Extracellular fatty acid binding protein (ex-FABP) is a stress protein expressed during chondrocyte and myoblast differentiation.

OBJECTIVE: We have isolated and characterized in our laboratory a lipocalin specifically binding unsaturated long chain fatty acids (Ex-FABP). In developing chicken embryo long bones, Ex-FABP first appears at the boundary of the cone of hypertrophic cartilage. 'In vitro' EX-FABP is highly expressed by differentiating hypertrophic chondrocytes. Ex-FABP is expressed also in the forming myotubes both 'in vivo' and 'in vitro'. In cultured chondrocytes, Ex-FABP expression is strongly induced by treatment with inflammatory agents such as the bacterial liposaccharide LPS or interleukin-6. The possible mechanism for this induction was investigated. Expression of Ex-FABP was studied in other stress conditions. DESIGN: To investigate a possible mechanism for Ex-FABP induction by LPS or interleukin-6, we have cultured the cells in the presence of either hydrogen peroxide or the NO donor SNAP (S-nitrosil-acetil-D, L-penicillamine), two agents known to produce cellular stresses through the activation of specific signalling pathways. To investigate Ex-FABP expression in other stress conditions, chondrocytes were cultured for 3 days in the presence of alpha,alpha-dipyridyl, an agent inhibiting prolyl hydroxylase activity and collagen secretion. Supplement of this agent to the culture medium results in an impairment of collagen secretion and assembly and the consequent altered interaction of the cell with the surrounding extracellular matrix. In addition Ex-FABP expression was studied also in chondrocytes cultured in the absence of serum, a stress condition activating cell defence mechanisms. RESULTS: We have excluded that induction of Ex-FABP expression by inflammatory agents is mediated by oxidative stress or NO production. Ex-FABP expression was induced also by changes in the hypertrophic chondrocyte microenvironment, considered either as extracellular matrix surrounding the cell in culture or as nature and concentration of growth factor in the culture medium. CONCLUSIONS: No definitive data are so far available on the possible role of Ex-FABP when induced by cellular stresses. The capacity of the protein to specifically bind and transport unsaturated long chain fatty acids suggests that lipid metabolism and fatty acid utilization by the cells may be involved. Based on literature data the NRL/N-GAL (neu-related lipocalin/neutrophil gelatinase-associated lipocalin) protein was proposed as a possible mammal counterpart of the chick Ex-FABP. We have suggested that Ex-FABP and NRL/NGAL expression in forming bones and muscles is part of a 'physiological' acute phase response. Interestingly the expression of Ex-FABP and NRL/NGAL is also activated in osteoarthritic cartilage and in the case of NRL/N-GAL during neoplastic transformation of chondrogenic lineage cells.

Modulation of commitment, proliferation, and differentiation of chondrogenic cells in defined culture medium.

The factors regulating the growth and development of mesenchymal precursor cells toward chondrogenesis are not well identified. We have developed a defined serum-free culture system that allows the proliferation of chick embryo chondrogenic cells and their maturation toward hypertrophic chondrocytes. Proliferation is obtained in adhesion in medium supplemented with insulin (Ins), Dexamethasone (Dex), and either basic fibroblast growth factor (FGF-2), platelet-derived growth factor bb, epithelial growth factor, or GH; the highest mitogenic response is induced by FGF-2 in synergy with Ins. Ins can be substituted by Ins-like growth factor I. When these cells are transferred into suspension culture in Ins/Dex and T3 without growth factor supplement, they undergo the complete chondrogenic development characterized by type X collagen synthesis and cellular hypertrophy. During differentiation, Ins cannot be substituted by Ins-like growth factor I. Chondrogenesis is also evidenced by the formation of hypertrophic cartilage when the medium is supplemented with ascorbic acid. If T3 is introduced in the proliferation phase, the cells fail to differentiate to hypertrophy in suspension unless bone morphogenetic protein-2 is added. Assays of ectopic tissue formation in nude mice, with cells implanted sc after adsorption on collagen sponge or porous hydroxyapatite ceramics, indicate that cells grown in Ins/FGF-2 reform mainly cartilage in vivo, whereas expansion in Ins/T3/Dex/FGF-2 leads to the formation of cartilage, bone, and adipose tissue.

Thyroid hormone, insulin, and glucocorticoids are sufficient to support chondrocyte differentiation to hypertrophy: a serum-free analysis.

Chondrocytes from chicken embryo tibia can be maintained in culture as adherent cells in Coon's modified Ham's F-12 medium supplemented with 10% FCS. In this condition, they dedifferentiate, losing type II collagen expression in favor of type I collagen synthesis. Their differentiation to hypertrophy can be obtained by transferring them to suspension culture. Differentiation is evidenced by the shift from type I to type II and type IX collagen synthesis and the following predominant expression of type X collagen, all markers of specific stages of the differentiation process. To identify the factors required for differentiation, we developed a serum-free culture system where only the addition of triiodothyronine (T3; 10(-11) M), insulin (60 ng/ml), and dexamethasone (10(-9) M) to the F-12 medium was sufficient to obtain hypertrophic chondrocytes. In this hormonal context, chondrocytes display the same changes in the pattern of protein synthesis as described above. For proper and complete cell maturation, T3 and insulin concentrations cannot be modified. Insulin cannot be substituted by insulin-like growth factor-I, but dexamethasone concentration can be decreased to 10(-12) M without chondrogenesis being impaired. In the latter case, the expression of type X collagen and its mRNA are inversely proportional to dexamethasone concentration. When ascorbic acid is added to the hormone-supplemented medium, differentiating chondrocytes organize their matrix leading to a cartilage-like structure with hypertrophic chondrocytes embedded in lacunae. However, this structure does not present detectable calcification, at variance with control cultures maintained in FCS. Accordingly, in the presence of the hormone mixture, the differentiating chondrocytes have low levels of alkaline phosphatase activity. This report indicates that T3 and insulin are primary factors involved in the onset and progression of chondrogenesis, while dexamethasone supports cell viability and modulates some differentiated functions.