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Estrogen receptor-related orphan receptor alpha 1 is a member of the steroid/thyroid nuclear receptor superfamily. We have previously cloned the human estrogen receptor-related orphan receptor alpha 1 (hERR alpha 1) cDNA and demonstrated that it enhances estrogen responsiveness of the lactoferrin gene promoter in transfected human endometrial carcinoma cells. In the present study, we used the hERR alpha 1 cDNA as a probe and isolated the mouse homologue of ERR alpha 1 from the cDNA libraries of the brain and kidney. Sequence comparison between human and mouse ERR alpha 1 (mERR alpha 1) revealed that the homologies are 89% in nucleotides and 97% in amino acids. By electrophoresis mobility shift assay, we showed that the glutathione S-transferase-mERR alpha 1 fusion protein produced in a bacterial system bound to the human ERR alpha 1 DNA-binding element. Mouse uterine nuclear extract also interacted with this DNA element and produced three complexes in the mobility shift assay, one of which was supershifted by the hERR alpha 1 antiserum. A 2.2 kbp transcript was detected by Northern analysis in all adult mouse tissues tested; however, large variations in the amount of ERR alpha 1 mRNA were found among them. Multiple immunoreactive forms of mouse ERR alpha 1 were detected by Western analysis in non-reproductive tissues, whereas a major 53 kDa protein was found in reproductive tissues such as uterus, cervix and vagina. Diethylstilbestrol (DES) stimulated the expression of ERR alpha 1 mRNA in the uterus of 19-day-old mouse. We showed that DES and estradiol, but not progesterone or dexamethasone, enhanced the level of immunoreactive ERR alpha 1 in the mouse uterus. These results demonstrated that the ERR alpha 1 is an estrogen-responsive gene in the mouse uterus and provides a model system with which to study the biological roles of this nuclear orphan receptor.

ABSTRACT

Prolactin is a member of the growth hormone family and is required for the growth and terminal differentiation of the mammary gland. Ectopic production of this hormone has been reported in several species, including rat, sheep, goat and human mammary tissues. In this study, mouse mammary cell lines, xenographs in the mammary gland from these cell lines and from hyperplastic alveolar nodules, spontaneous tumors, and normal tissues were studied for de novo production of this growth factor. Prolactin transcripts were found by reverse transcriptase PCR in some neoplastic and preneoplastic tissues and in mouse mammary cell lines, NOG8 and CDNR4, but were not detected in the normal mouse mammary gland. Northern analysis revealed a 1 kb transcript for both cell lines that co-migrated with the prolactin pituitary transcript. Conditioned medium from NOG8 cells was positive for prolactin bioactivity by the Nb2 rat lymphoma cell proliferation assay, and Western analysis revealed the presence of immunoreactive proteins at M _r 14 000 and 60 000. Prolactin-like bioactivity was not detected in conditioned medium from CDNR4 cells, but an immunoreactive protein of M _r 60 000 was detected by Western analysis. The mouse mammary cell line, Comma D, was negative for prolactin transcripts; however, adenocarcinomas derived from inoculation of Comma D cells into the cleared mammary fat pad were positive by reverse transcriptase PCR in two of four cases. Hyperplastic outgrowths maintained in the cleared mammary fat pad as well as spontaneous tumors were positive for prolactin transcripts in one of four cases. These results suggest that prolactin can be produced ectopically by the neoplastic mouse mammary gland.