Michael H. Melner

DAX-1 blocks steroid production at multiple levels.

DAX-1 is an unusual member of the nuclear hormone receptor superfamily whose expression is mainly, but not uniquely, restricted to steroidogenic tissues. We have recently shown that DAX-1 can block the first and rate-limiting step in steroid biosynthesis by repressing StAR (steroidogenic acute regulatory protein) expression. Here we show that DAX-1 blocks steroid production at multiple levels in the Y-1 mouse adrenocortical tumor cell line. Expression of DAX-1 in Y-1 cells significantly impairs both basal and cAMP-stimulated steroid production, without affecting the functionality of the cAMP-responsive PKA pathway. Experiments using an hydroxylated cholesterol derivative show that biochemical steps in steroidogenesis subsequent to cholesterol delivery to mitochondria are also impaired in Y-1 cells expressing DAX-1. This is explained by the repression of P450scc and 3β-HSD expression, in addition to StAR. DAX-1 expression in Y-1 cells results in the inhibition of the activity of the StAR, P450scc and 3β-HS...

Synergistic Activation of the Human Type II 3β-Hydroxysteroid Dehydrogenase/Δ5-Δ4 Isomerase Promoter by the Transcription Factor Steroidogenic Factor-1/Adrenal 4-binding Protein and Phorbol Ester

Steroidogenic factor-1/adrenal 4-binding protein (SF-1/Ad4BP) is an orphan nuclear receptor/transcription factor known to regulate the P450 steroid hydroxylases; however, mechanisms that regulate the activity of SF-1/Ad4BP are not well defined. In addition, little is known about the mechanisms that regulate the human steroidogenic enzyme, type II 3β-hydroxysteroid dehydrogenase (3β-HSD II), the major gonadal and adrenal isoform. Regulation of the 3β-HSD II promoter was examined using human adrenal cortical (H295R; steroidogenic) and cervical (HeLa; non-steroidogenic) carcinoma cells. H295R cells were transfected with a series of 5′ deletions of 1251 base pairs (bp) of the 3β-HSD II 5′-flanking region fused to a chloramphenicol acetyltransferase (CAT) reporter gene followed by treatment with or without phorbol ester (phorbol 12-myristate 13-acetate; PMA). CAT assay data indicated that the region from −101 to −52 bp of the promoter was required for PMA-induced expression. A putative SF-1/Ad4BP regulatory element, TCAAGGTAA, was identified by sequence homology at −64 to −56 bp of the promoter. Cotransfection of HeLa cells with the −101 3β-HSD-CAT construct and an expression vector for SF-1/Ad4BP increased CAT activity 49-fold. Subsequent treatment with PMA induced an unexpected synergistic increase in transcriptional activity 540-fold over basal. Mutation of the putative response element (TCAATAA to TCAATAA) abolished SF-1-induced CAT activity and the synergistic response to PMA. Gel mobility shift assays confirmed that SF-1/Ad4BP interacts with the putative element and transcripts for SF-1/Ad4BP were detected in H295R cells by Northern analysis. These data are the first to demonstrate 1) regulation of a non-cytochrome P450 steroidogenic enzyme promoter by SF-1/Ad4BP, 2) a powerful synergistic effect of PMA on SF-1/Ad4BP-induced transcription, and 3) the importance of the SF-1/Ad4BP regulatory element in the regulation of the 3β-HSD II promoter.

Paracrine Regulation of Matrix Metalloproteinase Expression in the Normal Human Endometrium

Endometrial expression of matrix metalloproteinase (MMP)-3, MMP-7 and MMP-11 occurs during menstrual breakdown and subsequent estrogen-mediated growth, but not during the secretory phase. These enzymes are suppressed by progesterone treatment. Paracrine factors, including transforming growth factor-β (TGF-β) and retinoic acid, are also critical for MMP regulation in the endometrium. In contrast, inflammatory cytokines such as interleukin-1α may block or interfere with steroid-mediated MMP regulation at ectopic sites of growth. Using in vitro models, our laboratory has investigated the complex interactions between progesterone and locally produced cytokines that may affect MMP expression during the development of endometriosis. Our results indicate that targeting the regulation of MMPs may represent an appropriate therapeutic strategy for the treatment of endometriosis.

The regulation of 3β-hydroxysteroid dehydrogenase expression

3 beta-Hydroxysteroid dehydrogenasel delta 5-->4-isomerase (3 beta-HSD) catalyzes the formation of delta 4-3-ketosteroids from delta 5-3 beta-hydroxysteroids, an obligate step in the biosynthesis not only of androgens and estrogens but also of mineralocorticoids and glucocorticoids. The enzyme is expressed in the adrenal cortex and in steroidogenic cells of the gonads, consistent with this role. However, 3 beta-HSD is also expressed in many other tissues, such as the liver and kidney, where its function is not entirely clear. It is established that a family of closely related genes encode for 3 beta-HSD. The various 3 beta-HSD isoforms are expressed in a tissue-specific manner involving separate mechanisms of regulation. The human type I 3 beta-HSD is expressed at high levels in syncytial trophoblast and in sebaceous glands, and the type II isoform is almost exclusively expressed in the adrenal cortex and gonads. An important feature in liver and kidney (at least of hamster, mouse, rabbit, and rat) is the sexual dimorphic nature of 3 beta-HSD expression. We briefly review studies on the regulation of the human 3 beta-HSD I and II genes in human trophoblast and adrenal cortex and extend this to discuss the rat 3 beta-HSD I gene expressed in adrenals and gonads. The complexity of 3 beta-HSD expression through multiple signaling pathways acting on a multigene family of enzymes may contribute importantly to the diverse patterns and locations of steroid hormone biosynthesis.

Characterization of macaque 3β-hydroxy-5-ene steroid dehydrogenase/Δ5-Δ4 isomerase: structure and expression in steroidogenic and peripheral tissues in primate

Abstract The conversion of 3β-hydroxy-5-ene steroids by the enzyme complex 3β-hydroxysteroid dehydrogenase/Δ 5 -Δ 4 isomerase (3β-HSD) is an obligatory step in the biosynthesis of all classes of hormonal steroids in classical steroidogenic as well as in peripheral tissues. To develop a model more closely related to the human, we have isolated and characterized cDNA clones encoding macaque 3β-HSD by screening a rhesus monkey ovary κgt11 cDNA library using a human 3β-HSD cDNA probe. Nucleotide sequence of 1629 bp from overlapping cDNA clones predicts a protein of 372 amino acids with a calculated molecular mass of 41,874 (excluding the first Met). The deduced amino acid sequence of macaque 3β-HSD displays 79.4% and 93.9% similarity with that of bovine and human 3β-HSD, respectively. RNA blot analysis performed under high stringency conditions of macaque poly(A) + RNA samples using full-length 32 P-labeled macaque 3β-HSD cDNA revealed the presence of an approximately 1.7 kb mRNA species in classical steroidogenic tissues, namely the ovary, testis and adrenal glands as well as in several peripheral tissues including the liver, kidney and epididymis. Computer analysis of the deduced macaque 3/gb-HSD protein sequence predicts the presence of an NH 2 -terminal membrane-associated segment as well as four additional membrane-spanning segments, thus suggesting that 3β-HSD is an integral protein. The availability of macaque cDNA should permit detailed studies concerning the tissue-specific expression as well as the hormonal regulation of 3β-HSD mRNA in classical steroidogenic glands as well as in peripheral tissues which are an important site of Steroidogenesis in primates.

Region-specific Expression and Secretion of the Fibrinogen-related Protein, fgl2, by Epithelial Cells of the Hamster Epididymis and Its Role in Disposal of Defective Spermatozoa

The cauda epididymidis functions in the storage and protection of mature, fertile spermatozoa. We previously identified a region-specific secretory glycoprotein (termed HEP64) of the hamster proximal cauda epididymidis that specifically bound and coated the nonviable, but not the viable, spermatozoa within the epididymal lumen. In this study we employed expression screening of a hamster epididymal cDNA library to obtain the full-length sequence of HEP64 and to identify it as the fibrinogen-like protein fgl2. Northern blot analysis demonstrated that fgl2 mRNA is highly expressed by the proximal cauda epididymidis in comparison to other hamster tissues examined, and, in situ hybridization analysis of the epididymis revealed that fgl2 mRNA exhibited a region- and principal cell-specific expression pattern. Immunohistochemistry confirmed the association of fgl2 with abnormal spermatozoa in the cauda epididymidis and revealed smaller fgl2-containing particles. Immunoelectron microscopy revealed that fgl2 was distributed throughout an amorphous, “death cocoon,” complex assembled onto abnormal spermatozoa and that the smaller fgl2 aggregates consisted of the amorphous material with embedded sperm fragments, organelles, and membrane vesicles. A protocol was developed to isolate an enriched death cocoon fraction. SDS-PAGE and microsequence analyses revealed that the Mr 64,000 fgl2 monomer was assembled into two disulfide-linked oligomers of Mr 260,000 and 280,000. These data demonstrate that the epididymis possesses a specific mechanism to identify and envelop defective spermatozoa with a protein complex containing the fibrinogen-like protein fgl2. We propose that this represents an important protective mechanism not only to shield the viable sperm population from potentially deleterious enzymes released by dying spermatozoa but also to prevent the release of sperm proteins that could initiate an immune response if they escaped the epididymal environment.

Characterization of protein tyrosine kinase activity in murine Leydig tumor cells

The activity of protein tyrosine kinase (EC 2.7.1.37) was characterized from Leydig tumor cells (M5480A) using the synthetic peptide NH2-Glu-Asp-Ala-Glu-Tyr-Ala-Ala-Arg-Arg-Arg-Gly-COOH as a substrate. Relatively high tyrosine-specific protein kinase activity (about 135 pmol/mg protein per min) was detected in a particulate fraction (30 000 X g pellet) and was found to be linear as a function of time and protein concentration. The enzymic activity in the particulate fraction was stimulated 1.4-fold by 0.02% Nonidet P-40 as judged by 32PO4 incorporated into the peptide. Phosphorylation of endogenous proteins in M5480A particulate fractions with [gamma-32P]ATP resulted in several alkali-resistant radiolabeled bands in polyacrylamide gels in the presence of sodium dodecyl sulfate. Included in this group was a major radiolabeled doublet with an apparent molecular-weight in the range of 50 000-54 000. Phosphoamino acid analysis of hydrolysates of these eluted proteins indicated the presence of phosphotyrosine. Several alkali-resistant radio-labeled bands, including a major doublet with an apparent molecular-weight of 32 000, were also detected after culturing M5480A cells in the presence of 32PO4. These studies demonstrate the presence of high levels of protein tyrosine kinase activity in Leydig tumor cells and of endogenous protein substrates for this enzyme activity.

Demonstration of Ubiquitin Thiolester Formation of UBE2Q2 (UBCi), a Novel Ubiquitin-Conjugating Enzyme with Implantation Site-Specific Expression

Abstract We recently identified a differentially expressed gene in implantation stage rabbit endometrium encoding a new member of the ubiquitin-conjugating enzyme family designated UBE2Q2 (also known as UBCi). Its unusually high molecular mass, novel N-terminus extension, and highly selective pattern of mRNA expression suggest a specific function in implantation. This study analyzes its relationship to the E2 ubiquitin-conjugating enzyme superfamily, investigates its enzymatic activity, and examines its localization in implantation site endometrium. Construction of a dendrogram indicated that UBE2Q2 is homologous to the UBC2 family of enzymes, and isoforms are present in a broad range of species. In vitro enzymatic assays of ubiquitin thiolester formation demonstrated that UBE2Q2 is a functional ubiquitin-conjugating enzyme. The Km for transfer of ubiquitin thiolester from E1 to UBE2Q2 is 817 nM compared to 100 nM for other E2 paralogs; this suggests that the unique amino terminal domain of UBE2Q2 confers specific functional differences. Affinity-purified antibodies prepared with purified recombinant UBE2Q2 showed that the protein was undetectable by immunoblot analysis in endometrial lysates from estrous and Day 6¾ pregnant (blastocyst attachment stage) rabbits but was expressed in both mesometrial and antimesometrial implantation site endometrium of Day 8 pregnant animals. No expression was detected in adjacent interimplantion sites. Immunohistochemistry demonstrated UBE2Q2 expression exclusively in mesometrial and antimesometrial endometrial luminal epithelial cells of the Day 8 implantation chamber. Immunohistochemical localization of ubiquitin mirrored UBE2Q2 expression, with low-to-undetectable levels in implantation sites of Day 6¾ pregnant endometrium but high levels in luminal epithelial cells of Day 8 pregnant endometrium. This implantation site-specific expression of UBE2Q2 in luminal epithelial cells could play major roles in orchestrating differentiation events through the modification of specific protein substrates.

Editorial: AutoImmune Premature Ovarian Failure—Endocrine Aspects of a T Cell Disease1

Premature ovarian failure (POF) is the loss of ovarian function in women less than 40 yr of age (reviewed in Ref. 1). It is associated with sex steroid deficiency, amenorrhea, infertility, and elevated serum gonadotropins. While there are multiple etiologies of POF including the exposure to iatrogenic treatments (chemotherapy, radiation), viral agents, and rare genetic disorders, in most patients no etiology can be identified (idiopathic POF). Significant evidence suggests that autoimmunity is a cause of some forms of ovarian failure although specific ovarian antigens are not known and the mechanisms of autoimmune disease development are unclear. Autoimmune POF in humans is frequently associated with other manifestations of autoimmune disease. For example, POF can precede the onset of Addison’s disease or adrenal autoimmunity leading to a deficiency of adrenocortical hormones (1). Autoimmune POF is characterized by inflammatory infiltration of developing follicles, production of antiovarian antibodies, atrophy, and sparing of primordial follicles (1‐3). Autoantibodies in these diseases sometimes react with common antigens in steroid-producing cells of the ovary and adrenal cortex. Common antigens identified have been steroidogenic enzymes including P450 side-chain cleavage (P450scc), 17a-hydroxylase, and 3b-hydroxysteroid dehydrogenase (4 ‐ 6). The identification of specific antigens involved in POF is important for multiple reasons. First, the development of appropriate reagents to screen for the presence of antibodies to these antigens could provide an analytical tool for diagnosing the disease, identifying patients at risk for developing the disease, and detecting patients who may respond to immune-modulating therapies. Second, these tools could be used in research to further understand the mechanisms of disease development and the mechanisms of ovarian pathology associated with the disease. Lastly, the identification of these antigens provides new information on novel proteins in the ovary and their potential function. Animal models of autoimmune premature ovarian failure have yielded important insight into both potential mechanisms of autoimmune disease development and ovarian antigens that may affect disease progression. These models for autoimmune ovarian failure can be induced by multiple methods such as immunization with specific ovarian antigens or neonatal thymectomy in specific genetic strains of mice. A detailed review of the findings will not be repeated here but some major developments will be summarized. The most important development in these animal models of autoimmune ovarian failure comes from multiple studies, all suggesting that the basis of the disease is a cell-mediated autoimmune reaction caused by an alteration in T cell regulation (1, 3). This is most evident in the neonatal thymectomy animal model. The removal of the thymus in specific genetic strains of mice (e.g. BALB/c or A/J) between postnatal days 2 and 5 results in autoimmune ovarian failure. There is a progressive onset of the disease that is potentiated by puberty and the most severe inflammation occurs between 4 ‐14 weeks after thymectomy (3). The proposed mechanism of the disease (3) is that autoreactive T cells (CD41) are generated during normal processes such as apoptosis of follicles in the ovary. These autoreactive cells are normally controlled by CD41 T cells with suppressor activity. However, because these cells are generated in the thymus after the first week of life, neonatal thymectomy results in a dramatic loss in T cells with suppressor function. This animal model strongly implicates T cell regulation in the disease process.

Differential Expression of Genes in the Endometrium at Implantation: Upregulation of a Novel Member of the E2 Class of Ubiquitin-Conjugating Enzymes

Abstract The process of embryo attachment and implantation is accompanied by dramatic cellular and functional changes in the endometrium, the control and mechanisms of which are not clearly understood. The cDNA cloning of differentially expressed genes, specifically at implantation sites in the rabbit endometrium, was used to identify genes controlling functional and remodeling changes. Tissue from the endometrium of Day 6¾ (preimplantation) and Day 8 (implantation initiation) pregnant rabbits was used to screen for differentially expressed genes by combined cDNA subtraction/suppressive hybridization. Twenty-nine differentially expressed genes were identified encoding protein modification enzymes, signaling proteins, structural proteins, and enzymes. One of these is a novel member of the E2 ubiquitin-conjugating enzyme family we have designated UBCi (i for implantation), which displayed dramatic nucleotide and deduced amino acid sequence conservation between rabbits, humans, and mice. In situ hybridization indicated UBCi expression exclusively in the luminal epithelium of the endometrium while glandular epithelium, trophoblast, and myometrium were negative. Expression was specific for epithelial cells at implantation sites and was not detected in non-implant-site endometrium. UBCi mRNA was detected in both the mesometrial and antimesometrial epithelial cells of the implantation sites, sites undergoing both differentiation and/or apoptosis. These results identify a group of differentially expressed genes in the endometrium including UBCi and provide new focal targets for studying processes controlling cellular remodeling during implantation. The important roles of ubiquitination in controlling the activities and turnover of key signaling proteins suggest potential roles in controlling critical aspects of implantation.