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Endocrinology Vol. 144, No. 6 2380-2387
Copyright © 2003 by The Endocrine Society

The FK506-Binding Immunophilin FKBP51 Is Transcriptionally Regulated by Progestin and Attenuates Progestin Responsiveness

Tina R. Hubler, Wesley B. Denny, Donna L. Valentine, Joyce Cheung-Flynn, David F. Smith and Jonathan G. Scammell

Departments of Pharmacology (T.R.H., W.B.D., D.L.V., J.G.S.) and Comparative Medicine (J.G.S.), University of South Alabama College of Medicine, Mobile, Alabama 36688; and Department of Biochemistry and Molecular Biology (J.C.-F., D.F.S.), Mayo Clinic, Scottsdale, Arizona 85259

Address all correspondence and requests for reprints to: Jonathan G. Scammell, Ph.D., Department of Pharmacology, MSB 3370, University of South Alabama, Mobile, Alabama 36688. E-mail: jscammel{at}jaguar1.usouthal.edu.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
FKBP51 and FKBP52 are large molecular weight FK506-binding immunophilins that have diverse biochemical functions. Best studied is the role that they play as components of steroid hormone receptors. Differential display and gene array screens have identified FKBP51 as a progestin-inducible gene. Here we demonstrate progestin enhancement of FKBP51 mRNA and protein in T-47D cells. FKBP51 mRNA and protein levels were increased 3-fold by 20 nM R5020. Induction of FKBP51 mRNA was unaffected by 1 µg/ml cycloheximide but was blocked by the progestin receptor (PR) antagonist RU486 (1 µM). Reporter plasmids containing 3.4 kb and 427 bp of 5'-flanking sequences of the human FKBP51 protein gene (FKBP5) exhibited regulation by progestin in T-47D cells. A construct containing 19 bp of upstream sequence demonstrated diminished basal activity and no stimulation by R5020. To test whether elevated FKBP51 affects progestin responsiveness, HepG2 cells were transfected with human FKBP51, PR, and mouse mammary tumor virus-luciferase plasmids, and treated with R5020 (0.03–10 nM). Expression of FKBP51 increased the EC50 for PR transactivation by 3.2-fold. Expression of FKBP51 from squirrel monkey, a New World primate with naturally occurring progestin resistance, increased the EC50 more dramatically (11.7-fold vs. control). Expression of FKBP51 bearing a double-point mutation in the tetratricopeptide repeat domain had no effect on PR transactivation. These results suggest that increased expression of FKBP51 by progestin may attenuate progestin responsiveness in hormone-conditioned cells. Furthermore, overexpression of FKBP51 in the squirrel monkey may be a contributing cause of progesterone resistance in this species.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IMMUNOPHILINS ARE PROTEINS so named because they bind the immunosuppressant drugs FK506 and cyclosporin A. The large molecular weight immunophilins are the FK506-binding FKBP51 and FKBP52, and the cyclosporin A-binding cyclophilin-40 (Cyp40; Refs.1, 2, 3). They are distinct from the small molecular weight immunophilins such as FKBP12 and cyclophilin A (CypA), which are the targets for the immunosuppressive activities of FK506 and cyclosporin A, respectively (4). The large molecular weight immunophilins exhibit sequence homology and similar structural organization. The N-terminal domains of these proteins possess peptidyl-prolyl isomerase activity, which converts prolyl peptide bonds within target proteins from cis to trans-proline (5). The C-terminal domains contain three tetratricopeptide repeats (TPR) that are highly degenerate 34 amino acid repeats involved in protein-protein interactions.

The large molecular weight immunophilins are capable of a number of diverse interactions and functions. The most well studied is their association with native steroid receptor complexes. The immunophilins and the phosphatase PP5 compete through their TPR domains for a TPR acceptor site on hsp90 in steroid receptor complexes where they may modulate receptor activity (6, 7). FKBP51 is a normal component of the unliganded high affinity glucocorticoid receptor (GR) complex (8, 9), although the presence of squirrel monkey FKBP51 in the GR complex lowers binding affinity and is the likely cause of glucocorticoid resistance in this species (10, 11). FKBP52 is found associated with the hormone-activated receptor and through interaction with the microtubule motor dynein is thought to facilitate the translocation of steroid receptors along the cytoskeleton to the nucleus (12, 13).

In roles unrelated to steroid receptor function, the FKBP52 homolog, PAS-1, in Arabidopsis thaliana plays a critical role in growth and development (14), whereas in Drosophila the FKBP52 homolog regulates the TRPL calcium entry channel (15). Additional functions in higher organisms suggest that the immunophilins may be neuroprotective (16), may act as transcriptional repressors (17, 18, 19), and may mediate the cardiotrophic effect of cardiotrophin-1 (20).

The functional diversity of the immunophilins has prompted interest in their differential expression and regulation. Initially thought to be quite restricted in their distribution, it is now clear that these proteins are expressed in a wide range of tissues (21, 22). However, evidence is mounting that these proteins are differentially regulated. The expression of Cyp40 mRNA is stimulated by heat stress and estrogen in MCF-7 breast cancer cells (23, 24), and recently the human Cyp40 promoter was isolated and characterized (25). FKBP52 mRNA expression also appears to be regulated by heat stress and estrogen (23, 24, 26). On the other hand, FKBP51 appears to be regulated by other steroid hormones. We and others have demonstrated that glucocorticoids increase FKBP51 mRNA in human B-lymphoblast, C7TK.4 human leukemia, and MCF-7 breast cancer cells (27, 28, 29). Gene array analysis of androgen-dependent CWR22 prostate tumors identified FKBP51 as an androgen-responsive gene (30, 31), a finding later confirmed in LNCaP cells (32, 33). FKBP51 may also be regulated by progesterone. Using differential display, Kester et al. (29) identified 57 clones, which were regulated by progestin in estrogen-primed T-47D breast cancer cells, of which FKBP51 was one. Very recently, gene array analysis identified FKBP51 as one of 94 genes regulated by progesterone in T-47D cells (34). However, except for the observed changes in the levels of FKBP51 and FKBP52 transcripts, little is known of how hormones regulate these immunophilins. Neither the human FKBP51 gene (FKBP5) nor FKBP52 gene (FKBP4) promoters have been isolated, and there are few studies on the functional consequences of increased expression of these proteins.

The goal of the work presented here is to confirm and expand the initial observations of FKBP5 regulation by progestin. We report that 1) progestin directly regulates the expression of FKBP51 mRNA and protein; 2) the activity of the FKBP5 promoter is stimulated by progestin; 3) overexpression of human FKBP51 reduces progestin responsiveness in HepG2 cells transfected with the progestin receptor (PR); and 4) squirrel monkey FKBP51 exhibits an inhibitory activity 3-fold greater than human FKBP51. These results suggest that FKBP51 may serve as part of a short feedback loop to attenuate progestin responses in hormone-conditioned cells and may contribute to naturally occurring progestin resistance in squirrel monkeys.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
Culture medium was obtained from Life Technologies, Inc. (Grand Island, NY). Defined and charcoal-dextran-treated fetal bovine serum (FBS) was purchased from Hyclone Laboratories, Inc. (Logan, UT). Gelding horse serum (GHS) was purchased form Central Biomedia, Inc. (Irwin, MI). R5020 was purchased from NEN Life Science Products (Boston, MA). RU486, mifepristone, was provided by Roussel-Uclaf (Romainville, France). The antibodies to human FKBP51 and Cyp40 were purchased from Affinity BioReagents, Inc. (Golden, CO), the antibody to hsp90 was from StressGen Biotechnologies Corp. (Victoria, British Columbia, Canada), and the ANTI-FLAG M2 antibody was from Stratagene (La Jolla, CA). Protease inhibitor cocktail, cycloheximide, and 5,6-dichlorobenzimidazole riboside were purchased from Sigma (St. Louis, MO). Human genomic DNA was obtained from CLONTECH Laboratories, Inc. (Palo Alto, CA). Construction of the expression plasmid containing human FKBP51 and FKBP52 complementary cDNAs have been described previously (10, 22). The plasmid BKCMV-hPR-B was a gift from Dr. D. P. McDonnell (Duke University Medical Center, Durham, NC). The mouse mammary tumor virus (MMTV) promoter-luciferase reporter vector was provided by Dr. R.M. Evans (The Salk Institute, La Jolla, CA).

Cell cultures
T-47D human breast cancer and HepG2 hepatoma cells were obtained from American Type Culture Collection (Manassas, VA). T-47D cells were grown in monolayer cultures in RPMI 1640 medium supplemented with 10% FBS, 50 U/ml penicillin G, and 0.05 mg/ml streptomycin, whereas HepG2 cells were grown in monolayers in DMEM with 10% FBS and antibiotics. Cells were grown at 37 C in a humidified atmosphere of 5% CO2-95% air. When cells were treated with hormone, they were first transferred to medium containing 10% GHS, which has low levels of progesterone, either 24 h before hormone treatment (protein and mRNA induction) or 4 h before hormone treatment (reporter gene assays).

Northern and Western blot analysis
For Northern blot analysis, cells were washed with cold PBS and total RNA was isolated using RNA STAT-60 (Tel-Test, Inc., Friendswood, TX). RNA was size fractionated on 1.4% agarose-2.2 M formaldehyde gels, transferred to Nytran (Schleicher \|[amp ]\| Schuell, Inc., Keene, NH), and cross-linked to the membrane in a GS Gene-Linker UV Chamber (Bio-Rad Laboratories, Inc., Richmond, CA). RNA was also prepared for slot blot analysis which was performed as described previously (27). The blots were hybridized with biotin-labeled FKBP51, FKBP52, or CypA cDNAs using the Random Primer Biotin Labeling Kit (NEN Life Science Products), and the signals were visualized using the CDP-Star Nucleic Acid Chemiluminescence Reagent (NEN Life Science Products).

For Western blot analysis, cells were washed with cold PBS and scraped into HEM buffer [10 mM HEPES (pH 7.4), 2 mM EDTA, 20 mM sodium molybdate] plus protease inhibitor, and lysed by sonication. The lysates were centrifuged at 13,500 x g for 15 min at 4 C and the supernatants collected. Protein concentrations were quantified by the method of Bradford (35). Aliquots of soluble extract were dissolved in 2x concentrated sample buffer, separated by SDS-PAGE, and transferred to nitrocellulose. Incubation with primary antibodies was carried out at 4 C overnight. After washing, blots were incubated with secondary antibody and developed using a WesternBreeze Immunodetection Kit (Invitrogen Corp., Carlsbad, CA) and quantified by densitometry.

FKBP5 promoter analysis
The organization of FKBP5 was previously reported with the exception of the position of the gene promoter (36). BLAST 2 alignment (37) of human FKBP51 cDNA sequence (GenBank accession no. NM_004117.2) with a working draft of a segment from human chromosome 6 (NT_007592.9) now indicate that exon 1 and exon 2 of FKBP5 are separated by approximately 46 kb of DNA. A fragment of 5'-flanking DNA was amplified by PCR using primers 5'-GCT ACG CGT ACC TGG AGA AAG TTG-3' and 5'-GAC AGA TCT ACT CTC CGC CTT GCC-3' corresponding to sequence approximately 3.4 kb upstream of exon 1 and positions 104–90 of the human FKBP51 cDNA (28) within exon 1, flanked by MluI and BglII sites (underlined sequences), respectively. The amplified fragment (-3436 to +104) was subcloned into the pGL3-Basic vector (Promega Corp., Madison, WI). Plasmid constructions were confirmed by restriction enzyme analysis and DNA sequencing of the 5' and 3' ends. A 531-bp FKBP5 promoter construct was generated using primers 5'-GAT ACG CGT CTA ACC TGG CCA G-3' and 5'-GAC AGA TCT ACT CTC CGC CTT GCC-3' (MluI and BglII sites, underlined) and the 3.5-kb promoter construct as the template. This promoter fragment (-427 to +104) was subcloned into pGL3-Basic, sequenced across both strands, and the sequence deposited in GenBank (accession no. AY114286). A 123-bp FKBP5 promoter construct was generated by introducing an MluI site into the proximal promoter of the 3.5-kb promoter construct using the primers 5'-GCG GGA CG A CGC GTG GCG CTG C-3' and 5'-GCA GCG CC A CGC GTC GTC CCG C-3' (MluI site underlined) and the QuikChange Site-Directed Mutagenesis Kit (Stratagene). The construct was digested with MluI, religated using the newly engineered MluI site, and the resulting construct (-19 to +104) sequenced.

A fragment of 5'-flanking DNA upstream of the mouse FKBP5 promoter was isolated from a bacterial artificial chromosome clone by Genome Systems Inc. (St. Louis, MO) using the following primers: 5'-ATG ACT ACT GAT GAG G-3' and 5'-CCT CTG TCT TTC TTC G-3', corresponding to positions 148–163 and 242–227 of the mouse FKBP51 cDNA (38), respectively. The genomic insert was digested and positive fragments identified with primers 5'-GCT GGG CGG GCT GAG CGG CGC GCG CGG-3' and 5'-CGG AGA GAC GCG GAG CGA GGG ACG C-3', corresponding to positions 49–75 and 79–103 in the 5'-untranslated region of the mouse FKBP51 cDNA (38). Two positive DNA fragments containing exon 1 and 5'-flanking DNA of the mouse FKBP5 gene were sequenced across both strands and the sequence deposited in GenBank (accession no. AY113161).

For analysis of hormonal regulation of FKBP5 promoter constructs, cells were plated in six-well tissue culture dishes at 3 x 105 cells per well and incubated overnight. Cells were transfected with 2 µg DNA/well of either the promoterless pGL3-Basic vector, or pGL3-Basic vector containing various FKBP51 promoter fragments, using Superfect Transfection Reagent (QIAGEN, Valencia, CA) in complete medium for 2 h. The medium was then replaced with fresh medium supplemented with 10% GHS, followed 4 h later by the addition of R5020 (20 nM). After 24 h, cells were lysed and assayed for luciferase activity as described (39).

Transactivation of PR in HepG2 cells
HepG2 cells were transfected with plasmids expressing PR and human FKBP51 using a modification of the method of Bodwell et al. (40). Cells were washed in PBS and suspended in electroporation buffer [10 mM piperazine-N,N'-bis(2-ethanesulfonic acid) (pH 7.4), 137 mM NaCl, 2.7 mM KCl, 2.7 mM EGTA, 5.6 mM glucose, 1 mM ATP] at a density of 6–7 x 107 cells/ml on ice for 15 min. Aliquots (300 µl) of cell suspension were electroporated in 50 µl TE buffer [10 mM Tris (pH 7.5), 1 mM EDTA] containing 10 µg BKCMV-hPR-B plasmid, 20 µg MMTV-luciferase reporter vector, and 40–80 µg of either empty pCI-neo vector, pCI-neo containing FLAG-tagged human FKBP51 or squirrel monkey FKBP51 cDNA, (h51FLAG-pCI-neo and sm51FLAG-pCI-neo, respectively), or h51-pCI-neo cDNA containing a double mutation in the TPR domain (h51mFLAG-pCI-neo). h51FLAG-pCI-neo was constructed by site-directed mutagenesis using the QuikChange Site-Directed Mutagenesis Kit (Stratagene), h51-pCI-neo (10) as the template, and primers CCT GAG GGC CAC GTC GAC TAC AAG GAC GAT GAT GAC AAG TAA GTC GAC CCG GGC GGC CG (sense) and CGG CCG CCC GGG TCG ACT TAC TTG TCA TCA TCG TCC TTG TAG TCG ACG TGG CCC TCA GG (antisense), corresponding to positions 1510–1551 in the human FKBP51 cDNA sequence (GenBank accession no. NM_004117, nucleotide mutations underlined). sm51FLAG-pCI-neo was constructed in the same manner using sm51-pCI-neo (10) as template and primers CCT GAA GGC CAC GTA GAC TAC AAG GAC GAC GAC GAC AAA TAG CTA ATG AAC TCG GCC (sense) and GGC CGA GTT CAT TAG CTA TTT GTC GTC GTC GTC CTT GTA GTC TAC GTG GCC TTC AGG (antisense), corresponding to positions 1377–1433 in the squirrel monkey FKBP51 cDNA sequence (GenBank accession no. AF140759, nucleotide mutations underlined). h51mFLAG-pCI-neo (Lys352 ->Ala, Arg356 -> Ala) was constructed as above using h51FLAG-pCI-neo as template and primers CTG GAC AGT GCC AAT GAG GCG GGC CTG TAT AGG AGA GGG (sense) and CCC TCT CCT ATA CAG GCC CGC CTC ATT GGC ACT GTC CAG (antisense), followed by GTG CCA ATG AGG CAG GCC TGT ATG CGA GAG GGG AAG CCC (sense) and GGG CTT CCC CTC TCG CAT ACA GGC CTG CCT CAT TGG CAC (antisense), nucleotide mutations underlined. The mutagenesis primers used to construct h51mFLAG-pCI-neo correspond to positions 1056–1094 and 1063–1101, respectively, in the squirrel monkey cDNA (AF140759), which codes for the same amino acids as human FKBP51 in this region. Plasmid mutations were confirmed by sequencing across both strands.

Electroporation was carried out using a Gene Pulser II (Bio-Rad Laboratories, Inc., Hercules, CA) with the Pulse Trac system activated to deliver a pulse of 180 V. Capacitance (1.4–1.8 mF) was adjusted to give a time constant of approximately 110 msec. Electroporated cells were diluted in culture medium and plated in 60-mm dishes at a density of 3 x 106 cells/dish. After 8 h, the medium was replaced with DMEM containing charcoal/dextran-treated FBS, and 12 h later the cells were treated with R5020 (0.03–10 nM). After 24 h, cell extracts were prepared for determination of luciferase activity and Western blot of FLAG-tagged FKBP51 using the M2 antibody.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Regulation of FKBP51 by progestin in T-47D cells
Differential display and gene array screens identified FKBP51 as one of more than fifty genes that are regulated by progestin in T-47D human breast cancer cells (29, 34). FKBP51 transcript levels were widely affected (3- to 100-fold) by progestin in the analyses. The initial goal of this study was to confirm these findings and to determine whether progestin regulation of FKBP51 is a direct, transcriptional event. First, we confirmed the results of the original screening analysis by rigorously quantifying the regulation of FKBP51 mRNA and protein levels by the progesterone analog R5020. The steady-state levels of FKBP51 mRNA were determined in T-47D cells after treatment with 20 nM R5020 for 4 h. The levels of mRNA of the structurally related immunophilin FKBP52 and CypA were also measured as an internal control. Figure 1Go shows the level of FKBP51 mRNA, represented by a band of approximately 3.8 kb, was increased by R5020, whereas neither the level of FKBP52 (2.3 kb) nor CypA (0.8 kb) mRNA was affected by the treatment. Slot blot analysis was used in subsequent experiments to accommodate the large number of samples and to ensure linearity of the chemiluminescent signal. We demonstrated that maximum induction (3- to 4-fold) of FKBP51 mRNA with R5020 occurred after 8 h and the levels of FKBP51 mRNA remained elevated for at least 24 h (Fig. 2AGo). The effect of R5020 was dose-dependent with an EC50 of approximately 1 nM (Fig. 2BGo). As dramatic changes in immunophilin mRNA levels have been observed without changes in corresponding protein expression (23), it was important to document that the changes in FKBP51 mRNA levels were reflected in alterations in FKBP51 protein. The effect of R5020 on the levels of FKBP51, FKBP52, and Cyp40 in T-47D cells was determined by Western blot (Fig. 3AGo). R5020 increased the levels of FKBP51 by approximately 3-fold but had no effect on the levels of FKBP52 and Cyp40 (Fig. 3BGo).



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  		Figure 1. The effect of R5020 on the steady-state level of FKBP51 and FKBP52 mRNA in human breast cancer T-47D cells. Total RNA was isolated from control T-47D cells or T-47D cells treated with 20 nM R5020 for 4 h. Northern blots were hybridized with biotin-labeled FKBP51, FKBP52, or CypA cDNAs, and the signals were visualized by an enhanced chemiluminescence detection system.

 


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  		Figure 2. Time course (A) and dose-response curve (B) of induction of FKBP51 mRNA by R5020 in T-47D cells. T-47D cells were treated with (A) 20 nM R5020 for periods up to 24 h or (B) R5020 (0.1 nM to 1 µM) for 24 h. Total RNA was isolated, immobilized by slot blot, and hybridized with biotin-labeled FKBP51 or CypA cDNAs. The hybridization intensity of FKBP51 mRNA was normalized to the intensity of CypA mRNA. Each point represents the mean ± SEM of three separate experiments.

 


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  		Figure 3. Western blots of FKBP51, FKBP52, Cyp40, and hsp90 in T-47D cells. A, T-47D cells were treated with 20 nM R5020 and 1 µM RU486 either alone or together for 24 h and cell extracts were prepared for Western blot analysis. B, Quantitation of the changes in immunophilins, which were analyzed by densitometry and corrected for the level of hsp90 used as an internal control. Each bar represents the mean ± SEM of three separate experiments.

 
To further define the effect of R5020 on the induction of FKBP51 in T-47D cells we tested the effect of the PR antagonist RU486 (41). Coincubation of T-47D cells with RU486 blocked the induction of FKBP51 by R5020 [Fig. 3AGo; R5020 (20 nM), 257 ± 13%; RU486 (1 µM), 97 ± 9%; R5020 + RU486, 103 ± 3% of control, respectively; n = 3], verifying that the effect of R5020 on FKBP51 is mediated by PR. The next experiments were to show that progestin causes a direct activation of FKBP51 transcription. One-half of the cells were treated with 1 µg/ml cycloheximide for 2 h, sufficient to inhibit the incorporation of [35S]methionine into protein by 90%, and the levels of FKBP51 mRNA were determined after R5020 treatment by slot blot analysis. The presence of cycloheximide had no effect on the accumulation of FKBP51 mRNA in R5020-treated T-47D cells (Fig. 4AGo) consistent with the initial screens showing that a progesterone-mediated increase in FKBP51 mRNA persisted in the presence of cycloheximide (29, 34). On the other hand, the induction of FKBP51 mRNA by R5020 was completely inhibited by preincubation of cells with the transcription inhibitor 5,6-dichlorobenzimidazole riboside (Fig. 4BGo). These results suggest that the effect of R5020 on FKBP51 mRNA in T-47D cells is a direct, PR-mediated transcriptional event that does not involve the synthesis of additional proteins.



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  		Figure 4. The effect of inhibitors of protein synthesis (A) or transcription (B) on the induction of FKBP51 mRNA by R5020 in T-47D cells. T-47D cells were treated for 2 h with (A) 1 µg/ml cycloheximide (CHX) or (B) 20 µg/ml 5,6-dichlorobenzimidazole riboside (DRB) before one half of the plates were treated with 20 nM R5020 for 4 h. Total RNA was collected, immobilized by slot blot, and hybridized with biotin-labeled FKBP51 or CypA cDNAs. The hybridization intensity of FKBP51 mRNA was normalized to the intensity of CypA mRNA. Each point represents the mean ± SEM of three separate experiments.

 
Isolation and regulation of the human FKBP51 promoter
To further substantiate the role of transcription in induction of FKBP51 by progestin, a 3.4-kb fragment of the FKBP5 promoter was amplified from human genomic DNA by PCR and subcloned into a luciferase reporter vector (p3540Luc, -3436 to +104). Approximately 500 bp of the 3'-end of this fragment was sequenced (Fig. 5Go) and the sequence deposited in GenBank with accession no. AY114286. The sequence of the complete DNA fragment is available as part of a working draft of chromosome 6 (NT_007592.9). The promoter sequence of FKBP5 was aligned with that of mouse FKBP5 (AY113161), which we sequenced from a bacterial artificial chromosome clone. The GC-rich nature of this region prevented an accurate determination of the transcription start site which we inferred from the most extensive mRNA sequence deposited in GenBank (NM_004117.2). The proximal regions of the human and mouse FKBP5 promoters share significant homology and putative transcription factor binding sites are highly conserved (Fig. 5Go). Sequences more than 250 bp upstream of the transcription start site show greater divergence between the human and mouse promoters. Additional reporter gene constructs were made which contained 427 bp and 19 bp of FKBP5 promoter sequence (p531Luc, -427 to +104, and p123Luc, -19 to +104, respectively; Fig. 5Go).



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  		Figure 5. Nucleotide sequences of the human and mouse FKBP51 gene promoters. Nucleotides are numbered from the transcription start site of the human FKBP51 gene (+1). Bases conserved between human and mouse sequences are indicated with dashed lines. Gaps are indicated by asterisks. Putative Sp1 and Oct-1 binding sites are underlined. The 5'-ends (-427 and -19) of two reporter gene constructs are shown. Sequences of the human and mouse gene promoters have been deposited in GenBank with accession nos. AY114286 and AY113161, respectively.

 
The responsiveness of each of the three human FKBP51 promoter constructs to progestin was tested in T-47D cells. Cells were transfected with p3540Luc, p531Luc, or p123Luc and incubated in the presence and absence of 20 nM R5020 for 24 h. The cells were collected and assayed for luciferase activity. In each experiment, a parallel set of cells was transfected with the promoterless pGL3-Basic vector to account for experiment-to-experiment differences in transfection efficiency, and the data in each experiment were expressed as fold induction over the activity of pGL3-Basic as described previously (39). The construct p3540Luc exhibited a constitutive activity in T-47D cells which was 19-fold higher than that of pGL3-Basic (Fig. 6Go). The activity of p3540Luc was stimulated 2.5-fold when the cells were treated with R5020. Deletion of 3 kb of 5'-flanking sequence had little effect on promoter activity as p531Luc exhibited an activity 24-fold higher than the promoterless vector, and the activity increased 2.7-fold when the cells were treated with R5020. However, deletion of an additional 0.4 kb (p123Luc) not only reduced the basal activity to 30% of p531Luc, but also eliminated the response to R5020. Thus, we have isolated the human FKBP51 gene promoter and shown that it exhibits progestin responsiveness in T-47D cells. These results indicate that a change in the rate of transcription of FKBP5 contributes to the induction of FKBP51 mRNA and protein levels by progestin.



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  		Figure 6. Regulation of the FKBP51 promoter by progestin. T-47D cells were transfected with the promoter-luciferase reporter gene constructs p3539Luc (-3436 to +104), p531Luc (-427 to +104), p123Luc (-19 to +104), or the promoterless pGL3-Basic vector and incubated in the absence or presence of 20 nM R5020. After 24 h, the cells were collected for assay of luciferase activity, and the data were expressed as fold induction over that achieved with pGL3-Basic. Each bar represents the mean ± SEM of four separate experiments.

 
Effect of overexpression of FKBP51 on progestin responsiveness in HepG2 cells
Having demonstrated here that FKBP51 levels are increased by progestin and having shown previously that elevated FKBP51 decreases glucocorticoid responsiveness (10, 11), the question arose whether elevated FKBP51 affects progestin responsiveness. To answer this question, we determined the effect of increased expression of FKBP51 on progestin responsiveness in human liver HepG2 cells transfected with the PR. HepG2 cells, which normally express relatively low levels of FKBP51, are an excellent model system to study the regulation of progestin activity (42, 43). We found that the R5020 dose-response curve for transactivation of the PR was shifted 3-fold to the right in HepG2 cells transfected with human FKBP51 compared with the response in control cells transfected with empty vector (Fig. 7AGo). The EC50 for R5020 was 0.23 ± 0.08 nM in control HepG2 cells and 0.75 ± 0.05 nM in cells transfected with FKBP51. We compared the inhibitory activity of human FKBP51 with that of FKBP51 from squirrel monkey, a New World primate with elevated FKBP51 and naturally occurring progesterone resistance (10, 36, 44). Squirrel monkey FKBP51 increased the EC50 for R5020 almost 12-fold (EC50 2.7 ± 0.4 nM) being 3.7-fold more potent than human FKBP51. We also determined whether interaction with hsp90 is important for the inhibitory activity of FKBP51. The large molecular weight immunophilins, including FKBP51, contain a TPR domain that mediates interaction with hsp90 within the PR heterocomplex (45). We tested the activity of a mutant FKBP51 (h51m) in which basic residues within the TPR critical for immunophilin binding to hsp90 (46) were converted to alanine (Lys352 -> Ala, Arg356 -> Ala). When expressed in HepG2 cells, h51m had no inhibitory activity on the activation of the reporter gene by R5020 (Fig. 7AGo), suggesting that FKBP51 interaction with hsp90 mediates the inhibitory effect of the immunophilin. The levels of expression of the FKBPs were similar in each of the experiments (Fig. 7BGo).



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  		Figure 7. The effect of expression of FKBP51 on transactivation of PR by R5020 in HepG2 cells. A, HepG2 cells were electroporated with BKCMV-hPR-B, MMTV-luciferase, and empty pCI-neo (CTL), or pCI-neo containing either hFKBP51 (h51), squirrel monkey FKBP51 (sm51), or human FKBP51 TPR mutant (h51m) cDNA and treated with the indicated concentrations of R5020. After 24 h, the cells were collected for assay of luciferase activity. Each point represents the mean ± SEM of three separate experiments. –{bullet}–, Control; –{square}–, human FKBP51; –{blacksquare}–, squirrel monkey FKBP51; –{circ}–, human FKBP51 TPR mutant; B, Western blot of FKBP51 and hsp90 in cytosol from HepG2 cells expressing h51, sm51, or h51m demonstrating similar expression of FKBP51 and equal loading of the lanes.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
These results indicate that transcription of the immunophilin FKBP51 is directly regulated by progestin. We have also demonstrated that increased expression of FKBP51 leads to an attenuated responsiveness to progestin. Furthermore, we propose that increased expression of FKBP51 in squirrel monkeys contributes to progesterone resistance in this primate species. These conclusions are based on the following findings: 1) induction of FKBP51 by progestin was independent of protein synthesis but is blocked by the PR antagonist RU486; 2) a human FKBP51 promoter-reporter construct containing approximately 0.4 kb of upstream sequence exhibited progestin responsiveness; 3) transfection of HepG2 cells with a human FKBP51 cDNA expression plasmid shifted the progestin dose-response curve to the right; and 4) squirrel monkey FKBP51 is almost 4-fold more potent than human FKBP51 in reducing progestin responsiveness.

Potential roles for FK506-binding proteins in the regulation of PR activity have been suggested by previous studies. In 1994, Tai and colleagues (47) reported that FK506 potentiated PR-mediated transcription in yeast. However, it was later demonstrated that the potentiation is a consequence of inhibition of an FK506-sensitive steroid efflux mechanism (48). More recently, Le Bihan and colleagues (49) showed that FK506 inhibits progestin-induced transcription in T-47D cells. FK506 has similar inhibitory effects in transactivation studies involving glucocorticoid-induced transcription in T-47D cells (49) and aldosterone-mediated transcription in RC.SV3 kidney tubular cells (50), suggesting that the effect of FK506 is not specific to a single receptor type. The mechanism of action of FK506 in this respect remains elusive, but the sum of these data suggests that it does not appear to be related to large molecular weight immunophilins or to a change in receptor binding. On the other hand, we show here that overexpression of the FK506-binding protein FKBP51 leads to decreased responsiveness to progestin. The effect of FKBP51 is likely mediated by interaction with hsp90 within the PR complex as expression of mutant FKBP51, altered to abrogate binding to hsp90, had no inhibitory activity. These results suggest that, through PR-mediated induction of FKBP51, prolonged elevation of progesterone may modulate subsequent response to progesterone, thereby constituting a short negative feedback loop.

This physiological mechanism appears to be naturally exaggerated in squirrel monkeys. Squirrel monkeys are New World primates that have naturally occurring progesterone resistance (44). To compensate for a receptor-mediated decrease in sensitivity to progesterone, circulating progesterone levels are 10- to 20-fold higher in squirrel monkeys than in Old World primates including man. The density of PR was lower in uterine tissue from estrogen-treated squirrel monkeys compared with tissue from similarly treated cynomolgus monkeys (44, 51), suggesting that progestin resistance results simply from low expression of PR. However, these findings are confounded by the blunted in vivo response of squirrel monkeys to estrogen (51). Our results suggest that expression of FKBP51 in squirrel monkeys contributes to end-organ resistance to progestin. Not only do squirrel monkeys express high levels of FKBP51 (10, 36) but, relative to human FKBP51, which is itself preferred over FKBP52 and Cyp40 in PR complexes (8), squirrel monkey FKBP51 is incorporated with even greater affinity (1). GR signaling is modulated by FKBP51 in a similar manner, and overexpression of FKBP51 is a contributing cause of glucocorticoid resistance in squirrel monkeys (10, 11). However, we do not know if this comprises a common mechanism responsible for resistance to other steroid hormones in this species (51, 52).

FKBP51 mRNA is induced by glucocorticoid, progestin, and androgen. Until now, the FKBP51 promoter had not been isolated, and studies to analyze regulation of the gene at the transcriptional level were not possible. We showed an approximately 3-fold induction of the human FKBP51 promoter activity in T-47D cells transfected with a FKBP51 promoter-reporter fusion gene and treated with progestin. This level of induction was similar to the increase in FKBP51 mRNA we observed with R5020 but was significantly lower than the 96-fold induction by progestin previously reported in T-47D cells (29). The differences in the findings are not likely a result of priming cells with estrogen because we found that pretreatment of the cells with 1 nM E2 for 2 d had no effect on the level of induction (data not shown). Recently, Richer and colleagues (34) also reported a more modest induction of FKBP51 mRNA in T-47D cells by progesterone. We isolated a DNA fragment containing 427 bp of human FKBP51 gene promoter sequence, which was responsive to progestin stimulation. This fragment was as responsive as a FKBP51 promoter-reporter construct containing 3.4 kb of upstream sequence. Deletion of all but 19 bp of the 5'-sequence of this fragment eliminated progestin responsiveness suggesting that progestin-response elements (PRE) are contained within this region. We have yet to identify the specific cis-acting elements that constitute the PRE within the human FKBP51 promoter. There are no classic palindromic PRE consensus sequences (53), suggesting that progestin transactivation of FKBP51 promoter activity is mediated by a nonclassical mechanism. In this regard, we identified several Sp1 sites in the proximal promoters of the human and mouse FKBP51 genes. PR-dependent induction of several genes is thought to occur through interaction with Sp1 sites (54, 55).

In summary, our work confirms that FKBP51 is regulated by progestin in T-47D cells. We have isolated the human FKBP51 gene promoter and demonstrated that the regulation by progestin is at the transcriptional level. We also showed that increased expression of FKBP51 blunts progestin responsiveness in HepG2 cells and that squirrel monkey FKBP51 is more potent than human FKBP51. Thus, FKBP51 may serve as part of a short feedback loop regulating progestin responsiveness in hormone-conditioned cells and in squirrel monkeys may contribute to progestin resistance.


   Footnotes
 
This work was supported by Grants 13200 and 01254 from the National Center for Research Resources (to J.G.S.) and Grant DK-48218 from the NIH (to D.F.S.). T.R.H. was supported by a predoctoral fellowship from the American Heart Association, Southeast Affiliate.

Abbreviations: Cyp40, Cyclophilin-40; CypA, cyclophilin A; FBS, fetal bovine serum; GHS, gelding horse serum; GR, glucocorticoid receptor; MMTV, mouse mammary tumor virus; PR, progestin receptor; PRE, progestin-response element; TPR, tetratricopeptide repeat.

Received January 17, 2003.

Accepted for publication February 25, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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