Abstract
Expression of the sex hormone-binding globulin gene (SHBG) in the liver produces SHBG, which transports sex steroids in the blood. In rodents, the SHBG gene is also expressed in Sertoli cells giving rise to the testicular androgen-binding protein, which is secreted into the seminiferous tubule where it presumably controls testosterone action. Evidence that the SHBG gene functions in this way in the human testis is lacking, and mice containing a human SHBG transgene (shbg4) under the control of its own promoter sequence are characterized by SHBG gene expression in the liver but not in the testis. A potential cis-element, defined as footprint 4 (FP4) within the human SHBG promoter, is absent in SHBG promoters of mammals that produce the testicular androgen-binding protein, and we have produced mice harboring a shbg4 transgene in which FP4 was deleted to evaluate its functional significance. Remarkably, these mice express the modified human SHBG transgene in the testis as well as the liver. Human SHBG transcripts were found within their Sertoli cells, primary cultures of which secrete human SHBG, and this was increased by treatment with follicle-stimulating hormone, retinoic acid, and estradiol but not testosterone. We have also found that the upstream stimulatory factors (USF-1 and USF-2) bind FP4 in vitro by electromobility shift assay of Sertoli cell nuclear extracts and in vivo by chromatin immunoprecipitation assay and conclude that USF transcription factors repress human SHBG transcription in Sertoli cells through an interaction with FP4 within its proximal promoter.
Highlights
Plasma sex hormone-binding globulin (SHBG)1 is produced by hepatocytes, and it transports sex steroids in the blood and regulates their access to target tissues [1]
This sequence is highly conserved in the chimpanzee sex hormone-binding globulin gene (SHBG) proximal promoter and contains a region that we have previously found contains a putative USF binding site by electrophoretic mobility shift assay (EMSA) and supershift assays of mouse liver nuclear protein extracts
Because the SHBG genes in rabbits [31] and sheep2 are expressed in the livers of adult animals, we assumed that the absence of this putative USF binding site in their SHBG promoters does not account for the lack of SHBG expression in the livers of adult rodents
Summary
Animals—Transgenic mice (line shbg 4-a) containing a 4.3-kb region of the human SHBG gene have been characterized previously [14, 16]. To confirm the presence or absence of human SHBG transcripts in separated testicular cell types we performed a standard reverse transcription-PCR based assay [13] using total RNA extracts with human SHBG-specific primer pairs (forward, 5Ј-GTTGCTACTACTGCGTCACAC; reverse, 5Ј-AAGAGGTGGAAGAGTCTTCTC) as well as mouse cyclophilin A-specific primer pairs Time Resolved Immunofluorometric Assay—Culture medium samples (100 l) from primary Sertoli cultures were added to the wells of a Nunc MaxiSorpTM (Nalge Nunc International, Rochester, NY) plate previously coated with a rabbit polyclonal anti-serum against human SHBG and blocked with a buffer (20 mM Tris-HCl, pH 8, 150 mM NaCl) containing 1% casein. After three washes with 50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 0.01% Tween-20, the wells were incubated with 100 l of europium-labeled monoclonal antibody against human SHBG [22] diluted 1/500 in assay buffer (PerkinElmer Life Sciences) for 1 h at room temperature. In EMSA and supershift EMSA experiments, proteinDNA complexes were separated from free probe by 6% PAGE, and the gel was dried and exposed to Biomax MR film (Eastman Kodak Co.) against an intensifying screen at Ϫ80 °C
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