phe (FSL-1) was synthesized. The lipopeptide FSL-1 activated human gingival fibroblasts and macrophages to produce inflammatory cytokines as LP44 did. Experiments using FSL-1 and its derivatives revealed that the diacylglyceryl and peptide portions of FSL-1 are indispensable for the activation of macrophages and for the recognition by Toll-like receptor 2 (TLR2) and TLR6. We have recently demonstrated that each of several leucine residues located at a leucine-rich repeat of TLR2 play a key role in the recognition of the diacylated lipopeptide FSL-1, mycoplamal LP and S. aureus peptidoglycan.
It has been reported that heat shock protein (HSP) 90 is involved in the regulation of signaling cascades including those resulting in cell proliferation and apoptosis. However, little is known about how HSP90 affects apoptosis signaling. In this study, using a specific inhibitor of HSP90, geldanamycin (GDM), we investigated the relationship between HSP90 and anti-Fas antibody-induced apoptosis in HSG cells and concomitantly examined the apoptosis-inducing ability of GDM. We also sought to identify the target proteins of HSP90. When HSG cells were treated with GDM, a time-dependent increase in cell death was found. From the morphological features, the positive TdT-mediated dUTP nick end labeling (TUNEL) staining, and the cleavage of poly (ADP-ribose) polymerase (PARP), we concluded that the induced cell death was apoptotic. The pretreatment with GDM prior to that with anti-Fas antibody (CH-11) significantly increased the cell death as compared with that obtained with GDM or CH-11 alone. Further, prolonged incubation with GDM prominently enhanced the cell death. The induced cell death was also apoptotic. The transfection of HSG cells with recombinant HSP90α significantly inhibited the CH-11- and GDM-induced apoptosis. From the inhibition and overexpression experiments on HSP90 using GDM treatment and transfection with HSP90, respectively, we showed that HSP90 has an anti-apoptotic activity toward HSG cells. Immunoprecipitation with antihuman HSP90 antibody and subsequent Western blotting analysis of the precipitates detected bands for Caspase-8 and FADD-like ICE inhibitory protein (FLIP), which is known to regulate Fas cascade. Therefore, Caspase-8 and FLIP were shown to be target proteins of HSP90. These results suggest that HSP90 inhibits apoptosis by associating with Caspase-8 and FLIP and negatively regulating their functions.
The temporomandibular joint (TMJ) is a multicomponent organ including the condyle, mandibular fossa, articular disc and clefts, and surrounding tissues. In the present study, we reported the chronology of TMJ development and associated tissue-marker expression in C57BL/6 mice. Wild-type mice of embryonic day 12.5 (E12.5) through postnatal day 7 were examined by histochemistry (HE, metachromasia by toluidine blue), enzyme-histochemistry for alkaline and acid phosphatases (ALP and TRAP) and immunohistochemistry for BrdU and type II collagen. The results confirmed that : 1) the condylar blastema was first designated as a troop of ALP-positive cells at E13.5, and some of those cells also displayed BrdU incorporation, 2) after differentiation of the blastema, condylar morphogenesis progressed from the anterior to the posterior direction, while the mandibular fossa of discrete origin developed inversely, and 3) development of the articular disk and superior/inferior clefts appeared to link intimately with the articular function. Histomorphometric analysis, in conjunction with BrdU administration, further revealed that condylar growth during development can be divided into three stages : the initial slow growth due to cell proliferation by E13.5, the second rapid growth between E14.5 and E17.5 due to production of extracellular matrices instead of acceleration of cell proliferation, and the last functional adaptation after birth that progresses at a steady growth rate coupled with systemic body growth. The current observation also supported the spatial linkage of the condylar blastema with a group of the mesenchymal cell population that is involved in mandibular bone development. Further analysis in conjunction with three-dimensional reconstruction of ALP-positive cell condensation is required to clarify the origin and spatio-temporal development of condylar blastema in detail.