ABSTRACT
Here we examine the roles of two isoforms of glycogen synthase kinase-3 (GSK-3), GSK-3α and GSK-3ß, in skeletal development. Both isoforms were unphosphorylated and active in chondrocyte differentiation stages during SOX9 and type II collagen (COL2A1) expression. Although knock-out of both alleles of Gsk3a (Gsk3a(-/-)) or a single allele of Gsk3b (Gsk3b(+/-)) in mice did not significantly affect skeletal development, compound knock-out (Gsk3a(-/-);Gsk3b(+/-)) caused dwarfism with impairment of chondrocyte differentiation. GSK-3α and GSK-3ß induced differentiation of cultured chondrocytes with functional redundancy in a cell-autonomous fashion, independently of the Wnt/ß-catenin signal. Computational predictions followed by SOX9 and COL2A1 transcriptional assays identified RelA (NF-κB p65) as a key phosphorylation target of GSK-3. Among several phosphorylation residues in RelA, Thr-254 was identified as the critical phosphorylation site for GSK-3 that modulated chondrocyte differentiation. In conclusion, redundant functions of GSK-3α and GSK-3ß through phosphorylation of RelA at Thr-254 play a crucial role in early stages of chondrocyte differentiation.
Subject(s)
Cell Differentiation , Chondrocytes/enzymology , Glycogen Synthase Kinase 3/metabolism , Transcription Factor RelA/metabolism , Animals , Bone Development/genetics , Cell Line , Chondrocytes/pathology , Collagen Type II/biosynthesis , Dwarfism/enzymology , Dwarfism/genetics , Glycogen Synthase Kinase 3/genetics , Glycogen Synthase Kinase 3 beta , Mice , Mice, Knockout , Phosphorylation/genetics , SOX9 Transcription Factor/genetics , SOX9 Transcription Factor/metabolism , Transcription Factor RelA/genetics , Wnt Proteins/genetics , Wnt Proteins/metabolism , Wnt Signaling Pathway/genetics , beta Catenin/genetics , beta Catenin/metabolismABSTRACT
The present study attempted to identify and characterize the embryonic promoter of Sox6, a determinant regulator of chondrogenic differentiation. A common transcription start region for human and mouse Sox6 was initially identified, which contained a highly conserved sequence, A-box. Tandem repeats of A-box had a strong transcriptional activity both at the basal level and in response to Sox9. Cells carrying the 4xA-box-DsRed2 reporter fluoresced only upon chondrogenic differentiation. The 46-bp core enhancer region (CES6) was then identified in the 3' half of A-box, within which a C/EBP-binding motif was identified. Overexpressed C/EBPbeta activated the Sox6 promoter, and mutant 4xCES6 constructs lacking the C/EBP motif lost their basal activity. CES6 and nuclear extracts formed a specific complex, which was supershifted by anti-C/EBPbeta antibody, and in vitro translated C/EBPbeta specifically bound to CES6. Thus, we successfully identified the Sox6 promoter and its core enhancer and characterized the interactions with regulatory transcription factors.
