Nicotine treatment induces expression of matrix metalloproteinases in human osteoblastic Saos-2 cells

Tobacco smoking is an important risk factor for the development of severe periodontitis.Recently,we showed that nicotine affected mineralized nodule formation,and that nicotine andlipopolysaccharide stimulated the formation of osteoclast-like cells by increasing production of macrophagecolony-stimulating factor (M-CSF) and prostaglandin E_2 (PGE_2) by human osteoblastic Saos-2 cells.In thepresent study,we examined the effects of nicotine on the expression of matrix metalloproteinases (MMPs),tissue inhibitors of matrix metalloproteinases (TIMPs),the plasminogen activation system including thecomponent of tissue-type plasminogen activator (tPA),urokinase-type PA (uPA),and PA inhibitor type 1(PAI-1),α7 nicotine receptor,and c-fos.We also examined the effect of the nicotine antagonist D-tubocurarineon nicotine-induced expression of MMP-1.Gene expression was examined using real-time polymerase chainreaction (PCR) to estimate mRNA levels.In addition,expression of the MMP,TIMP,uPA,tPA,and PAI-1proteins was determined by Western blotting analysis.Nicotine treatment caused expression of MMP-1,2,3,and 13,but not MMP-14,to increase significantly after 5 or 10 d of culture;MMP-14 expression did notchange through day 14.Enhancement of MMP-1 expression by nicotine treatment was eliminated bysimultaneous treatment with D-tubocurarine.In the presence of nicotine,expression of uPA,PAI-1,orTIMP-1,2,3,or 4 did not change over 14 d of culture,whereas expression of tPA increased significantly byday 7.Nicotine also increased expression of the α7 nicotine receptor and c-fos genes.These results suggestthat nicotine stimulates bone matrix turnover by increasing production of tPA and MMP-1,2,3,and 13,thereby tipping the balance between bone matrix formation and resorption toward the latter process.     

Dysfunction of regulatory volume increase is a key component of apoptosis

Sustained cell shrinkage is a major hallmark of apoptotic cell death. In apoptotic cells, whole cell volume reduction, called apoptotic volume decrease (AVD), proceeds until fragmentation of cells. Under non-apoptotic conditions, human epithelial HeLa cells exhibited a slow regulatory volume increase (RVI) after osmotic shrinkage induced by exposure to hypertonic solution. When AVD was induced by treatment with a Fas ligand, TNF-alpha or staurosporine, however, it was found that HeLa cells failed to undergo RVI. When RVI was inhibited by combined application of Na+/H+ exchanger (NHE) and anion exchanger blockers, hypertonic stress induced prolonged shrinkage followed by caspase-3 activation in HeLa cells. Hypertonicity also induced apoptosis in NHE1-deficient PS120 fibroblasts, which lack the RVI response. When RVI was restored by transfection of these cells with NHE1, hypertonicity-induced apoptosis was completely prevented. Thus, it is concluded that RVI dysfunction is indispensable for the persistence of AVD and induction of apoptosis.     

Complete genome sequence of the alkaliphilic bacterium Bacillus halodurans and genomic sequence comparison with Bacillus subtilis

The 4 202 353 bp genome of the alkaliphilic bacterium Bacillus halodurans C-125 contains 4066 predicted protein coding sequences (CDSs), 2141 (52.7%) of which have functional assignments, 1182 (29%) of which are conserved CDSs with unknown function and 743 (18.3%) of which have no match to any protein database. Among the total CDSs, 8.8% match sequences of proteins found only in Bacillus subtilis and 66.7% are widely conserved in comparison with the proteins of various organisms, including B.subtilis. The B.halodurans genome contains 112 transposase genes, indicating that transposases have played an important evolutionary role in horizontal gene transfer and also in internal genetic rearrangement in the genome. Strain C-125 lacks some of the necessary genes for competence, such as comS, srfA and rapC, supporting the fact that competence has not been demonstrated experimentally in C-125. There is no paralog of tupA, encoding teichuronopeptide, which contributes to alkaliphily, in the C-125 genome and an ortholog of tupA cannot be found in the B.subtilis genome. Out of 11 σ factors which belong to the extracytoplasmic function family, 10 are unique to B.halodurans, suggesting that they may have a role in the special mechanism of adaptation to an alkaline environment.     

Interleukin-17F affects cartilage matrix turnover by increasing the expression of collagenases and stromelysin-1 and by decreasing the expression of their inhibitors and extracellular matrix components in chondrocytes

Interleukin (IL)-17, a proinflammatory cytokine, is produced primarily by activated Th17 cells. IL-17 consists of six ligands that signal through five receptors (IL-17Rs); IL-17A and IL-17F share the highest homology in the family. Matrix metalloproteinases (MMPs) degrade the extracellular matrix during cartilage remodeling whereas tissue inhibitor of metalloproteinases (TIMPs) inhibit the action of MMPs. In the present study, we examined the effect of IL-17F on the degradation and synthesis of the extracellular matrix in cartilage using human articular chondrocytes. We examined the effect of IL-17F on the expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and cyclooxygenases (COXs), as well as on prostaglandin E₂ (PGE₂) production. We also examined the indirect effect of PGE₂ on the above IL-17F-induced/reduced components using NS-398, a specific inhibitor of COX-2. Cells were cultured with or without IL-17F in the presence or absence of either an IL-17R antibody or NS-398 for up to 28 days. Expression of IL-17Rs, MMPs, TIMPs, type II collagen, aggrecan, link protein, and COXs at mRNA and protein levels was determined using real-time polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA), respectively. PGE₂ production was determined by ELISA. The expression of all types of IL-17Rs was detected in chondrocytes. However, IL-17RE expression was extremely low, compared with other IL-17Rs. The expression of MMP-1, MMP-3, MMP-13, and COX-2 as well as PGE₂ production were increased by addition of IL-17F, whereas the expression of IL-17RD, TIMP-2, TIMP-4, type II collagen, aggrecan, link protein, and COX-1 was decreased. The expression of IL-17RA, IL-17RB, IL-17RC, MMP-2, MMP-14, TIMP-1, and TIMP-3 was unaffected by addition of IL-17F. The IL-17R antibody blocked the stimulating/reducing effect of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, aggrecan, and link protein. NS-398 blocked the reducing effect of IL-17F on aggrecan expression, whereas it did not completely block the stimulating/reducing effects of IL-17F on the expression of MMP-1, MMP-3, MMP-13, TIMP-2, TIMP-4, type II collagen, and link protein. Our results suggest that IL-17F stimulates cartilage degradation by increasing the expression of collagenases (MMP-1 and -13) and stromelysin-1 (MMP-3) and by decreasing expression of their inhibitors (TIMP-2 and -4), type II collagen, aggrecan, and link protein in chondrocytes. Furthermore, our results suggest that the expression of aggrecan, link protein, and TIMP-4 decrease through the autocrine action of PGE₂ in chondrocytes.     

Analysis of the Inhibitory Effect of Canavanine on the Replication of Influenza RI/5+ Virus: II. Interaction of M Protein with the Plasma Membrane

When influenza A/RI/5⁺ virus-infected cells were incubated in medium to which 2 μg of canavanine (arginine analog) per ml had been added 4 hr after infection, all viral polypeptides were synthesized but the budding-like process with the appearance of extracellular virus was completely inhibited. The plasma membrane isolated from these cells contained exclusively hemagglutinin (HA), and membrane (M) protein and nucleoprotein (NP) appeared to be associated with the nucleus, in contrast to untreated cells whose plasma membrane contained abundant HA, M protein, and NP. Disruption of canavanine-treated cells by freeze-thawing generated a number of hemagglutinating membranous vesicles or fragments containing exclusively HA. By isotope labeling it was found that the M protein synthesized in the presence of canavanine, together with HA and NP, is a canavanine-substituted polypeptide. It is suggested that canavanine inhibits the formation of the mature envelope of influenza RI/5⁺, because of the inability of M protein to associate with the plasma membrane.     

Extracellular ATP activates Ca2(+)-dependent K+ conductance via Ca2+ influx in mouse macrophages

1. Using the perforated patch recording, the effects of ATP on membrane current were investigated in mouse peritoneal macrophages. 2. Extracellularly applied ATP induced a biphasic current consisting of a initial inward current [Ii(ATP)] followed by an outward current [Io(ATP)]. These currents were associated with a marked increase in conductance at their peaks. 3. Ii(ATP) reversed close to 0 mV and was attenuated by removal of external Na+. 4. Io(ATP) reversed near -80 mV and was increased by decreasing the external concentration of K+. 5. Io(ATP) was completely abolished by removal of external Ca2+, treatment with an intracellular Ca2+ chelator, the acetoxymethyl ester of 1,2-bis (2-aminophenoxy) ethane-N,N,N',N'-tetra acetic acid (BAPTA-AM) and bath applied quinidine but not tetraethylammonium (TEA) or apamin. 6. These results suggest that Ii(ATP) and Io(ATP) are due to an activation of nonspecific cationic and Ca2(+)-dependent K+ conductances, respectively, and raise the possibility that the putative ATP receptor may be important in regulating macrophage functions, motility, phagocytosis and cytokines secretion.