Development of a Panel of Monoclonal Antibodies against the Mineralocorticoid Receptor
http://www.100md.com
《内分泌学杂志》
Endocrinology, G.V. (Sonny) Montgomery VA Medical Center (C.E.G.-S., E.P.G.-S.) and University of Mississippi Medical Center (C.E.G.-S., A.F.d.R., D.G.R., J.E., M.P.W., E.P.G.-S.), Jackson, Mississippi 39216
Research Mississippi, Inc. (M.T.G.-S.), Jackson, Mississippi 39216
Abstract
Mineralocorticoid receptors (MR) bind both mineralocorticoids and glucocorticoids. They are expressed in multiple tissues and mediate diverse functions. Less is known about MR regulation and function compared with other major steroid receptors, although its importance has become increasingly apparent. A significant obstacle to such studies has been the dearth of specific high-affinity MR antibodies. We have produced monoclonal antibodies against 10 different peptide conjugates, six from the N terminus (A/B domain) and four from the C terminus (steroid binding domain), with the anticipation that their individual affinities for the MR would differ depending upon its conformation, which in turn, is dependent upon the location of the receptor within the cell and the proteins associated with it. Hybridoma clones with high titers to the cognate peptide ELISA were analyzed by Western blots using protein from Chinese hamster ovary cells transfected with enhanced green fluorescent protein-rat MR cDNA and from hippocampal cytosol from adrenalectomized rats. Immunohistochemistry was done on kidney, heart, colon, and brain. Antibodies that proved to be most useful for Western blot analysis and immunohistochemistry include those raised against peptides comprising amino acids 1–18, 64–82, 79–97, and 365–381. The intensity of immunoreactivity in the cytosol compared with nucleus in the same cells differed between antibodies, suggesting that certain receptor epitopes were more or less exposed depending on the location of the receptor within the cell. In summary, several antibodies are described that recognize different parts of the MR that should facilitate the study of this important mediator of two classes of steroid hormone action.
Introduction
THE MINERALOCORTICOID receptor (MR) is a member of the steroid receptor superfamily of ligand-regulated transcription factors (1, 2). Receptors from this family comprise several functional domains including a ligand binding, DNA binding, hinge, and N-terminal region (A/B) where coactivators and corepressors bind to modulate activity (3). The most closely related receptor to the MR is the glucocorticoid receptor, which is 94% homologous in the DNA binding domain and 57% homologous in the ligand binding domain, but only 15% homologous in the N-terminal region (3). Members of the steroid receptor family mediate gene expression by binding to hormone response elements as dimers in a ligand-dependent manner. The MR has a similar affinity for the mineralocorticoid aldosterone and the glucocorticoids corticosterone and cortisol (1, 4, 5). Although rats and mice synthesize only corticosterone, cortisol is the predominant glucocorticoid in humans and many other mammals, including other rodents.
MR are expressed in many tissues and modulate a variety of functions, the best known being the stimulation of electrolyte and water transfer by transporting epithelia in the kidney and colon. MR are also expressed in select areas of the central nervous system where they modulate multiple cell processes, including trophic effects in hippocampal neurons involved in learning and memory (6) and modulation of sodium appetite control (7) and blood pressure (8) through actions within the amygdala and circumventricular organs.
Several polyclonal antibodies against the MR have been described that were raised against peptides (9, 10, 11), recombinant MR protein fragments (12, 13), or the receptor purified from rat kidney (14). The commercial antibodies currently available have not been well characterized by the vendor; however, their usefulness for the detection of MR in the brain has been published (15). An auto-anti-idiotipic monoclonal antibody generated in animals immunized against an aldosterone-3-carboxymethoxyoxime-BSA conjugate (16) has also been described. This auto-anti-idiotypic antibody presumably recognizes the binding domain of the unbound MR, so aldosterone competes with the antibody for binding to the receptor (17). Because it recognizes the specific conformation of the unbound receptor, the auto-anti-idiotypic antibody has been used for immunohistochemistry (ihc) and does not work for Western blots in which the protein is denatured.
We have produced antibodies to different epitopes of the MR with the anticipation that they would recognize different conformational states of the receptor determined by the steroid ligands and/or cytosolic proteins to which it is bound. The latter presumably depend upon cell type, the location within the cell. and physiological state. In this study, we describe the production of a panel of monoclonal antibodies against various regions of the MR using peptide antigens comprising different portions of the MR molecule conjugated to strong immune-responsive proteins and the patterns of immunoreactivity when these are used for ihc in tissues from normal rats on a standard diet.
Materials and Methods
All animal use was approved by the Institutional Animal Care and Use Committee of the G.V. Montgomery VA Medical Center under Animal Component of Research Protocol approved by the Central Office of the Department of Veterans Affairs.
Preparation of peptide antigens and immunization
Hydropathy plots were used to select peptides (14–20 residues) corresponding to various regions of the rat MR (rMR) that have a high probability of being exposed when the molecule is folded normally (18, 19). These peptides were synthesized commercially with a cysteine at the N-terminal, middle, or C-terminal end (Table 1 and Fig. 1). Peptides were conjugated to keyhole limpet hemocyanin or Blue immunogenic protein, and casein (Pierce Biotechnology, Rockford, IL). For conjugation, 5 mg of protein was dissolved in 1 ml of 0.1 M sodium phosphate buffer (pH 7.4), then 2 mg of 6-(iodoacetamido)caproic acid N-hydroxysuccinimide ester dissolved in 0.1 ml of DMSO was added. After 60 min, the mixture was purified using a 20-ml spin column packed with Sephadex G-25 equilibrated with 0.05 M phosphate, 1 mM EDTA, and 0.5% SDS (pH 7.6), and centrifuged at 800 x g for 10 min. The peptide (3–4 mg) was dissolved in 1 ml of water, added to the conjugation protein, and stirred for 3–4 h. After the reaction, the conjugates were dialyzed in 4 liters of PBS overnight. Swiss-Webster female mice were immunized with 50 μg of the keyhole limpet hemocyanin conjugates emulsified in complete Freund’s adjuvant, divided into six sc sites, as the primary, then boosted twice, 3 wk apart, with the corresponding conjugates emulsified with incomplete Freund’s adjuvant. Two weeks after the last sc inoculation, the mice were injected ip with an aqueous solution of the peptides conjugates, and 3 d later, blood was obtained by cardiac puncture under deep isofluorane anesthesia and the spleens were removed under sterile conditions. Splenocytes were mechanically separated and frozen in two aliquots in freezing media containing 5% DMSO. Sera were screened by ELISA using 96-well plates onto which the peptide conjugated to casein (100 ng) was immobilized. Sera that were positive by ELISA were screened by ihc using rat kidney slices. Hybridomas were produced using splenocytes of mice whose sera were positive by ihc.
Fusions
Cells from half a spleen were thawed and fused with SP2-mIL6 or HL-1 mouse myelomas (American Type Culture Collection, Rockville, MD) using polyethylene glycol 1500 (Roche Diagnostics, Indianapolis, IN) and placed in eight six-well plates in methylcellulose media prepared as described (20) with some modifications. The following was added to Iscove medium: 10% FetalClone I (Hyclone, Provo, UT), hypoxanthine/aminopterin/thymidine (Sigma-Aldrich Chemical Company, St. Louis, MO), Glutamax I, -mercaptoethanol, 20 ng/ml human IL-6 (PeproTech, Rocky Hill, NJ), 50 μg/ml endothelial cell growth factor (Upstate Biotechnology, Lake Placid, NY), and 5% methylcellulose (Methocel MC high viscosity; Sigma-Aldrich Chemical Company). After 10–21 d, individual clones were transferred by micropipette into 96-well plates and grown 2–7 d, and the medium was screened by ELISA. Positive clones were expanded in 24-well plates, and then frozen while the antibodies were further characterized using ihc and Western blot. The most promising of these clones were subcloned using the methylcellulose media. Using this technique, approximately 90% of clones picked initially were monoclonal.
Western blots
Western blot analysis was used to screen the media from the clones positive by ELISA. MR protein sources were whole cell extracts of Chinese hamster ovary (CHO)-K1 cells transfected with enhanced green fluorescent protein (EGFP)-rMR [kindly provided by Drs. A. Naray-Fejes-Toth (Dartmouth University, Lebanon, NH) and D. Pearce (University of California San Francisco, San Francisco, CA)] (21) and CHO cells infected with a lentivirus expressing the complete sequence of the rMR, as well as cytosol from hippocampus of adrenalectomized rats. The 7.5% PAGE gels were transferred to polyvinylidene difluoride membranes using a tank technique, blocked with 1% Carnation dry skim milk in Tris-buffered saline with 0.05% Tween 20. The membranes were incubated with the primary antibody overnight, washed, and developed using a screened affinity purified goat antimouse IgG peroxidase-labeled antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). The membranes were developed using a chemiluminescence substrate (Pierce WestPico Super Signal; Pierce Biotechnology) and recorded using auto-radiographic film (Fujifilm; Fisher Scientific).
Immunohistochemistry
Tissues were collected from intact rats, anesthetized, then perfused with saline, followed by Streck Tissue Fixative (Streck Tissue Fixative, Omaha, NE). The kidneys, hearts, and brains were further fixed in Streck Tissue Fixative overnight, then embedded in paraffin. Six-micrometer sections were cut, deparaffinized, treated with 0.1% phenylhydrazine for 30 min to inhibit peroxidases, then blocked with 0.05 M Tris, 5% dry milk, 2% normal donkey serum, and 0.2% SDS for 1 h. The slides were then incubated overnight with the antibody in a similar buffer containing 0.05% Tween 20. After washing, the sections were incubated for 1 h with an affinity purified donkey antimouse IgG biotin-labeled antibody, washed, and incubated with peroxidase-labeled streptavidin (Zymed, Invitrogen, Carlsbad, CA) in Superblocker (Pierce Biotechnology) for 30 min. The slides were developed using diaminobenzidine and counterstained with hematoxylin (22). Negative controls included incubation with no primary antibody and with primary antibody preincubated with an excess of immunizing peptide.
Results
Monoclonal antibodies were generated against 10 different peptide conjugates, six from the N terminus (A/B domain) and four from the C terminus (steroid binding domain) (Fig. 1). All 10 peptides elicited high serum antibody titers in most of the inoculated mice as determined by ELISA. All positive sera were also evaluated using ihc with rat kidney slices. The splenocytes from the mice with the best titer and characteristics were used for fusion and generation of hybridomas. Sera from animals injected with the peptide conjugates: rMR 366–384, rMR 411–424, rMR 846–859, and rMR 833–853 were highly positive by ELISA, but had weak specific staining by ihc. The splenocytes from the animals with the best titers and ihc characteristics were fused with myeloma cells, yielding many positive clones by ELISA; however, all were negative by ihc, so were not pursued further. Similarly, the three positive hybridoma clones derived from the mouse inoculated with the rMR 963–981 (C-terminal end) conjugate with the highest titer, were not sensitive for Western blots and were negative for ihc staining, so were not evaluated further.
Five monoclonal antibodies against the rMR 1–18 peptide were studied; 6G1 and 1D5 were evaluated fully and seemed to be very similar. Figure 2 shows the ihc using the rMR 1–18 6G1 antibody in kidney, heart, colon, and central nervous system of intact rats. rMR 1–18 6G1 staining was primarily nuclear with lighter cytoplasmic staining in all tissues. Immunoreactivity in kidneys of intact rats was seen in connecting tubules, distal convoluted tubules, and cortical collecting tubules (Fig. 2, B and C), hippocampus (Fig. 2E), choroid plexus (Fig. 2G), cerebellum (Fig. 2H), and colon (Fig. 2I). Staining of the heart was also primarily nuclear with cytosolic staining (Fig. 2K) and strong staining of cytosol and nuclei of the vascular smooth muscle layer of the coronary arteries (Fig. 2L). Western blot analysis of hippocampal cytosol from adrenalectomized animals using rMR 1–18 antibodies produces a single band at a molecular mass of approximately 107 kDa, the expected molecular mass of the rMR (Fig. 3). The sequence of the rMR 1–18 peptide is identical in the rat, human, and mouse, so this antibody should be useful for ihc and Western blots in these species.
Antibodies against rMR 64–82 (clones 2B7 and 2D6) lightly stained nuclei from the connecting, distal and cortical collecting tubules in the kidney (Fig. 2, M and N) in areas similar to those recognized by the antibody raised against rMR 1–18; however, all of the rMR 64–82 antibodies also strongly stained the luminal membrane of proximal convoluted tubular cells of the inner cortex (Fig. 2O). Staining in the hippocampus was primarily nuclear (Fig. 4A), whereas staining in the heart and choroid plexus was both cytosolic and nuclear (Fig. 4, B and C). Western blots of homogenates of CHO cells transfected with either EGFP-rMR or rMR-EGFP gave a single band at a molecular mass of approximately 140 kDa and a single band using hippocampal cytosol at a molecular mass of approximately 107 kDa (Fig. 3). The sequence in this region differs between the rat and human (amino acid 68 a histidine vs. a glutamine and in positions 73–74 a tyrosine-isoleucine vs. a cysteine-leucine). The mouse sequence only differs from that of the rat at amino acids 73–74, where it is like the human sequence.
Monoclonal antibodies elicited by the rMR 79–97 peptides (3F10) produced excellent ihc cytosolic and nuclear staining in the expected kidney cells (Fig. 4, D and E). They also stained cytosol and nuclei of the heart (Fig. 4, H and I), hippocampus (Fig. 4F), and choroid plexus (Fig. 4G) well, but the rMR 79–97 antibody did not work for Western blots. In the colon, both the cytosol and nuclei of epithelial cells and cells of the smooth muscle layer were stained (Fig. 4I). Goblet cells were not stained. The amino acid sequence of this region of the MR is identical in the rat and mouse, but differs at amino acids 79, a serine vs. asparagine, and 81, a serine vs. proline, in the human. Because the conjugation was done by adding a cysteine before the first amino acid, the epitopes recognized by the antibody are probably distal to the conjugation site. Thus it was not unexpected that this antibody also works well with human tissues (data not presented).
Antibodies from the rMR 365–381 (4D6) recognized nuclei of distal and cortical collecting tubular cells of the kidney (Fig. 4J), hippocampus (Fig. 4K), cerebellum (Fig. 4L), in the heart (Fig. 4, M and N) and vascular smooth muscle of coronary vessels (Fig. 4O). They produced a Western blot band at approximately 107 kDa, as did the other antibodies, but were less sensitive (Fig. 3). The sequence of this region is identical between the rat and mouse, but differs from the human at amino acids 366, a histidine vs. glutamine, and amino acids 371–372, histidine-aspartic acid vs. glutamine-glutamic acid. Because the conjugation was done through a cysteine added to the C-terminal portion, one expects that the antibody might not cross-react with the human MR (not tested).
Monoclonal antibodies against the peptide 832–846 (n = 4) produced weak ihc staining that was similar to that of the other antibodies, but the monoclonal antibodies were negative by Western blots. Therefore these were not studied further.
Discussion
The only commercially available monoclonal antibody against the MR is an auto-anti-idiotypic antibody raised against an aldosterone conjugate. It stains both cytosol and nuclei of the distal convoluted tubules of the kidney (17). Because this antibody recognizes the binding epitope of the receptor, and thus reacts with the unoccupied receptor, it has limited use. Several polyclonal antibodies have been described (9, 10, 11, 12, 13, 14). The only commercially available polyclonal antibodies at this time are three antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), one against the first 300 amino acids, the other two against vaguely described portions of the N- and C-terminal regions of the receptor. The description of these antibodies provided by the company is very incomplete; however, there are more thorough descriptions in the literature of one of these identified as MRC N-17 (15), as well as of an unspecified one of two antibodies against a portion of the N-terminal end of the MR (23).
Our monoclonal antibodies were generated against epitopes located at both the N and C termini of the molecule. Several of the N-terminal antibodies are useful for both ihc and Western blots (rMR 1–18, rMR 64–82, rMR 365–381), although another produced excellent ihc staining, but did not work for Western blot analyses (rMR 79–87). No useful C-terminal monoclonal antibodies were obtained.
The antibody rMR 64–82 (2D6) primarily stained nuclei of cells in the distal, connecting, and cortical collecting tubules (Fig. 2, J and K). It also stained nuclei and, to a lesser extent, cytoplasm of cardiomyocytes and specific neurons in the brain in a pattern like that produced by tritiated aldosterone autoradiography, including the hippocampus where the highest density of MR occurs (24). Antibody rMR 64–82 (2D6) and several other clones from the same fusion also stained the luminal membrane of proximal tubular cells of nephrons in the inner cortex (Fig. 2, M and O). This luminal membrane staining is probably due to cross-reactivity with an epitope of an unknown protein and is not related to the MR, because no other antibody exhibited this pattern of staining. A more remote possibility is that this represents a membrane-attached configuration of the MR for which there is evidence but little information (25). This antibody was the most sensitive in Western blots and produced a single band with protein from hippocampal cytosol unless the gel was overloaded (Fig. 3).
The rMR 1–18 (6G1) antibody stained both nuclei and the cytosol, but not brush borders in the kidneys. Western blots of hippocampal cytosolic protein-using rMR 1–18 (6G1) also showed a single band at the predicted molecular mass (Fig. 3). A functional alternatively translated MR starting at methionine 15 has been demonstrated by in vitro transcription/translation studies of the human MR (26). This MR would not be detected by the rMR 1–18 (6G1) antibody if it exists in nature, but it would be detected by the other MR antibodies. Western blots using the rMR 1–18 (6G1) produced similar results as those using rMR 365–381 (4D6) and rMR 64–82, suggesting that if the alternatively spliced MR starting at amino acid 15 exists, the full-length MR predominates.
The rMR 79–97 (3F10) antibody stained both cytoplasm and nuclei of rat kidneys. It also gave excellent labeling in the heart and brain, including the choroid plexus (Fig. 4G). This antibody did not work for Western blots, producing only several faint bands when large amounts of protein were used.
It was originally thought that the unoccupied MR resided in the cytoplasm and moved into the nucleus upon occupation by an agonist (27); however, others using immunofluorescence staining have found three different patterns of subcellular distribution including diffuse, nuclear, and cytoplasmic (28). Similar cytoplasmic and nuclear staining has been found using the monoclonal auto-anti-idiotypic antibody (17, 29). In the absence of ligand, MR is located in both the cytosol and nucleus bound by a variety of chaperone proteins, including hsp90. Upon exposure to either aldosterone or corticosterone, most MR are found in the nucleus, where they form prominent clusters that are not seen in the absence of hormone (21, 30). It appears that several of our monoclonal antibodies have a strong preference for the MR in the nucleus. This could be due to masking of MR epitopes by binding to cytoplasmic proteins that dissociate before the receptor enters the nuclei. In vitro transcription translation experiments have demonstrated that the transcriptional activity of the MR is altered by the modification of the receptor by sumoylation (31); however, we found no evidence by Western blots of an alteration of molecular weight of the MR in hypothalamic cytosol.
In conclusion, monoclonal antibodies against several distinct regions of the rMR have been produced to facilitate its study. Although most of the conjugated peptides elicited the formation of specific antibodies that recognize the peptide in an ELISA, some of these antibodies do not recognize the native MR. Several of the best MR antibodies, while recognizing the protein in the same cells as the others, differ in their affinity for the receptor depending on its location within the cell, presumably due to conformational changes wrought by association with other proteins. These should be useful tools for the study of the MR biology.
Footnotes
This work was supported by Medical Research funds from the Department of Veterans Affairs and National Institutes of Health Grants HL27255 and HL75321.
First Published Online November 17, 2005
Abbreviations: CHO, Chinese hamster ovary; EGFP, enhanced green fluorescent protein; ihc, immunohistochemistry; MR, mineralocorticoid receptor; rMR, rat MR.
Accepted for publication November 10, 2005.
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Research Mississippi, Inc. (M.T.G.-S.), Jackson, Mississippi 39216
Abstract
Mineralocorticoid receptors (MR) bind both mineralocorticoids and glucocorticoids. They are expressed in multiple tissues and mediate diverse functions. Less is known about MR regulation and function compared with other major steroid receptors, although its importance has become increasingly apparent. A significant obstacle to such studies has been the dearth of specific high-affinity MR antibodies. We have produced monoclonal antibodies against 10 different peptide conjugates, six from the N terminus (A/B domain) and four from the C terminus (steroid binding domain), with the anticipation that their individual affinities for the MR would differ depending upon its conformation, which in turn, is dependent upon the location of the receptor within the cell and the proteins associated with it. Hybridoma clones with high titers to the cognate peptide ELISA were analyzed by Western blots using protein from Chinese hamster ovary cells transfected with enhanced green fluorescent protein-rat MR cDNA and from hippocampal cytosol from adrenalectomized rats. Immunohistochemistry was done on kidney, heart, colon, and brain. Antibodies that proved to be most useful for Western blot analysis and immunohistochemistry include those raised against peptides comprising amino acids 1–18, 64–82, 79–97, and 365–381. The intensity of immunoreactivity in the cytosol compared with nucleus in the same cells differed between antibodies, suggesting that certain receptor epitopes were more or less exposed depending on the location of the receptor within the cell. In summary, several antibodies are described that recognize different parts of the MR that should facilitate the study of this important mediator of two classes of steroid hormone action.
Introduction
THE MINERALOCORTICOID receptor (MR) is a member of the steroid receptor superfamily of ligand-regulated transcription factors (1, 2). Receptors from this family comprise several functional domains including a ligand binding, DNA binding, hinge, and N-terminal region (A/B) where coactivators and corepressors bind to modulate activity (3). The most closely related receptor to the MR is the glucocorticoid receptor, which is 94% homologous in the DNA binding domain and 57% homologous in the ligand binding domain, but only 15% homologous in the N-terminal region (3). Members of the steroid receptor family mediate gene expression by binding to hormone response elements as dimers in a ligand-dependent manner. The MR has a similar affinity for the mineralocorticoid aldosterone and the glucocorticoids corticosterone and cortisol (1, 4, 5). Although rats and mice synthesize only corticosterone, cortisol is the predominant glucocorticoid in humans and many other mammals, including other rodents.
MR are expressed in many tissues and modulate a variety of functions, the best known being the stimulation of electrolyte and water transfer by transporting epithelia in the kidney and colon. MR are also expressed in select areas of the central nervous system where they modulate multiple cell processes, including trophic effects in hippocampal neurons involved in learning and memory (6) and modulation of sodium appetite control (7) and blood pressure (8) through actions within the amygdala and circumventricular organs.
Several polyclonal antibodies against the MR have been described that were raised against peptides (9, 10, 11), recombinant MR protein fragments (12, 13), or the receptor purified from rat kidney (14). The commercial antibodies currently available have not been well characterized by the vendor; however, their usefulness for the detection of MR in the brain has been published (15). An auto-anti-idiotipic monoclonal antibody generated in animals immunized against an aldosterone-3-carboxymethoxyoxime-BSA conjugate (16) has also been described. This auto-anti-idiotypic antibody presumably recognizes the binding domain of the unbound MR, so aldosterone competes with the antibody for binding to the receptor (17). Because it recognizes the specific conformation of the unbound receptor, the auto-anti-idiotypic antibody has been used for immunohistochemistry (ihc) and does not work for Western blots in which the protein is denatured.
We have produced antibodies to different epitopes of the MR with the anticipation that they would recognize different conformational states of the receptor determined by the steroid ligands and/or cytosolic proteins to which it is bound. The latter presumably depend upon cell type, the location within the cell. and physiological state. In this study, we describe the production of a panel of monoclonal antibodies against various regions of the MR using peptide antigens comprising different portions of the MR molecule conjugated to strong immune-responsive proteins and the patterns of immunoreactivity when these are used for ihc in tissues from normal rats on a standard diet.
Materials and Methods
All animal use was approved by the Institutional Animal Care and Use Committee of the G.V. Montgomery VA Medical Center under Animal Component of Research Protocol approved by the Central Office of the Department of Veterans Affairs.
Preparation of peptide antigens and immunization
Hydropathy plots were used to select peptides (14–20 residues) corresponding to various regions of the rat MR (rMR) that have a high probability of being exposed when the molecule is folded normally (18, 19). These peptides were synthesized commercially with a cysteine at the N-terminal, middle, or C-terminal end (Table 1 and Fig. 1). Peptides were conjugated to keyhole limpet hemocyanin or Blue immunogenic protein, and casein (Pierce Biotechnology, Rockford, IL). For conjugation, 5 mg of protein was dissolved in 1 ml of 0.1 M sodium phosphate buffer (pH 7.4), then 2 mg of 6-(iodoacetamido)caproic acid N-hydroxysuccinimide ester dissolved in 0.1 ml of DMSO was added. After 60 min, the mixture was purified using a 20-ml spin column packed with Sephadex G-25 equilibrated with 0.05 M phosphate, 1 mM EDTA, and 0.5% SDS (pH 7.6), and centrifuged at 800 x g for 10 min. The peptide (3–4 mg) was dissolved in 1 ml of water, added to the conjugation protein, and stirred for 3–4 h. After the reaction, the conjugates were dialyzed in 4 liters of PBS overnight. Swiss-Webster female mice were immunized with 50 μg of the keyhole limpet hemocyanin conjugates emulsified in complete Freund’s adjuvant, divided into six sc sites, as the primary, then boosted twice, 3 wk apart, with the corresponding conjugates emulsified with incomplete Freund’s adjuvant. Two weeks after the last sc inoculation, the mice were injected ip with an aqueous solution of the peptides conjugates, and 3 d later, blood was obtained by cardiac puncture under deep isofluorane anesthesia and the spleens were removed under sterile conditions. Splenocytes were mechanically separated and frozen in two aliquots in freezing media containing 5% DMSO. Sera were screened by ELISA using 96-well plates onto which the peptide conjugated to casein (100 ng) was immobilized. Sera that were positive by ELISA were screened by ihc using rat kidney slices. Hybridomas were produced using splenocytes of mice whose sera were positive by ihc.
Fusions
Cells from half a spleen were thawed and fused with SP2-mIL6 or HL-1 mouse myelomas (American Type Culture Collection, Rockville, MD) using polyethylene glycol 1500 (Roche Diagnostics, Indianapolis, IN) and placed in eight six-well plates in methylcellulose media prepared as described (20) with some modifications. The following was added to Iscove medium: 10% FetalClone I (Hyclone, Provo, UT), hypoxanthine/aminopterin/thymidine (Sigma-Aldrich Chemical Company, St. Louis, MO), Glutamax I, -mercaptoethanol, 20 ng/ml human IL-6 (PeproTech, Rocky Hill, NJ), 50 μg/ml endothelial cell growth factor (Upstate Biotechnology, Lake Placid, NY), and 5% methylcellulose (Methocel MC high viscosity; Sigma-Aldrich Chemical Company). After 10–21 d, individual clones were transferred by micropipette into 96-well plates and grown 2–7 d, and the medium was screened by ELISA. Positive clones were expanded in 24-well plates, and then frozen while the antibodies were further characterized using ihc and Western blot. The most promising of these clones were subcloned using the methylcellulose media. Using this technique, approximately 90% of clones picked initially were monoclonal.
Western blots
Western blot analysis was used to screen the media from the clones positive by ELISA. MR protein sources were whole cell extracts of Chinese hamster ovary (CHO)-K1 cells transfected with enhanced green fluorescent protein (EGFP)-rMR [kindly provided by Drs. A. Naray-Fejes-Toth (Dartmouth University, Lebanon, NH) and D. Pearce (University of California San Francisco, San Francisco, CA)] (21) and CHO cells infected with a lentivirus expressing the complete sequence of the rMR, as well as cytosol from hippocampus of adrenalectomized rats. The 7.5% PAGE gels were transferred to polyvinylidene difluoride membranes using a tank technique, blocked with 1% Carnation dry skim milk in Tris-buffered saline with 0.05% Tween 20. The membranes were incubated with the primary antibody overnight, washed, and developed using a screened affinity purified goat antimouse IgG peroxidase-labeled antibody (Jackson ImmunoResearch Laboratories, West Grove, PA). The membranes were developed using a chemiluminescence substrate (Pierce WestPico Super Signal; Pierce Biotechnology) and recorded using auto-radiographic film (Fujifilm; Fisher Scientific).
Immunohistochemistry
Tissues were collected from intact rats, anesthetized, then perfused with saline, followed by Streck Tissue Fixative (Streck Tissue Fixative, Omaha, NE). The kidneys, hearts, and brains were further fixed in Streck Tissue Fixative overnight, then embedded in paraffin. Six-micrometer sections were cut, deparaffinized, treated with 0.1% phenylhydrazine for 30 min to inhibit peroxidases, then blocked with 0.05 M Tris, 5% dry milk, 2% normal donkey serum, and 0.2% SDS for 1 h. The slides were then incubated overnight with the antibody in a similar buffer containing 0.05% Tween 20. After washing, the sections were incubated for 1 h with an affinity purified donkey antimouse IgG biotin-labeled antibody, washed, and incubated with peroxidase-labeled streptavidin (Zymed, Invitrogen, Carlsbad, CA) in Superblocker (Pierce Biotechnology) for 30 min. The slides were developed using diaminobenzidine and counterstained with hematoxylin (22). Negative controls included incubation with no primary antibody and with primary antibody preincubated with an excess of immunizing peptide.
Results
Monoclonal antibodies were generated against 10 different peptide conjugates, six from the N terminus (A/B domain) and four from the C terminus (steroid binding domain) (Fig. 1). All 10 peptides elicited high serum antibody titers in most of the inoculated mice as determined by ELISA. All positive sera were also evaluated using ihc with rat kidney slices. The splenocytes from the mice with the best titer and characteristics were used for fusion and generation of hybridomas. Sera from animals injected with the peptide conjugates: rMR 366–384, rMR 411–424, rMR 846–859, and rMR 833–853 were highly positive by ELISA, but had weak specific staining by ihc. The splenocytes from the animals with the best titers and ihc characteristics were fused with myeloma cells, yielding many positive clones by ELISA; however, all were negative by ihc, so were not pursued further. Similarly, the three positive hybridoma clones derived from the mouse inoculated with the rMR 963–981 (C-terminal end) conjugate with the highest titer, were not sensitive for Western blots and were negative for ihc staining, so were not evaluated further.
Five monoclonal antibodies against the rMR 1–18 peptide were studied; 6G1 and 1D5 were evaluated fully and seemed to be very similar. Figure 2 shows the ihc using the rMR 1–18 6G1 antibody in kidney, heart, colon, and central nervous system of intact rats. rMR 1–18 6G1 staining was primarily nuclear with lighter cytoplasmic staining in all tissues. Immunoreactivity in kidneys of intact rats was seen in connecting tubules, distal convoluted tubules, and cortical collecting tubules (Fig. 2, B and C), hippocampus (Fig. 2E), choroid plexus (Fig. 2G), cerebellum (Fig. 2H), and colon (Fig. 2I). Staining of the heart was also primarily nuclear with cytosolic staining (Fig. 2K) and strong staining of cytosol and nuclei of the vascular smooth muscle layer of the coronary arteries (Fig. 2L). Western blot analysis of hippocampal cytosol from adrenalectomized animals using rMR 1–18 antibodies produces a single band at a molecular mass of approximately 107 kDa, the expected molecular mass of the rMR (Fig. 3). The sequence of the rMR 1–18 peptide is identical in the rat, human, and mouse, so this antibody should be useful for ihc and Western blots in these species.
Antibodies against rMR 64–82 (clones 2B7 and 2D6) lightly stained nuclei from the connecting, distal and cortical collecting tubules in the kidney (Fig. 2, M and N) in areas similar to those recognized by the antibody raised against rMR 1–18; however, all of the rMR 64–82 antibodies also strongly stained the luminal membrane of proximal convoluted tubular cells of the inner cortex (Fig. 2O). Staining in the hippocampus was primarily nuclear (Fig. 4A), whereas staining in the heart and choroid plexus was both cytosolic and nuclear (Fig. 4, B and C). Western blots of homogenates of CHO cells transfected with either EGFP-rMR or rMR-EGFP gave a single band at a molecular mass of approximately 140 kDa and a single band using hippocampal cytosol at a molecular mass of approximately 107 kDa (Fig. 3). The sequence in this region differs between the rat and human (amino acid 68 a histidine vs. a glutamine and in positions 73–74 a tyrosine-isoleucine vs. a cysteine-leucine). The mouse sequence only differs from that of the rat at amino acids 73–74, where it is like the human sequence.
Monoclonal antibodies elicited by the rMR 79–97 peptides (3F10) produced excellent ihc cytosolic and nuclear staining in the expected kidney cells (Fig. 4, D and E). They also stained cytosol and nuclei of the heart (Fig. 4, H and I), hippocampus (Fig. 4F), and choroid plexus (Fig. 4G) well, but the rMR 79–97 antibody did not work for Western blots. In the colon, both the cytosol and nuclei of epithelial cells and cells of the smooth muscle layer were stained (Fig. 4I). Goblet cells were not stained. The amino acid sequence of this region of the MR is identical in the rat and mouse, but differs at amino acids 79, a serine vs. asparagine, and 81, a serine vs. proline, in the human. Because the conjugation was done by adding a cysteine before the first amino acid, the epitopes recognized by the antibody are probably distal to the conjugation site. Thus it was not unexpected that this antibody also works well with human tissues (data not presented).
Antibodies from the rMR 365–381 (4D6) recognized nuclei of distal and cortical collecting tubular cells of the kidney (Fig. 4J), hippocampus (Fig. 4K), cerebellum (Fig. 4L), in the heart (Fig. 4, M and N) and vascular smooth muscle of coronary vessels (Fig. 4O). They produced a Western blot band at approximately 107 kDa, as did the other antibodies, but were less sensitive (Fig. 3). The sequence of this region is identical between the rat and mouse, but differs from the human at amino acids 366, a histidine vs. glutamine, and amino acids 371–372, histidine-aspartic acid vs. glutamine-glutamic acid. Because the conjugation was done through a cysteine added to the C-terminal portion, one expects that the antibody might not cross-react with the human MR (not tested).
Monoclonal antibodies against the peptide 832–846 (n = 4) produced weak ihc staining that was similar to that of the other antibodies, but the monoclonal antibodies were negative by Western blots. Therefore these were not studied further.
Discussion
The only commercially available monoclonal antibody against the MR is an auto-anti-idiotypic antibody raised against an aldosterone conjugate. It stains both cytosol and nuclei of the distal convoluted tubules of the kidney (17). Because this antibody recognizes the binding epitope of the receptor, and thus reacts with the unoccupied receptor, it has limited use. Several polyclonal antibodies have been described (9, 10, 11, 12, 13, 14). The only commercially available polyclonal antibodies at this time are three antibodies from Santa Cruz Biotechnology (Santa Cruz, CA), one against the first 300 amino acids, the other two against vaguely described portions of the N- and C-terminal regions of the receptor. The description of these antibodies provided by the company is very incomplete; however, there are more thorough descriptions in the literature of one of these identified as MRC N-17 (15), as well as of an unspecified one of two antibodies against a portion of the N-terminal end of the MR (23).
Our monoclonal antibodies were generated against epitopes located at both the N and C termini of the molecule. Several of the N-terminal antibodies are useful for both ihc and Western blots (rMR 1–18, rMR 64–82, rMR 365–381), although another produced excellent ihc staining, but did not work for Western blot analyses (rMR 79–87). No useful C-terminal monoclonal antibodies were obtained.
The antibody rMR 64–82 (2D6) primarily stained nuclei of cells in the distal, connecting, and cortical collecting tubules (Fig. 2, J and K). It also stained nuclei and, to a lesser extent, cytoplasm of cardiomyocytes and specific neurons in the brain in a pattern like that produced by tritiated aldosterone autoradiography, including the hippocampus where the highest density of MR occurs (24). Antibody rMR 64–82 (2D6) and several other clones from the same fusion also stained the luminal membrane of proximal tubular cells of nephrons in the inner cortex (Fig. 2, M and O). This luminal membrane staining is probably due to cross-reactivity with an epitope of an unknown protein and is not related to the MR, because no other antibody exhibited this pattern of staining. A more remote possibility is that this represents a membrane-attached configuration of the MR for which there is evidence but little information (25). This antibody was the most sensitive in Western blots and produced a single band with protein from hippocampal cytosol unless the gel was overloaded (Fig. 3).
The rMR 1–18 (6G1) antibody stained both nuclei and the cytosol, but not brush borders in the kidneys. Western blots of hippocampal cytosolic protein-using rMR 1–18 (6G1) also showed a single band at the predicted molecular mass (Fig. 3). A functional alternatively translated MR starting at methionine 15 has been demonstrated by in vitro transcription/translation studies of the human MR (26). This MR would not be detected by the rMR 1–18 (6G1) antibody if it exists in nature, but it would be detected by the other MR antibodies. Western blots using the rMR 1–18 (6G1) produced similar results as those using rMR 365–381 (4D6) and rMR 64–82, suggesting that if the alternatively spliced MR starting at amino acid 15 exists, the full-length MR predominates.
The rMR 79–97 (3F10) antibody stained both cytoplasm and nuclei of rat kidneys. It also gave excellent labeling in the heart and brain, including the choroid plexus (Fig. 4G). This antibody did not work for Western blots, producing only several faint bands when large amounts of protein were used.
It was originally thought that the unoccupied MR resided in the cytoplasm and moved into the nucleus upon occupation by an agonist (27); however, others using immunofluorescence staining have found three different patterns of subcellular distribution including diffuse, nuclear, and cytoplasmic (28). Similar cytoplasmic and nuclear staining has been found using the monoclonal auto-anti-idiotypic antibody (17, 29). In the absence of ligand, MR is located in both the cytosol and nucleus bound by a variety of chaperone proteins, including hsp90. Upon exposure to either aldosterone or corticosterone, most MR are found in the nucleus, where they form prominent clusters that are not seen in the absence of hormone (21, 30). It appears that several of our monoclonal antibodies have a strong preference for the MR in the nucleus. This could be due to masking of MR epitopes by binding to cytoplasmic proteins that dissociate before the receptor enters the nuclei. In vitro transcription translation experiments have demonstrated that the transcriptional activity of the MR is altered by the modification of the receptor by sumoylation (31); however, we found no evidence by Western blots of an alteration of molecular weight of the MR in hypothalamic cytosol.
In conclusion, monoclonal antibodies against several distinct regions of the rMR have been produced to facilitate its study. Although most of the conjugated peptides elicited the formation of specific antibodies that recognize the peptide in an ELISA, some of these antibodies do not recognize the native MR. Several of the best MR antibodies, while recognizing the protein in the same cells as the others, differ in their affinity for the receptor depending on its location within the cell, presumably due to conformational changes wrought by association with other proteins. These should be useful tools for the study of the MR biology.
Footnotes
This work was supported by Medical Research funds from the Department of Veterans Affairs and National Institutes of Health Grants HL27255 and HL75321.
First Published Online November 17, 2005
Abbreviations: CHO, Chinese hamster ovary; EGFP, enhanced green fluorescent protein; ihc, immunohistochemistry; MR, mineralocorticoid receptor; rMR, rat MR.
Accepted for publication November 10, 2005.
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