CHARACTERIZATION OF A HUMAN-SPECIFIC REGULATOR OF PLACENTAL CORTICOTROPIN-RELEASING HORMONE

The hypothalamic hormone CRH is also expressed in the placentas of hum ans and higher primates and may play an important role in the regulati on of labor. In choriocarcinoma cell lines, activation of cAMP-depende nt pathways increases human (h)CRH reporter gene expression. A cAMP-re sponsive region distinct from the cAMP response element at -220 bp, ha s been identified between -200 and -99 bp, and a candidate transcripti on factor was identified in nuclear extracts of human, beet not rodent , choriocarcinoma cell lines. This region, which does not contain a ca nonical cAMP response element (CRE), transfers protein kinase A respon siveness to a heterorogous promoter. Electromobility shift assays sand methylation and uracil interference studies localized factor binding to a 20-bp region from -128 to -109 bp of the hCRH promoter. This 20-b p fragment exhibited a similar shift in nuclear extracts from both hum an term placenta and from human JEG-3 cells. Base contacts, identified ire interference studies, were confirmed as critical for binding, as a mutation of these bases abolished factor binding. Furthermore, a CRH promoter containing this mutation exhibited a diminished response to forskolin. UV cross-linking demonstrated the protein in nuclear extrac ts from human, but not rodent, chorio-carcinoma cell lines and estimat ed its size as 58 kDa. Although this factor participates in cAMP-regul ated gene expression, competition electrophoretic mobility assays demo nstrated that the factor does not bind to a CRE. Furthermore, neither anti-CREB nor anti-ATF2 antibodies alter factor binding. These data id entify this 58-kDa protein as the human-specific CRH activator previou sly identified as a candidate factor contributing to the species-speci fic expression of CRH in human placenta.