Antiestrogens specifically up-regulate bone morphogenetic protein-4 promoter activity in human osteoblastic cells.

Bone morphogenetic protein-4 (BMP-4) plays an important role in the onset of endochondral bone formation in humans, and a reduction in BMP-4 expression has been associated with a variety of bone diseases. Here we describe, by transient transfection assays in bone cells, that the human BMP-4 promoter recently characterized in our laboratory can be stimulated specifically by antiestrogens but not by estrogens or other steroid hormones. This activity is dependent on the presence of the estrogen receptor (ER)-alpha, although the promoter lacks a consensus estrogen-responsive element. No activity was observed in the presence of ERbeta, but synergy was observed when both ER subtypes were cotransfected. The observed stimulation of BMP-4 promoter activity by antiestrogens appeared bone cell specific and was reversed upon addition of estrogens. Since antiestrogens are known to be effective in hormone replacement therapies for postmenopausal women, this observation may help to develop new strategies for treatment and prevention of osteoporosis.

Human progesterone receptor A and B isoforms in CHO cells. I. Stable transfection of receptor and receptor-responsive reporter genes: transcription modulation by (anti)progestagens.

A hormone-dependent transcription modulation system was established on the basis of a two-step transfection procedure of the human progesterone receptor isoforms (hPR-A and hPR-B, respectively) and a progesterone receptor-responsive reporter (MMTV-Luc). In the first step, stable transfection of the hPR-A and hPR-B isoform-encoding cDNAs was performed in the steroid receptor-negative CHO K1 cell line. Individual clones were characterized for hPR-isoform expression with respect to Western immuno-blotting, transcriptional activation and hormone binding. With respect to the latter characteristic, individual hPR-isoforms demonstrated similar dissociation constants (Kd for hPR-A: 0.5 +/- 0.3 and hPR-B: 0.8 +/- 0.3 nM, respectively) irrespective of the amount of receptor isoform expressed (Bmax varying from 4.1 to 33.2 nM). The Kd values observed for individual hPR-isoforms were comparable to those found for human breast tumor MCF-7 cells (Kd for hPR-A + hPR-B: 0.6 +/- 0.3 nM). In the second step, hPR-isoform expressing CHO clones were supertransfected with a MMTV-Luc reporter construct resulting in permanent cell lines useful for testing the activity of natural and synthetic steroids in their ability to modulate gene transcription. Both isoform-specific reporter cell lines responded in a similar ranking order towards different progesterone reference compounds such as Org 2058, progesterone (Prog), R5020, norethisterone (NE), and medroxy progesterone acetate (MPA). Moreover, a good correlation was observed between the relative binding affinity (RBA) and the transcriptional activation potency of these compounds towards the individual hPR-isoforms. The latter correlation could not only be demonstrated for the progestagenic agonist reference compounds but was also observed for the progestagenic antagonist reference compounds like Org 33628, Org 31710, RU 38486 and ZK 98299. The major difference observed between the individual PR-isoforms was related to the degree of stimulation of the reporter gene (MMTV-based) within the cellular CHO context. Therefore, these cell lines can be used for the determination and quantitation of the activity of (anti)progestagenic compounds in vitro but may also be useful to predict the activity of compounds in vivo (see also II Comparison of binding, transactivation and ED50 values of several synthetic (anti) progestagens in vitro in CHO and MCF-7 cells and in vivo in rabbits and rats).

Human progesterone receptor A and B isoforms in CHO cells. II. Comparison of binding, transactivation and ED50 values of several synthetic (anti)progestagens in vitro in CHO and MCF-7 cells and in vivo in rabbits and rats.

The human progesterone receptor A and B isoforms (hPR-A and hPR-B) were stably transfected in Chinese Hamster Ovary (CHO) cells in the presence or absence of the mouse mamma tumor virus (MMTV) promoter and luciferase (LUC) reporter gene. In this way four stably transfected CHO cell lines, i.e. hPR-A, hPR-B, hPR-A-MMTV-LUC and hPR-B-MMTV-LUC cells, were prepared. hPR-A and -B isoforms were compared by binding and transactivation analysis for 14 progestagens and 7 antiprogestagens. Thereby Org 2058 was used as standard in both agonistic and binding assays and Org 31710 in antagonistic assays. The obtained data were compared with relative binding affinities (RBA) for both hPR-A and -B, which are present in human breast tumor MCF-7 cells, and with biopotency estimations with McPhail tests in rabbits and ovulation inhibition and pregnancy interruption tests in rats. The relative binding affinities of 14 progestagens and 7 antiprogestagens towards hPR-A, hPR-B or hPR-A/B of either CHO or MCF-7 cells were highly correlated with respect to ranking. This was also shown by the high correlation coefficients between the RBA's of hPR-B and hPR-A in CHO cells (r = 0.983) and between those of hPR-B of CHO and hPR A/B of MCF-7 cells (r = 0.957). The transactivation data of the 14 progestagens and 7 antiprogestagens for the hPR-B-MMTV-LUC cells were compared with those for hPR-A-MMTV-LUC cells and showed no differences between both cell lines with exception of the progestagens Org 32704 and 33766 and the antiprogestagen Org 33245. Therefore only the relative agonistic activities (RAA) and relative antagonistic activities (RANTA) of hPR-B-MMTV-LUC cells were compared with RBA values of hPR-B, showing a high similarity in ranking for the tested compounds, and high correlation coefficients of r = 0.91 and r = 0.97, respectively. Remarkably, RBA's were 1.6 fold higher than RAA's and RANTA's. These in vitro RBA, RAA and RANTA values for hPR-B were checked for their pharmacological relevance by in vivo biopotency measurements with the 14 progestagens and 7 antiprogestagens in rabbits and rats. The in vitro binding and transactivation potencies of progestagens appeared to be very predictive for in vivo analysis on endometrium proliferation in rabbits in the McPhail test with correlation coefficients of r = 0.81 and r = 0.87, while ovulation inhibition in rats correlated less well with r = 0.516 and r = 0.65. On the other hand, the antiprogestagenic potencies found with binding and transactivation assays had a good correlation with the potencies in the pregnancy interruption test in rats for all antiprogestagens tested, being r = 0.849 and r = 0.744, respectively. In conclusion, the binding and transactivation potencies for the tested compounds in hPR-A and -B containing cell lines showed in general a good resemblance. The transactivation studies with hPR-B-MMTV-LUC cells indicated that ranking of compounds was fairly identical to binding analysis and could be used for pre-screening of the (anti)-progestagenic bioactivity in the McPhail test in rabbits, the ovulation inhibition test and the pregnancy interruption test in rats. Therefore this transactivation assay can replace binding assays. Moreover, direct pre-screening of agonists, antagonists and partial antagonists is even possible in this in vitro bioassay, making transactivation assays for a particular class of chemical compounds to a valuable pre-screening tool for in vivo studies.

X-ray structure of antistasin at 1.9 A resolution and its modelled complex with blood coagulation factor Xa.

The three-dimensional structure of antistasin, a potent inhibitor of blood coagulation factor Xa, from the Mexican leech Haementeria officinalis was determined at 1.9 A resolution by X-ray crystallography. The structure reveals a novel protein fold composed of two homologous domains, each resembling the structure of hirustasin, a related 55-residue protease inhibitor. However, hirustasin has a different overall shape than the individual antistasin domains, it contains four rather than two beta-strands, and does not inhibit factor Xa. The two antistasin domains can be subdivided into two similarly sized subdomains with different relative orientations. Consequently, the domain shapes are different, the N-terminal domain being wedge-shaped and the C-terminal domain flat. Docking studies suggest that differences in domain shape enable the N-terminal, but not C-terminal, domain of antistasin to bind and inhibit factor Xa, even though both have a very similar reactive site. Furthermore, a putative exosite binding region could be defined in the N-terminal domain of antistasin, comprising residues 15-17, which is likely to interact with a cluster of positively charged residues on the factor Xa surface (Arg222/Lys223/Lys224). This exosite binding region explains the specificity and inhibitory potency of antistasin towards factor Xa. In the C-terminal domain of antistasin, these exosite interactions are prevented due to the different overall shape of this domain.

ER beta: identification and characterization of a novel human estrogen receptor.

A novel estrogen receptor (hereinafter referred to as ER beta) was cloned using degenerate PCR primers. A comparison of the amino acid sequence of ER beta with the "classical' ER (ER alpha) shows a high degree of conservation of the DNA-binding domain (96%), and of the ligand-binding domain (58%). In contrast, the A/B domain, the hinge region and the F-domain are not conserved. Northern blot analysis revealed that ER beta is expressed in human thymus, spleen, ovary and testis. Transient transfections of an ER beta expression construct together with an ERE-based reporter construct in CHO cells clearly demonstrated transactivation of ER beta by 17 beta-estradiol. In addition, the ER alpha antagonist ICI-164384 is a potent antagonist for ER beta as well. Interestingly, the level of transactivation by 17 beta-estradiol is higher for ER alpha than for ER beta, which may reflect suboptimal conditions for ER beta at the level of the ligand, responsive element or cellular context.

Pharmacological properties of a new selective antiprogestagen: Org 33628.

UNLABELLED: For antiprogestagens both selectivity (ratio of antiprogestational to antiglucocorticoid activity) and potency are important conditions for their applications in fertility regulation and correction of hormone-dependent irregularities. Org 33628 appears to fulfill both conditions most convincingly. The activities of this new antiprogestagen in various assays are compared with those of RU 38486 and a few other antiprogestagens. The binding of Org 33628 to the progesterone receptor is twice as high as that of RU 38486 whereas the binding to the glucocorticoid receptor is 25 times lower than that of RU 38486. The activity of Org 33628 in the pregnancy interruption test in rats is 16 times higher than that of RU 38486. The antiglucocorticoid activity of Org 33628 in rats is about eight times lower than that of RU 38486. In the ovulation inhibition test in rats Org 33628 is approximately 80 times more potent than RU 38486. For menses induction in the stumptail monkey activity observed for Org 33628 is only twice as high. IN CONCLUSION: Org 33628 is a very potent and selective antiprogestagen with a remarkably high ovulation-inhibitory activity. The magnitude of the potency difference with RU 38486 is species and/or target organ dependent.

Synergism between androgens and protein kinase-C on androgen-regulated gene expression.

Androgen (R1881) induced transcriptional activity of the human androgen receptor, stably expressed in CHO cells, can be stimulated an extra 2-fold by the addition of the protein kinase C activator, 4 beta-phorbol 12-myristate 13-acetate (PMA). This extra stimulation is not observed when the protein kinase A activator bromoadenosine 3':5'-cyclic monophosphate (8-BrcAMP) is used. The transcriptional activity was measured using a reporter plasmid containing the MMTV-promoter, coupled to the luciferase gene. The effect of PMA on R1881-induced transcription was not due to a higher expression level of the androgen receptor. Also, no extra phosphorylation of the androgen receptor could be measured after incubation with PMA. When GRE-tk-LUC and PSA-LUC reporters were used, the synergistic effect of PMA could not be observed. The findings on the composite MMTV-LTR promoter can be explained by either a direct synergistic interaction between occupied AP-1 like responsive elements and the androgen receptor or via an unknown transcription factor activated by the PKC pathway and interacting with the androgen receptor.

Mutational analysis of antistasin, an inhibitor of blood coagulation factor Xa derived from the Mexican leech Haementeria officinalis.

Antistasin is a Factor Xa inhibitor that is present in the salivary glands of the Mexican leech Haementeria officinalis. The antistasin protein consists of 119 amino acids, of which residues 1-55 (domain I) are 56% similar to residues 56-110 (domain II). Of the nine C-terminal amino acids (residues 111-119; domain III), four are positively charged. The reactive site for Factor Xa is located in domain I. In this study we assessed the role of separate domains and of individual amino acids in the reactive site for the inhibition of Factor Xa. A series of mutants was constructed and expressed in Chinese hamster ovary (CHO) cells. In vitro chromogenic assays for Factor Xa show that domain I is sufficient for inhibition of Factor Xa. Domains II and III neither contain any intrinsic Factor Xa inhibitory activity, nor contribute to the activity of domain I. Furthermore, domain II does not become a Factor Xa inhibitor by partially adaptating its sequence towards that of the reactive site in domain I. Mutation of the cysteine at position 33 is not crucial for Factor Xa inhibition, suggesting a relatively rigid reactive site loop structure.

The intact and cleaved human antithrombin III complex as a model for serpin-proteinase interactions.

Antithrombin is a member of the serine proteinase inhibitor (serpin) family which contain a flexible reactive site loop that interacts with, and is cleaved by the target proteinase. In cleaved and latent serpins, the reactive site loop is inserted into a large central beta-sheet in the same molecule, whereas in ovalbumin, a nonfunctional serpin, the reactive site loop is completely exposed and in an alpha-helical conformation. However, in neither conformation can the reactive site loop bind to target proteinases. Here we report the structure of an intact and cleaved human antithrombin complex. The intact reactive site loop is in a novel conformation that seems well suited for interaction with proteinases such as thrombin and blood coagulation factor Xa.

Eimeria refractile body proteins contain two potentially functional characteristics: transhydrogenase and carbohydrate transport.

cDNA encoding an immunogenic protein from partially sporulated oocysts of Eimeria acervulina was cloned and used to search for the homologous counterpart in Eimeria tenella. Monospecific antibodies were raised against the recombinant expression product. Using these antibodies, the parasite proteins were found to be localised in the refractile bodies. The derived amino-acid sequences were compared by computer using the SWISSPROT protein database. In addition to high homology between the Eimeria species, extensive similarity was found with pyridine nucleotide transhydrogenase from Escherichia coli. Comparison with the sugar signature database also resulted in a possible sugar binding domain present only in the Eimeria proteins. It is possible that the corresponding parasite proteins play a role in the recently discovered mannitol cycle of Eimeria.

Crystallization and preliminary crystallographic analysis of antistasin, a leech-derived inhibitor of blood coagulation factor Xa.

The salivary gland of the Mexican leech Haementeria officinalis contains a 15 kDa protein which is a potent and selective inhibitor of factor Xa. It inhibits not only blood coagulation, but also metastasis. A gene, coding for a sequence similar to published antistasin sequences, has been synthesized and expressed in Chinese hamster ovary (CHO) cells. The recombinant protein was purified and crystallized at pH 6.0, using 31% ammonium sulfate as a precipitant. The crystals diffract at least to 2.8 A. The spacegroup is I422 with a = b = 77.7 A and c = 88.4 A. The crystals contain 42% solvent and one protein molecule in the asymmetric unit. A search for heavy atom derivatives is in progress.

Dissociation of heparin-dependent thrombin and factor Xa inhibitory activities of antithrombin-III by mutations in the reactive site.

Antithrombin-III (AT-III) is a heparin-dependent inhibitor of thrombin and Factor Xa, two serine proteases that are crucial for blood coagulation. In order to assess whether it would be possible to target AT-III only towards Factor Xa, we replaced parts of the reactive site, or P region, of AT-III by sequences present in prothrombin, a substrate of Factor Xa in the coagulation cascade. We show that replacement of the P3 to P3' region generates the hypothesized phenotype. In fact, point mutation of the P1' site from Ser (present in AT-III) to Ile (present in prothrombin) is sufficient to dissociate heparin-dependent thrombin and Factor Xa inhibitory activities. Interestingly, a combined mutation at P3 and P3' brings about the same dissociation. We show that besides Ile, other amino acids at P1' can lead to the dissociation in inhibitory activity. Amino acids with small side chains (Gly, Ser, Ala, and Thr) have only a marginal effect on the inhibitory activity against either protease. However, larger residues at the P1' position abolish the heparin-dependent anti-thrombin activity, whereas the heparin-dependent anti-Factor Xa activity is not at all or only moderately affected. These results can be rationalized by a comparison of the x-ray structure and a three-dimensional model of the S1' binding pockets of thrombin and Factor Xa, respectively. It appears that the S1' pocket of Factor Xa leaves much more space for the P1' residue of AT-III than the S1' pocket of thrombin.

Crystallization and preliminary X-ray analysis of human antithrombin III.

Human antithrombin III has been crystallized from 18 to 21% (w/v) polyethylene glycol 4000 at pH 7.15. The spacegroup is P2(1) with cell parameters a = 89.8 A, b = 100.8 A, c = 70.0 A and beta = 106 degrees. The diffraction limit is 3.2 A. The asymmetric unit contains two protein molecules. Analysis of dissolved crystals for biological activity and by gel electrophoresis suggests that one protein molecule in the asymmetric unit is intact, while the other is cleaved.

Genomic organization, coding sequence and functional expression of human 5-HT2 and 5-HT1A receptor genes.

The family of serotonin receptors consists of at least eight distinct subtypes, divided into four classes based on their pharmacological and functional characteristics. Here we report the cloning and expression in Swiss 3T3 cells of the human 5-HT2 and 5-HT1A receptor subtypes. Both genes encode functional receptors for 5-HT, that differ considerably in genomic structure, primary amino acid sequence, pharmacology and signal transduction. The 5-HT1A receptor transfectants displayed a single high affinity site for the agonist [3H](+/-)-8-hydroxy-2-(di-n-propylamino)tetralin HBr ([3H]8-OH-DPAT) and a pharmacological profile specific for the 5-HT1A receptor. In these transfectants, 5-HT mediated a dose-dependent inhibition of forskolin-stimulated cAMP levels. Cells expressing the 5-HT2 receptor exhibited high affinity binding for the antagonist [3H]ketanserin with a 5-HT2 receptor specific pharmacological profile. In these cells 5-HT activated phospholipase C in a dose-dependent manner. The 5-HT2 receptor displayed a genomic organization quite different from the 5-HT1A, 5-HT1B and 5-HT1D receptor subtypes. While these receptors are encoded by one single exon, the 5-HT2 receptor is encoded by three exons separated by two introns. The latter finding adds and additional molecular criterion for receptor classification.

Sequence of the gene encoding an immunodominant microneme protein of Eimeria tenella.

A heterodisperse family of antigens, previously detected on sporozoites and merozoites of Eimeria tenella, has been localised to the microneme organelles within the sporozoite. Sequencing of genomic and cDNA clones shows that the gene for this antigen family contains 4 exons separated by 3 short (519, 226 and 156 nucleotides) intervening sequences and that the predicted polypeptide from the longest open reading frame has 4 structural domains. One of these contains 5 copies of the thrombospondin-like motif, previously identified in the partial sequence of the gene, which is conserved in a variety of molecules which have been demonstrated to have adhesive properties. A second domain of the polypeptide has strong similarity to a conserved region that occurs in another group of molecules which have adhesive properties, including the alpha subunits of several integrins, complement factor Bb and a number of extracellular matrix glycoproteins. Overall the antigen resembles the thrombospondin-related anonymous protein identified in the erythrocytic stage of Plasmodium falciparum. The structure of the gene supports a role for this microneme antigen in cell-cell or cell-matrix interactions.

The hepatitis delta (delta) virus possesses a circular RNA.

Hepatitis delta (delta) virus (HDV), a satellite virus of the hepatitis B virus (HBV), causes a severe form of viral hepatitis in humans. Here we present evidence based on electron microscopy and electrophoretic behaviour that HDV contains a single stranded circular RNA molecule. This is the first animal virus identified with a circular RNA genome. Circular RNAs have only been found in plant viruses. We have obtained a partial complementary DNA clone representing approximately 25% of the total genome of HDV. Analysis of this cDNA revealed similarity to two plant viruses that may explain the origin of the virus.

Cloning and expression of the chromosomal immune interferon gene of the rat.

The chromosomal immune interferon gene of the rat (IFN-gamma) was identified by screening a recombinant rat lambda phage library with a human IFN-gamma cDNA probe. In contrast to the genes of other rat IFNs, this rat IFN-gamma chromosomal gene contains introns and its structural organization closely resembles that of the human and murine IFN-gamma genes. The rat IFN-gamma gene encodes a signal sequence of 19 amino acids followed by the mature IFN-gamma protein of 137 amino acids. The gene was expressed under control of the simian virus 40 (SV40) early promoter in Chinese hamster ovary (CHO) cells deficient in dihydrofolate reductase (DHFR) after co-transformation with a plasmid containing the mouse DHFR gene. Initial transformants with a DHFR+ phenotype produced IFN-gamma titres ranging from 20 to 1600 units/ml. After stepwise increases in the concentration of methotrexate (MTX) in the growth medium of transformed CHO cells, MTX-resistant clones producing 80 000-100 000 units per ml were isolated. Protein analysis of supernatants of these MTX-resistant cells by polyacrylamide gel electrophoresis revealed a product with an apparent mol. wt. of 18 000 daltons which was not detectable in the growth medium of DHFR+ transformants that did not produce IFN. The product was identified as rat IFN-gamma and constituted approximately 5% of the proteins excreted from these cells.

Antiviral activity of interferon in rats and the effect of immune suppression.

The antiviral effect of interferon was studied in a number of experimental virus infections in the rat. Interferon was shown to protect rats infected with pseudorabies virus, herpes simplex virus or vesicular stomatitis virus. The antiviral activity was not inhibited by immune suppression induced by azothioprine, prednisolone or cyclosporin A treatment. Cyclophosphamide completely blocked the in vivo activity of interferon in rats.