Functional characterization of the human biglycan 5'-flanking DNA and binding of the transcription factor c-Krox

The transcriptional regulation of human biglycan expression under normal and pathological conditions nas studied, The 5'-flanking regions of the human and mouse genes mere isolated and analyzed; the two promoter regions share 81% identity, Both promoters are without a TATA and CAT box and contain multiple Spl sites, Human dermal fibroblasts were transiently transfected with progressive deletional human biglycan 5'-flanking DNA-CAT constructs, and a significant variation in activity among the individual constructs was found, A small deletion in several cases caused a more than 2-fold increase or decrease in promoter activity, thereby mapping the target sites for repressors or activators, Human biglycan expression is reduced in females with Ullrich-Turner syndrome (45,X) and increased in individuals with supernumerary sex chromosomes, and it has been speculated that biglycan plays a role in the short stature phenotype of Turner syndrome, Analysis of the transcriptional regulation of biglycan in individuals with sex chromosome anomalies showed that a -262 to -218 region of the biglycan promoter was differentially regulated, This region was extensively analyzed by DNAse footprinting and electrophoretic mobility shift assays, and a putative binding site for the transcription factor c-Krox was discovered, The binding of c-Krox to a site located at approximately -248 to -230 in the human biglycan promoter was confirmed by using extracts from COS cells expressing recombinant human c-Krox. The expression of c-Krox in bone was then examined by reverse-transcribed polymerase chain reaction and Northern blotting analysis; an similar to 3.4 kb transcript was detected in primary osteoblastic cells, in MG-63 cells, and in human bone marrow stromal cells, This is the first detection of c-Krox in bane cells, and it suggests that c-Krox, like another member of the Krox family, Krox-20, might play a regulatory role in bone.