OVEREXPRESSION OF A SINGLE HELIX-LOOP-HELIX-TYPE TRANSCRIPTION FACTOR, SCLERAXIS, ENHANCES AGGRECAN GENE-EXPRESSION IN OSTEOBLASTIC OSTEOSARCOMA ROS17 2.8 CELLS/

Cell differentiation is determined by a certain set of transcription f actors such as MyoD in myogenesis, However, transcription factors that play a positive role in phenotypic gene expression in skeletal cells are largely unknown, except the recently identified CBFA1. Scleraxis i s a helix-loop-helix-type transcription factor whose transcripts are e xpressed in sclerotome and in a certain set of skeletal cells; however , nothing is known about its function with regard to the regulation of cell function, To examine possible roles of scleraxis, we overexpress ed scleraxis in osteoblastic ROS17/2.8 cells, which express low levels of scleraxis, Scleraxis overexpression enhanced expression of the agg recan gene, which is not normally expressed at high levels in these os teoblastic cells, Overexpression of scleraxis also increased mRNA leve ls of type II collagen and osteopontin while suppressing expression of osteoblast phenotype-related genes encoding type I collagen and alkal ine phosphatase, Transient transfection experiments indicated that scl eraxis enhanced the chloramphenicol acetyltransferase activity of the reporter construct AgCAT-8, which contained an 8-kilobase pair (kb) fr agment of the aggrecan gene including both the promoter and its first intron, Deletion analysis identified a 1-kb region that is responsive to scleraxis within the aggrecan gene, This region contains two adjace nt E-box sequences, A 29-base pair DNA fragment (AgE) containing these E-box sequences bound to proteins in the ROS17/2.8 cell nuclear extra cts as well as to in vitro translated scleraxis, This binding was comp eted with unlabeled AgE, but not with a mutated E-box DNA sequence (mA gE), indicating the specificity of the binding activity, The AgE bindi ng activity in the ROS17/2.8 cell nuclear extracts was enhanced in the cells overexpressing scleraxis and was supershifted by the antiserum raised against scleraxis, Furthermore, AgE, but not mAgE, conferred re sponsiveness to scleraxis overexpression to a heterologous promoter, F inally, replacement mutation of the AgE sequence within the 2.5-kb AgC AT-1 construct significantly reduced its responsiveness to scleraxis, These results indicate that overexpression of a single helix-loop-heli x-type transcription factor, scleraxis, enhances aggrecan gene express ion via binding to the E box-containing AgE sequence in ROS17/2.8 cell s.