INDUCTION OF SP1 IN DIFFERENTIATING HUMAN EMBRYONAL CARCINOMA-CELLS TRIGGERS TRANSCRIPTION OF THE FIBRONECTIN GENE

Cells of the human embryonal carcinoma line NEC14 proliferate as dense ly packed clusters consisting of small, polygonal stem cells and do no t express a detectable level of fibronectin (FN). Upon induction of di fferentiation by treatment with N,N'-hexamethylene bisacetamide (HMBA) , the level of FN mRNA increased steeply within 24 h and FN began to b e accumulated, along with the organization of actin filaments in the c ells. The FN promoter elements required for the activation were analyz ed in reference to a cluster of GC boxes by using the chloramphenicol acetyltransferase (CAT) gene fused to 5' sequential-deletion derivativ es of the promoter and promoters carrying base substitutions in the GC boxes. Among four CC boxes, GC boxes 2 and 3 had the greatest effect on promoter activation, and base substitutions in these GC boxes resul ted in 80% reduction in promoter activity. The pattern of DNA-protein complex formation with these GC boxes changed drastically after induct ion of differentiation. The extract prepared from undifferentiated NEC 14 cells formed fast-migrating complexes (UnD complexes), while the ex tract prepared from NEC14 cells treated with HMBA for 24 h formed slow -migrating complexes containing Sp1. Both complexes were formed predom inantly with GC box 2. Base substitutions within the GC boxes complete ly abolished the formation of both Uno and Spl complexes. Consistent w ith these changes, the Sp1 level increased steeply within 24 h. Induct ion of Sp1 expression in NEC14 cells effectively stimulated the promot er activity of the transfected FN promoter-CAT constructs. These resul ts indicate that activation of the FN promoter in differentiating NEC1 4 cells occurs by the steep induction of Sp1, which prevents an undiff erentiated cell factor from binding to the Sp1 sites.