BIGLYCAN GENE-EXPRESSION IN THE HUMAN LEIOMYOSARCOMA CELL-LINE SK-UT-1 - BASAL AND PROTEIN-KINASE A-INDUCED TRANSCRIPTION INVOLVES BINDING OF SP1-LIKE SP3 PROTEINS IN THE PROXIMAL PROMOTER REGION/

In this study we demonstrate that the gene encoding the small leucine- rich proteoglycan biglycan is expressed in human myometrial tissue and in the human leiomyosarcoma cell line SK-UT-1. Treatment of SK-UT-1 c ells with forskolin or 8-bromo-cAMP strongly increased biglycan mRNA a nd this effect was transcriptional as shown by transient transfection experiments with biglycan promoter-luciferase reporter fusion genes. T he cAMP-mediated induction of the transfected biglycan promoter in SK- UT-1 cells was abolished by coexpression of a specific protein kinase A inhibitor, and was mimicked by overexpression of the catalytic subun it (CP) of protein kinase A. By 5' deletion analysis, part of the cAMP response was localized to the segment from residues -78 to -46 of the biglycan promoter. This region conferred strong cAMP responsiveness t o a heterologous promoter. Electrophoretic mobility shift and antibody supershift assays identified two specific complexes that contained nu clear proteins antigenically related to the ubiquitous transcription f actors Spl and Sp3, respectively. The binding site of these proteins w as mapped to a CT-rich sequence extending from -59 to -49 in the bigly can promoter. Mutating this sequence eliminated complex formation and markedly reduced basal and cAMP-dependent promoter activity of transfe cted reporter genes. In vitro binding studies using recombinant Spl re vealed that the nuclear factor binding to the CT element was not Spl b ut a Spl-like protein(s). Western blot analysis of SK-UT-1 nuclear pro teins confirmed expression of Sp3, Spl and nuclear proteins that cross reacted with Spl antibody but according to their molecular weight were not Spl. These results indicate that all cAMP-dependent as well as so me basal biglycan transcription in SK-UT-1 cells is mediated through a ctivated protein kinase A and that both functions are conferred at the promoter level through the interaction of Sp1-like/Sp3 factors with t he CT element at -59 in the biglycan promoter.