Biomedical subjects

N Pagé

Publications and source records attributed to N Pagé.

At least 19 recordsLinked to original sources

PubMed2004

Mechanical strain enhances proteoglycan message in fibroblasts from asthmatic subjects.

BACKGROUND: Remodelling of the asthmatic airway includes increased deposition of proteoglycan (PG) molecules. One of the stimuli driving airway remodelling may be excessive mechanical stimulation. OBJECTIVE: We hypothesized that fibroblasts from asthmatic patients would respond to excessive mechanical strain with up-regulation of message for PGs. METHODS: We obtained fibroblasts from asthmatic patients (AF) and normal volunteers (NF) using endobronchial biopsy. Cells were maintained in culture until the fifth passage and then grown on a flexible collagen-coated membrane. Using the Flexercell device, cells were then subjected to cyclic stretch at 30% amplitude at 1 Hz for 24 h. Control cells were unstrained. Total RNA was extracted from the cell layer and quantitative RT-PCR performed for decorin, lumican and versican mRNA. RESULTS: In unstrained cells, the expression of decorin mRNA was greater in AF than NF. With strain, NF showed increased expression of versican mRNA and AF showed increased expression of versican and decorin mRNA. The relative increase in versican mRNA expression with strain was greater in AF than NF. CONCLUSIONS: These data support the hypothesis that proteoglycan message is increased in asthmatic fibroblasts subject to mechanical strain. This finding has implications for the mechanisms governing airway wall remodelling in asthma.

Adult↗

PubMed2003

Regulation of procollagen I (alpha1) by interleukin-4 in human bronchial fibroblasts: a possible role in airway remodelling in asthma.

BACKGROUND: In bronchial mucosa, T cells are in close association with fibroblasts. This cell contact raises the possibility of cross-talk between the two cell types through cytokines, such as interleukin-4 (IL-4). OBJECTIVE: We postulated that IL-4 may modulate collagen synthesis and degradation in the fibroblasts of asthmatics. METHODS: Bronchial fibroblasts from asthmatics (BAF) and normal controls (BNF) were stimulated with IL-4. Procollagen I gene expression and protein production were measured by real-time PCR, RT-PCR, and radioimmunoassay. The effect of IL-4 on the regulation of procollagen I (alpha1) promoter was studied through transient cell transfections. The implication of Sp1 and AP-1 in regulating IL-4-induced procollagen I (alpha1) production was determined. The effect of IL-4 on metalloproteinase-2 (MMP-2) and tissue inhibitor of metalloproteinase-2 (TIMP-2) production and gene expression was evaluated. RESULTS: Following IL-4 stimulation, there was a significant increase in the expression of mRNA of procollagen I (alpha1) by human bronchial fibroblasts of asthmatics and controls. IL-4 has a dose-response effect on mRNA, with a maximal effect at 5 ng/mL, as determined by real-time PCR. The maximal increase in procollagen I (alpha1) was observed at 6 h after IL-4 stimulation in both BNF and BAF. BAFs have a greater increase in the procollagen I (alpha1)/beta2 microglobulin ratio after 6 h of IL-4 stimulation (4.1 x 10-2+/-0.03 to 20.8 x 10-2+/-0.1) compared with BNF (2.9 x 10-2+/-0.006 to 9.2 x 10-2+/-0.08) (P=0.001). In transient transfection experiments, IL-4 increased promoter activity by threefold in BAF and BNF. Sp1 was up-regulated after IL-4 stimulation and AP-1 was down-regulated as shown by electrophoretic mobility shift assay. IL-4 decreased MMP-2 protein and mRNA levels, and did not alter TIMP-2 production. CONCLUSIONS: IL-4 positively regulates procollagen I (alpha1) transcription by direct promoter activation and increases the TIMP-2/MMP-2 ratio, thereby supporting the profibrotic effect of this cytokine. Thus, this study emphasizes that IL-4 may be considered as a link between inflammation and collagen deposition observed in asthmatic airways.

Asthma↗

PubMed2003

Endothelial cells modulate eosinophil surface markers and mediator release.

Migration from blood to tissue modulates eosinophil function, possibly through interactions with endothelial cells. The effects of contact with and migration through endothelial cells on eosinophil expression of surface markers and release of leukotriene C4 were evaluated. A small proportion (2.6%) of eosinophils spontaneously migrated through endothelial cell monolayers. Activation of endothelial cells by interleukin (IL)-4 or IL-1beta slightly increased this migration (to 12.4%), which became much greater when a chemoattractant was placed in the lower chamber (84.3%). However, the chemotactic effect was downregulated by pretreating endothelial cells with interferon gamma (IFN-gamma; 63.1%). At baseline, 5% of eosinophils expressed CD69; this increased to 30.7% in culture on untreated endothelial cells and to 50.9% on IL-1beta-pretreated endothelial cells. This effect was mediated through intercellular adhesion molecule-1/CD11b interaction. Eosinophil migration through endothelial cells further increased CD69 expression to 63.9% and also increased CD35 expression from 83.3 to 91.3%. Upon stimulation, eosinophils that had migrated through endothelial cells produced more leukotriene C4 than control cells (872.4 and 103.9 pg x mL(-1), respectively). Endothelial cell pretreatment with IL-4 or IL-1beta further increased leukotriene C4 release (1,789.1 and 2,895.1 pg x mL(-1), respectively), whereas pretreatment with IFN-gamma decreased it (293.7 pg x mL(-1)). These data show that in vitro interactions with endothelial cells upregulate eosinophil membrane receptor expression and mediator release and that these effects are differently modulated by T-helper cell type 1 and 2 cytokines. These eosinophil modulations may play an important role in asthma pathogenesis.

Adjuvants, Immunologic↗

PubMed2001

Systematic genetic analysis with ordered arrays of yeast deletion mutants.

In Saccharomyces cerevisiae, more than 80% of the approximately 6200 predicted genes are nonessential, implying that the genome is buffered from the phenotypic consequences of genetic perturbation. To evaluate function, we developed a method for systematic construction of double mutants, termed synthetic genetic array (SGA) analysis, in which a query mutation is crossed to an array of approximately 4700 deletion mutants. Inviable double-mutant meiotic progeny identify functional relationships between genes. SGA analysis of genes with roles in cytoskeletal organization (BNI1, ARP2, ARC40, BIM1), DNA synthesis and repair (SGS1, RAD27), or uncharacterized functions (BBC1, NBP2) generated a network of 291 interactions among 204 genes. Systematic application of this approach should produce a global map of gene function.

Carrier Proteins↗

PubMed2001

Bronchial mucosa produced by tissue engineering: a new tool to study cellular interactions in asthma.

BACKGROUND: The use of fiberoptic bronchial biopsies has improved our understanding of the immunopathology of asthma. However, this approach offers a limited ability to perform mechanistic studies observing cell-cell and cell-matrix interactions, which are a key issue in the study of airway remodeling. Tissue engineering is a technique that combines the use of biology and engineering expertise to generate a limitless amount of tissue from small samples. This technology allows for the study of cell interactions under conditions as close as possible to the natural environment. OBJECTIVE: The aim of this study was to evaluate the feasibility of an engineered human bronchial mucosa as a model to study cellular interactions in asthma. METHODS: Human bronchial fibroblasts from normal and asthmatic donors were incorporated into collagen gel. Bronchial epithelial cells were seeded over this gel and then cultured in an air-liquid interface in the presence or the absence of T lymphocytes. Biopsy specimens from these engineered mucosa were taken for structural and ultrastructural analysis, and T lymphocytes were harvested and used to localize IL-5. RESULTS: Histologic analysis showed that engineered mucosa with normal bronchial cells presented a pseudostratified ciliated epithelium with the presence of mucus secretory cells. The electron microscopy analysis confirmed these histologic results. These features were comparable with those observed in normal bronchial tissues. However, in engineered mucosa from asthmatic subjects, the tissue structure was disorganized, particularly the epithelial cell arrangement. The percentage of IL-5(+) lymphocytes was significantly (P =.03) higher in engineered bronchial mucosa from asthmatic subjects (87% +/- 2%) compared with mucosa from normal volunteers (2% +/- 0.3%). CONCLUSION: Using tissue engineering, we produced an in vitro model of bronchial mucosa from normal and asthmatic subjects. These models could be a valuable tool to better understand key mechanisms involved in inflammation and airway repair.

Asthma↗

PubMed2001

IL-17 is increased in asthmatic airways and induces human bronchial fibroblasts to produce cytokines.

BACKGROUND: IL-17 is a cytokine that has been reported to be produced by T lymphocytes. In vitro, IL-17 activates fibro-blasts and macrophages for the secretion of GM-CSF, TNF-alpha, IL-1beta, and IL-6. A number of these cytokines are involved in the airway remodeling that is observed within the lungs of asthmatic individuals. OBJECTIVE: In this study, we investigated the expression of IL-17 in sputum and bronchoalveolar lavage specimens obtained from asthmatic subjects and from nonasthmatic control subjects. METHODS: IL-17 was detected through use of immunocytochemistry, in situ hybridization, and Western blot. Bronchial fibroblasts were stimulated with IL-17, and cytokine production and chemokine production were detected through use of ELISA and RT-PCR. RESULTS: Using immunocytochemistry, we demonstrated that the numbers of cells positive for IL-17 are significantly increased in sputum and bronchoalveolar lavage fluids of subjects with asthma in comparison with control subjects (P <.001 and P <.005, respectively). We demonstrated that in addition to T cells, eosinophils in sputum and bronchoalveolar lavage fluids expressed IL-17. Peripheral blood eosinophils were also positive for IL-17, and the level of IL-17 in eosinophils purified from peripheral blood was significantly higher in subjects with asthma than in controls (P <.01). To further investigate the mechanism of action of IL-17 in vivo, we examined the effect of this cytokine on fibroblasts isolated from bronchial biopsies of asthmatic and nonasthmatic subjects. IL-17 did enhance the production of pro-fibrotic cytokines (IL-6 and IL-11) by fibroblasts, and this was inhibited by dexamethasone. Similarly, IL-17 increased the level of other fibroblast-derived inflammatory mediators, such as the alpha-chemokines, IL-8, and growth-related oncogene-alpha. CONCLUSION: Our results, which demonstrate for the first time that eosinophils are a potential source of IL-17 within asthmatic airways, suggest that IL-17 might have the potential to amplify inflammatory responses through the release of proinflammatory mediators such as alpha-chemokines.

Adult↗

PubMed2001

Bud8p and Bud9p, proteins that may mark the sites for bipolar budding in yeast.

The bipolar budding pattern of a/alpha Saccharomyces cerevisiae cells appears to depend on persistent spatial markers in the cell cortex at the two poles of the cell. Previous analysis of mutants with specific defects in bipolar budding identified BUD8 and BUD9 as potentially encoding components of the markers at the poles distal and proximal to the birth scar, respectively. Further genetic analysis reported here supports this hypothesis. Mutants deleted for BUD8 or BUD9 grow normally but bud exclusively from the proximal and distal poles, respectively, and the double-mutant phenotype suggests that the bipolar budding pathway has been totally disabled. Moreover, overexpression of these genes can cause either an increased bias for budding at the distal (BUD8) or proximal (BUD9) pole or a randomization of bud position, depending on the level of expression. The structures and localizations of Bud8p and Bud9p are also consistent with their postulated roles as cortical markers. Both proteins appear to be integral membrane proteins of the plasma membrane, and they have very similar overall structures, with long N-terminal domains that are both N- and O-glycosylated followed by a pair of putative transmembrane domains surrounding a short hydrophilic domain that is presumably cytoplasmic. The putative transmembrane and cytoplasmic domains of the two proteins are very similar in sequence. When Bud8p and Bud9p were localized by immunofluorescence and tagging with GFP, each protein was found predominantly in the expected location, with Bud8p at presumptive bud sites, bud tips, and the distal poles of daughter cells and Bud9p at the necks of large-budded cells and the proximal poles of daughter cells. Bud8p localized approximately normally in several mutants in which daughter cells are competent to form their first buds at the distal pole, but it was not detected in a bni1 mutant, in which such distal-pole budding is lost. Surprisingly, Bud8p localization to the presumptive bud site and bud tip also depends on actin but is independent of the septins.

Actins↗

PubMed2000

Observations on topical ivermectin in the treatment of otoacariosis, cheyletiellosis, and toxocariosis in cats.

The purpose of this study was to observe the efficacy of a topical pour-on formulation of ivermectin in the treatment of otoacariosis, cheyletiellosis, and toxocariosis in cats. Forty-five cats were treated. All cats received 2 to 4 topical applications of ivermectin on the skin between the shoulder blades in a narrow strip, 14 days apart. This practical treatment was effective in 96% (23/24) of cases of feline otoacariosis and in 100% (20/20) of cats with toxocariosis. All cats with cheyletiellosis (16/16) received 4 treatments and had resolution of clinical signs, but one Cheyletiella egg could still be found 45 days after the last treatment. The viability of this egg could not be evaluated, but the cats were still free of clinical signs on follow-up 6 months later. The treatment was well tolerated in all the animals. A few cats developed a transient small alopecic area and mild scaling at the site of application of the drug.

Administration, Topical↗

PubMed1997

Topical (pour-on) ivermectin in the treatment of canine scabies.

The efficacy of a pour-on formulation of ivermectin at 500 micrograms/kg body weight applied on the dorsum on days 1 and 15 was evaluated in 90 dogs from a shelter, naturally infested with Sarcoptes scabiei. This very practical form of treatment was successful in eradicating scabies from this shelter.

Administration, Topical↗

PubMed1996

Expression of human glucocorticoid receptor gene and interaction of nuclear proteins with the transcriptional control element.

We have identified sequences responsible for the expression of the human glucocorticoid receptor gene (GR gene) using a set of 5' promoter deletion mutants in HeLa, human placenta, and human breast tumor (MCF-7) cells. The chimeric gene construct -892 5'-GAAGTGACACACTTC3' -878-CAT was sufficient for high level of expression in HeLa and placenta cells in culture. Deletion of palindromic sequences decreased levels of GR expression in these cells. By oligonucleotide-affinity chromatography with the palindromic glucocorticoid receptor enhancing factor-binding element (GREFE), we have isolated from human placenta nuclear extract two novel proteins glucocorticoid receptor enhancing factors 1 and 2 (GREF1 and GREF2), with apparent molecular masses of 80 and 62 kDa, respectively. These proteins, similar to the DNA-binding autoantigen Ku are, like Ku, heterodimers of polypeptide subunits p80 and p62, immunologically related to factors binding to proximal sequence element 1 in the promoter of small nuclear RNA (PSE1) and transferrin receptor enhancing factors. Both Ku80 and Ku70 polypeptides were present in high concentrations in human placenta and HeLa cells. In MCF-7 cells, however, only a high level of p62 was detected. While cotransfection of pcDNA-Ku80 with pHGR(-892 to -878)-CAT potentiated the expression of CAT, introduction of pcDNA-Ku70 did not affect the expression of CAT in transfected MCF-7 cells. UV cross-linking analysis showed that only GREF1 contacted DNA directly. Supershift assays with monoclonal antibodies Ab 111 (Ku80) or Ab N3H10 (Ku70) showed a direct interaction of GREF1 and GREF2 heterodimers with the palindrome. Partial peptide fingerprinting of GREF1 and GREF2 using alpha-chymotrypsin and immunoblotting with Ab 111 and Ab N3H10 confirmed their identities as Ku80 and Ku70, respectively.

Antibodies, Monoclonal↗

PubMed1996

Increased xylanase yield in Streptomyces lividans: dependence on number of ribosome-binding sites.

The Streptomyces lividans xylanase A (XlnA) signal peptide (sp) was replaced with the signal peptides of either mannanase A (ManA) or cellulase A (CelA), two enzymes secreted by S. lividans. Depending on the location of the ribosome binding sites (RBS) with respect to a potential initiation codon, the length of the putative sps of ManA and CelA is either 34 or 43 amino acids and 27 or 46 amino acids, respectively. The sequence encoding these sps were fused to the xylanase A gene (xlnA). Clones harboring the short sps of ManA and CelA produced as much xylanase as the clone with the control wild-type sp sequence of XlnA. In clones containing the long sps of ManA and CelA, the XlnA production was enhanced 1.5- and 2.5-fold, respectively. These XlnA yields are reduced by half and one third respectively when the internal initiation codons of the long sp sequences of ManA and CelA are mutated. Since these clones exhibited the same transcription levels, the results indicate that both RBSs are used concomitantly in S. lividans to increase the enzyme production at the translational level. However, when the short and long sps of ManA were fused to the long CelA sp sequence, giving constructs containing respectively 3 and 4 RBSs, a decrease in xylanase production was observed.

Amino Acid Sequence↗

PubMed1996

Effect of signal peptide alterations and replacement on export of xylanase A in Streptomyces lividans.

Starting from its translation initiation site, the Streptomyces lividans xylanase A signal peptide consists of 41 amino acids. This signal peptide was deleted and successively replaced with one of six signal peptides from other enzymes secreted by S. lividans and by a signal peptide from the outer membrane protein (LamB) of Escherichia coli. Deletion of the xylanase A signal peptide or modification of its cleavage site abolished secretion of the enzyme. Replacement with the signal peptides of either xylanase B, cellulase A, mannanase, or acetylxylan esterase produced equivalent amounts of xylanase A, while the signal peptides of cellulase B, xylanase C, and LamB secreted less enzyme than did the wild type. All the clones exhibited the same transcription levels, which indicated that the variations in xylanase production were due to the natures of the signal sequences.

Amino Acid Sequence↗

PubMed1996

Serum-free thyroxine concentrations, measured by chemiluminescence assay before and after thyrotropin administration in healthy dogs, hypothyroid dogs, and euthyroid dogs with dermathopathies.

The purpose of this study was to determine the usefulness of free thyroxine (FT4) measured by chemiluminescence in evaluating thyroid function in dogs. Total thyroxine (TT4) concentration measured by radioimmunoassay (RIA) and FT4 measured by chemiluminescence were evaluated in 30 healthy dogs, 60 euthyroid dogs with concurrent dermatopathies, and 30 hypothyroid dogs before and after intravenous stimulation with 1 or 2 IU of thyrotropin (TSH). Median basal TT4 and median TT4 concentrations at 4 h post-TSH administration were not significantly different (P < 0.0001) between healthy dogs and euthyroid dogs with dermatopathies, but were significantly higher than those in hypothyroid dogs. In healthy dogs, the median TT4 concentrations at 4 and 6 h post-TSH administration were not significantly different. Median basal FT4 and median FT4 concentrations at 4 h post-TSH administration in healthy dogs were significantly lower (P < 0.0001) than those in euthyroid dogs with dermatopathies, but significantly higher than the same parameters in hypothyroid dogs. There was a significant difference between the median FT4 concentrations at 4 h post-TSH administration and median basal FT4 concentrations for healthy dogs and euthyroid dogs with dermatopathies, but not for hypothyroid dogs. Lastly, in healthy dogs, median FT4 concentrations at 4 and 6 h post-TSH administration were not significantly different. Free thyroxine measured by chemiluminescence was highly correlated (P < 0.0001; Spearman r = 0.91) with FT4 measured by the reference method for free hormone analysis, namely, equilibrium dialysis, when sera from 56 dogs were used.

Animals↗

PubMed1994

Substitution of Cys-560 by Phe, Trp, Tyr, and Ser in the first zinc finger of human androgen receptor affects hormonal sensitivity and transcriptional activation.

We have established and characterized four human androgen receptor (AR) mutants, AR C560F, C560W, C560Y, C560S). To assess the functional significance of these substitutions, we compared the transcriptional activation, hormone binding affinity, receptor-DNA interaction, and the subcellular distribution of the hormone-receptor complexes. Binding studies showed that all mutants bound methyltrienolone (R1881) with wild type affinity (Kd = 0.5 nM). Transactivation efficiency, as compared to wild type AR, increased 150% with C560F and decreased to 70% with C560W and C560Y and to 40% with mutant C560S. Subcellular receptor distribution showed that 85% of C560F bound with hormone was extracted from the nuclear fraction and 15% in the cytosol. Gel mobility shift assays showed that C560F expressed in CV-1 cells bound to an androgen responsive element (ARE) with equal efficiency as the wild type human AR. The mutants C560W and C560Y demonstrated a lower ability to bind to ARE, whereas C560S showed a significantly lower ability to interact with ARE. We propose that the change in polarity introduced into the loop structure by C560S leads to a shorter period of contact between the mutant receptor and DNA resulting in decreased transcriptional activation levels.

Amino Acids↗

PubMed1994

Transcription activation of mouse mammary tumor virus-chloramphenicol acetyltransferase: a model to study the metabolism of cortisol.

The human 11 beta-hydroxysteroid dehydrogenase (h11 beta-HSD) inactivates the active corticosteroid cortisol to its inactive metabolite cortisone. We have developed transactivation analyses of the reporter chimeric gene mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) to study the catalytic activity of h11 beta-HSD introduced by cotransfection into receptor and 11 beta-HSD deficient CV-1 cells. Assay of 11 beta-HSD expressed in CV-1 cells by cotransfection showed that the catalyzed dehydrogenation of cortisol to cortisone was 2-fold higher in the presence of NADP. The reductase activity was dependent on the coenzyme NADPH. The addition of increasing concentrations of the inhibitor carbenoxolone (CBX) in the incubates blocked the enzyme activity in a dose dependent fashion. In CV-1 cells cotransfected with expression vectors of either human glucocorticoid (hGR1-777) or mineralocorticoid (hMR1-984) and the reporter plasmid MMTV-CAT, dexamethasone (DEX), aldosterone (ALDO), cortisol, and corticosterone induction of CAT activity was dose dependent. Cotransfection of CV-1 cells transfected with 10 micrograms of 11 beta-HSD expression vector reduced the transactivation of MMTV-CAT by hGR or hMR in the presence of either cortisol or corticosterone to basal values. The concomitant addition of 100 nM cortisone and 1 microM NADPH to these transfectants elevated CAT activity. These data show that transactivation analyses can be used to study the 11 beta-HSD-catalyzed regulation of corticosteroid levels, which triggers physiological processes and in certain cases provides an alternative to animal experimentation.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1994

Antiandrogens inhibit human androgen receptor-dependent gene transcription activation in the human prostate cancer cells LNCaP.

Human androgen receptor (hAR) is a ligand-dependent transcription factor that mediates androgen-induced actions on target tissues. Transfection studies in the human prostate cancer cell line LNCaP examine the ability of dihydrotestosterone (DHT), hydroxyflutamide (HO-FLU), cyproterone acetate (Cypro.A), and RU 23908-10 to stimulate or to inhibit the transcription activation of mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase (MMTV-CAT). DHT stimulated transcription activation of MMTV-CAT gene in LNCaP cells in a dose-dependent manner. HO-FLU, Cypro.A, and RU 23908-10, though only partially, also stimulated the transcription activation of MMTV-CAT. Despite this, 100- to 1,000-fold molar excess of all antiandrogens inhibited the agonistic activity of 10 nM DHT in this system. Receptor binding assays confirmed that HO-FLU, Cypro.A, and RU 23908-10 competed with DHT for AR binding in LNCaP cells. Western blot analysis using AR antipeptide antibodies raised in rabbits revealed the presence of two AR protein bands in LNCaP cells, following treatment with antiandrogens. Increasing doses of HO-FLU stimulated the expression of the 114-kDa AR by 2.5-fold, but did not affect the 108-kDa AR. Increasing doses of Cypro.A and RU 23908-10 decreased the levels of both the 114-kDa and the 108-kDa AR. Although the exact nature of 108-kDa and 114-kDa AR in LNCaP cells is still unknown, these data suggest that the regulatory actions of each individual antiandrogen on AR expression in LNCaP cells may be different.

Amino Acid Sequence↗

PubMed1994

11 beta-Hydroxysteroid dehydrogenase and tissue specificity of androgen action in human prostate cancer cell LNCaP.

Incubation of whole LNCaP cells in suspension with tritium labeled cortisol revealed two major and one minor radioactive product. Of the major products, one migrated with an Rf value identical to cortisol (Kendall's compound "F"), and the second migrated with an Rf value similar to nonradioactive cortisone (Kendall's compound "E"); the third minor product comigrated with 21-acetylated cortisol. The conversion of cortisol to cortisone was linear with respect to cell number, and conversion reached a plateau after 120 min of incubation at 37 degrees C. One half of the cortisol was converted to cortisone within 2 h of incubation at 37 degrees C. This conversion was nicotine amide dinucleotide (NAD) dependent. Low levels of transcription activation by cortisol were documented in LNCaP cells transfected with glucocorticoid and androgen responsive mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase chimeric gene (MMTV-CAT). Hormone binding assay and transactivation analysis revealed the presence of a functional mineralocorticoid receptor in LNCaP cells. Treatment of transfectants with F in the presence of carbenoxolone, a potent inhibitor of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD), resulted in a two orders of magnitude increase in measurable CAT activity. The addition of the reduced form of nicotine amide dinucleotide (NADH) in the presence of 10(-7) M E stimulated measurable CAT activity in LNCaP cells. In conferring aldosterone specificity in mineralocorticoid target tissues, 11 beta-HSD may have an important role as "gate keeper" in allowing a specific androgen response in hormone responsive LNCaP prostate cancer cells.

11-beta-Hydroxysteroid Dehydrogenases↗

PubMed1994

11 beta-hydroxysteroid dehydrogenase activity in human lung cells and transcription regulation by glucocorticoids.

Selectivity to aldosterone (Aldo) in mineralocorticoid target tissues has been suggested to be due to the activity of 11 beta-hydroxysteroid dehydrogenase (11 beta-HSD). This enzyme inactivates the endogenous glucocorticoid cortisol, thus permitting the unhindered access of Aldo to the mineralocorticoid receptor. The 11 beta-HSD activity was measured by the conversion of cortisol to cortisone and vice versa. Concomitant treatment of the cells with either cortisone or cortisol in the presence of the glycyrrhetinic acid derivative carbenoxolone (CBX) blocked both activities of 11 beta-HSD. Dexamethasone and Aldo activated the transcription of transiently transfected mouse mammary tumor virus-bacterial chloramphenicol acetyltransferase chimeric gene in LU-19 cells. The transcription activation by cortisol was synergized by concomitant treatment of the transfectants with CBX. Transactivation with Aldo was inhibited by spironolactone. The enzyme 11 beta-HSD in LU-19 cells is similar to the cloned liver isoform and catalyzes both reduction and dehydrogenation.

11-beta-Hydroxysteroid Dehydrogenases↗