Kap promoter analysis in vivo: a regulatory role for a truncated L1 repeat.

Reporter gene expression directed by a 1542-base pair (bp) fragment of the Kap promoter is specific to the proximal tubules of the kidney and androgen-regulated. In the present study, the characteristics of the androgen response from the 1542-bp promoter were examined in vivo. The estrogen response in the kidney and uterus was also examined. The reporter gene expression was assayed in lines of transgenic mice generated from a truncated promoter construct in which the L1 repeat, present at the distal portion of the 1542-bp, had been deleted. The pattern of androgen response of the reporter gene is similar to that of the endogenous Kap. Reporter gene expression in the 1542-bp promoter does not respond to estrogen in the kidney, while perinatal expression in the uterus does occur. Truncation of the L1 results in loss of reporter gene expression. We conclude that L1 sequences present near the Kap promoter have a regulatory function.

Cell-specific and hormonal regulation of the rat kidney androgen-regulated protein (KAP) gene.

In mouse kidney, the kidney androgen-regulated protein (KAP) gene is regulated in a sex-dependent manner by a complex tissue- and cell-specific multihormonal system. KAP is also found in mouse uterus during the period surrounding birth. We describe here for the first time the existence of KAP in a species other than mouse. The rat cDNA sequence was determined and the derived peptide sequence displayed only 53% identity with murine KAP, although the genomic organization of the genes was identical. Expression of rat KAP was restricted to kidney and uterus, but was constitutive in the latter and drastically induced at parturition. The renal expression of the rat KAP gene was sexually dimorphic and regulated by physiological levels of steroid hormones. The effects of castration, hypophysectomy, thyroidectomy, and castration plus thyroidectomy on KAP mRNA levels in both kidney and uterus were determined. Constitutive expression of the protein was strictly dependent on thyroid hormone in female kidneys where it was modulated by estrogens and other ovarian factor(s). In the uterus, KAP mRNA was mainly under estrogen control. In males, expression of the KAP gene was under the dual regulation of thyroid hormone and androgens. Its complex regulation suggests a carefully delineated role for KAP in the kidney and uterus, but its physiological function remains unknown.

Isolation and characterization of a full-length cDNA encoding the 55-kDa rabbit zona pellucida protein.

A full-length cDNA (rc55) encoding the major rabbit zona pellucida (ZP) glycoprotein (55 kDa) has been cloned and sequenced. A lambda gt11 expression library was constructed using poly(A)+ mRNA isolated from sexually immature rabbit ovaries which contain large numbers of developing follicles. The rc55 cDNA was identified using affinity purified polyclonal antibodies specific to ZP antigens which are shared among mammalian species. The deduced amino acid sequence of the full-length rc55 clone was matched to the NH2-terminal 25-amino acid sequence obtained for this protein. The predicted amino acid sequence consists of 540 amino acids including a putative signal peptide of 18-24 residues and six potential N-glycosylation sites. The cDNA hybridizes to a 2000-base species of mRNA from rabbit ovary which is not detected in other rabbit tissues. The message is present early in ovarian follicular development and is approximately 600-fold greater in sexually immature as compared with sexually mature rabbit ovaries. This cDNA was expressed as a cro-beta-galactosidase fusion protein using the pEX expression vector. Antibodies against native rabbit ZP, affinity-purified on the recombinant 55-kDa ZP protein, were found to recognize the native rabbit ZP glycoprotein, indicating partial conservation of native epitopes in the expressed recombinant protein.

Genomic organization and DNA sequence of the mouse kidney androgen-regulated protein (KAP) gene.

The gene for kidney androgen-regulated protein (KAP) is expressed under androgenic control in the epithelial cells of the renal cortical proximal tubules. However, there is an androgen-independent component of the expression of this gene that occurs specifically in the outermedullary S3 segments of the proximal tubules. In these cells, the KAP gene is estrogen responsive and its expression is dependent on pituitary function. As a first step in correlating its interesting cell-specific and hormonal regulation with the structure of the gene, the genomic organization of the KAP gene was described and sequence of the gene and the proximal 1 kb of 5'-flanking DNA was determined. Sequence motifs were identified in the 5'-flanking DNA that may function in the regulation KAP gene expression by androgen, estrogen, and pituitary glycoprotein hormones. The gene is present in a single copy in the mouse genome and is 3,807 nucleotides in length. It contains 4 exons of 120, 177, 63, and 251 nucleotides and three intervening sequences of 1,450, 126, and 1,620 nucleotides. The gene exhibits a high degree of a genetic polymorphism as revealed by comparison of restriction digests of DNA from two highly inbred strains, BALB/c and C57BL/6.

Use of immunoaffinity purified antibodies to zona pellucida to compare alloimmunization of male and female rabbits.

In order to study the immunogenicity as well as tissue specificity of zona pellucida (ZP) antigens, the present studies have been designed to examine the effects of alloimmunization of male and female rabbits with rabbit zonae pellucidae. These studies are the first to demonstrate that high titers of antibodies to homologous ZP antigens are developed in male rabbits while no detectable antibodies are developed in females. As demonstrated using the ELISA assay, the antibodies from these males immunized with rabbit ZP, have a greater reactivity against rabbit ZP antigens than do antibodies from female rabbits heteroimmunized with porcine ZP. The antibodies from the male rabbits immunized with rabbit ZP also recognize antigenic determinants of porcine ZP. Methods for the immunoaffinity purification of antibodies from serum were developed to determine whether low levels of antibodies against ZP are present in sera of alloimmunized female rabbits. They also allow more detailed analysis of antibodies used to detect antigenic determinants which are cross-reactive between different mammalian species. Although this method was effective in isolating low levels of antibodies from male alloimmunized rabbits or from female rabbits heteroimmunized with porcine ZP proteins, no specific antibodies could be isolated from the serum of females alloimmunized with rabbit ZP. These studies more clearly demonstrate that zona pellucida antigens are specific to the ovary in that female rabbits do not develop significant antibody levels against rabbit ZP antigens, even following active immunization with adjuvant, while male rabbits develop high titers of antibodies.

"Defective" receptors in steroid-resistant conditions may be proteolytic artifacts.

The specific question addressed in this report is whether the resistance to steroid treatment of certain tissues or tumors which appear to contain a normal quantity of steroid-binding sites may be due to structural defects in the receptors. This question may be seen as part of the more general question of whether there are intrinsic variations in the structures of receptors for a given class of steroids in different healthy tissues, in healthy vs. malignant tissues or in different types of tumors. Our experimental approach to these questions has involved the stabilization and precise physicochemical characterization of the receptors. To date, we have studied the estrogen and progestin receptors from human breast cancers and benign and malignant gynecologic specimens and the glucocorticoid receptors from several healthy and malignant rodent tissues and from normal human lymphocytes and various types of leukemic cells. Chromatographic and ultracentrifugal analyses in buffers of low ionic strength, containing 20 mM Na2MoO4 as the stabilizer, have revealed each of these receptors to be a large, oligomeric complex, characterized by remarkably similar values of the Stokes radius, sedimentation coefficient, molecular weight and axial ratio. In the absence of adequate stabilization, however, we found that the receptors for three classes of steroids in extracts of some healthy, steroid-responsive tissues, such as rat kidney and human uterine endometrium, are invariably degraded by endogenous proteinases. The extent of such cleavage is increased considerably by freezing the tissues prior to homogenization. Studies designed to distinguish the intact receptors from the products of proteolysis have included the characterization of receptors in cytosols prepared from mixtures of rat liver and kidney. The results strongly support the interpretation that the smaller size of the receptors detected in kidney cytosol reflects their cleavage by the more active proteinases in that tissue. The sizes and shapes of the receptors in cytosols from various tissues were found to be correlated with the activities of specific endopeptidases, assayed fluorometrically with peptidyl derivatives of 7-amino-4-methylcoumarin (AMC). These studies suggested that the receptors are vulnerable to cleavage by "lysine-specific" endopeptidases, detected with t-butyloxycarbonyl-L-valyl-L-leucyl-L-lysyl-AMC. An enzyme of this specificity was partially purified from rat kidney cytosol and tested for its ability to digest the glucocorticoid receptors from rat liver cytosol.(ABSTRACT TRUNCATED AT 400 WORDS)

[Cytoplasmic estradiol and progesterone receptors in human endometrium with normal menstrual cycles: 116 cases].

PIP: Endomentrial samples were obtained from 116 patients who underwent hysterectomy or dilatation and currettage. The normal menstrual cycles were determined on the basis of clinical and histological features. They were divided into 6 categories: early, middle and late proliferative and secretory phases. The highest progesterone receptor level was present in the late profilerative phase and the lowest in the late secretory phase. Expect for the fact that the lowest level of estrogen receptor was present in the late secretory phase, differences in the other phases were not significant. The data was split into younger and older groups and analyzed separately. In the proliferative phase, the estrogen and progesterone receptor levels were higher in the perimenopausal group than in the premenopausal group, but no significant differences were found in the secretory phase. On the whole, the estrogen and progesterone receptor levels were higher in proliferative than in the secretory samples, and the progesterone receptor level was much higher than the estrogen receptor level in both phases. These findings were in agreement with data from the literature. Simultaneously, the estradiol and progesterone levels in serum and cytosol were measured in some cases. The progesterone receptor level was closely correlated with progesterone level in serum and tissues (negative correlation) and also closely correlated with estradiol in tissues (positive correlation). The variations in the estrogen and progesterone receptors in endometrial cytosol during the normal menstrual cycle of Chinese women may offer some useful information for further basic or clinical research in the fields of planned parenthood and gynecology. (author's modified)^ieng

Human breast tumor estrogen receptor: effects of molybdate and electrophoretic analyses.

The largest and smallest discrete forms of the estrogen receptor in human breast tumor cytosol were characterized by competitive steroid binding, ultracentrifugation, gel filtration, and electrophoresis in polyacrylamide gels of several concentrations. Incubation of cytosol with [3H]estradiol and centrifugation in glycerol gradients containing 20 mM Na2MoO4 and 0 or 150 mM KCl revealed a 9-10S form of the receptor. It resembles the molybdate-stabilized complexes in cytosols of other human and rodent, malignant and healthy tissues, and the complex detected in breast tumor cytosol containing leupeptin, a bacterial protease inhibitor. Preservation of receptor integrity during purification and discrimination from serum steroid-binding components are facilitated by inclusion of molybdate in all buffers. Possible mechanisms of action of molybdate include the inhibition of ribonuclease action on RNA-associated receptor forms and protection against specific proteolytic cleavage by stabilization of a phosphate group on the vulnerable residue or a neighboring one. During fractionation of tumor cytosol in the absence of molybdate, the receptor is converted to a mixture of fragments. The smallest that retains the bound steroid, the mero-receptor, resembles the products of endogenous and exogenous protease action on receptors for all classes of steroids in a wide range of tissues. The similarities between both the largest and the smallest known forms of the breast tumor estrogen receptor and corresponding forms of other receptors support the notion of the common architecture of steroid receptors in normal and malignant tissues of diverse origins.