Regulation Rules Of HoxC8 In Mouse Osteoblast Differentiation

The Hox genes, members of a family of essential developmental regulators, have the intriguing property that their expression in the developing animal embryo is colinear with their chromosomal organization. Members of the Hox gene family share a conserved DNA binding domain termed the Homeodomain, which mediates interactions of Hox proteins with DNA regulatory elements in the transcriptional control regions of target genes.Hox genes are expressed in all metazoan even in some plants. The vertebrate Hox genes have been shown to confer regional identity along the anteroposterior axis of the developing embryo, especially within the central nervous system (CNS) and the paraxial mesoderm.We are interested in investigating the function of HoxC8 in skeletogenesis. Previous studies have shown that deregulation of HoxC8 expression in the mouse leads to several skeletal defects, such as Homeotic transformation in the thoracic vertebrae, abnormal development of the rib cage, and over proliferation of chondrocytes in the hypertrophic area. And, precise temporal expression of HoxC8 is important for determining the correct identity of the vertebral column in early embryos. It seems that HoxC8 has multiple roles in normal skeletal development. So, according to these data, we presume that HoxC8 is a regulator in skeletogenesis.In this study, a MC3T3-E1 subclone 4 osteogenic cell differentiation model was used to examine the expression of HoxC8 at multiple stages of osteogenesis. We found that HoxC8 expression levels do not change in the early stage but increase in the middle stage and decrease in the late stage of osteogenesis. Knockdown of HoxC8 by small-interfering RNA transfection in C2C12 cells indicated that HoxC8 is a negative regulator of osteogenesis. Similarly, expression of HoxC8 in C2C12 cells decreases alkaline phosphatase levels induced by bone morphogenetic protein-2 (BMP2). Moreover, we also identified downstream targets of HoxC8 relevant to osteoblast differentiation and BMP2 signaling pathway. As a result, we find that ectogenic HoxC8 and Smad1 can colocalized in the nucleus after inducing by BMP2, and HoxC8 can bind Smad1 directly and that results in suppressing Smad6 which induced by BMP2, these results indicate that Smad1 and Smad6 are important transcription factor in BMP2 signaling pathway.In addition, we also confirmed a specific HoxC8 binding cluster existing at the upstream of ALP promoter, which indicates HoxC8 controls osteoblast marker genes at both early and late stage(ALP and OCN)by binding HBE directly.The results of this study showed that HoxC8 is involved in BMP2-induced osteogenesis by binding with Smad1 directly and suppressing Smad6, and osteoblast differentiation in vitro is negatively regulated by HoxC8, suggesting that HoxC8 regulation is essential for osteoblast differentiation.