Functional analysis of newly collagen (mouse Co124a1) expressed in bone tissues

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Mouse col24al encodes a polypeptide chain of 1733 amino acids, and the overall structure is conserved. Oligo-CAP Race indicated that mouse col24al gene has at least two major splicing variants. Cell transfection experiments in combination with DNA-binding assays demonstrated that Col24al promoter activity is under the control of an upstream cis-acting element, which is shared by both transcripts and is recognized by specific combinations of c-Jun, CREB1, ATF1 and ATF2 dimers. Consistent with these results, overexpression of c-Jun, ATF1, ATF2 or CREB1 in transiently transfected osteoblastic cells stimulated transcription from reporter gene constructs driven by the Col24al promoter to different degrees. Moreover, chromatin immunoprecipitation experiments showed that these nuclear factors bind the same upstream sequence of the endogenous Co124a1 gene.In situ hybridization on Neonatal (p4) tibia confirmed that col24al is expressed in bone shaft, but it is not expressed in cartilage or growth plates. In accordance with these in vivo observations, several osteoblast lineage cell lines, such as ROS17 and MC3T3-E1, and primary calvarial osteoblast, were found to express col24al. Furthermore, col24al expression was activated during BMP-2 induced differentiation of C3H10T1/2 mesenchymal cells.Collectively these data provide new information about transcriptional control of collagen fibrillogenesis, in addition to implicating for the first time CREB/AP1 protein complexes in the regulation of collagen gene expression in osteoblasts.