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.     

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.     

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.     

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.     

Ex-FABP: a fatty acid binding lipocalin developmentally regulated in chicken endochondral bone formation and myogenesis

Extracellular fatty acid binding protein (Ex-FABP) is a 21 kDa lipocalin specifically binding fatty acids, expressed during chicken embryo development in hypertrophic cartilage, in muscle fibers and in blood granulocytes. 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 chickens. The possible mammalian counterpart is the Neu-related lipocalin (NRL), a lipocalin overexpressed in rat mammary cancer; NRL is homologous to the human neutrophil gelatinase associated lipocalin (NGAL) expressed in granulocytes and in epithelial cells in inflammation and malignancy and to the Sip24 (super-inducible protein 24), an acute phase lipocalin expressed in mouse after turpentine injection. Immunolocalization and in situ hybridization showed that NRL/NGAL is expressed in hypertrophic cartilage, in forming skeletal muscle fibers and in developing heart. In adult cartilage NRL/NGAL was expressed in articular cartilage from osteoarthritic patients and in chondrosarcoma. Moreover, NRL was induced in chondrocyte and myoblast cultures by an inflammatory agent. We propose that these lipocalins (Ex-FABP, NRL/NGAL, Sip24) represent stress proteins physiologically expressed in tissues where active remodeling is taking place during development and also present in tissues characterized by an acute phase response due to pathological conditions.     

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.     

Expression of anchorin CII mRNA by cultured chondrocytes

The establishment of a cell culture system promoting chondrocyte differentiation has been utilized to better characterize phenotypic stages of chondrogenesis at the cellular level. Although the expression of the type II collagen gene has been studied during “in vitro” chondrocyte differentiation, little is known about the expression of the gene coding for its receptor: anchorin CII. The modulation of the anchorin mRNA steady state level in chick embryo chondrocytes at different developmental stages is described here.The anchorin mRNA level was low in dedifferentiated chondrocytes, progressively increased after the cell transfer into suspension (a condition promoting differentiation), reached its maximal value after 4 weeks and decreased after 5 weeks.Therefore anchorin CII mRNA reaches its maximum level in hypertrophic stage II chondrocytes.     

Chondrocyte protein with a poly-proline region is a novel protein expressed by chondrocytes in vitro and in vivo

Chondrogenic differentiation is a multistep process entailing the sequential activation and inhibition of the expression of a number of genes. To identify genes preferentially expressed at the hypertrophic stage rather than early differentiation stages of chicken chondrocyte differentiation, a subtracted cDNA library was generated. Here we describe the characterization of a cDNA isolated from this library and that of the encoded protein referred to as Chondrocyte Protein with a Poly-proline Region (CHPPR).The cDNA coding for CHPPR hybridizes with a 3.0-kb mRNA expressed at extremely low levels in dedifferentiated chondrocytes, cultured in adherent conditions, at low levels in differentiating chondrocytes and at very high levels in hypertrophic chondrocytes in suspension culture. The Parathyroid Hormone peptide [PTH (1-34)] enhances accumulation of CHPPR mRNA in cultured chondrocytes. This 3.0-kb mRNA is also detectable in several chick embryo tissues but at a lower extent when compared to that present in cartilage and in hypertrophic chondrocytes. The CHPPR cDNA has a complete open reading frame coding for a polypeptide with a calculated mass of 35.6 kDa containing a proline-rich region with a PPLP motif (single-letter amino acid code). We demonstrate by Western blot analysis that two CHPPR isoforms are detected in the cell lysates from cultured chondrocytes when they are not in the culture medium; furthermore, we find that the CHPPR gene is expressed in vivo by chick embryo chondrocytes at higher levels in the prehypertrophic and hypertrophic zones.     

Expression of anchorin CII mRNA by cultured chondrocytes

The establishment of a cell culture system promoting chondrocyte differentiation has been utilized to better characterize phenotypic stages of chondrogenesis at the cellular level. Although the expression of the type II collagen gene has been studied during in vitro chondrocyte differentiation, little is known about the expression of the gene coding for its receptor: anchorin CII. The modulation of the anchorin mRNA steady state level in chick embryo chondrocytes at different developmental stages is described here.The anchorin mRNA level was low in dedifferentiated chondrocytes, progressively increased after the cell transfer into suspension (a condition promoting differentiation), reached its maximal value after 4 weeks and decreased after 5 weeks.     

Expression ofruntBIs Modulated during Chondrocyte Differentiation

Theruntlocus inDrosophilaencodes a nuclear protein involved in embryo segmentation, sex determination/X dosage compensation, and neurogenesis.runthomologues have been identified in higher vertebrates. The encoded proteins share a domain of 128 amino acids called the runt domain. It has been reported that this domain mediates DNA binding and heterodimerization. Here, we analyzeruntBexpression during chondrocyte differentiation “in vitro” and “in vivo.” We have first isolated, from a chondrocyte library, a cDNA clone coding for aruntBchicken homologue and containing a complete open reading frame. The predicted protein product is 84% identical to the mouse PEBP2αB2 isoform. By RT-PCR analysis we have also cloned the chicken cDNA fragment coding for ΔαB2, the exon sequence included in the B1 isoform mRNA. On Northern blot analysis of cultured chondrocytes, runtB mRNA levels increase dramatically with the transition from stage 0 (dedifferentiated) to stages I and II (hypertrophic chondrocytes). Moreover, runt polypeptides were demonstrated in chondrocytes bothin vivoandin vitro.These results suggest thatruntplays a role in chondrogenic differentiation.     

Expression of serum amyloid A in chondrocytes and myoblasts differentiation and inflammation: possible role in cholesterol homeostasis.

Serum amyloid A (SAA) is synthesized by the liver during the acute phase. Local expression of SAA mRNA has been reported also in non-liver cells, a potential local source of SAA protein not related to the systemic acute phase response. SAA function has not been established yet. In the present study, we identified SAA as a protein expressed by chondrocytes and myoblasts in response to inflammatory stimula. In both cell systems, SAA mRNA and protein expression is strongly stimulated by bacterial lipopolysaccharide treatment. SAA mRNA expression is also enhanced during terminal differentiation of cells of the chondrogenic and myogenic lineage; mRNA is barely detectable in prechondrogenic cells and is highly expressed in differentiated hyperthrophic chondrocytes. An increased level of SAA mRNA was also observed in vivo when we compared mRNA extracted from tibiae of 10 day embryos, still fully cartilaginous, with tibiae from 18 day embryos, a stage when the endochondral ossification process has already started. p38 activation, a well-known event of the chondrogenesis signaling cascade, controls expression of SAA in cartilage following inflammatory stimuli. SAA secreted by stimulated chondrocytes is associated with cholesterol. Cholesterol is synthesized by the same chondrocytes and is also increased in inflammatory conditions. A role of SAA in cholesterol homeostasis in chondrocytes is proposed.     

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.     

Inhibition of RhoA but not ROCK induces chondrogenesis of chick limb mesenchymal cells

Cell shape change and cytoskeletal reorganization are known to be involved in the chondrogenesis. Negative role of RhoA, a cytoskeleton-regulating protein, and its downstream target, Rho-associated protein kinase (ROCK) in the chondrogenesis has been studied in many different culture systems including primary chondrocytes, chondrogenic cell lines, dedifferentiated chondrocytes, and micromass culture of mesenchymal cells. To further investigate the role of RhoA and ROCK in the chondrogenesis, we examined the RhoA-ROCK-myosin light chains (MLC) pathway in low density culture of chick limb bud mesenchymal cells. We observed for the first time that inhibition of RhoA by C3 cell-permeable transferase, CT04, induced chondrogenesis of undifferentiated mesenchymal single cells following dissolution of actin stress fibers. Inhibition of RhoA activity by CT04 was confirmed by pull down assay using the Rho-GTP binding domain of Rhotekin. CT04 also inhibited ROCK activity. In contrast, inhibition of ROCK by Y27632 neither altered the actin stress fibers nor induced chondrogenesis. In addition, inhibition of RhoA or ROCK did not affect the phosphorylation of MLC. Inhibition of myosin light chain kinase (MLCK) by ML-7 or inhibition of myosin ATPase with blebbistatin dissolved actin stress fibers and induced chondrogenesis. ML-7 reduced the MLC phosphorylation. Taken together, our current study suggests that RhoA uses other pathway than ROCK/MLC in the modulation of actin stress fibers and chondrogenesis. Our data also imply that, irrespective of mechanisms, dissolution of actin stress fibers is crucial for chondrogenesis.     

Regulation of chicken ccn2 gene by interaction between RNA cis-element and putative trans-factor during differentiation of chondrocytes

CCN2/CTGF is a multifunctional growth factor. Our previous studies have revealed that CCN2 plays important roles in both growth and differentiation of chondrocytes and that the 3'-untranslated region (3'-UTR) of ccn2 mRNA contains a cis-repressive element of gene expression. In the present study, we found that the stability of chicken ccn2 mRNA is regulated in a differentiation stage-dependent manner in chondrocytes. We also found that stimulation by bone morphogenetic protein 2, platelet-derived growth factor, and CCN2 stabilized ccn2 mRNA in proliferating chondrocytes but that it destabilized the mRNA in prehypertrophic-hypertrophic chondrocytes. The results of a reporter gene assay revealed that the minimal repressive cis-element of the 3'-UTR of chicken ccn2 mRNA was located within the area between 100 and 150 bases from the polyadenylation tail. Moreover, the stability of ccn2 mRNA was correlated with the interaction between this cis-element and a putative 40-kDa trans-factor in nuclei and cytoplasm. In fact, the binding between them was prominent in proliferating chondrocytes and attenuated in (pre)hypertrophic chondrocytes. Stimulation by the growth factors repressed the binding in proliferating chondrocytes; however, it enhanced it in (pre)hypertrophic chondrocytes. Therefore, gene expression of ccn2 mRNA during endochondral ossification is properly regulated, at least in part, by changing the stability of the mRNA, which arises from the interaction between the RNA cis-element and putative trans-factor.