CALbeta, a novel lipocalin associated with chondrogenesis and inflammation.

We have previously demonstrated the association of the chicken lipocalin Ex-FABP with cartilage formation and inflammatory responses as a marker of these processes (Descalzi Cancedda et al., Biochim. Biophys. Acta 1482, 127-135, 2000). Here we report the isolation and characterisation of a new lipocalin gene laying upstream the Ex-FABP, thus representing the second member of a possible genomic cluster. This gene contains an open reading frame coding for a polypeptide of about 19 kDa. The amino-acid sequence revealed a conserved lipocalin secondary structure. Tissue distribution of the protein in developing embryos showed a preferential expression in the heart although mRNA transcripts could be detected also in muscle, lung and liver. The lowest expression was observed in the stomach, brain and skin. During endochondral formation of long bones, the protein is differentially distributed, as the transcripts, evidenced in the tibia by in situ hybridisation, are present in the hypertrophic cone of the cartilage and mostly absent in the area of the proliferating chondrocytes. Such developmental regulation was observed also in vitro in cultured chondrocytes where the transcripts were barely detectable in dedifferentiated cells but highly expressed in hypertrophic chondrocytes. The protein was also significantly induced by lipopolysaccharide stimulation of chondrocytes, indicating a possible involvement in acute phase response. Raising specific antibodies in a rabbit allowed validating, at the protein level, all the transcriptional data. Moreover, we gained evidence that the protein is actively secreted in the extracellular matrix surrounding the chondrocytes. Because of its peculiar expression in cartilage, this new protein was named chondrogenesis-associated lipocalin beta (thereafter referred to as CAL beta). The close similarity between Ex-FABP and CAL beta expression patterns supports the hypothesis of a genomic organisation in a cluster where both genes could be co-ordinately regulated.

Phylogeny and regulation of four lipocalin genes clustered in the chicken genome: evidence of a functional diversification after gene duplication.

A novel lipocalin gene is here reported that represents the fourth member of a cluster we have identified in the chicken genome. This cluster also includes Chondrogenesis-Associated Lipocalins beta and gamma (CAL beta, CAL gamma) and Extracellular Fatty Acid Binding Protein (Ex-FABP). The new gene codes for a 22-kDa secreted protein with three cysteine residues and a series of sequence features well conserved in the lipocalin family. All the genes in the cluster are structurally similar presenting comparable exon/intron boundary positions and exon sizes. A phylogenetic analysis indicates the monophyletic grouping of these genes, and their relationship with the lipocalins alpha-1-microglobulin (A1mg), complement factor 8 gamma chain (C8GC), prostaglandin D synthase (PGDS), and neutrophil-gelatinase-associated lipocalin (NGAL). The new cluster gene appears to be the ortholog of the mammalian C8GC and was thus named Ggal-C8GC. This orthology also suggests that this lipocalin was present in the ancestor common to reptiles and mammals. In addition to other expressing tissues, Ex-FABP, CAL beta and CAL gamma genes are highly transcribed in chondrocytes at late stages of chondrogenesis during endochondral bone formation and/or upon inflammatory stimulation. Here, we show that they are also transcriptionally induced when chondrocytes are subjected to various biological events as cell quiescence, cell shape transition, and hormonal stimulation. By contrast, Ggal-C8GC transcripts are only barely detectable in chondrocytes, but are more abundant in liver, kidney, brain, heart, skeletal muscle and particularly in skin. Moreover, no expression induction was observed neither during chondrocyte differentiation, nor upon any of the stimulations mentioned above. This indicates that the Ggal-C8GC gene was co-opted for a novel function after the duplication events that gave rise to the cluster. The peculiar coordinated regulation of Ex-FABP, CAL beta and CAL gamma, and the apparent divergent role of Ggal-C8GC suggest that these gene duplications may have been maintained during evolution by a sub-functionalization mechanism where some common function(s) are shared by several members of the cluster and some other specialized function(s) are unique to other members.

Characterization of an osteoblast-specific enhancer element in the CBFA1 gene.

Cbfa1 is a critical regulator of cell differentiation expressed only in the osteochondrogenic lineage. To define the molecular basis of this cell-specific expression we analyzed the murine Cbfa1 promoter. Here we show that the first 976 bp of this promoter are specifically active in osteoblastic cells. Within this region DNase I footprinting delineated a 40-bp area (CE1) protected differently by nuclear extracts from osteoblastic cells and from non-osteoblastic cells. When multimerized, CE1 conferred an osteoblast-specific activity to a heterologous promoter in DNA transfection experiments; this enhancing ability was conserved between mouse, rat, and human CE1 present in the respective Cbfa1 promoters. CE1 site-specific mutagenesis determined that it binds NF1- and AP1-like activities. Further analyses revealed that the NF1 site acts as a repressor in non-osteoblastic cells due to the binding of NF1-A, a NF1 isoform not expressed in osteoblastic cells. In contrast, the AP1 site mediates an osteoblast-specific activation caused by the preferential binding of FosB to CE1 in osteoblastic cells. In summary, this study identified an osteoblast-specific enhancer in the Cbfa1 promoter whose activity is achieved by the combination of an inhibitory and an activatory mechanism.

Differentiation-dependent activation of the extracellular fatty acid binding protein (Ex-FABP) gene during chondrogenesis.

Chicken hypertrophic chondrocytes secrete the extracellular fatty acid binding protein (Ex-FABP), a lipocalin not expressed by their undifferentiated precursors. Genomic clones coding for the full protein are here structurally and functionally analyzed. We first determined that the promoter sequence markedly differs from that reported for the homologous p20K, and we confirmed by genomic DNA Southern analysis the exactness of our sequence. This is of relevance since we have identified another lipocalin gene within the region of discrepancy, indicating thereby the existence of a lipocalin cluster within the same chromosomal locus. By transient transfections with 5'-deletions and the chloramphenicol acetyl transferase (CAT) reporter gene, the region between nt -926 and nt -629 was shown to be strongly active, specifically in hypertrophic chondrocytes and not in dedifferentiated cells. Responsive elements for several potential transcription factors lay within this sequence. Among those, activating protein-1 (AP-1) was shown to be involved in the regulation of the Ex-FABP gene during chondrocyte differentiation, as indicated by electrophoretic mobility shift assay, AP-1 site mutagenesis and functional interference assays.