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Original Studies |
Departments of Pharmacology (P.D.R., J.G.S.), and Comparative Medicine (J.G.S.), University of South Alabama, Mobile, Alabama 36688; and Department of Pharmacology (Y.R., D.F.S.), University of Nebraska Medical Center, Omaha, Nebraska 68198
Address all correspondence and requests for reprints to: Jonathan G. Scammell, Ph.D., Department of Pharmacology, MSB 3130, University of South Alabama, Mobile, Alabama 36688. E-mail: jscammel{at}jaguar1.usouthal.edu
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Abstract |
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Introduction |
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Our recent studies have focused on the mechanisms of glucocorticoid insensitivity in species of neotropical primates. Squirrel monkeys, cotton-top tamarins, and owl monkeys have markedly elevated plasma cortisol levels, but they show no signs of glucocorticoid excess (5, 6). They have been proposed as models for the in vivo study of glucocorticoid sensitivity (3, 5). Chrousos et al. (5) demonstrated a greater than 20-fold higher apparent dissociation constant for the GR in squirrel monkey mononuclear leukocytes than in human cells, suggesting that a decreased binding affinity contributes to glucocorticoid resistance. To determine whether changes in the primary structure contribute to the decreased affinities of neotropical primate GRs, we cloned the GR from squirrel monkey liver and compared the sequence with receptor sequences obtained from cotton-top tamarin B95–8 cells and human lymphocytes (HL) (7). The ligand-binding domain of the squirrel monkey GR contained four amino acid differences from the human GR, all of which were present in the cotton-top tamarin receptor. We determined, however, that these substitutions are not the cause of the decreased binding affinity of the squirrel monkey GR. When squirrel monkey GRs were expressed in reticulocyte lysate, they exhibited high-affinity binding similar to that of human GR (7). We concluded that the low binding affinity of squirrel monkey GR is more likely a result of the expression of cytosolic factors that affect GR function. A similar conclusion was reached by Brandon et al., who showed that cotton-top tamarin cells express a protein that affects GR binding (8). However, neither the nature of this factor nor the manner in which it affects GR binding was determined. We report here that the hsp90-associated immunophilin, FKBP51, is overexpressed in squirrel monkeys and is responsible for the low binding affinity of the squirrel monkey GR.
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Materials and Methods |
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Culture medium was obtained from Life Technologies (Grand Island, NY). FBS and horseradish peroxidase-labeled goat antirabbit and goat antimouse IgGs were from Hyclone Laboratories, Inc. (Logan, UT). [3H]Dexamethasone (82–86 Ci/mmol) was from Amersham Life Sciences (Arlington Heights, IL). hsp90 and hsp70 antibodies were purchased from StressGen Biotechnologies Corp. (Victoria, BC, Canada). Cyclophilin 40 (CyP-40), human GR (clone 59), and mouse GR (BuGR2) antibodies were from Affinity BioReagents, Inc. (Golden, CO). p23 and protein phosphatase 5 (PP5) antibodies were gifts from Drs. David Toft (Mayo Clinic, Rochester, MN) and Michael Chinkers (University of South Alabama, Mobile, AL), respectively. Antibodies to human FKBP51 (Hi51), human FKBP52 (Hi52c), Hip, and Hop have been described previously (9, 10, 11).
Cell cultures
B95–8, COS-7, COS-1, and L929 cells were obtained from American Type Culture Collection (Rockville, MD). HL cells, transformed with Epstein-Barr virus (EBV), were kindly provided by Dr. David Brandon (Oregon Health Sciences University, Portland, OR). Squirrel monkey and owl monkey B-lymphoblast (SML and OML, respectively) cell lines were transformed with EBV (12, 13). HL, SML, OML, and B95–8 cells were grown in suspension cultures in RPMI 1640 media supplemented with 10% FBS, 4 mM glutamine, 50 U/mL penicillin G, and 0.05 mg/mL streptomycin. L929, COS-7, and COS-1 cell lines were grown in monolayers in DMEM supplemented with 10% FBS and antibiotics. Cells were grown at 37 C in a humidified atmosphere of 5% CO2-95% air.
Cytosol preparation
Cells were collected in culture media, washed with PBS, and resuspended in cold TEDGM [10 mM Tris (pH 7.4), 2 mM EDTA, 1 mM dithiothreitol, 10% glycerol, 10 mM sodium molybdate] or HEM [10 mM HEPES (pH 7.4), 2 mM EDTA, 20 mM sodium molybdate] and lysed by sonication (HL, SML, COS-7, and COS-1 cells) or repeated strokes in a glass dounce homogenizer (L929 cells). A soluble fraction (hereafter referred to as cytosol) was isolated by centrifugation at 100,000 x g for 1 h at 4 C. Protein concentrations were quantified by the method of Bradford (14). In cytosol mixing studies, cytosols from each cell line were adjusted to equal protein concentrations and mixed 1:1 for 2 h on ice.
Western blot analysis
Cytosolic proteins were dissolved in 2 x concentrated sample buffer, the proteins separated by SDS-PAGE, and transferred to nitrocellulose. The blots were blocked in TBS-T [20 mM Tris (pH 7.6), 137 mM NaCl, 0.1% Tween-20], containing 5% nonfat milk (blocking buffer), for 1 h at room temperature. Incubation with primary antibody (1:1000) was carried out at 4 C in blocking buffer overnight. After washing, blots were incubated with secondary antibody (1:10,000), developed using the Enhanced Chemiluminescent system (Amersham Life Sciences), and the resulting bands were quantified by densitometry.
GR binding analysis
Fifty-microliter aliquots of cytosol were added to a mix containing [3H]dexamethasone, in the absence and presence of 20 µM radioinert dexamethasone. After 4 h at 4 C, free dexamethasone was removed by the addition of dextran-coated charcoal, and the radioactivity in the supernatants was determined. Specific binding was determined by subtracting nonspecific counts from total counts. Data were analyzed by nonlinear regression analysis and visualized by the method of Scatchard (15) using GraphPad PRISM software (GraphPad Software, Inc., San Diego, CA).
Immunoadsorption
Immunoadsorption of hsp90 was performed as described (16). Twenty-five microliters of anti-hsp90 rat IgM were preadsorbed to antirat IgM-coupled Sepharose in 100 mM phosphate buffer, pH 8. After washing the Sepharose pellet in HEM buffer, 0.5-mL aliquots of cytosol (1 mg/mL) from HL or SML cells were added and mixed at 4 C for 2 h. Immunoadsorption of mouse GR was performed as described (17). Four micrograms of BuGR2 was preadsorbed to protein A-Sepharose in 100 mM phosphate buffer, pH 8. After washing in HEM buffer, 0.5-mL aliquots of L929 cytosol, mixed with either COS-7 or SML cell cytosol (4 mg/mL), were added and mixed at 4 C for 2 h. The pellets were washed with HEM buffer, boiled in sample buffer, and subjected to SDS-PAGE and Western blot analysis.
Construction of plasmids for transfection
A full-length squirrel monkey FKBP51 complementary DNA (cDNA) was obtained by RT-PCR of poly(A)+ RNA from SML using primers: 5'-CGA ATT CGA CAG GTT CTC TAC-3' and 5'-ACG GAT CCT GTT CTG TCC TGA-3', positions 129–143 and 1605–1592 in the human FKBP51 cDNA (18) flanked by EcoRI and BamHI sites (underlined sequences), respectively. This fragment was subcloned into pGEM-7Z for sequencing and later subcloned into the pCI-neo Mammalian Expression Vector (Promega Corp., Madison, WI) with EcoRI and SmaI. The human FKBP51 cDNA was generated by PCR using human FKBP51 cDNA in pSPUTK as the template and primers: 5'-TAT ATA GCT AGC ACC ATG GAG ACT GAT GAA GG-3' and 5'-A TAT GAA TTC TCA TAC GTG GCC CTC AGG-3', positions 154–170 and 1527–1510 flanked by NheI and EcoRI sites (underlined sequences), respectively. The 1.4-kilobase fragment containing the full open reading frame of FKBP51 was sequenced and subcloned into the pCI-neo vector. Twenty-four hours before transfection, COS-1 cells were trypsinized and plated in 100-mm dishes at 5 x 106 cells per dish. The cells were transfected with 4 µg/dish of either pCI-neo (control) or pCI-neo containing squirrel monkey or human FKBP51 cDNA (COSsm51 and COSh51, respectively) using the LipofectAMINE PLUS reagent (Life Technologies). After 24 h, the medium was replaced, and the cells were incubated for 24 h before cytosol was isolated.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Previous results suggested that the low binding affinity of GRs in
squirrel monkey cells is not a result of substitutions in the receptor
protein but might be because of the expression of cytosolic factors
that affect GR binding (7). To demonstrate that squirrel monkey cells
express soluble inhibitory factors, GR binding was determined in
cytosol from mouse L929 cells, mixed 1:1 with cytosol from either SML
or COS-7 cells. GR binding is undetectable in SML cytosol under these
conditions. COS-7 and COS-1 cells, which were used in a later study,
express very low levels of GR and were used as a control. The addition
of SML cytosol to L929 cell cytosol resulted in an 11-fold decrease in
binding affinity without a change in receptor number (Fig. 1
), suggesting that SML cells contain a
soluble factor that inhibits GR binding. Similar results were obtained
with mixes of cytosols of SML and HL, another source of high-affinity
GR (data not shown).
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, top
panel), including hsp90, hsp70, Hop, and p23 (17). Altered
expression of hsp90 or expression of mutant forms of hsp90 is
associated with reduced responsiveness to glucocorticoid (19, 20, 21). As a
first step in identifying inhibitory factors in squirrel monkey cells,
we compared the levels of components of the GR heterocomplex in SML and
HL cells. The apparent molecular weights and levels of expression of
most of the chaperone proteins were the same in HL and SML cells
(i.e. hsp90, hsp70, Hop, CyP-40, and PP5; Fig. 2, lower panel). Hip was 2-fold higher in SML than in HL cells,
but the greatest changes were seen in the immunophilins FKBP51
and FKBP52. Compared with HL cell cytosol, FKBP51 was 13-fold higher in
SML cell cytosol, whereas FKBP52 was 42% of that in HL cells.
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). The high levels of FKBP51 in SML and
B95–8 cells, relative to the modest elevation in OML, are consistent
with the level of glucocorticoid resistance of these animals in
vivo (5). We next investigated whether the elevated cytosolic
levels of FKBP51 were reflected in an abundance of FKBP51 associated
with hsp90. Hsp90 complexes were immunoadsorbed from SML or HL cell
cytosols, and the immunophilin components of the complex were analyzed
by Western blot. No consistent differences were observed in the amounts
of PP5, CyP-40, or FKBP52 precipitated in a complex with hsp90 from SML
and HL (Fig. 4). However, FKBP51 was very
prominent in hsp90-complexes in SML cells, compared with that in HL
cells.
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We next asked: does the inhibitory effect of squirrel monkey
cytosol on GR binding activity require the incorporation of FKBP51 into
the GR heterocomplex? We mixed cytosol from SML cells with cytosol from
mouse L929 cells and determined the level of incorporation of FKBP51
into the mouse GR heterocomplex. L929 cells were used because they
express GR, which can be selectively immunoadsorbed with the mouse GR
antibody, BuGR2 (22). No antibodies suitable for quantitative
immunoprecipitation of human GR are available. Western blots of GR
heterocomplexes showed that FKBP51 was incorporated into the complexes
when cytosol from SML cells was mixed with L929 cytosol (Fig. 5). No FKBP51 was detected in GR
heterocomplexes when L929 cytosol was mixed with cytosol from COS-7
cells (used as a control). COS-7 cells, and COS-1 cells used below,
express FKBP52 at levels comparable with HL and L929 cells but very
little, if any, FKBP51.
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). There was no effect of
either FK506 or rapamycin on the binding in mixes of L929 and COS-7
cell cytosols. Second, we demonstrated that the inhibitory effect of
SML cytosol on GR binding is reproduced using cytosol from COS-1 cells
expressing FKBP51. Cytosols from COS-1 cells expressing either squirrel
monkey or human FKBP51 decreased GR binding by at least 40% (Fig. 7). The greater effect of cytosol from
cells expressing squirrel monkey FKBP51 is even more striking, because
the level of expression of FKBP51 in these cells was less than 50% of
that in cells expressing human FKBP51 (data not shown). Thus, squirrel
monkey FKBP51 may preferentially incorporate into the GR heterocomplex
or, once incorporated, it may have greater influence on GR binding than
human FKBP51.
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To determine whether the sensitivity of the effect of SML cytosol
to FK506 resulted from failure of FKBP51 to incorporate into the
GR heterocomplex or from inhibition of PPIase activity, the level
of FKBP51 in the GR heterocomplex was analyzed in the presence of
FK506. L929 cell cytosol was mixed 1:1 with cytosol from either SML or
COS-7 cells, in the presence or absence of FK506, and the relative
amount of FKBP51 was determined in the GR heterocomplex. We found a
marked reduction in the amount of FKBP51 in GR heterocomplexes in mixes
of L929 and SML cell cytosols when FK506 was present (Fig. 8, compare lanes 3 and 4).
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Discussion |
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FKBP51 and FKBP52 are FK506-binding immunophilins that, with PP5 and Hop, possess tetratricopeptide repeat motifs and compete for a common binding site on hsp90 (reviewed in Ref. 4). The make-up of hsp90 complexes is determined by the levels and affinities of these proteins for the hsp90 binding site (24, 25). Once formed, the hsp90 complexes associate with steroid receptors in a manner which is also determined by the receptor. The work of Silverstein et al. (26) and our results demonstrate that high-affinity GR heterocomplexes in L929 cells contain either FKBP52 or PP5, but little FKBP51. However, L929 cells, like HL and COS cells, express very little FKBP51. When FKBP51 is present during receptor assembly, at a level comparable with other immunophilins, it is preferentially incorporated into GR complexes (27). However, once incorporated into the complex, the role of any of these proteins in regulating receptor function has been less clear. FKBP52 has PPIase activity, a property shared with FKBP51 and CyP-40 (23); but inhibition of this activity with FK506 had no effect on GR binding activity in either L929 cell cytosol (28) or in mixes of cytosols from L929 and COS-7 cells. Recent studies have demonstrated not only that Cpr7 (the homolog of CyP-40) is necessary for full GR activity in Saccharomyces cerevisiae (29) but also that Cpr7 lacking the PPIase domain supports hsp90-mediated GR activity as efficiently as wild-type (30). Thus, the role of the immunophilins in enhancing steroid receptor function may be related more to protein-protein interaction through the tetratricopeptide repeat domain than to intrinsic PPIase activity.
But here we show that the incorporation of another immunophilin, FKBP51, into the GR heterocomplex has an inhibitory effect on GR function. The mechanism by which FKBP51 effects low GR binding is not known. Nor do we know why squirrel monkey FKBP51, which shares 94% amino acid identity with human FKBP51 (data not shown), is even more effective in reducing GR binding than the human protein. Our results suggest that FKBP51 acts in a competitive manner. Although the way that FKBP51 interacts with the GR is not fully understood, studies with the avian progesterone receptor (PR) have shown that hormone displaces FKBP51, but not FKBP52, from the receptor complex, under conditions that stabilize the receptor heterocomplex (31, 32). Furthermore, FKBP51 cannot be adsorbed from cell extracts using an FK506-affinity matrix, unless it is dissociated from the hsp90 heterocomplex; whereas FK506 binds to FKBP52, whether in the heterocomplex or not (33). FK506 will also bind to FKBP52 in GR complexes (34). Thus, FKBP51 and FKBP52 associate differently with steroid receptors; FKBP51 may have more direct (FK506-inaccessible) interactions with the hormone binding pocket than FKBP52 and may sterically hinder hormone binding. The intrinsic PPIase activity of FKBP51 likely does not contribute to its effect on GR or other steroid receptor function. First, a mutant of human FKBP51 that lacks PPIase activity was shown to maintain its preferential association with PR heterocomplexes (27). And we showed that the sensitivity of the inhibitory effect of FKBP51 to FK506 results from inhibiting its association with the GR heterocomplex.
The incorporation of FKBP51 into the GR heterocomplex may affect the GR indirectly, by causing the loss of functionally important immunophilins. We have yet to quantify changes in GR-associated FKBP52 and PP5 in mixes of cytosols from SML and L929 cells. Alternatively, FKBP51 may recruit accessory proteins, into the GR heterocomplex, that affect GR function. Chambraud et al. (35) identified a protein, termed FAP48, that forms a complex with FKBP52. This association is blocked by FK506 but not cyclosporin A. The role that FAP48 plays in immunophilin function is not known, nor is it known whether this or other cochaperones associate with FKBP51. If the activity of an FKBP51-associated factor is responsible for the inhibitory effect of FKBP51, the expression of such a cochaperone is not limited to squirrel monkey cells, because cytosol from COS cells expressing FKBP51 was also inhibitory.
Based on the data presented here, FKBP51 overexpression and perhaps specific FKBP51 sequence differences seem to be the major causes of glucocorticoid resistance in neotropical primates. Although two alternatively spliced forms of the GR (GRArg452 and GRß) have been described in cotton-top tamarin B95–8 cells (36, 3), we do not favor the theory that they contribute significantly to glucocorticoid resistance. First, we were unable to demonstrate the presence of the Arg452 isoform of the GR in either B95–8 cells or in squirrel monkey or owl monkey liver (7). Even if this isoform is expressed under certain conditions, it exhibits more than 50% of the transcriptional activity of wild-type GR (37, 38) and would have only a modest effect on glucocorticoid signaling. Second, although GRß was initially thought to inhibit transactivation by wild-type GR (39, 40), this effect has subsequently been shown to be possibly an artifact of the transfection protocol (41).
The finding that altered expression (and perhaps sequence) of FKBP51 in squirrel monkeys influences GR function, has a number of implications. The expression of FKBP51 messenger RNA is increased by glucocorticoids (13, 18). This regulation of FKBP51 levels may represent a previously unrecognized short-feedback loop in which the sensitivity of a tissue to glucocorticoids may be influenced by previous exposure to hormone. Changes in immunophilins may also affect the activity of other steroid hormone receptors in neotropical primates. For example, squirrel monkeys have markedly elevated levels of progesterone, apparently to compensate for receptor-mediated end-organ resistance (42). Though it is established that PRs are complexed with immunophilins (reviewed in 4), the role of differential expression of FKBP51 and FKBP52, in regulating hormone binding to PR in squirrel monkeys, has yet to be investigated.
The molecular events that led to the reciprocal changes in FKBP51 and FKBP52 expression in neotropical primates are unknown. Although these immunophilins may affect the sensitivity to glucocorticoids and progestins, other factors are likely responsible for the resistance to vitamin D observed in these species. Adams et al. demonstrated that a protein with homology to hsp70 acts as a competitive inhibitor of vitamin D in B95–8 cells (43). Thus, during the course of independent evolution on the South American subcontinent, presumably in response to different dietary and environmental stresses, survival and successful reproduction of several genera of neotropical primates was dependent on their abilities to alter the sensitivity to certain steroid hormones. In so doing, they have provided an intriguing experiment of nature, to gain insight into factors which influence steroid receptor activity.
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Acknowledgments |
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Footnotes |
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Received August 13, 1998.
Revised September 30, 1998.
Accepted October 16, 1998.
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L. C. Russell, S. R. Whitt, M.-S. Chen, and M. Chinkers Identification of Conserved Residues Required for the Binding of a Tetratricopeptide Repeat Domain to Heat Shock Protein 90 J. Biol. Chem., July 16, 1999; 274(29): 20060 - 20063. [Abstract] [Full Text] [PDF]
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A. J. Ramsey, L. C. Russell, S. R. Whitt, and M. Chinkers Overlapping Sites of Tetratricopeptide Repeat Protein Binding and Chaperone Activity in Heat Shock Protein 90 J. Biol. Chem., June 2, 2000; 275(23): 17857 - 17862. [Abstract] [Full Text] [PDF]
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H. Chen, B. Hu, E. A. Allegretto, and J. S. Adams The Vitamin D Response Element-binding Protein. A NOVEL DOMINANT-NEGATIVE REGULATOR OF VITAMIN D-DIRECTED TRANSACTIVATION J. Biol. Chem., November 3, 2000; 275(45): 35557 - 35564. [Abstract] [Full Text] [PDF]
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R. Kumar, N. Grammatikakis, and M. Chinkers Regulation of the Atrial Natriuretic Peptide Receptor by Heat Shock Protein 90 Complexes J. Biol. Chem., March 30, 2001; 276(14): 11371 - 11375. [Abstract] [Full Text] [PDF]
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