INTEGRATION OF MULTIPLE SIGNALS THROUGH A COMPLEX HORMONE RESPONSE UNIT IN THE PHOSPHOENOLPYRUVATE CARBOXY GENE PROMOTER

Transcription of the phosphoenolpyruvate carboxykinase gene is stimula ted by glucocorticoids, retinoic acid, and cAMP and is dominantly inhi bited by insulin and phorbol esters. The glucocorticoid response is me diated by a complex regulatory unit that consists of two glucocorticoi d receptor (GR) binding sites (GR1 and GR2) and two adjacent accessory factor elements (AF1 and AF2). Deletion of either the AF1 or the AF2 element results in a 50-75% reduction of the glucocorticoid response. In addition to their accessory role in glucocorticoid action, the AF1 and AF2 elements mediate retinoic acid and insulin/phorbol ester effec ts, respectively. Site-directed mutagenesis was performed on AF1 and A F2 to precisely locate the sequences responsible for accessory activit y in each element. The glucocorticoid accessory activity of the AF1 el ement maps to the same 12-base pair sequence (TGACCTTTGGCC) involved i n the response of the PEPCK gene to retinoic acid. The glucocorticoid accessory activity of the AF2 region maps to the same 10-base pair seq uence (TGGTGTTTTG) responsible for mediating the insulin and phorbol e ster responses through this element. The AF1 and AF2 elements bind dif ferent sets of nuclear proteins, and this binding is not qualitatively or quantitatively affected by treatment of the rat H4IIE hepatoma cel ls with retinoic acid (AF1) or insulin (AF2). AF2 functions in a heter ologous context (a consensus glucocorticoid response element and the t hymidine kinase promoter), whereas AF1 functions in this context only if the retinoic acid receptor is overexpressed in the cells. These res ults show that the AF1 and AF2 elements affect the glucocorticoid resp onse through different protein DNA interactions, and that a small sequ ence in each serves multiple functions. Together with GR1 and GR2, the y form a complex hormone response unit which provides an integrated re sponse of the phosphoenolpyruvate carboxykinase gene to a variety of p ositive and negative signals.