Because upregulation of gene expression by BMP-2 is much higher than that of other homeodomain genes. Alx3 is a homeobox gene that belongs to a group of Aristaless-related genes that includes Alx4 and Cart1. Alx3, Alx4, and Cart1 encode highly related proteins, and their expression patterns are highly similar. Alx3, originally isolated from the hamster insulinoma cell line HIT-T15, participates in regulation of insulin gene expression in pancreatic ��-cells through transactivation of the insulin promoter by acting on the E2A3/4 enhancer in cooperation with E47/Pan1. Alx3 is also expressed in mesenchyme of developing limbs and craniofacial regions. The physiological roles of Alx3 have been studied using Alx3-null mice, a number of which died during embryogenesis. Alx3null mice exhibit increased failure of cranial neural tube closure and increased cell death in the craniofacial region as embryos in the absence of folic acid. It has also been revealed that ALX3 is essential for normal facial development in humans and that its deficiency causes frontonasal malformation. Furthermore, Alx3 has been linked to developmental functions in craniofacial structures. However, little is known regarding its direct relationship with bone formation, especially with AbMole 4-Demethylepipodophyllotoxin osteoblast differentiation. In the present study, we focused on the mechanisms of Alx3 gene expression and function during osteoblast differentiation induced by BMP-2. C2C12 cells, one of the mouse myoblast cell lines, were used as a model of BMP-2-induced osteoblast differentiation. Cells were differentiated into osteoblasts or myotubules using low mitogen medium with or without BMP-2. First, we checked the gene expressions of Alp, Osteocalcin, and Myogenin after treating C2C12 cells with or without BMP-2. Time course analysis revealed that C2C12 cells differentiated into myotubules without BMP-2, as the expression of Myogenin was increased in a time-dependent manner. On the other hand, the cells differentiated into osteoblasts with BMP-2, as the expressions of Alp and Osteocalcin were increased in a time-dependent manner. Furthermore, analyses of ALP activity and ��-MHC immunohistochemistry also revealed that C2C12 cells were differentiated into osteoblasts or myotubules with or without BMP-2 treatment, respectively. These results are consistent with the previous report. To elucidate the transcriptional regulator for BMP-2-induced osteoblast differentiation, we performed cDNA microarray analysis to compare between BMP-2-treated and -untreated C2C12 cells. Among them, we selected Alx3, a homeobox gene that belongs to a group of Aristalessrelated genes, as it was clearly induced during osteoblast differentiation. Previous studies have indicated that homeobox genes including Dlx3, Dlx5, and Msx2 are important for regulation of osteoblast differentiation, while some are upregulated in C2C12 cells during BMP-2-induced osteoblast differentiation. Furthermore, the expressions of Cart1and Alx4, which belong to the same protein family as Alx3, were examined during BMP-2-induced osteoblast differentiation.