An adipogenic basic helix-loop-helix-leucine zipper type transcription factor (ADD1) mRNA is expressed and regulated by retinoic acid in osteoblastic cells

Abstract

ADD1 is a recently identified basic helix-loop-helix leucine zipper-type transcription factor that acts as a positive regulator of adipocyte-specific gene expression. Since adipocytes may share their precursor with osteoblasts, we examined the expression of ADD1 mRNA in osteoblast-like cells. In osteoblastic MC3T3-E1 cells, the level of the ADD1 mRNA expression was low at the early period of cultures while it subsequently increased with time up to more than 10-fold in the later period of cultures along with the expression of alkaline phosphatase, a differentiation marker of these cells. In ROS17/2.8 cells, which represent mature osteoblasts, ADD1 mRNA was expressed constitutively. Treatment with retinoic acid (RA) enhanced the ADD1 mRNA expression several fold in these cells within 4 h in a dose-dependent manner. This RA effect on the ADD1 mRNA expression was blocked by dichloro-D-ribofuranosylbenzimidazole but not by cycloheximide. RA treatment did not affect the ADD1 mRNA stability, suggesting the involvement of transcriptional control. Electrophoretic mobility shift assay revealed that proteins in the crude nuclear extracts prepared from ROS17/2.8 cells were bound to the E box-containing ADD1 recognition DNA sequence, E/C, and that this binding activity was enhanced by the RA treatment. Neither the E2A protein recognition sequence nor the Myo-D/E12 recognition sequence competed against the E/C sequence for the binding, indicating the sequence specificity of the binding activity. Furthermore, RA treatment enhanced the transactivation activity of the chloramphenicol acetyltransferase construct containing the E/C sequence in the transient transfection assay in ROS17/2.8 cells. RA treatment also enhanced the ADD1 mRNA expression in another rat calvaria-derived cell line, RCT1, and in the primary cultures of newborn rat calvaria cells. Overexpression of ADD1 in ROS17/2.8 enhanced the level of the osteocalcin mRNA expression. These results indicated that the adipogenic basic helix-loop-helix leucine zipper-type transcription factor (ADD1) mRNA was expressed in osteoblastic cells and that its expression was associated with the expression of an osteoblastic phenotype-related gene.