Tumor necrosis factor α induction in human monocytes

The present study was undertaken to assess the presence of tumor necrosis factor (TNF)-α mRNA and protein in circulating human blood monocytes and to study the TNF-α gene expression in human monocytes isolated by continuous Percoll gradient fractionation. The technique of RNA isolation directly from the blood samples was used to study TNF-α mRNA expression in circulating human blood leukocytes. It was shown that human blood leukocytes of healthy donors contained no presynthesized pool of TNF-α mRNA as well as no TNF-α protein. It was found that early pretreatment with cycloheximide interfers with TNF-α mRNA induction by Staphylococcus aureus.     

Amyloid precursor protein expression is induced by tumor necrosis factor α in 3T3‐L1 adipocytes

Amyloid precursor protein (APP) has been characterized as an adipocyte‐secreted protein that might contribute to obesity‐related insulin resistance, inflammation, and dementia. In the current study, regulation of APP by the proinflammatory and insulin resistance‐inducing cytokine tumor necrosis factor (TNF) α was determined in 3T3‐L1 adipocytes. Interestingly, APP protein synthesis and mRNA expression were significantly increased by TNFα in a time‐dependent manner with maximal induction observed after 24 h of treatment. Furthermore, TNFα induced APP mRNA expression dose‐dependently with maximal 6.4‐fold upregulation seen at 100 ng/ml effector. Moreover, inhibitor experiments suggested that TNFα‐induced APP expression was mediated by nuclear factor κ B. Taken together, we show for the first time a potent upregulation of APP by TNFα suggesting a potential role of this adipocyte‐secreted protein in TNFα‐induced insulin resistance and inflammatory disease. J. Cell. Biochem. 108: 1418–1422, 2009. © 2009 Wiley‐Liss, Inc.     

Effects of tumor necrosis factor α (TNFα) on the phospholipid metabolism of Tetrahymena pyriformis

The effect of (0·05 ng ml⁻¹ and 0·1 ng ml⁻¹) TNFα on the phospholipid metabolism of Tetrahymena pyriformis was studied. The amount of phosphatidyl choline (PC), phosphatidyl inositol (PI), phosphatidic acid (PA), phosphatidyl ethanolamine (PE), diacylglycerol (DAG), arachidonic acid (AA) and ceramide was higher, but the phosphatidyl inositol 4 phosphate (PIP) and phosphatidyl inositol bis-phosphate (PIP₂) as well, as sphingomyelin (SM) content was lower in TNFα-treated cells than in the controls. In the culture medium (secreted forms) this situation was reversed. There were differences in the results gained by incorporation of [³H]-palmitic acid or ³²P into the phospholipids. To control the functional effects of TNFα in Tetrahymena, the rate of cell division, the condensation of chromatin, the viability of cells and morphometrical values have been studied. The cytokine reduced cell growth, altered morphometric indices and increased chromatin condensation, however cell viability was not influenced. The results demonstrate the effects of TNFα at a low level of evolution, what is realized by changes in the phospolipid metabolism participating in signalling pathways. © 1998 John Wiley & Sons, Ltd.     

Impaired integrin α5/β1‐mediated hepatocyte growth factor release by stellate cells of the aged liver

Hepatic blood flow and sinusoidal endothelial fenestration decrease during aging. Consequently, fluid mechanical forces are reduced in the space of Disse where hepatic stellate cells (HSC) have their niche. We provide evidence that integrin α₅/β₁ is an important mechanosensor in HSC involved in shear stress‐induced release of hepatocyte growth factor (HGF), an essential inductor of liver regeneration which is impaired during aging. The expression of the integrin subunits α₅ and β₁ decreases in liver and HSC from aged rats. CRISPR/Cas9‐mediated integrin α₅ and β₁ knockouts in isolated HSC lead to lowered HGF release and impaired cellular adhesion. Fluid mechanical forces increase integrin α₅ and laminin gene expression whereas integrin β₁ remains unaffected. In the aged liver, laminin β2 and γ1 protein chains as components of laminin‐521 are lowered. The integrin α₅ knockout in HSC reduces laminin expression via mechanosensory mechanisms. Culture of HSC on nanostructured surfaces functionalized with laminin‐521 enhances Hgf expression in HSC, demonstrating that these ECM proteins are critically involved in HSC function. During aging, HSC acquire a senescence‐associated secretory phenotype and lower their growth factor expression essential for tissue repair. Our findings suggest that impaired mechanosensing via integrin α₅/β₁ in HSC contributes to age‐related reduction of ECM and HGF release that could affect liver regeneration.     

Structural classification of αββ and ββα supersecondary structure units in proteins

We present a fully automatic structural classification of supersecondary structure units, consisting of two hydrogen-bonded β strands, preceded or followed by an α helix. The classification is performed on the spatial arrangement of the secondary structure elements, irrespective of the length and conformation of the intervening loops. The similarity of the arrangements is estimated by a structure alignment procedure that uses as similarity measure the root mean square deviation of superimposed backbone atoms. Applied to a set of 141 well-resolved nonhomologous protein structures, the classification yields 11 families of recurrent arrangements. In addition, fragments that are structurally intermediate between the families are found; they reveal the continuity of the classification. The analysis of the families shows that the α helix and β hairpin axes can adopt virtually all relative orientations, with, however, some preferable orientations; moreover, according to the orientation, preferences in the left/right handedness of the α–β connection are observed. These preferences can be explained by favorable side by side packing of the α helix and the β hairpin, local interactions in the region of the α–β connection or stabilizing environments in the parent protein. Furthermore, fold recognition procedures and structure prediction algorithms coupled to database-derived potentials suggest that the preferable nature of these arrangements does not imply their intrinsic stability. They usually accommodate a large number of sequences, of which only a subset is predicted to stabilize the motif. The motifs predicted as stable could correspond to nuclei formed at the very beginning of the folding process. Proteins 30:193–212, 1998. © 1998 Wiley-Liss, Inc.     

Membrane lymphotoxin-α2β is a novel tumor necrosis factor (TNF) receptor 2 (TNFR2) agonist

In the early 1990s, it has been described that LTα and LTβ form LTα₂β and LTαβ₂ heterotrimers, which bind to TNFR1 and LTβR, respectively. Afterwards, the LTαβ₂–LTβR system has been intensively studied while the LTα₂β–TNFR1 interaction has been ignored to date, presumably due to the fact that at the time of identification of the LTα₂β–TNFR1 interaction one knew already two ligands for TNFR1, namely TNF and LTα. Here, we show that LTα₂β interacts not only with TNFR1 but also with TNFR2. We furthermore demonstrate that membrane-bound LTα₂β (memLTα₂β), despite its asymmetric structure, stimulates TNFR1 and TNFR2 signaling. Not surprising in view of its ability to interact with TNFR2, LTα₂β is inhibited by Etanercept, which is approved for the treatment of rheumatoid arthritis and also inhibits TNF and LTα.Subject terms: Cytokines, Signal transduction     

Regulation of β-amyloid stimulated proinflammatory responses by peroxisome proliferator-activated receptor α

Amyloid deposition within the brains of Alzheimer's Disease patients results in the activation of microglial cells and the induction of a local inflammatory response. The interaction of microglia or monocytes with β-amyloid (Aβ) fibrils elicits the activation a complex tyrosine kinase-based signal transduction cascade leading to stimulation of multiple independent signaling pathways and ultimately to changes in proinflammatory gene expression. The Aβ-stimulated expression of proinflammatory genes in myeloid lineage cells is antagonized by the action of a family of ligand-activated nuclear hormone receptors, the peroxisome proliferator-activated receptors (PPARs). We report that THP-1 monocytes express predominantly PPARγ isoform and lower levels of PPARα and PPARδ isoforms. PPAR mRNA levels are not affected by differentiation of the cells into a macrophage phenotype, nor are they altered following exposure to the classical immune stimulus, lipopolysaccharide. Previous studies have found that PPARγ agonists act broadly to inhibit inflammatory responses. The present study explored the action of the PPARα isoform and found that PPARα agonists inhibited the Aβ-stimulated expression of TNFα and IL-6 reporter genes in a dose-dependent manner. Moreover, the PPARα agonist WY14643 inhibited macrophage differentiation and COX-2 gene expression. However, the PPARα agonists failed to inhibit Aβ-stimulated elaboration of neurotoxic factors by THP-1 cells. These findings demonstrate that PPARα acts to suppress a diverse array of inflammatory responses in monocytes.     

Selective inhibition of 11β-hydroxysteroid dehydrogenase 1 by 18α-glycyrrhetinic acid but not 18β-glycyrrhetinic acid

Elevated cortisol concentrations have been associated with metabolic diseases such as diabetes type 2 and obesity. 11β-hydroxysteroid dehydrogenase (11β-HSD) type 1, catalyzing the conversion of inactive 11-ketoglucocorticoids into their active 11β-hydroxy forms, plays an important role in the regulation of cortisol levels within specific tissues. The selective inhibition of 11β-HSD1 is currently considered as promising therapeutic strategy for the treatment of metabolic diseases. In recent years, natural compound-derived drug design has gained considerable interest. 18β-glycyrrhetinic acid (GA), a metabolite of the natural product glycyrrhizin, is not selective and inhibits both 11β-HSD1 and 11β-HSD2. Here, we compare the biological activity of 18β-GA and its diastereomer 18α-GA against the two enzymes in lysates of transfected HEK-293 cells and show that 18α-GA selectively inhibits 11β-HSD1 but not 11β-HSD2. This is in contrast to 18β-GA, which preferentially inhibits 11β-HSD2. Using a pharmacophore model based on the crystal structure of the GA-derivative carbenoxolone in complex with human 11β-HSD1, we provide an explanation for the differences in the activities of 18α-GA and 18β-GA. This model will be used to design novel selective derivatives of GA.     

Enzymatic resolutions of α- and β-hydroxypropionic acid esters

The synthesis of some acyloxy-methoxy-cinnamic acid derivatives, azidohydroxy butanoates, and azidohydroxy butanedioates in enantiomerically pure form is presented. Racemic diastereomerically pure educts were prepared in few steps. These racemates are resolved with lipases from Candida cylindracea (CC) and Pseudomonas fluorescens (P).     

Studies on the 5α-androstane-3β, 17β-diol-6α-hydroxylase and on the 5α-androstane-3β, 17β-diol-7α-hydroxylase in anterior hypophysis of male rats.

In anterior pituitaries from male rats, it appeared that 5α-androstane-3β, 17β-diol was quickly metabolized into 5α-androstane-3β,6α-17β-triol and 5α-androstane-3β,7α, 17β-triol by action of 6α- and 7α-hydroxylases. Hydroxysteroid hydroxylases were located in endoplasmic reticulum and were dependent on NADPH⁺. Their optimum pH was 8.0, optima temperature, 37°C, and their apparent Km was 2.7 μM. Hydroxylative reactions were not reversible and not modified by gonadectomy. Hydroxylation seemed an efficient control of the pituitary level of 5α-andros-tane-3β, 17β-diol.     

Cyclic strain induces reorganization of integrin α5β1 and α2β1 in human umbilical vein endothelial cells

Cyclic strain has been shown to modulate endothelial cell (EC) morphology, proliferation, and function. We have recently reported that the focal adhesion proteins focal adhesion kinase (pp125^FAK) and paxillin, are tyrosine phosphorylated in EC exposed to strain and these events regulate the morphological change and migration induced by cyclic strain. Integrins are also localized on focal adhesion sites and have been reported to induce tyrosine phosphorylation of pp125^FAK under a variety of stimuli. To study the involvement of different integrins in signaling induced by cyclic strain, we first observed the redistribution of α and β integrins in EC subjected to 4 h cyclic strain. Human umbilical vein endothelial cells (HUVEC) seeded on either fibronectin or collagen surfaces were subjected to 10% average strain at a frequency 60 cycles/min. Confocal microscopy revealed that β₁ integrin reorganized in a linear pattern parallel with the long axis of the elongated cells creating a fusion of focal adhesion plaques in EC plated on either fibronectin (a ligand for α₅β₁) or collagen (a ligand for α₂β₁) coated plates after 4 h exposure to cyclic strain. β₃ integrin, which is a vitronectin receptor, did not redistribute in EC exposed to cyclic strain. Cyclic strain also led to a reorganization of α₅ and α₂ integrins in a linear pattern in HUVEC seeded on fibronectin or collagen, respectively. The expression of integrins α₅, α₂, and β₁ did not change even after 24 h exposure to strain when assessed by immunoprecipitation of these integrins. Cyclic strain-induced tyrosine phosphorylation of pp125^FAK occurred concomitant with the reorganization of β₁ integrin. We concluded that α₅β₁ and α₂β₁ integrins play an important role in transducing mechanical stimuli into intracellular signals. J. Cell. Biochem. 64:505–513. © 1997 Wiley-Liss, Inc.     

Platelet-derived growth factor and inflammatory cytokines have differential effects on the expression of integrins α1β1 and α5β1 by human dermal fibroblasts in vitro

Dermal fibroblasts are essential for the repair of cutaneous wounds. Fibroblasts presumably use cell surface receptors of the integrin family during migration into a wound from the adjacent uninjured tissue and for the subsequent matrix repairs. We have investigated the possible roles of platelet-derived growth factor and inflammatory cytokines in the regulation of integrin expression on wound fibroblasts using a porcine cutaneous wound model and cultured human cells. Tissue specimens collected from 4-day pig wounds were stained with antibodies specific for the α1 and α5 integrin subunits. Staining for α1 was markedly decreased on fibroblasts adjacent to the wound and in the granulation tissue, while staining for α5 was clearly enhanced in both locations. Normal adult human dermal fibroblasts in culture express the integrins α1β1, a collagen receptor, and α5β1, a fibronectin receptor. Quantitative flow cytometry was used to measure cell surface integrin expression after treatment with platelet-derived growth factor (PDGF)-AA, PDGF-AB, or PDGF-BB. Each isoform of PDGF produced a significant decrease in the level of α1 present on the cell surface and an increase in the level of α5. Furthermore, PDGF-BB produced a corresponding decrease in α1 mRNA and an increase in α5 mRNA. In contrast, treatment with three inflammatory cytokines, IL-1β, TNF-α, and IFN-γ, produced clear increases in the levels of α1 and α5 present on the cell surface. Our observations suggest that the differential effects of PDGF and inflammatory cytokines may be part of the mechanism regulating the expression of α1 and α5 integrins by dermal fibroblasts during wound repair. © 1996 Wiley-Liss, Inc.     

Role of the I-domain in collagen binding specificity and activation of the integrins α1β1 and α2β1

Adhesion to collagens by most cell types is mediated by the integrins α1β1 and α2β1. Both integrin α subunits belong to a group which is characterized by the presence of an I domain in the N-terminal half of the molecule, and this domain has been implicated in the ligand recognition. Since purified α1β1 and α2β1 differ in their binding to collagens I and IV and recognize different sites within the major cell binding domain of collagen IV, we investigated the potential role of the α1 and α2 I domains in specific collagen adhesion. We find that introducing the α2 I domain into α1 results in surface expression of a functional collagen receptor. The adhesion mediated by this chimeric receptor (α1-2-1β1) is similar to the adhesion profile conferred by α2β1, not α1β1. The presence of α2 or α1-2-1 results in preferential binding to collagen I, whereas α1 expressing cells bind better to collagen IV. In addition, α1 containing cells bind to low amounts of a tryptic fragment of collagen IV, whereas α2 or α1-2-1 bearing cells adhere only to high concentrations of this substrate. We also find that collagen adhesion of NIH-3T3 mediated by α2β1 or α1-2-1β1, but not by α1, requires the presence of Mn²⁺ ions. This ion requirement was not found in CHO cells, implicating the I domain in cell type-specific activation of integrins. J. Cell. Physiol. 176:634–641, 1998. © 1998 Wiley-Liss, Inc.     

Interleukin-1β and tumor necrosis factor-α have opposite effects on fibroblasts and epithelial cells during basement membrane formation

Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) are typical proinflammatory cytokines that influence various cellular functions, including metabolism of the extracellular matrix. We examined the roles of IL-1beta and TNF-alpha in basement membrane formation in an in vitro model of alveolar epithelial tissue composed of alveolar epithelial cells and pulmonary fibroblasts. Formation of the basement membrane by immortalized rat alveolar type II epithelial (SV40-T2) cells, which ordinarily do not form a continuous basement membrane, was dose-dependently upregulated in the presence of 2 ng/ml IL-1beta or 5 ng/ml TNF-alpha. IL-1beta or TNF-alpha alone induced increased secretion of type IV collagen, laminin-1, and nidogen-1/entactin, all of which contributed to this upregulation. In contrast, while SV40-T2 cells cultured with a fibroblasts-embedded type I collagen gel were able to form a continuous basement membrane, they failed to form a continuous basement membrane in the presence of IL-1beta or TNF-alpha. Fibroblasts treated with IL-1beta or TNF-alpha secreted matrix metalloproteinase (MMP)-9 and MMP-2, and these MMPs inhibited basement membrane formation and degraded the basement membrane architecture. Neither IL-1beta- nor TNF-alpha-treated SV40-T2 cells increased the secretion of MMP-9 and MMP-2. These results suggest that IL-1beta participates in basement membrane formation in two ways. One is the induction of MMP-2 and MMP-9 secretion by fibroblasts, which inhibits basement membrane formation, and the other is induction of basement membrane component secretion from alveolar epithelial cells to enhance basement membrane formation.     

Human α1β3γ2L gamma‐aminobutyric acid type A receptors: High‐level production and purification in a functional state

Gamma‐aminobutyric acid type A receptors (GABA_ARs) are the most important inhibitory chloride ion channels in the central nervous system and are major targets for a wide variety of drugs. The subunit compositions of GABA_ARs determine their function and pharmacological profile. GABA_ARs are heteropentamers of subunits, and (α1)₂(β3)₂(γ2L)₁ is a common subtype. Biochemical and biophysical studies of GABA_ARs require larger quantities of receptors of defined subunit composition than are currently available. We previously reported high‐level production of active human α1β3 GABA_AR using tetracycline‐inducible stable HEK293 cells. Here we extend the strategy to receptors containing three different subunits. We constructed a stable tetracycline‐inducible HEK293‐TetR cell line expressing human (N)–FLAG–α1β3γ2L–(C)–(GGS)₃GK–1D4 GABA_AR. These cells achieved expression levels of 70–90 pmol [³H]muscimol binding sites/15‐cm plate at a specific activity of 15–30 pmol/mg of membrane protein. Incorporation of the γ2 subunit was confirmed by the ratio of [³H]flunitrazepam to [³H]muscimol binding sites and sensitivity of GABA‐induced currents to benzodiazepines and zinc. The α1β3γ2L GABA_ARs were solubilized in dodecyl‐d ‐maltoside, purified by anti‐FLAG affinity chromatography and reconstituted in CHAPS/asolectin at an overall yield of ～30%. Typical purifications yielded 1.0–1.5 nmoles of [³H]muscimol binding sites/60 plates. Receptors with similar properties could be purified by 1D4 affinity chromatography with lower overall yield. The composition of the purified, reconstituted receptors was confirmed by ligand binding, Western blot, and proteomics. Allosteric interactions between etomidate and [³H]muscimol binding were maintained in the purified state.     

Involvement of integrins α3β1 and α5β1 and glycoprotein IIb in megakaryocyte‐induced osteoblast proliferation

As the prevalence of osteoporosis is expected to increase over the next few decades, the development of novel therapeutic strategies to combat this disorder becomes clinically imperative. These efforts draw extensively from an expanding body of knowledge pertaining to the physiologic mechanisms of skeletal homeostasis. To this body of knowledge, we contribute that cells of hematopoietic lineage may play a crucial role in balancing osteoblastic bone formation against osteoclastic resorption. Specifically, our laboratory has previously demonstrated that megakaryocytes (MKs) can induce osteoblast (OB) proliferation in vitro, but do so only when direct cell‐to‐cell contact is permitted. To further investigate the nature of this interaction, we have effectively neutralized several adhesion molecules known to function in the analogous interaction of MKs with another cell type of mesenchymal origin—the fibroblast (FB). Our findings implicate the involvement of fibronectin/RGD‐binding integrins including α3β1 (VLA‐3) and α5β1 (VLA‐5) as well as glycoprotein (gp) IIb (CD41), all of which are known to be expressed on MK membranes. Furthermore, we demonstrate that interleukin (IL)‐3 can enhance MK‐induced OB activation in vitro, as demonstrated in the MK–FB model system. Taken together, these results suggest that although their physiologic and clinical implications are very different, these two models of hematopoietic–mesenchymal cell activation are mechanistically analogous in several ways. J. Cell. Biochem. 109: 927–932, 2010. © 2010 Wiley‐Liss, Inc.     

The Muscle-Specific Laminin Receptor α7β1 Integrin Negatively Regulates α5β1 Fibronectin Receptor Function

α7β1 is the major integrin complex expressed in differentiated muscle cells where it functions as a laminin receptor. In this work we have expressed the α7 integrin subunit in CHO cells to investigate the functional properties of this receptor. After transfection with α7 CHO cells acquired the ability to adhere and spread on laminin 1 consistent with the laminin receptor activity of the α7β1. α7 transfectants, however, showed a 70% reduction in the ability to adhere to fibronectin and were unable to assemble a fibronectin matrix. The degree of reduction was inversely related to the level of α7 expression. To define the mechanisms underlying this adhesive defect we analyzed surface expression and functional properties of the α5β1 fibronectin receptor. Although cell surface expression of α5β1 was reduced by a factor of 20–25% in α7 transfectants compared to control untransfected cells, this slight reduction was not sufficient to explain the dramatic reduction in cell adhesion (70%) and matrix assembly (close to 100%). Binding studies showed that the affinity of¹²⁵I-fibronectin for its surface receptor was decreased by 50% in α7 transfectants, indicating that the α5β1 integrin is partially inactivated in these cells. Inactivation can be reversed by Mn²⁺, a cation known to increase integrin affinity for their ligands. In fact, incubation of cells with Mn²⁺restored fibronectin binding affinity, adhesion to fibronectin, and assembly of fibronectin matrix in α7 transfectants. These data indicate that α7 expression leads to the functional down regulation of α5β1 integrin by decreasing ligand binding affinity and surface expression. In conclusion, the data reported establish the existence of anegative cooperativitybetween α7 and α5 integrins that may be important in determining functional regulation of integrins during myogenic differentiation.