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We have dissected the mouse H‐2Kb gene promoter in order to define the sequences responsible for induction by tumour necrosis factor (TNF‐alpha). An enhancer element (‐187 to ‐158) composed of two imperfect direct palindromic repeats has been shown to be necessary and sufficient for TNF‐alpha induction of a heterologous promoter. A multimer of either repeat is also responsive, while a single copy is not: this is the situation in the beta 2‐microglobulin (beta 2‐m) promoter which contains a single palindrome and does not respond to TNF‐alpha. We had previously found that the two repeats can bind a factor named KBF1. We show here that in the uninduced state the transcription factor AP2 binds to the interpalindromic region, while in TNF‐treated cells an NF kappa B‐like activity is induced which displaces both KBF1 and AP2 and binds to the two palindromes. This strongly suggests that induction of an NF kappa B‐like activity is responsible for TNF‐alpha stimulation of mouse MHC class I genes.

The Epstein‐Barr virus (EBV) protein EBNA2, which is essential for the immortalization of human primary B cells by EBV, acts as a transcriptional activator of cellular and viral genes. Specific responsive elements have been characterized in several of the promoters activated by EBNA2. They all share the core sequence GTGGGAA. EBNA2 does not, however, bind to these sequences directly, but appears to be targeted to them by a cellular protein. A similar core sequence has recently been identified as a high‐affinity binding site for the human recombination signal sequence binding protein RBP‐J kappa. Here we provide evidence that RBP‐J kappa binds to specific sequences in EBNA2‐responsive elements. Our results also demonstrate that RBP‐J kappa makes direct physical contact with EBNA2 in solution and recruits EBNA2 to its cognate DNA sequences, suggesting that RBP‐J kappa may mediate EBNA2 transactivation of both cellular and viral genes.

The KBF1 factor, which binds to the enhancer A located in the promoter of the mouse MHC class I gene H‐2Kb, is indistinguishable from the p50 DNA binding subunit of the transcription factor NF‐kappa B, which regulates a series of genes involved in immune and inflammatory responses. The KBF1/p50 factor binds as a homodimer but can also form heterodimers with the products of other members of the same family, like the c‐rel and v‐rel (proto)oncogenes. The dimerization domain of KBF1/p50 is contained between amino acids 201 and 367. A mutant of KBF1/p50 (delta SP), unable to bind to DNA but able to form homo‐ or heterodimers, has been constructed. This protein reduces or abolishes in vitro the DNA binding activity of wild‐type proteins of the same family (KBF1/p50, c‐ and v‐rel). This mutant also functions in vivo as a trans‐acting dominant negative regulator: the transcriptional inducibility of the HIV long terminal repeat (which contains two potential NF‐kappa B binding sites) by phorbol ester (PMA) is inhibited when it is co‐transfected into CD4+ T cells with the delta SP mutant. Similarly the basal as well as TNF or IL1‐induced activity of the MHC class I H‐2Kb promoter can be inhibited by this mutant in two different cell lines. These results constitute the first formal demonstration that these genes are regulated by members of the rel/NF‐kappa B family.

Progesterone receptor from rabbit uterine cytosol was purified to a specific activity of approximately 2 nmol of bound hormone per mg of protein. A goat was immunized with this preparation and, after two injections of 0.7-0.8 nmol, yielded antireceptor antibodies. The antiserum reacted with both cytosolic and nuclear rabbit progesterone receptor and also with progesterone receptor from other rabbit tissues (vagina and pituitary). A crossreaction was observed with progesterone receptors from other mammalian, especially human, tissues (cytosolic receptor from rat and guinea pig uterus, cytosolic receptor from human breast cancer, and nuclear receptor from human endometrium). On the contrary, there was no interaction with a nonmammalian receptor (chicken oviduct progesterone receptor). The antibodies did not crossreact with other rabbit steroid receptors (uterine estradiol receptor and liver glucocorticoid receptor) or with nonreceptor progesterone-binding proteins (transcortin from plasma and uteroglobin from uterine fluid).

Monoclonal antibodies were used to study the structure and the biosynthesis of the rabbit progesterone receptor. Proteins in nonfractionated uterine cytosol were submitted to gel electrophoresis in denaturing conditions, transferred onto nitrocellulose, and reacted with monoclonal antireceptor antibodies and 125I-protein A. A single 110,000-dalton protein was observed when precautions were taken during homogenization of the uteri and protease inhibitors used. Smaller forms of receptor (essentially of 79,000 daltons but also of 72,000 and in some experiments of 64,000 daltons) were present when these precautions were not observed and thus probably arose from artifactual proteolysis of receptor. When poly(A)+ RNA from rabbit uterus was translated in a reticulocyte lysate and the radioactive proteins precipitated by the antireceptor monoclonal antibodies, a radioactive protein of 110,000 daltons was also observed. Further evidence that this protein was the product of the translation of progesterone receptor mRNA was obtained by precipitation and immunoaffinity purification with several antireceptor monoclonal and polyclonal antibodies, inhibition of immunoprecipitation by purified receptor and its absence in a receptor-poor tissue (liver). Estrogen treatment is known to increase the concentration of progesterone receptor. RNA translation experiments showed that this effect is due to an increase in the concentration of receptor mRNA. The size of this messenger RNA was studied by sucrose gradient ultracentrifugation, followed by mRNA translation, and specific immunoprecipitation: progesterone receptor mRNA was found by this method to sediment at 20 S.

The concentration of progesterone receptors in rat uterine cytosol and nuclei was measured during the oestrous cycle and pregnancy. The method used allowed the measurement of the total concentration of binding sites (unbound and hormone-bound).During the oestrous cycle, the concentration of receptors in the cytosol peaked at pro-oestrus, low concentrations were observed at oestrus and metoestrus, and an increase was seen at dioestrus. In the nuclei, maximum concentrations occurred at pro-oestrus and dioestrus. The number of receptors in the cytosol was very low during the first half of pregnancy, but the concentration increased progressively after day 15 to attain a very high level (about 26 000 binding sites/cell) on day 22.In the nuclei, the concentration of receptors was low at the beginning of pregnancy. On day 5 (day of implantation) there was a slight increase, which corresponded to a decrease in the number of cytosolic receptors and a small peak in the level of progesterone in the plasma. Maximum concentrations were attained during a 'plateau' period between days 9 and 15. Thereafter, there was a decrease in the concentration of nuclear receptors and on day 22, the mean value was very low; in some animals, probably on the verge of parturition, no receptors were detectable in the nuclei.

The study of the regulation of uteroglobin gene in the rabbit endometrium constitutes a model for analyzing the mechanism of action of progesterone in mammals. The gene has been cloned into lambda phage and sequenced. Comparison of the sequence of the gene with the amino acid sequence of preuteroglobin and the three-dimensional structure of uteroglobin established by crystal x-ray diffraction showed that the 3 exons correspond to different functional domains of the protein and that at least one of the splice junctions does not map at the surface of the protein. S1 mapping allowed us to define the RNA polymerase initiation site. No difference was observed when analyzing premessengers from the endometrium, where the gene is controlled by progesterone and estradiol, and from lung where the gene is constitutively expressed and not controlled by these hormones. In addition, S1 mapping revealed the existence of several minor transcription initiation sites. In the 5' flanking region between positions -33 and -24 there is the sequence AATACAAAAA which may correspond to a Goldberg-Hogness box. Two other A- and T-rich sequences were found further upstream from the gene, one of these preceding by about 30 nucleotides a minor start of transcription. No obvious feature, possibly related to steroid regulation, was observed in the nucleotide sequence. A fragment of the gene containing the “promoter” region (from nucleotide +10 to nucleotide -394) was preferentially retained on nitrocellulose filters after incubation with purified rabbit uterine receptor. A competitive binding assay was used to compare the affinity for the receptor of various DNA fragments. Labeled “promoter” region DNA was incubated with receptor and various concentrations of nonlabeled competing DNA, and the nitrocellulose-bound radioactivity was measured. This method showed the existence of several high affinity binding sites in the 5' part of the gene and in adjacent regions. However, no high affinity binding sites were observed in the 3' part of the gene. Also, within the “promoter” region there were at least two high affinity binding sites for the receptor.

A mouse was immunized with purified rabbit uterine cytosolic progesterone receptor (specific activity: 3 nmol of steroid bound per mg of protein). After fusion of its spleen cells with Sp2-OAg myeloma cells, supernatants of 11 hybrid cultures were found to react in both an immunoenzymatic test and a double-immunoprecipitation test with the progesterone receptor. Clones were obtained from the five hybrid cells that gave the strongest response in both tests. Antibodies from cell culture supernatants and ascitic fluids were characterized. Three are of the IgG1 and two of the IgG2a isotype. Their apparent affinity for the progesterone receptor was measured by immunoprecipitation in physiological salt conditions. The equilibrium dissociation constants were between 0.1 and 4 nM. All five monoclonal antibodies crossreacted with the rabbit nuclear receptor, the human cytosolic receptor, and other mammalian (rat, guinea pig) but not avian (chicken) cytosolic progesterone receptors. There was no interaction with the glucocorticoid receptor and corticosteroid binding globulin.

The p105 Rel protein has dual functions; it is the precursor of the p5O subunit of NF-kappaB, and it acts as an IkappaB-like inhibitor to retain other Rel subunits in the cytoplasm. We have investigated the posttranslational regulation of p105 following activation of Jurkat T cells and find that a rapid and sustained phosphorylation of p105 is induced. The inducible phosphorylation occurs on multiple serines in the C-terminal-most 150 amino acids of the molecule, a region rich in Pro, Glu, Ser, and Thr residues. Phosphorylation of p105 in Jurkat cells treated with phorbol 12-myristate 13-acetate/ionomycin or with okadaic acid, another activator of NF-kappaB, is correlated with an increase in proteolytic processing to p5O. Intact PEST sequences are required for the phorbol 12-myristate 13-acetate/ionomycin-induced p105 processing, as a 68-amino acid C-terminal deletion abolishes the response to stimulation. When compounds that block Ikappa B alpha phosphorylation and degradation were tested, the serine protease inhibitors L-1-tosylamido-2-phenylethyl chloromethyl ketone and 1-chloro-3-tosyl-amido-7-amino-2-heptanone blocked inducible p105 phosphorylation, but the antioxidants pyrrolidine dithiocarbamate and butylated hydroxyanisol did not. Thus, while regulation of the p105 IkappaB resembles that of lkappaBa, involving inducible serine phosphorylation and proteolysis of the inhibitory ankyrin repeat domain, it depends on a different, redox-insensitive, signaling pathway.

The progesterone receptor has been localized in the rabbit uterus by immunocytochemistry at the electron microscopic level, using monoclonal antibodies and the protein A-gold technique. The progesterone receptor in uterine stromal cells was mainly localized in the nucleus; however, a small fraction of antigen was present in the cytoplasm, where it was associated with the rough endoplasmic reticulum and with free ribosomes. The plasma membrane was not labeled. In the nucleus, the receptor was always associated with condensed chromatin or areas surrounding condensed chromatin, whereas the nuceolus was not labeled. In the chromatin, receptor distribution varied according to the hormonal state: in the absence of progesterone, the receptor was randomly scattered over the clumps of condensed chromatin; after administration of the progestin R5020, it was mainly detected in the border regions between condensed chromatin and nucleoplasm and, to a lesser extent, over dispersed chromatin in the nucleoplasm. These areas have been shown to be the most active sites of gene transcription.

A radioimmunoassay method to measure progesterone receptor in rabbit and human tissues was devised and applied to human breast cancer. A specific progesterone receptor antibody was prepared by purifying rabbit receptor by immunoaffinity chromatography, sodium dodecylsulphate-polyacrylamide gel electrophoresis and injection of the isolated 110 000 dalton receptor band into a goat. Immunoblot studies of progesterone target and non-target tissues showed the specificity of the antibody, which was used at a dilution of 1/45 000. The tracer consisted of 125I-labelled electroeluted 110 000 dalton receptor.The sensitivity of the method was 1 fmol/tube for the rabbit receptor and 3 fmol/tube for the human receptor. The intra-assay coefficient of variation was 11% for tumours positive for the progesterone receptor and 9·9% for those on the borderline (10–30 fmol receptor/mg protein). The interassay coefficients of variation were 20 and 19% respectively. The correlation between the radioimmunoassay and a steroid-binding assay was studied in 40 tumour biopsies. In 39 cases, very good correlation was found (r= 0·99); in a single case an immunoreactive protein was detected which apparently bound steroid poorly.One important feature of this method was that receptor immunoreactivity remained unchanged when either the tissue or the cytosol was exposed to a temperature of 20 °C for relatively long periods of time. Under the same conditions the steroid-binding capacity declined rapidly. This characteristic of the radioimmunoassay may prevent errors due to improper handling of tissue samples. Such stability was not observed for oestrogen receptors when measured by a sandwich immunoenzymatic method after incubation of tissue at 20 °C.J. Endocr.(1988) 116,427–434

Two lambda gt11 clones containing fragments of cDNA encoding the rabbit progesterone receptor were isolated with the aid of monoclonal and monospecific polyclonal antireceptor antibodies. RNA gel blot analysis showed that the corresponding mRNA was approximately equal to 5900 nucleotides in size and present in the uterus, where its concentration was increased by estrogen treatment, and in the vagina. This mRNA was not detected in liver, in spleen, in intestine, and in kidney where the receptor protein is known to be absent or present in very small concentration. Cross-hybridizing clones were isolated from a lambda 10 library. The DNA was sequenced, and the primary structure of the progesterone receptor was deduced. It consists of 930 amino acids and contains a basic, cysteine-rich region (residues 568-645) with extensive homology to the glucocorticoid and estrogen receptors and the v-erbA oncogene protein. This region is followed by a C-terminal domain that is similar in size to the corresponding domains of the other steroid receptors and v-erbA and shows striking amino acid homology with the glucocorticoid receptor and significant homology with the estrogen receptor. In contrast, the region extending from the cysteine-rich segment toward the N terminus differed in size and amino acid sequence from that of the other receptors and v-erbA. This region had a high proline content in the progesterone receptor.