Retinoic acid-inducible gene-I is induced in gingival fibroblasts by lipopolysaccharide or poly IC: possible roles in interleukin-1beta, -6 and -8 expression.

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH family of proteins, and little is known of its biological function in the oral region. We previously reported that interleukin 1beta (IL-1beta) induced RIG-I expression in gingival fibroblasts. In this study, we studied the mechanism of RIG-I expression induced by lipopolysaccharide (LPS) or double-stranded RNA (dsRNA) in gingival fibroblasts. We also addressed the role of RIG-I in the expression of IL-1beta, IL-6 and IL-8 in gingival fibroblasts stimulated with LPS or dsRNA. We stimulated cultured human gingival fibroblasts with LPS or dsRNA, and examined the expression of RIG-I mRNA and protein. The effect of cycloheximide, a protein synthesis inhibitor, on RIG-I induction by these stimuli was examined. The expression of IL-1beta, IL-6 and IL-8 in gingival fibroblasts transfected with RIG-I cDNA stimulated with LPS or dsRNA was examined. LPS or dsRNA induced the expression of mRNA and protein for RIG-I in concentration- and time-dependent manners. We also examined the localization of RIG-I, and found that it was expressed in cytoplasm. Cycloheximide did not suppress the LPS or dsRNA-induced RIG-I expression. Introduction of RIG-I cDNA into gingival fibroblasts resulted in enhanced expression of IL-1beta, IL-6 and IL-8; moreover, overexpression of RIG-I stimulated with LPS or dsRNA synergistically increased expression of IL-1beta, IL-6 and IL-8. RIG-I may have important roles in the innate immune response in the regulation of IL-1beta, IL-6 and IL-8 expression in gingival fibroblasts in response to LPS and dsRNA.

Retinoic acid-inducible gene-I is induced by interleukin-1beta in cultured human gingival fibroblasts.

Retinoic acid-inducible gene-I (RIG-I) is a member of the DExH box family protein, and details of its biological function are not known. We have studied the mechanism of the interleukin-1beta (IL-1beta)-induced RIG-I expression in human gingival fibroblasts in culture. We also addressed the possibility of enhanced expression of COX-2, RANTES and galectin-9 in fibroblasts overexpressed RIG-I. We stimulated cultured human gingival fibroblasts with IL-1beta and examined the expression of RIG-I mRNA and protein by reverse transcriptase-mediated polymerase chain reaction and Western blot analysis. The effect of cycloheximide, a protein synthesis inhibitor, on the IL-1beta-induced expression of RIG-I was examined. The expression of COX-2, RANTES, galectin-9 and monocyte chemoattractant protein-1 in gingival fibroblasts transfected with RIG-I cDNA was also examined. IL-1beta stimulated the expressions of mRNA and protein for RIG-I, in cultured fibroblasts, in a time- and concentration-dependent manner. Cycloheximide did not suppress the IL-1beta-induced RIG-I expression. Introduction of RIG-I cDNA into fibroblasts resulted in enhanced expression of COX-2 mRNA, and slightly enhanced the expression of mRNA for RANTES and galectin-9. In contrast, RIG-I overexpression did not alter the level of mRNA for monocyte chemoattractant protein-1. We conclude that IL-1beta stimulates RIG-I expression in human gingival fibroblasts.

High IL-6 synthesis in cultured fibroblasts isolated from radicular cysts.

OBJECTIVE: Inflammatory cytokines have been reported to be related with inflammation and expansion of jaw cysts. In this study, to examine the relationship between radicular cysts and inflammatory cytokines, it was found that there was notable unique evidence on cytokine synthesis from fibroblasts isolated from radicular cysts. METHODS: The expression of such cytokines, namely, interleukin-1beta, IL-1beta, IL-6, IL-8, IL-10, tumour necrosis factor-alpha (TNF-alpha), interferon-gamma (IFN-gamma), transforming growth factor-beta1 (TGF-beta1), and granulocyte-macrophage colony-stimulating (GM-CSF) mRNA, in nine radicular cysts was examined and compared with that detected in six specimens of healthy gingival mucosa. Furthermore, separating all fibroblasts from their respective radicular cysts, healthy gingival mucosa, and healthy periodontal ligaments, these fibroblast groups were cultured without stimulators and a supernatant for each was obtained to analyse IL-1beta, IL-6, IL-8, TNF-alpha, and IFN-gamma by ELISA. RESULTS: Differences between radicular cysts and healthy gingival mucosa were not clearly shown by the expression of cytokine mRNA. Analysing inflammatory cytokine synthesis in fibroblast groups from these three kinds of tissues, surprisingly, the levels of IL-6 mRNA and protein were recognised to be higher in fibroblasts of radicular cysts than in those of control tissues by ELISA and a real-time RT-PCR. Significant differences in the cultured supernatants of these fibroblast groups were not recognised in the release of IL-1beta, IL-8, TNF-alpha, and IFN-gamma by ELISA. CONCLUSIONS: From these results, it was suggested that fibroblasts inducing IL-6 production might play important roles in the expansion of radicular cysts. It is considered that fibroblasts around radicular cysts may lead to high IL-6 synthesis over time in chronic inflammation.

Interleukin-1beta induces matrix metalloproteinase-1 expression in cultured human gingival fibroblasts: role of cyclooxygenase-2 and prostaglandin E2.

OBJECTIVE: Matrix metalloproteinases (MMPs) degrade extracellular matrices and are responsible for excessive connective tissue breakdown in inflammatory disorders. We investigated the mechanism of MMP-1 expression in human gingival fibroblasts in response to the stimulation with interleukin-1beta (IL-1beta), and the role of inducible-type cyclooxygenase-2 (COX-2) and prostaglandin E2 (PGE2) in the regulation of MMP-1 expression. MATERIALS AND METHODS: We stimulated cultured human gingival fibroblasts with r(h)IL-1beta, and examined the expression of MMP-1 mRNA and protein by quantitative polymerase chain reaction and enzyme-linked immunosorbent assay. The effect of indomethacin, dexamethasone, or cycloheximide (CHX) on the IL-1beta-induced expression of MMP-1 was examined. The expression of MMP-1 in gingival fibroblasts stimulated with PGE2 was also examined. RESULTS: IL-1beta stimulated the expressions of mRNA and protein for MMP-1, in cultured fibroblasts, in time- and concentration-dependent manners. Pretreatment of the cells with indomethacin or dexamethasone inhibited the IL-1beta-induced MMP-1 expression. CHX, a protein synthesis inhibitor, also suppressed the MMP-1 expression. IL-1beta also induced COX-2 expression in gingival fibroblasts, and PGE2, a major COX-2 product, was found to enhance MMP-1 expression. CONCLUSION: The IL-1beta-induced MMP-1 expression in gingival fibroblasts may be mediated, at least in part, by COX-2 and its product PGE2.

Single molecule probe scanning optical force imaging microscopefor viewing live cells.

We have developed an imaging system that combines the soft compliance of an optical trap with the sensitivity of single particle tracking to image forces on/in live cells using a single molecule probe. The probe used is a single (or few) molecule of interest that is conjugated with a single 40 nm colloidalgold probe. The colloidal gold/membrane protein complex, freely diffusing on a live cell, is held in a laser trap while the cell is scanned underneath. Computer control allows for synchronization of the cell scan and capture of the probe position. Resistance to the dragging of the probe images a fine structure of barriers in the membrane of live cells.

A polyalanine-based peptide cannot form a stable transmembrane alpha-helix in fully hydrated phospholipid bilayers.

The conformation and amide proton exchangeability of the peptide acetyl-K(2)-A(24)-K(2)-amide (A(24)) and its interaction with phosphatidylcholine bilayers were examined by a variety of physical techniques. When dissolved in or cast from methanol as a dried film, A(24) is predominantly alpha-helical. In aqueous media, however, A(24) exists primarily as a mixture of helical (though not necessarily alpha-helical) and random coiled structures, both of which allow rapid H-D exchange of all amide protons. When incorporated into phospholipids in the absence of water, A(24) also exists primarily as a transmembrane alpha-helix. However, upon hydration of that system, rapid exchange of all amide protons also occurs along with a marked change in the amide I absorption band of the peptide. Also, when dispersed with phosphatidylcholine in aqueous media, the conformation and thermal stability of A(24) are not significantly altered by the presence of the phospholipid or by its gel/liquid-crystalline phase transition. Differential scanning calorimetric and electron spin resonance spectroscopic studies indicate that A(24) has relatively minor effects on the thermodynamic properties of the lipid hydrocarbon chain-melting phase transition, that it does not abolish the lipid pretransition, and that its presence has no significant effect on the orientational order or rates of motion of the phospholipid hydrocarbon chains. We therefore conclude that A(24) has sufficient alpha-helical propensity, but insufficient hydrophobicity, to maintain a stable transmembrane association with phospholipid bilayers in the presence of water. Instead, it exists primarily as a dynamic mixture of helices and other conformers and resides mostly in the aqueous phase where it interacts weakly with the bilayer surface or with the polar/apolar interfacial region of phosphatidylcholine bilayers. Thus, polyalanine-based peptides are not good models for the transmembrane alpha-helical segments of natural membrane proteins.

Fast lipid disorientation at the onset of membrane fusion revealed by molecular dynamics simulations.

Membrane fusion is a key event in vesicular trafficking in every cell, and many fusion-related proteins have been identified. However, how the actual fusion event occurs has not been elucidated. By using molecular dynamics simulations we found that when even a small region of two membranes is closely apposed such that only a limited number of water molecules remain in the apposed area (e.g., by a fusogenic protein and thermal membrane fluctuations), dramatic lipid disorientation results within 100 ps-2 ns, which might initiate membrane fusion. Up to 12% of phospholipid molecules in the apposing layers had their alkyl chains outside the hydrophobic region, lying almost parallel to the membrane surface or protruding out of the bilayer by 2 ns after two membranes were closely apposed.

Single molecule imaging of green fluorescent proteins in living cells: E-cadherin forms oligomers on the free cell surface.

Single green fluorescent protein (GFP) molecules were successfully imaged for the first time in living cells. GFP linked to the cytoplasmic carboxyl terminus of E-cadherin (E-cad-GFP) was expressed in mouse fibroblast L cells, and observed using an objective-type total internal reflection fluorescence microscope. Based on the fluorescence intensity of individual fluorescent spots, the majority of E-cad-GFP molecules on the free cell surface were found to be oligomers of various sizes, many of them greater than dimers, suggesting that oligomerization of E-cadherin takes place before its assembly at cell-cell adhesion sites. The translational diffusion coefficient of E-cad-GFP is reduced by a factor of 10 to 40 upon oligomerization. Because such large decreases in translational mobility cannot be explained solely by increases in radius upon oligomerization, an oligomerization-induced trapping model is proposed in which, when oligomers are formed, they are trapped in place due to greatly enhanced tethering and corralling effects of the membrane skeleton on oligomers (compared with monomers). The presence of many oligomers greater than dimers on the free surface suggests that these greater oligomers are the basic building blocks for the two-dimensional cell adhesion structures (adherens junctions).

The Ste20 group kinases as regulators of MAP kinase cascades.

Ste20p (sterile 20 protein) is a putative yeast mitogen-activated protein kinase kinase kinase kinase (MAP4K) involved in the mating pathway. Its homologs in mammals, Drosophila, Caenorhabditis elegans and other organisms make up a large emerging group of protein kinases including 28 members in human. The Ste20 group kinases are further divided into the p21-activated kinase (PAK) and germinal center kinase (GCK) families. They are characterized by the presence of a conserved kinase domain and a noncatalytic region of great structural diversity that enables the kinases to interact with various signaling molecules and regulatory proteins of the cytoskeleton. This review describes the phylogenetic relationships of the Ste20 group kinases based on discussions with many researchers in this field. With the newly established phylogenetic relationships, crucial arguments can be advanced regarding the functions of these kinases as upstream activators of the MAPK pathways and possible activity as MAP4Ks. Their involvement in apoptosis, morphogenesis and cytoskeletal rearrangements is also discussed.

Pulse EPR detection of lipid exchange between protein-rich raft and bulk domains in the membrane: methodology development and its application to studies of influenza viral membrane.

A pulse saturation-recovery electron paramagnetic resonance (EPR) method has been developed that allows estimation of the exchange rates of a spin-labeled lipid between the bulk domain and the protein-rich membrane domain, in which the rate of collision between the spin label and molecular oxygen is reduced (slow-oxygen transport domain, or SLOT domain). It is based on the measurements of saturation-recovery signals of a lipid spin label as a function of concentrations of both molecular oxygen and the spin label. Influenza viral membrane, one of the simplest paradigms for the study of biomembranes, showed the presence of two membrane domains with slow and fast collision rates with oxygen (a 16-fold difference) at 30 degrees C. The outbound rate from and the inbound rate into the SLOT domain (or possibly the rate of the domain disintegration and formation) were estimated to be 7.7 x 10(4) and 4.6 x 10(4) s(-1), (15 micros residency time), respectively, indicating that the SLOT domain is highly dynamic and that the entire SLOT domain represents about one-third of the membrane area. Because the oxygen transport rate in the SLOT domain is a factor of two smaller than that in purple membrane, where bacteriorhodopsin is aggregated, we propose that the SLOT domain in the viral membrane is the cholesterol-rich raft domain stabilized by the trimers of hemagglutinin and/or the tetramers of neuraminidase.

Molecular dynamics generation of nonarbitrary membrane models reveals lipid orientational correlations.

This report addresses the following problems associated with the generation of computer models of phospholipid bilayer membranes using molecular dynamics simulations: arbitrary initial structures and short equilibration periods, an Ewald-induced strong coupling of phospholipids, uncertainty regarding which value should be used for surface tension to alleviate the problem of the small size of the membrane, and simultaneous realization of both order parameters and the surface area. We generated a computer model of the liquid-crystalline L-alpha-dimyristoylphosphatidylcholine (DMPC) bilayer, starting from a configuration based on a crystal structure (rather than from an arbitrary structure). To break the crystalline structure, a 20-ps high-temperature pulse of 510 K (but not 450 or 480 K) was effective. The system finally obtained is an all-atom model, with Ewald summation to evaluate Coulombic interactions and a constant surface tension of 35 dynes/cm/water-membrane interface, equilibrated for 12 ns (over 50 ns total calculation time), which reproduces all of the experimentally observed parameters examined in this work. Furthermore, this model shows the presence of significant orientational correlations between neighboring alkyl chains and between shoulder vectors (which show the orientations of the lipids about their long axes) of neighboring DMPCs.

Molecular cloning of MINK, a novel member of mammalian GCK family kinases, which is up-regulated during postnatal mouse cerebral development.

A new germinal center kinase (GCK) family kinase, Misshapen/NIKs-related kinase (MINK), has been cloned and its expression has been characterized in several tissues and various developmental stages of the mouse brain. MINK encodes a 1300 amino acid polypeptide, consisting of an N-terminal kinase domain, a proline-rich intermediate region, and a C-terminal GCK homology region. The expression of MINK is up-regulated during the postnatal development of the mouse brain. MINK activates the cJun N-terminal kinase and the p38 pathways.

Cholesterol effects on the phosphatidylcholine bilayer polar region: a molecular simulation study.

A molecular dynamics (MD) simulation of a fully hydrated, liquid-crystalline dimyristoylphosphatidylcholine (DMPC)-Chol bilayer membrane containing approximately 22 mol% Chol was carried out for 4.3 ns. The bilayer reached thermal equilibrium after 2.3 ns of MD simulation. A 2.0-ns trajectory generated during 2.3-4.3 ns of MD simulation was used for analyses to determine the effects of Chol on the membrane/water interfacial region. In this region, 70% of Chol molecules are linked to DMPC molecules via short-distance interactions, where the Chol hydroxyl group (OH-Chol) is 1) charge paired to methyl groups of the DMPC choline moiety ( approximately 34%), via the hydroxyl oxygen atom (Och); 2) water bridged to carbonyl ( approximately 19%) and nonester phosphate ( approximately 14%) oxygen atoms, via both Och and the hydroxyl hydrogen atom (Hch); and 3) directly hydrogen (H) bonded to carbonyl ( approximately 11%) and nonester phosphate ( approximately 5%) oxygen atoms, via Hch ( approximately 17% of DMPC-Chol links are multiple). DMPC's gamma-chain carbonyl oxygen atom is involved in 44% of water bridges and 51% of direct H bonds formed between DMPC and Chol. On average, a Chol molecule forms 0.9 links with DMPC molecules, while a DMPC molecule forms 2.2 and 0.3 links with DMPC and Chol molecules, respectively. OH-Chol makes hydrogen bonds with 1.1 water molecules, preferentially via Hch. The average number of water molecules H bonded to the DMPC headgroup is increased by 7% in the presence of Chol. These results indicate that inclusion of Chol decreases interlipid links and increases hydration in the polar region of the membrane.

Mobility and cytoskeletal interactions of cell adhesion receptors.

Clustering of cell adhesion receptors and their interactions with the cytoskeleton are key events in the formation and function of cell adhesion structures. On the free cell surface, cadherin molecules interact with the cytoskeleton/membrane skeleton by being bound or corralled, and such interactions are greatly enhanced by the formation of cadherin oligomers. Corralled cadherin molecules undergo hop diffusion from one compartment to an adjacent one (membrane skeleton fence model), which prompts the initial formation of small adhesion clusters at cell-cell contact sites, but larger-scale assemblies of cadherin and actin filaments might require a further co-ordinated recruitment of these molecules.

Charge pairing of headgroups in phosphatidylcholine membranes: A molecular dynamics simulation study.

Molecular dynamics simulation of the hydrated dimyristoylphosphatidylcholine (DMPC) bilayer membrane in the liquid-crystalline phase was carried out for 5 ns to study the interaction among DMPC headgroups in the membrane/water interface region. The phosphatidylcholine headgroup contains a positively charged choline group and negatively charged phosphate and carbonyl groups, although it is a neutral molecule as a whole. Our previous study (Pasenkiewicz-Gierula, M., Y. Takaoka, H. Miyagawa, K. Kitamura, and A. Kusumi. 1997. J. Phys. Chem. 101:3677-3691) showed the formation of water cross-bridges between negatively charged groups in which a water molecule is simultaneously hydrogen bonded to two DMPC molecules. Water bridges link 76% of DMPC molecules in the membrane. In the present study we show that relatively stable charge associations (charge pairs) are formed between the positively and negatively charged groups of two DMPC molecules. Charge pairs link 93% of DMPC molecules in the membrane. Water bridges and charge pairs together form an extended network of interactions among DMPC headgroups linking 98% of all membrane phospholipids. The average lifetimes of DMPC-DMPC associations via charge pairs, water bridges and both, are at least 730, 1400, and over 1500 ps, respectively. However, these associations are dynamic states and they break and re-form several times during their lifetime.

Regulation mechanism of the lateral diffusion of band 3 in erythrocyte membranes by the membrane skeleton.

Mechanisms that regulate the movement of a membrane spanning protein band 3 in erythrocyte ghosts were investigated at the level of a single or small groups of molecules using single particle tracking with an enhanced time resolution (0.22 ms). Two-thirds of band 3 undergo macroscopic diffusion: a band 3 molecule is temporarily corralled in a mesh of 110 nm in diameter, and hops to an adjacent mesh an average of every 350 ms. The rest (one-third) of band 3 exhibited oscillatory motion similar to that of spectrin, suggesting that these band 3 molecules are bound to spectrin. When the membrane skeletal network was dragged and deformed/translated using optical tweezers, band 3 molecules that were undergoing hop diffusion were displaced toward the same direction as the skeleton. Mild trypsin treatment of ghosts, which cleaves off the cytoplasmic portion of band 3 without affecting spectrin, actin, and protein 4.1, increased the intercompartmental hop rate of band 3 by a factor of 6, whereas it did not change the corral size and the microscopic diffusion rate within a corral. These results indicate that the cytoplasmic portion of band 3 collides with the membrane skeleton, which causes temporal confinement of band 3 inside a mesh of the membrane skeleton.

Structure of the erythrocyte membrane skeleton as observed by atomic force microscopy.

The structure of the membrane skeleton on the cytoplasmic surface of the erythrocyte plasma membrane was observed in dried human erythrocyte ghosts by atomic force microscopy (AFM), taking advantage of its high sensitivity to small height variations in surfaces. The majority of the membrane skeleton can be imaged, even on the extracellular surface of the membrane. Various fixation and drying methods were examined for preparation of ghost membrane samples for AFM observation, and it was found that freeze-drying (freezing by rapid immersion in a cryogen) of unfixed specimens was a fast and simple way to obtain consistently good results for observation without removing the membrane or extending the membrane skeleton. Observation of the membrane skeleton at the external surface of the cell was possible mainly because the bilayer portion of the membrane sank into the cell during the drying process. The average mesh size of the spectrin network observed at the extracellular and cytoplasmic surfaces of the plasma membrane was 4800 and 3000 nm2, respectively, which indicates that spectrin forms a three-dimensionally folded meshwork, and that 80% of spectrin can be observed at the extracellular surface of the plasma membrane.

Cytoplasmic regulation of the movement of E-cadherin on the free cell surface as studied by optical tweezers and single particle tracking: corralling and tethering by the membrane skeleton.

The translational movement of E-cadherin, a calcium-dependent cell-cell adhesion molecule in the plasma membrane in epithelial cells, and the mechanism of its regulation were studied using single particle tracking (SPT) and optical tweezers (OT). The wild type (Wild) and three types of artificial cytoplasmic mutants of E-cadherin were expressed in L-cells, and their movements were compared. Two mutants were E-cadherins that had deletions in the COOH terminus and lost the catenin-binding site(s) in the COOH terminus, with remaining 116 and 21 amino acids in the cytoplasmic domain (versus 152 amino acids for Wild); these are called Catenin-minus and Short-tailed in this paper, respectively. The third mutant, called Fusion, is a fusion protein between E-cadherin without the catenin-binding site and alpha-catenin without its NH2-terminal half. These cadherins were labeled with 40-nm phi colloidal gold or 210-nm phi latex particles via a monoclonal antibody to the extracellular domain of E-cadherin for SPT or OT experiments, respectively. E-cadherin on the dorsal cell surface (outside the cell-cell contact region) was investigated. Catenin-minus and Short-tailed could be dragged an average of 1.1 and 1.8 micron by OT (trapping force of 0.8 pN), and exhibited average microscopic diffusion coefficients (Dmicro) of 1.2 x 10(-10) and 2.1 x 10(-10) cm2/s, respectively. Approximately 40% of Wild, Catenin-minus, and Short-tailed exhibited confined-type diffusion. The confinement area was 0.13 micron2 for Wild and Catenin-minus, while that for Short-tailed was greater by a factor of four. In contrast, Fusion could be dragged an average of only 140 nm by OT. Average Dmicro for Fusion measured by SPT was small (0.2 x 10(-10) cm2/s). These results suggest that Fusion was bound to the cytoskeleton. Wild consists of two populations; about half behaves like Catenin- minus, and the other half behaves like Fusion. It is concluded that the movements of the wild-type E-cadherin in the plasma membrane are regulated via the cytoplasmic domain by (a) tethering to actin filaments through catenin(s) (like Fusion) and (b) a corralling effect of the network of the membrane skeleton (like Catenin-minus). The effective spring constants of the membrane skeleton that contribute to the tethering and corralling effects as measured by the dragging experiments were 30 and 5 pN/micron, respectively, indicating a difference in the skeletal structures that produce these two effects.