Distal Sp3 binding sites in the hIGBP-1 gene promoter suppress transcriptional repression in decidualized human endometrial stromal cells: identification of a novel Sp3 form in decidual cells.

Abstract

The purpose of this study was to identify the nuclear components that account for a dramatic transcriptional induction of the human insulin-like growth factor binding protein-1 (IGFBP-1) gene in decidualized human endometrial stromal cells. The study focused on the functional analysis and binding characteristics of the two distal promoter regions, 132 bp -2.732 to -2.6 kb [C/al/Xbal, (CX) fragment] and 30 bp [-2830 to -2800 bp, Hincll/ Sphl [HS] fragment]. In the primary culture of endometrial stromal cells, we have examined the three cis-elements, I, II and III, in the 58-bp BsaHl/ Rsal (BR) fragment (a promoter fragment that resides in CX) previously identified in HEC-1B cells. When pBRPL (BR 5'-linked to -2600 to +68 bp IGFBP-1 promoter/chloramphenicol acetyl transferase gene) was transfected into cells, only cis-element II (-2660 to -2638 bp) mediated the activation, whereas I and III (-2675 to -2666 bp and -2637 to -2628 bp) were inactive in both unstimulated and hormone-stimulated endometrial stromal cells. Promoter activity derived from pCXPL was repressed in unstimulated cells. During decidualization of endometrial stromal cells, the repression was gradually diminished, which was mediated by cis-element I and III. The binding pattern of the BR fragment analyzed by electrophoretic mobility shift assay using nuclear extracts of decidual tissue or cells showed three major specific binding complexes, C1, C2, and C3, which were abolished by a complementary oligonucleotide of the Sp1-binding motif dose-dependently. Only two complexes, C2 and C3, were formed by nuclear extracts of unstimulated stromal cells. A binding study with mutated BR fragment showed that the binding sites of C1, C2, and C3 resided at cis-elements I and III. The Sp3 antibody abolished complexes C1, C2, and C3. The Sp1 and Sp4 antibodies, however, had little effect. The other distal promoter region, 30-bp HS fragment, which contains a core sequence homologous to the Sp1-binding motif, formed a complex with mobility similar to C1. It is transcriptionally active only in decidualized stromal cells. The binding protein of this complex recognized the Sp3 antibody. Immunoblot analysis of the nuclear extracts of stromal and decidual cells showed that Sp3 antibody identified 120-, 82-, and 80-kDa Sp3 proteins. An additional 28-kDa Sp3 protein was present only in the extract of decidual cells. These findings suggest that decidual Sp3 protein may be responsible, at least in part, for the transcriptional regulation in decidualized endometrial stromal cells.