Natural chondroitin sulphates increase aggregation of proteoglycan complexes and decrease ADAMTS-5 expression in interleukin 1 beta-treated chondrocytes.

OBJECTIVE: To assess the effect of natural chondroitin sulphate (CS) on the ability of neosynthesized sulphated proteoglycans (PGs) to aggregate in cultured chondrocytes treated with interleukin (IL)1 beta. METHODS: Primary cultured rabbit articular chondrocytes were treated or not with IL1 beta alone or with concentrations of CS for 20 h. Neosynthesized PGs were labelled by incorporation of [35SO(4)]-sulphate and analysed by chromatography on Sepharose 2B columns. Gelatinolytic activity was measured by zymography, and matrix metalloproteinase (MMP)1 mRNA level in chondrocytes underwent real-time PCR. Expression of ADAMTS (for "a disintegrin and metalloproteinase with thrombospondin motifs") -4 and -5 was analysed by real-time PCR and western blotting. RESULTS: The production of [35SO(4)]-labelled PGs was significantly increased with 10 microg/ml CS in the cellular pool rather than in the incubation medium. The addition of CS to IL1 beta-treated cells inhibited in part the disaggregation of sulphated PGs induced by IL1 beta. This inhibitory effect of CS is associated with a significant decrease in ADAMTS-5 expression at the mRNA and protein levels. No effect of CS was observed on IL1 beta-induced gelatinolytic activity, MMP1 mRNA expression or ADAMTS-4 expression. CONCLUSION: CS increases the production of functional sulphated PGs in the direct environment of chondrocytes in vitro. This beneficial effect of CS in IL1 beta-treated cells is associated with decreased expression of ADAMTS-5.

Monoclonal antibodies against native ant denatured forms of estrogen-induced breast cancer protein (BCEI/pS2) obtained by expression in Escherichia coli.

Several vectors were used to express the complementary DNA for breast cancer estrogen-induced protein BCEI (also called pS2) in Escherichia coli. The best results were obtained by using the pUR 290 expression vector after deletion of the sequence encoding the signal peptide of the protein. In these conditions, beta-galactosidase-BCEI/pS2 fusion protein accounted for approximately 20% of total proteins in bacterial extracts. It was purified by chromatography on DEAE-Trisacryl or by gel electrophoresis and electroelution. Polyclonal antibodies were obtained by immunization of rabbits and goats, and monoclonal antibodies were raised in mice. Two types of monoclonal antibodies were obtained: one class recognized the native protein and was very efficient for the immunoprecipitation and immunopurification of the protein from breast cancer cells; a second class recognized the denatured protein and was especially effective for immunoblot studies. BCEI/pS2 could be detected by immunocytochemistry in breast cancer biopsies using monoclonal antibodies on frozen or paraffin-embedded sections. One of the antibodies (mBCEI11) exhibited high affinity for the protein and could be used at 1.9 micrograms/ml concentration for immunolabeling of histological sections. The mBCEI11 antibody was used in immunoaffinity chromatography to purify the peptide in a single step from culture media of estrogen-treated MCF-7 cells.

Effect of PML and PML-RAR on the transactivation properties and subcellular distribution of steroid hormone receptors.

PML (promyelocytic leukemia) is a protein involved in the t (15;17) translocation of promyelocytic leukemia and is mainly localized in nuclear bodies. Here we show that PML exerts a very powerful enhancing activity (up to 20-fold) on the transactivating properties of the progesterone receptor (PR) and has a similar effect on several other steroid hormone receptors. There is probably a direct or indirect interaction between PR and PML, because when the latter was expressed at high concentrations it shifted PR into the nuclear bodies. The use of deletion mutants showed that both activation functions (AF1 and AF2) of PR as well as the coiled coil and His-Cys-rich domains of PML were required for transcriptional enhancement. The fusion protein PML-RAR which is not localized in nuclear bodies, also enhanced the transactivating activity of PR, but this effect was totally suppressed by the administration of retinoic acid. PML, which is ubiquitously expressed, may thus be involved in the transactivation properties of steroid hormone receptors. This mechanism may also play a role in the oncogenic properties of PML-RAR and in their suppression by retinoic acid.

Interaction of triiodothyronine-receptor complexes with simian virus 40 minichromosomes in monkey kidney CV-1 cells.

Thyroid hormone-responsive tissues contain chromatin-localized receptors that bind to DNA and may associate preferentially with actively transcribed chromatin. To study such receptor-chromatin localization, we have used cultured CV-1 cells permissive for simian virus 40 (SV40), in which viral minichromosomes can be separated from the cellular chromatin. CV-1 cells were found to contain intranuclear thyroid hormone-binding sites with an affinity for T3 and T4 and a site concentration similar to those in other thyroid hormone-responsive tissues. When these cells were infected with SV40 or an SV40-human GH gene recombinant, T3 did not affect SV40 replication, early or late gene transcription, or human GH gene expression. However, in both cases, these infections resulted in the association of about 7.5% of the total specific T3-binding activity with the SV40 minichromosome, representing about 1 receptor molecule/65 minichromosomes and a 10-fold enrichment over the cellular chromatin-associated activity (4.3 fmol/micrograms SV40 minichromosomal DNA vs. 0.43 fmol/micrograms chromosomal DNA); 30% of this could be covalently cross-linked to the minichromosome with dissuccinimidyl suberate. The minichromosomes were also found to be transcriptionally active. Thus, thyroid hormone receptors interact preferentially with the SV40 minichromosome, possibly owing to their tendency to associate with transcriptionally active chromatin. This system provides an alternate approach to study the association of thyroid hormone receptors with defined chromosomal segments.

Promegestone (R5020) and mifepristone (RU486) both function as progestational agonists of human glycodelin gene expression in isolated human epithelial cells.

One of the most abundant protein products of human secretory endometrium is glycodelin, a glycoprotein previously referred to as PP14. Although the precise function of this protein is unknown, its unique glycosylation pattern is believed to affect immunomodulatory activity during human embryonic implantation and inhibition of sperm-egg binding after ovulation. Having confirmed the expression of glycodelin in secretory endometrial glands, we used purified endometrial epithelial cell cultures to demonstrate the hormonal regulation of glycodelin synthesis and secretion. The findings were corroborated by transiently transfecting glycodelin gene promoter-reporter constructs into human epithelioid HeLa and Ishikawa cells. Our results indicate that glycodelin protein production by endometrial epithelial cells is directly up-regulated 4- to 9-fold by progestins and antiprogestins in vitro. Transcriptional regulation of the glycodelin gene promoter expressed in HeLa cells is progesterone receptor-dependent. As observed in the primary endometrial cells, progestins and antiprogestins both act as agonists on the in vitro expression of this endometrial gene. Our findings provide insight into the regulation of this abundant endometrial protein and raise interesting questions about the physical nature of the interaction of agonist- and antagonist-bound progesterone receptors with the glycodelin gene promoter.

Inhibition of dioxin effects on bone formation in vitro by a newly described aryl hydrocarbon receptor antagonist, resveratrol.

Aryl hydrocarbon receptor (AhR) ligands are environmental contaminants found in cigarette smoke and other sources of air pollution. The prototypical compound is TCDD (2,3,7, 8-tetrachlorodibenzo-p-dioxin), also known as dioxin. There is an increasing body of knowledge linking cigarette smoking to osteoporosis and periodontal disease, but the direct effects of smoke-associated aryl hydrocarbons on bone are not well understood. Through the use of resveratrol (3,5,4'-trihydroxystilbene), a plant antifungal compound that we have recently demonstrated to be a pure AhR antagonist, we have investigated the effects of TCDD on osteogenesis. It was postulated that TCDD would inhibit osteogenesis in bone-forming cultures and that this inhibition would be antagonized by resveratrol. We employed the chicken periosteal osteogenesis (CPO) model, which has been shown to form bone in vitro in a pattern morphologically and biochemically similar to that seen in vivo, as well as a rat stromal cell bone nodule formation model. In the CPO model, alkaline phosphatase (AP) activity was reduced by up to 50% (P<0.01 vs control) in the presence of 10(-9) M TCDD and these effects were reversed by 10(-6) M resveratrol (P<0.05 vs TCDD alone). TCDD-mediated inhibition of osteogenesis was restricted primarily to the osteoblastic differentiation phase (days 0-2) as later addition did not appear to have any effects. Message levels for important bone-associated proteins (in the CPO model) such as collagen type I, osteopontin, bone sialoprotein and AP were inhibited by TCDD, an effect that was antagonized by resveratrol. Similar findings were obtained using the rat stromal bone cell line. TCDD (at concentrations as low as 10(-10)M) caused an approximately 33% reduction in AP activity, which was abrogated by 3. 5x10(-7) M resveratrol. TCDD also induced a marked reduction in mineralization ( approximately 75%) which was completely antagonized by resveratrol. These data suggest that AhR ligands inhibit osteogenesis probably through inhibition of osteodifferentiation and that this effect can be antagonized by resveratrol. Since high levels of AhR ligands are found in cigarette smoke, and further since smoking is an important risk factor in both osteoporosis and periodontal disease, it may be postulated that AhR ligands are the component of cigarette smoke linking smoking to osteoporosis and periodontal disease. If so, resveratrol could prove to be a promising preventive or therapeutic agent for smoking-related bone loss.

Origin of the high constitutive level of progesterone receptor in T47-D breast cancer cells.

The T47-D breast cancer cell line constitutively expresses high levels of progesterone receptor (PR). This does not appear to be related to an anomaly in the estrogen receptor (ER) as shown by cloning of the ER cDNA from T47-D cells and its insertion into the expression vector pKSV-10. When transfected into heterologous Cos-7 and L cells this receptor exerts a normal biological activity, stimulating the transcription of a reporter gene only in the presence of estrogen. Moreover, normal estrogen regulation of the transcription of the reporter gene was also observed in situ in T47-D cells. Southern blot experiments showed the presence of four copies of the progesterone receptor gene in T47-D cells. This was related to the existence of four copies of chromosome 11 in these cells. The most likely explanation of the anomalous regulation of progesterone receptor expression in T47-D cells is thus the presence of at least one copy of the PR gene bearing an anomaly in its regulatory region(s).

Resveratrol decreases hyperalgesia induced by carrageenan in the rat hind paw.

The effect of resveratrol, an aryl hydrocarbon receptor (AhR) antagonist, known to inhibit inducible cyclooxygenase-2 (COX2) and its transcription were examined in a model of hyperalgesia induced by carrageenan in the rat. Pretreatment with resveratrol did not reverse swelling and edema, but reversed the hyperalgesia induced by local tissue injury provoked by carrageenan. This reversal, occurring at resveratrol concentrations as low as 2 mg/kg, lasted for at least 48 hours. The link with COX2 activity inhibition and COX2 gene transcription, as well as a potential AhR inhibitory effect, remain to be established.

In vitro molecular assessment of the mechanisms of action of 19-nor progestins used as contragestational agents.

Norethisterone (NET) and levonorgestrel (LNG) are synthetic progestins used as contragestational agents. Both compounds are biotransformed at target tissues into A-ring reduced metabolites which possess different pharmacological properties. The aim of this study was to determine the molecular mechanisms of the progestational and antiprogestational effects of NET, LNG and their metabolites by using a highly efficient, sensitive in vitro molecular assay based on the detection of a reporter gene expression (the bacterial chloramphenicol acetyltransferase (CAT) inserted downstream of a minimal promoter containing two progesterone responsive elements (PRE2) and the TATA box. For this purpose we used CV-1 monkey kidney cells, which do not possess steroid receptors. These cells were cotransfected with a progesterone receptor expression vector and the reporter vector PRE2-TATA-CAT. Data obtained using this model showed that NET and LNG induced CAT activity in a manner similar to that of the potent progestin R5020. NET and LNG metabolites exhibited a weak progestational activity; however, when 5 alpha-NET metabolite was simultaneously administered with R5020, a clear antiprogestational effect similar to that of the antiprogestin RU486 was observed. Therefore, the results clearly demonstrate that the use of the reporter CAT vector containing hormone responsive elements is a suitable assay for the screening and evaluation of new synthetic steroids with agonist or antagonist progestational activities in transfected CV-1 cell line.

Resveratrol and cancer: a review.

The various properties of the stilbene phytoalexin Resveratrol provide interesting new avenues of research in the field of chemoprevention and chemotherapy. A particular emphasis is given on xenobiotic-related carcinogenesis.

Resveratrol, a natural aryl hydrocarbon receptor antagonist, protects sperm from DNA damage and apoptosis caused by benzo(a)pyrene.

Benzo(a)pyrene (BaP), an aryl hydrocarbon receptor (AhR) ligand present in cigarette smoke and car exhaust, is thought to have negative effects on male reproduction. We hypothesized that BaP damages sperm through AhR activation, phase I enzyme induction, DNA adduct formation, and increased germ cell apoptosis in the testis, and that resveratrol, a natural competitive inhibitor of the AhR found in some red wines, could prevent the adverse effects of BaP on sperm. Male Balb C mice were injected subcutaneously (s.c.) for 5 weeks with a range of BaP doses (0.5 mg/kg to 50 mg/kg). Live sperm were obtained from the vas deferens, counted, and stained to measure annexin-V positive (apoptotic) cells. In a subsequent study, mice were injected for 5 weeks with corn oil (control), BaP (5 mg/kg/week), or BaP plus resveratrol (50 mg/kg/week) (n = 3 per group). Immunohistochemistry (IHC) was performed on testis sections for the determination of CYP1A1, BaP diol epoxide (BPDE) DNA adducts, and apoptosis and the results quantified by using the HSCORE, a semiquantitative scoring system. Our results demonstrated that sperm counts after 5 weeks were inversely correlated to BaP dosage. BaP (0.5 to 5 mg/week) positively correlated with sperm apoptosis while higher doses increased sperm necrosis. CYP1A1 protein was observed mainly in interstitial cells of some testis sections, but there was no significant induction by BaP. BPDE DNA adducts were induced in all components of the seminiferous tubules by BaP and suppressed by resveratrol: median HSCORE (interquartile range) control 61 (52-71.5); BaP 213 (192-248), P = 0.01 compared to control; BaP plus resveratrol 83 (70-90). BaP significantly increased apoptosis, mainly in spermatogonia: medain HSCORE (interquartile range) BaP 189 (161-223) versus control 83 (57-93), P < 0.01; and this effect was abrogated by resveratrol. Median HSCORE for BaP plus resveratrol was 112 (range 99-121). In summary, BaP caused increased sperm cell BPDE DNA adduct formation and apoptosis in the mouse. The natural AhR antagonist, resveratrol diminished BaP-induced DNA adducts and apoptosis in seminiferous tubules.

[Effect of PML and PML-RAR on the transactivation properties and subcellular localization of steroid hormone receptors]. / Effet de PML et PML-RAR sur les propriétés transactivatrices et la localisation sub-cellulaire des récepteurs stéroïdiens.

PML is a protein involved in the t (15, 17) translocation of promyelocytic leukemia and is mainly localized in nuclear bodies. Here we show that PML exerts a very powerful enhancing activity (up to 20-fold) on the transactivating properties of the progesterone receptor (PR) and has a similar effect on several other steroid hormone receptors. There is probably a direct or indirect interaction between PR and PML since when the latter was expressed at high concentrations it shifted PR into the nuclear bodies. Use of deletion mutants showed that both activation functions (AF1 and AF2) of PR as well as the coiled coil and His-Cys rich domains of PML were required for transcriptional enhancement. The fusion protein PML-RAR, which is not localized in nuclear bodies, also enhanced the transactivating activity of PR but this effect was totally suppressed by the administration of retinoic acid. PML, which is ubiquitously expressed, may thus be involved in the transactivation properties of steroid hormone receptors. This mechanism may also play a role in the oncogenic properties of PML-RAR and in their suppression by the retinoic acid.

Uteroglobin: a model for the sutyd of progesterone action in mammals.

Uteroglobin, a progesterone-binding protein, is expressed in several organs, principally endometrium and lung, of the rabbit and other rodents. The phasic activation of the uteroglobin gene in the endometrium during early pregnancy is regulated by progesterone, which contrasts with the constitutive, nonregulated expression of this gene in the lung. Thus, uteroglobin provides a useful model for the study of differential gene regulation by hormones as well as for the study of steroid-protein interactions.

Control of biosynthesis and post-transcriptional modification of the progesterone receptor.

The rabbit progesterone receptor undergoes dual regulation at the level of transcription: positive by estrogens and negative by progestins. The two aspects of this regulation are mediated by a single intragenic estrogen-responsive element. Estrogen receptor binding to this element has been demonstrated but progestin down-regulation does not proceed through DNA binding of the progesterone receptor. This result suggests some kind of protein-protein interaction--direct or indirect--between estrogen and progesterone receptors. At the post-transcriptional level, the progesterone receptor undergoes a hormone-dependent hyperphosphorylation of serine residues localized in the N-terminal region. Studies of progesterone receptor mutants have determined the influence of the different receptor domains in the phosphorylation mechanism. A casein kinase copurifies with the receptor. The role of this phosphorylation remains to be determined.

The aryl hydrocarbon receptor and its xenobiotic ligands: a fundamental trigger for cardiovascular diseases.

This review reconsiders a major cause of cardiovascular diseases, tobacco smoking, as the activation of the Aryl hydrocarbon Receptor (AhR), also known as the dioxin receptor, by aryl hydrocarbons from the tar fraction of tobacco in various organs of the cardiovascular domain. This concept sheds new light on well-known albeit controversial epidemiological concepts such as the Mediterranean diet and the French paradox. We also review the discovery that resveratrol, a natural AhR antagonist, may be of interest in the prevention and treatment of cardiovascular diseases.

Characterization of phenformin and metabolites in plasma.

A colorimetric assay of biguanides was adapted for small volumes of plasma and its specificity was improved. This method is based on the reaction of guanidine groups with alpha-naphtol-diacetyl. Interference of endogenous guanidine derivatives and of the water-soluble metabolites of phenformin can be excluded by the extraction procedure. Counting of plasma fractions from 14C-phenformin-injected rats and thin-layer chromatography, before and after treatment with beta-glucuronidase, were also performed: the results suggest that after adequate extraction of plasma, the colorimetric assay measures specifically the biologically active phenformin. Results of this assay in plasma from biguanide-induced lactic acidotic patients and rats are given and compared with controls : results are consistent with the hypothesis of an accumulation of biologically active biguanide in such cases.

Factors modifying equilibrium between activated and non-activated forms of steroid-receptor complexes.

Steroid-receptor complexes formed in concentrated cytosol at low temperature, low ionic strength and neutral pH are unable to bind to nuclei. Various procedures are known to promote their 'activation'. In the present work it is shown that an increase in temperature only enhances the rate of the reaction whereas no change in the equilibrium between activated and non-activated complexes is observed. On the contrary an increase in ionic strength or pH, as well as a removal of a low-molecular-weight inhibitor, not only accelerate the reaction but also increase the concentration of activated complexes at equilibrium. Using two steroids differing 3-fold in their affinity for the receptor, no difference was seen in the effect of the bound steroid on receptor activation. When combining various activation procedures it was observed that they acted independently of each other and additively. In all cases they retained their property of either modifying only the rate of the reaction or both its rate and equilibrium. Using changes in pH, it was also possible to induce shifts in the equilibrium between activated and non-activated complexes. After activation at pH 6.5, a first equilibrium was attained. When the pH was increased to 8 the equilibrium was displaced towards higher concentrations of activated complexes. A lowering of the pH resulted in a reversal of steroid-receptor complexes from the activated to the non-activated state. To clearly establish that this was not due to irreversible damage of the receptor, which would render it unable to bind to nuclei, it was shown that the complexes which had reverted to the non-activated state were still susceptible to activation. Regulatory events may thus exist which, for a given level of hormone and receptor, modulate the concentration of activated steroid-receptor complexes.

Interaction of small nuclear ribonucleoproteins with simian virus 40 in CV-1 cells: is U2 snRNA involved in regulating replication?

The small nuclear RNAs (snRNAs) in African Green Monkey kidney cells (CV-1 cells) were examined by polyacrylamide gel electrophoresis. Methodology was developed to improve their extraction from enriched fractions. Cellular fractionation studies and subsequent analysis of these RNAs indicate that they are tightly associated with chromatin. Treatment of cells with alpha-amanitin totally suppressed transcription of U1, U2, U4, U5, and partially suppressed transcription of U6, suggesting that these snRNAs are transcribed by RNA polymerase II. Upon infection of the cells by simian virus 40 (SV40), overall transcription of these and other cellular RNAs was stimulated. Gel filtration and formaldehyde crosslinking studies indicated that the ribonucleoproteins (snRNPs) containing snRNAs are associated with the viral minichromosome. Nucleotide sequence comparisons show extensive sequence complementarity between the 5' end of U2 RNA, the replication origin of SV40, and a prokaryotic RNA (RNA I) that is involved in control of plasmid replication. The clustered homologies between these RNAs and the association of snRNAs with the SV40 chromosome suggest that snRNAs may be evolutionarily related to small RNAs from plasmids and are consistent with an hypothesis that U2 RNA may be involved in DNA replication.