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

小鼠col 24 al编码一条1733个氨基酸的多肽链，整体结构保守。Oligo-CAP Race表明小鼠col 24 al基因至少有两个主要的剪接变异体。细胞转染实验结合DNA结合试验表明，Col 24 al启动子活性的上游顺式作用元件的控制下，这是共享的两个转录本，并被识别的c-Jun，CREB 1，ATF 1和ATF 2二聚体的特定组合。与这些结果一致，c-Jun，ATF 1，ATF 2或CREB 1在瞬时转染成骨细胞中的过表达刺激了由Col 24 al启动子驱动的报告基因构建体的转录到不同程度。此外，染色质免疫沉淀实验表明，这些核因子结合的内源性Co 124 a1基因的相同上游序列。在新生儿（p4）胫骨原位杂交证实，col 24 a1在骨干中表达，但它不表达在软骨或生长板。根据这些体内观察，发现几种成骨细胞谱系细胞系，如ROS 17和MC 3 T3-E1，以及原代颅骨成骨细胞表达col 24 al。此外，在BMP-2诱导的C3 H10 T1/2间充质细胞分化过程中，col 24 a1的表达被激活，这些数据为CREB/AP 1蛋白复合物参与成骨细胞胶原基因表达调控提供了新的信息。

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.