Reconstitution of the N-terminal transcription activation function of human mineralocorticoid receptor in a defective human glucocorticoid receptor.

N-terminal sequences involved in transcription activation by the human mineralocorticoid receptor (hMR) have yet to be defined. We have addressed this issue and generated overlapping internal deletion mutants hMRDelta59-162, hMRDelta59-247, hMRDelta59-328, hMRDelta162-247, hMRDelta247-328, hMRDelta247-382, and hMRDelta328-382 with intact DNA-binding and hormone-binding domains. A second set of mutant receptors with unique BglII sites was generated to facilitate the isolations of fragments. Immunodetection with anti-hMR peptide antibodies and hormone-binding assays showed that the mutations did not affect the expression of the receptors or ability to bind aldosterone. Distribution of aldosterone binding activity of wild type and deletion mutants expressed in HeLa cells was predominantly nuclear. Furthermore, deletion of sequences between 59 and 390 did not affect DNA binding activity. Transfection studies with HeLa cells revealed a region around residue 247 that was crucial for normal receptor function. Deletion of amino acids 59-162 did not affect the transcriptional activity of the hMR. However, deletion of sequences 247-382 and 328-382 markedly decreased the transcription activation function. The induction of the reporter gene by the chimera hGRDelta71-262/hMR328-382 was 2-fold higher than with the wild type hGR, but 200-fold when compared with hGRDelta71-262, indicating that the AF-1 domain is located between positions 328 and 382 in the hMR.

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.

Cysteines 638 and 665 in the hormone binding domain of human glucocorticoid receptor define the specificity to glucocorticoids.

To understand the function of cysteines, we have substituted cysteines 638, 643, and 665 by serine in the hormone-binding domain (HBD) of the human glucocorticoid receptor (hGR). In hormone-binding assays using [3H]dexamethasone, hGR C643S and hGR C665S exhibited wild type receptor Kd of 2.5 nM and hGR C665SM666L displayed a Kd of 3.7 nM, while hGR C638S exhibited a Kd of 162 pM, a 15-fold higher affinity. The affinity of hGR C638S for RU486 was 10-fold higher, and the mutants C643S and C665S bound RU486 with a 10-fold lower affinity when compared to wild type GR. While C665S bound aldosterone with very high relative affinity, the double mutant C665SM666L failed to bind aldosterone. The expression of wild type, mutant, and truncated hGRs in vitro showed an identical level of expression of the cloned receptors. Similar levels of expression of the receptors were observed in transfected cells, both by immunoprecipitation and by Western blotting. Transcription activation of the chimeric reporter gene mouse mammary tumor virus-chloramphenicol acetyltransferase (MMTV-CAT) with hGR C638S was 4-fold higher than the level observed with wild type hGR in the presence of dexamethasone. In the presence of RU486, hGR C638S induced MMTV-CAT 25-fold compared to the highest levels observed with wild type hGR and RU486. Even though the hGR C665S stimulated transcription with aldosterone, hGR C665SM666L did not. DNA-receptor interaction analyses by gel mobility shift assay demonstrated that the increased transactivation potential of hGR C638S was due to its intense interaction with DNA. These findings suggest that C638 and C665 are involved in maintaining specificity to glucocorticoids.

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.

Hormone binding domain of human glucocorticoid receptor. Enhancement of transactivation function by substitution mutants M565R and A573Q.

To determine the importance of specific amino acids in the hormone-binding domain of the human glucocorticoid receptor (hGR), we have generated mutants M565R, G567A, and A573Q. In hormone binding assays using [3H]cortisol, half-maximal saturation of dexamethasone competition was achieved at 10 pM with hGR M565R and hGRA573Q compared to 10 nM with wild type hGR. Similar results were obtained in competition assays with [3H]dexamethasone and the glucocorticoid antagonist RU 486. The substitution mutants M565R and A573Q demonstrated a higher relative affinity for aldosterone compared to the wild type hGR. In CV-1 cells cotransfected with the mutant receptors, hGR M565R and A573Q showed a remarkable 6-fold elevated transcription activation of the chimeric reporter gene mouse mammary tumor virus-chloramphenicol acetyl-transferase (MMTV-CAT). The mutant hGR G567A failed to bind agonists and antagonists efficiently. Immunoblotting with hGR specific antibodies of the whole cell extract from transfected CV-1 cells revealed that these differences in hormone binding and transcription activation were not due to the decreased levels of expression. These data support that idea that Gly567 in the hGR hormone-binding domain lies in a region crucial to ligand binding and transactivation function.

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.

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 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.

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.

Interaction of antiandrogen-androgen receptor complexes with DNA and transcription activation.

Human androgen receptor (hAR) is a ligand-dependent transcription factor that mediates androgen-induced actions on target tissues. Transfection studies in receptor deficient monkey kidney cells CV-1 in culture examine the ability of dihydrotestosterone (DHT) and of the antiandrogens hydroxylflutamide (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). CV-1 cells cotransfected with wild type hAR (hAR1-910) and MMTV-CAT, were treated with varying concentrations of DHT. DHT stimulated transcription activation of MMTV-CAT gene in a dose-dependent fashion. Cypro.A though only partially, also stimulated the transcription activation of MMTV-CAT. In the absence of steroids, HO-FLU induced the MMTV-CAT transcription in transfectants only 4% above the basal level. RU 23908-10 revealed the least agonistic activity at concentrations between 10 nM and 1 microM. Despite this, 100- to 1000-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 [3H]DHT for AR binding with hAR expressed in CV-1 cells. Western blot analysis using AR antipeptide antibodies raised in rabbits revealed the presence of two AR protein bands in extracts prepared from hAR1-910 transfected CV-1 cells. Incubation of labeled synthetic palindromic androgen responsive element (ARE) with the hAR containing CV-1 cell extracts followed by u.v. cross-linking demonstrated the specificity of AR-DNA interaction. Analysis by gel mobility shift assays showed that the interaction of AR-antiandrogen complexes with labeled ARE was specific.