Extracellular fatty acid binding protein (Ex-FABP) modulation by inflammatory agents: "physiological" acute phase response in endochondral bone formation

Ex-FABP, extracellular fatty acid binding protein, is a 21 kDa lipocalin expressed in hypertrophic cartilage, muscle and heart during chick embryo development and in granulocytes. Ex-FABP synthesis was increased in chondrocyte and myoblast cultures by inflammatory agents (LPS; IL6) and repressed by antiinflammatory agents. Expression of Ex-FABP and specific gelatinases is paralleled in hypertrophic cartilage; LPS specifically induced high molecular weight gelatinase ( > 200 kDa). LPS-treated hypertrophic chondrocytes showed increased chemotactic activity for endothelial cells paralleled by increased expression of transferrin. A high amount of Ex-FABP was expressed in adult pathological cartilage both in dyschondroplastic and osteoarthritic chickens. Controls were negative. Ex-FABP could represent a stress protein physiologically expressed in tissues where active remodelling is taking place during development and in tissues characterized by an acute phase response due to pathological conditions. We also suggest that during endochondral bone formation other responses characteristic of a local inflammatory status, such as gelatinase production and angiogenic factor secretion, are "physiologically" activated.     

Ex-FABP,extracellular fatty acid binding protein,is a stress lipocalin expressed during chicken embryo development

Extracellular Fatty Acid Binding Protein (Ex-FABP) is a 21 kDa lipocalin, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibres and in blood granulocyte. The protein selectively binds with high affinity fatty acids, preferably long chain unsaturated fatty acids in chondrocyte and myoblast cultures Ex-FABP expression is increased by inflammatory-agents and repressed by anti-inflammatory-agents. In adult cartilage, Ex-FABP is expressed only in pathological conditions such as in dyschondroplastic and osteoarthritic chicken cartilage. We propose that lipocalin Ex-FABP represents a stress protein physiologically expressed in tissues where active remodelling is taking place during development and also present in tissues characterized by a stress response due to pathological conditions.     

Inhibition of cell proliferation and induction of apoptosis by ExFABP gene targeting

Ex-FABP, an extracellular fatty acid binding lipocalin, is physiologically expressed by differentiating chicken chondrocytes and myoblasts. Its expression is enhanced after cell treatment with inflammatory stimuli and repressed by anti-inflammatory agents, behaving as an acute phase protein. Chicken liver fragments in culture show enhanced protein expression after bacterial endotoxin treatment. To investigate the biological role of Ex-FABP, we stably transfected proliferating chondrocytes with an expression vector carrying antisense oriented Ex-FABP cDNA. We observed a dramatic loss of cell viability and a strong inhibition of cell proliferation and differentiation. When chondrocytes were transfected with the antisense oriented Ex-FABP cDNA we observed that Ex-FABP down-modulation increased apoptotic cell number. Myoblasts transfected with the same expression vector showed extensive cell death and impaired myotube formation. We suggest that Ex-FABP acts as a constitutive survival protein and that its expression and activation are fundamental to protect chondrocytes from cell death.     

NGAL基因在永生化食管上皮细胞恶性转化中过表达的研究

为研究NGAL（neutrophil gelatinase-associated lipocalin）基因在永生化食管上皮细胞恶性转化中的表达情况,以永生化食管上皮细胞系SHEE和食管癌细胞系SHEEC互为对照,用cDNA微列阵进行筛选,用RNA印迹和RT-PCR进行鉴定,cDNA克隆测序后与GenBank进行BLAST分析比较.结果表明NGAL基因在SHEEC中出现显著差异过表达,其cDNA序列与小鼠24p3、大鼠NRL（neu-related lipocalin）、人中性粒细胞NGAL和卵巢癌NGAL具有较高的相似性.这提示NGAL基因在永生化食管上皮细胞恶性转化中可能发挥着重要作用,可能是一种新的癌基因或促癌基因.     

Expression of the Extracellular Fatty Acid Binding Protein (Ex-FABP) during Muscle Fiber Formationin Vivoandin Vitro

We report that Ex-FABP, an extracellular protein belonging to the lipocalin family and involved in the extracellular transport of long-chain fatty acids, is expressed in the forming myotubes bothin vivoandin vitro.The presence of the protein and of the mRNA was observed in newly formed myotubes at early stages of chick embryo development by immunohistochemistry and byin situhybridization. At later stages of development myofibers still expressed both the mRNA and the protein. Ex-FABP expression was observed also in the developing myocardium and the muscular layer of large blood vessels. In agreement with these findings, an initial expression of the mRNA and protein secretion by cultured chicken myoblasts were observed only after the onset of myoblast fusion. Double-immunofluorescence staining of these cultured cells revealed that multinucleate myotubes were stained by antibodies directed against both the Ex-FABP and the sarcomeric myosin, whereas immature myotubes and single myoblasts were not. When added to cultured myoblasts, antibodies against the Ex-FABP induced a strong enhancement of the production of the same protein. In all experiments some cell sufferance and a transient impairment of myotube formation were also observed. The finding that the continuous removal of the Ex-FABP from the culture medium of myoblasts, due to the formation of immune complexes, resulted in an overproduction of the protein suggests a feedback (autocrine) control during myotube differentiation and maturation. We propose that the requirement for increased transport and metabolism of free fatty acid released from the membrane phospholipids and storage lipids, mediated by Ex-FABP, may be essential during differentiation of multinucleated myotubes or that an increased local demand of fatty acids and metabolites may act as a local hormone in tissues differentiating and undergoing morphogenesis.     

Transglutaminase 2 as a biomarker of osteoarthritis: an update

Osteoarthritis is a progressive joint disease characterized by cartilage degradation and bone remodelling. Under physiologic conditions, articular cartilage displays a stable chondrocyte phenotype, whereas in osteoarthritis a chondrocyte hypertrophy develops near the sites of cartilage surface damage and associates to the pathologic expression of type X collagen. Transglutaminases (TGs) include a family of Ca²⁺-dependent enzymes that catalyze the formation of γ-glutamyl cross-links. Their substrates include a variety of intracellular and extracellular macromolecular components. TGs are ubiquitously and abundantly expressed and implicated in a variety of physiopathological processes. TGs activity is modulated by inflammatory cytokines. TG2 (also known as tissue transglutaminase) mediates the hypertrophic differentiation of joint chondrocytes and interleukin-1-induced calcification. Histomorphometrical and biomolecular investigations document increased TG2 expression in human and experimental osteoarthritis. Consequently, the level of TG2 expression may represent an adjuvant additional marker to monitor tissue remodelling occurring in osteoarthritic joint tissue. Experimental induction of osteoarthritis in TG2 knockout mice is followed from reduced cartilage destruction and increased osteophyte formation compared to wild-type mice, suggesting a different influence on joint bone and cartilage remodelling. The capacity of transamidation by TG2 to regulate activation of latent TGF-β seems to have a potential impact on the regulation of inflammatory response in osteoarthritic tissues. Additional studies are needed to define TG2-regulated pathways that are differently modulated in osteoblasts and chondrocytes during osteoarthritis.     

Ligand preference inferred from the structure of neutrophil gelatinase associated lipocalin

Neutrophil gelatinase associated lipocalin (NGAL), a constituent of neutrophil granules, is a member of the lipocalin family of binding proteins. NGAL can also be highly induced in epithelial cells in both inflammatory and neoplastic colorectal disease. NGAL is proposed to mediate inflammatory responses by sequestering neutrophil chemoattractants, particularly N-formylated tripeptides and possibly leukotriene B(4) and platelet activating factor. The crystal structures of NGAL display a typical lipocalin fold, albeit with an unusually large and atypically polar binding site, or calyx. The fold of NGAL is most similar to the epididymal retinoic acid-binding protein, another lipocalin, though the overall architecture of the calyces are very different. The crystal structures also reveal either sulfate ions or an adventitiously copurified fatty acid bound in the binding site. Neither ligand is displaced by added N-formylated tripeptides. The size, shape, and character of the NGAL calyx, as well as the low relative affinity for N-formylated tripeptides, suggest that neither the copurified fatty acid nor any of the proposed ligands are likely to be the preferred ligand of this protein. Comparisons between the crystal structures and the recently reported solution structure of NGAL reveal significant differences, in terms of both the details of the structure and the overall flexibility of the fold.     

Osteopontin is expressed by adult human osteoarthritic chondrocytes: protein and mRNA analysis of normal and osteoarthritic cartilage.

Osteopontin, a sulfated phosphoprotein with cell binding and matrix binding properties, is expressed in a variety of tissues. In the embryonic growth plate, osteopontin expression was found in bone-forming cells and in hypertrophic chondrocytes. In this study, the expression of osteopontin was analyzed in normal and osteoarthritic human knee cartilage. Immunohistochemistry, using a monoclonal anti-osteopontin antibody was negative on normal cartilage. These results were confirmed in Western blot experiments, using partially purified extracts of normal knee cartilage. No osteopontin gene expression was observed in chondrocytes of adult healthy cartilage, however, in the subchondral bone plate, expression of osteopontin mRNA was detected in the osteoblasts. In cartilage from patients with osteoarthritis, osteopontin could be detected by immunohistochemistry, Western blot analysis, in situ hybridization, and Northern blot analysis. A qualitative analysis indicated that osteopontin protein deposition and mRNA expression increase with the severity of the osteoarthritic lesions and the disintegration of the cartilaginous matrix. Osteopontin expression in the cartilage was limited to the chondrocytes of the upper deep zone, showing cellular and territorial deposition. The strongest osteopontin detection was found in deep zone chondrocytes and in clusters of proliferating chondrocytes from samples with severe osteoarthritic lesions. These data show the expression of osteopontin in adult human osteoarthritic chondrocytes, suggesting that chondrocyte differentiation and the expression of differentiation markers in osteoarthritic cartilage resembles that of epiphyseal growth plate chondrocytes.     

A common pathway in differentiation and inflammation: p38 mediates expression of the acute phase SIP24 iron binding lipocalin in chondrocytes

SIP24 is an acute phase iron binding lipocalin physiologically expressed in vivo in developing cartilage by prehypertrophic/hypertrophic chondrocytes. Taking advantage of the chondrocytic cell line MC615 and using SIP24 as a marker we investigated the pathways active in cartilage differentiation and inflammation. MC615 cells were cultured as: (i) proliferating prechondrogenic cells expressing type I collagen (ii) differentiated hyperconfluent cells expressing Sox9 and type II collagen. In proliferating cells the pathway PKC/ERK1, ERK2 was activated and SIP24 was not expressed while in differentiated cells the pathway p38/NF-kappaB was activated and SIP24 was expressed. Proliferating cells treated with inflammatory agents expressed a large amount of SIP24 and showed activation of p38/NF-kappaB pathway and inhibition of PKC/ERK1, ERK2 pathway indicating that in inflammation and differentiation the same factors are activated (p38, NF-kappaB) or inactivated (PKC, ERKs). Treatment of proliferating cells with the p38 specific inhibitor SB203580 inhibited the inflammation induced activation of p38 and the synthesis of SIP24. PMA treatment induced activation of PKC, inactivation of p38 and suppression of SIP24 synthesis, suggesting that PKC activation inhibits p38 activation. In differentiated hyperconfluent cells the same factors (p38/NF-kappaB/SIP24) are constitutively activated: treatment with inflammatory agents does not increase synthesis of SIP24 while treatment with SB203580 and with PMA does not repress activation of p38 nor synthesis of SIP24. We propose that the SIP24 stress related protein is expressed via p38 activation/NF-kappaB recruitment both in chondrocyte differentiation and inflammation and that a signaling pathway active in the acute phase response is physiologically activated in differentiation.     

Expression patterns of chondrocyte genes cloned by differential display in tibial dyschondroplasia

Tibial dyschondroplasia (TD) appears to involve a failure of the growth plate chondrocytes within growing long bones to differentiate fully to the hypertrophic stage, resulting in a mass of prehypertrophic chondrocytes which form the avascular TD lesion. Many biochemical and molecular markers of chondrocyte hypertrophy are absent from the lesion, or show reduced expression, but the cause of the disorder remains to be identified. As differentiation to the hypertrophic state is impaired in TD, we hypothesised that chondrocyte genes that are differentially expressed in the growth plate should show altered expression in TD. Using differential display, four genes, B-cadherin, EF2, HT7 and Ex-FABP were cloned from chondrocytes stimulated to differentiate to the hypertrophic stage in vitro, and their differential expression confirmed in vivo. Using semi-quantitative RT-PCR, the expression patterns of these genes were compared in chondrocytes from normal and TD growth plates. Surprisingly, none of these genes showed the pattern of expression that might be expected in TD lesion chondrocytes, and two of them, B-cadherin and Ex-FABP, were upregulated in the lesion. This indicates that the TD phenotype does not merely reflect the absence of hypertrophic marker genes, but may be influenced by more complex developmental mechanisms/defects than previously thought.     

Galline Ex-FABP is an antibacterial siderocalin and a lysophosphatidic acid sensor functioning through dual ligand specificities

Galline Ex-FABP was identified as another candidate antibacterial, catecholate siderophore binding lipocalin (siderocalin) based on structural parallels with the family archetype, mammalian Siderocalin. Binding assays show that Ex-FABP retains iron in a siderophore-dependent manner in both hypertrophic and dedifferentiated chondrocytes, where Ex-FABP expression is induced after treatment with proinflammatory agents, and specifically binds ferric complexes of enterobactin, parabactin, bacillibactin and, unexpectedly, monoglucosylated enterobactin, which does not bind to Siderocalin. Growth arrest assays functionally confirm the bacteriostatic effect of Ex-FABP in?vitro under iron-limiting conditions. The 1.8?? crystal structure of Ex-FABP explains the expanded specificity, but also surprisingly reveals an extended, multi-chambered cavity extending through the protein and encompassing two separate ligand specificities, one for bacterial siderophores (as in Siderocalin) at one end and one specifically binding copurified lysophosphatidic acid, a potent cell signaling molecule, at the other end, suggesting Ex-FABP employs dual functionalities to explain its diverse endogenous activities.     

The neutrophil lipocalin NGAL is a bacteriostatic agent that interferes with siderophore-mediated iron acquisition

First identified as a neutrophil granule component, neutrophil gelatinase-associated lipocalin (NGAL; also called human neutrophil lipocalin, 24p3, uterocalin, or neu-related lipocalin) is a member of the lipocalin family of binding proteins. Putative NGAL ligands, including neutrophil chemotactic agents such as N-formylated tripeptides, have all been refuted by recent biochemical and structural results. NGAL has subsequently been implicated in diverse cellular processes, but without a characterized ligand, the molecular basis of these functions remained mysterious. Here we report that NGAL tightly binds bacterial catecholate-type ferric siderophores through a cyclically permuted, hybrid electrostatic/cation-pi interaction and is a potent bacteriostatic agent in iron-limiting conditions. We therefore propose that NGAL participates in the antibacterial iron depletion strategy of the innate immune system.     

Human neutrophil gelatinase-associated lipocalin and homologous proteins in rat and mouse

Neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa lipocalin originally purified from human neutrophils. It exists in monomeric and homo- and heterodimeric forms, the latter as a dimer with human neutrophil gelatinase. It is secreted from specific granules of activated human neutrophils. Homologous proteins have been identified in mouse (24p3/uterocalin) and rat (alpha(2)-microglobulin-related protein/neu-related lipocalin). Structural data have confirmed a typical lipocalin fold of NGAL with an eight-stranded beta-barrel, but with an unusually large cavity lined with more polar and positively charged amino acid residues than normally seen in lipocalins. Chemotactic formyl-peptides from bacteria have been proposed as ligands of NGAL, but binding experiments and the structure of NGAL do not support this hypothesis. Besides neutrophils, NGAL is expressed in most tissues normally exposed to microorganisms, and its synthesis is induced in epithelial cells during inflammation. This may indicate either a microbicidal activity of NGAL or a role in regulation of inflammation or cellular growth, putative functions yet to be demonstrated.