A study of the transcriptional regulation of the mouse Indian Hedgehog gene in ATDC5 and COS7 cells /

The signaling peptide Indian Hedgehog (Ihh) is secreted by prehypertrophic chondrocytes of the growth plate, the growth center of long bones. It functions to negatively regulate differentiation and positively regulate proliferation of chondrocytes. Thus, Ihh ultimately controls the rate of bone growth.The transcriptional regulation of the Ihh gene, however, remains uncharacterized. In order to study the gene's regulation, the genomic Ihh sequences from several species were aligned to identify conserved regions that may contain regulatory sites. Two putative Stat transcription factor binding sites were identified, one of which is conserved across all species studied while the other is rodent-specific.In addition, an in vitro system was established to test the upstream region of the gene for transcriptional activity. ATDC5 chondrogenic cells were stably transfected with a plasmid containing 5kb of sequence located upstream of Ihh as well as a luciferase reporter gene. The presence of the Ihh sequence induced expression of the luciferase reporter 50 fold above expression from a control plasmid. COS7 and ATDC5 cells transiently transfected with similar Ihh-luciferase constructs resulted in unique induction patterns. Thus, the Ihh upstream genomic region contains sequences that regulate expression in a tissue-specific fashion