Putative intestinal estrogen receptor: evidence for regional differences

Abstract

Previously we demonstrated that 17β-estradiol (E 2) stimulates intestinal calcium absorption and enhances uptake of calcium by intestinal mucosal cells in vitro, and that the latter contains estrogen receptor (ER)-like proteins and mRNAs. The current study was undertaken to further characterize the putative intestinal ERs using RT-PCR analysis, Western blot analysis, Southern blot analysis, ligand binding assays and gel shift assays. RT-PCR analysis using primers directed at the terminal end of the A B domain of the uterine ER confirmed our previous finding that intestinal ER mRNAs are present throughout the intestine, with the exception that the duodenum lacked the amplified region. Western blot analysis, using ER-715 antibody, detected the expected 68- to 70-kDa ER protein and additional 58-, 46- and 41-kDa proteins in the uterus and colon, while duodenal extract contained only a faint 46-kDa and strong 28-kDa protein bands. Southern blot analysis performed on enzyme-digested genomic DNAs demonstrated the presence of ER-like sequences in genomic DNA from the uterus, duodenum and colon. However, enzyme restriction maps of genomic DNAs from intestinal segments were different from the map for uterine genomic DNA digested with similar enzymes. In ligand binding assays, 125I-labeled E 2 bound specifically to 46-kDa protein in duodenal, colonic and uterine extracts and the binding was competitively inhibited by excess cold E 2. Gel mobility shift assays using 32P-labeled vitellogenin derived ERE demonstrated that jejunal, colonic and uterine ER proteins contain the classical DNA binding domain. In addition, ERE bound in a specific fashion to sites in genomic DNAs from the uterus, colon and jejunum. In contrast, protein extracts and genomic DNAs from the duodenum failed to bind to 32P-labeled ERE and, consequently, appear to be devoid of the classical DNA binding domain. These findings establish the presence of ER-like proteins and genes in intestinal mucosal cells of rats and suggest that the duodenum contains a variant ER gene that encodes a variant ER protein.