AN ADIPOGENIC BASIC HELIX-LOOP-HELIX-LEUCINE ZIPPER TYPE TRANSCRIPTION FACTOR (ADD1) MESSENGER-RNA IS EXPRESSED AND REGULATED BY RETINOIC ACID IN OSTEOBLASTIC CELLS

ADD1 is a recently identified basic helix-loop-helix leucine zipper-ty pe 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 osteoblas t-like cells. in osteoblastic MC3T3-E1 cells, the level of the ADD1 mR NA expression was low at the early period of cultures while it subsequ ently increased with time up to more than 10-fold in the later period of cultures along with the expression of alkaline phosphatase, a diffe rentiation 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-o-ribofuranosylben zimidazole but not by cycloheximide. RA treatment did not affect the A DD1 mRNA stability, suggesting the involvement of transcriptional cont rol. Electrophoretic mobility shift assay revealed that proteins in th e crude nuclear extracts prepared from ROS17/2.8 cells were bound to t he E box-containing ADD1 recognition DNA sequence, E/C, and that this binding activity was enhanced by the RA treatment. Neither the E2A pro tein recognition sequence nor the Myo-D/E12 recognition sequence compe ted against the UC sequence for the binding, indicating the sequence s pecificity 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 a ssay in ROS17/2.8 cells. RA treatment also enhanced the ADD1 mRNA expr ession in another rat calvaria-derived cell line, RCT1, and in the pri mary cultures of newborn rat calvaria cells. Overexpression of ADD1 in ROS17/2.8 enhanced the level of the osteocalcin mRNA expression. Thes e results indicated that the adipogenic basic helix-loop-helix leucine zipper-type transcription factor (ADD1) mRNA was expressed in osteobl astic cells and that its expression was associated with the expression of an osteoblastic phenotype-related gene.