Molecular and Cellular Endocrinology, 0 1992 Elsevier Scientific Publishers
MOLCEL
21
85 (1992) 21-32 Ireland, Ltd. 0303-7207/92/$05.00
02738
Type I corticosteroid receptor-like immunoreactivity in the rat salivary glands and distal colon: modulation by corticosteroids Zygmunt Krozowski a, Kelli Wendell a, Rexford Ahima b and Richard Harlan b aPrince Henry’s Institute of Medical Research, Prince Henry’s Hospital, Melbourne, Vie. 3205, Australia and ’ Department of Anatomy, Tulane Unicersity Medical Center, New Orleans, LA 70112, USA (Received
Key words: Receptor,
type I; Mineralocorticoid
20 August
receptor;
1991; accepted
Colon rat; Parotid
2 January
gland;
1992)
Fusion
protein;
Antibody
Summary A 167 amino acid fragment of the N-terminal domain of the human type I corticosteroid (mineralocorticoid) receptor was fused to the glutathione S-transferase gene using the Gex expression plasmid and the fusion protein used to raise the monospecific polyclonal antibody, MINREC4. Immunostaining experiments showed that MINREC4 specifically bound type I receptor in the distal tubule of the kidney, the ductal elements of the salivary glands and the epithelium of the distal colon in the rat. Adrenalectomy abolished staining in the parotid and colon, and reduced immunoreactivity in the submandibular gland. Administration of corticosterone or aldosterone resulted in partial restoration of immunostaining in the parotid, and a complete restoration of staining to intact levels in the submandibular gland and colon. These results suggest that adrenocorticoid binding to the type I receptor may result in tissue specific conformational changes in the binding protein and that the MINREC4 antibody may be used to study these effects.
Introduction The type I adrenocorticoid (mineralocorticoid) receptor displays equivalent affinity for corticosterone and aldosterone (Beaumont and Fanestil, 1983; Krozowski and Funder, 1983). It is thought that the presence of lip-hydroxysteroid dehydrogenase in target cells serves to reduce the high level of glucocorticoid and allows aldosterone access to the type I receptor in mineralocorticoid selective target tissues (Edwards et al., 1988; Funder et al., 1988). However, other evidence sug-
Correspondence to: Dr. Z. Krozowski, Baker Institute of Medical Research, P.O. Box 348, Prahran, Vie. 3181, Australia. Fax 61-3-521.1362.
gests that the specificity conferring mechanism may be far more complex (Rundle et al., 1989; Krozowski et al., 1990). The availability of a panel of monospecific antibodies to the type I receptor may aid the resolution of this conundrum. We have previously raised a polyclonal antibody to the type I receptor by immunizing with a synthetic peptide corresponding to a region between the DNA and hormone binding domains of the human mineralocorticoid receptor (Krozowski et al., 1989). The overall pattern of staining obtained in that study has recently been confirmed by use of an auto-anti-idiotypic antibody which recognizes the unbound form of the receptor (Lombes et al., 1990). However, the subcellular distribution of staining between the two studies appeared to differ, possibly reflecting the de-
22
tection of bound and unbound forms of the receptor. These observations suggest that different antisera may be useful in discriminating between various conformations of the receptor and may aid the elucidation of the specificity conferring mechanism in mineralocorticoid selective target tissues. In our continuing efforts to raise a battery of polyclonal antibodies to the type I receptor we have constructed a fusion protein comprising part of the N-terminus of the receptor fused to glutathione S-transferase. The full-length fusion protein can be expressed in Escherichia coli and can be purified by a single step on a glutathione affinity column. Antibodies raised against the fusion protein were used to map type I receptor distribution in various tissues in the presence and absence of corticosteroids. The results obtained with this antiserum were then compared with those obtained using the previously described MINREC2 antibody. Materials
and methods
Production of bacterial proteins A 497 base pair BamHI (1273 bp) to EcoRI (1770 bp) fragment of the human type I adrenocorticoid receptor cDNA (Arriza et al., 19871, corresponding to G1y-&er5i9, was cloned into the multiple cloning site of the pGEX2 expression vector (Smith and Johnson, 1988) to produce the pGTMR500 recombinant plasmid. Either pGEX2 or pGTMR500 was transfected into Escherichia coli. JM109 cells, an overnight culture of the cells diluted 1: 10 in culture medium and grown for 1 h at 37°C before induction of protein synthesis with 0.1 mM isopropylthiolgalatoside. After a further 3 h of growth the cells were pelleted and resuspended in phosphate-buffered saline (PBS) containing 1% Triton X-100 and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). E. coli were lysed by freezing at -20°C overnight and thawing at room temperature. Centrifugation of the lysate at 10,000 X g for 5 min yielded a clear supernatant which was mixed at room temperature for 30 min with preswollen agarose beads. The beads were pelleted at 500 X g for 10 s and washed 3 times with PBS containing 0.1% Triton X-100. Protein was eluted from the affinity
beads by competition with free glutathione in 50 mM Tris pH 8.0 containing 5 mM reduced glutathione. The fusion protein derived from pGTMR500 was designated GTMR4 while the parent plasmid pGEX2 produced glutathione Stransferase (GST). Bacterial proteins were freeze-dried (Speed-Vat) and stored at - 20°C. Immunization and screening protocol Three New Zealand White rabbits were injected subcutaneously along the spine with 100 pg GST protein in complete adjuvant and were boosted 4 weeks later with 500 pg GST in complete adjuvant. Rabbits were bled every 2-3 weeks. Serum was separated by incubating the blood at 37°C for 1 h, storing overnight at 4°C and then spinning down the red blood cells. AntiGST antibodies appeared 2 weeks after the booster injection of GST, were of maximum titre by 7 weeks and then showed a decline in antibody titre by week 10. All rabbits were then injected with 5 pg GTMR4 in complete adjuvant and boosted 4 weeks later with 50 pg GTMR4 in complete adjuvant. The appearance of anti-GST and anti-GTMR4 antibodies was monitored by dot blot analysis of GST and GTMR4 proteins using a 1: 1000 dilution of antiserum preincubated for 1 h at room temperature in the presence or absence of 10 pg GST or 10 pg GST plus 10 pg GTMR4. GST (0.1 pg) and GTMR4 (0.1 pg> were spotted onto nitrocellulose filters in 1 ~1 PBS and the filters processed as described for Western blot analysis. Rabbit C showed high levels of anti-GTMR4 antibodies and was bled out 1 month after injection of the booster dose of GTMR4; the antiserum thus obtained was designated MINREC4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis was performed as previously described (Krozowski, 1989) except that gel electrophoresis was performed using a 10% polyactylamide gel and Western blot analysis was performed using a 1: 200 dilution of anti-GST antiserum. Animals Adult male Sprague-Dawley rats were used throughout the study. Rats were kept under standard lighting conditions (12 h light and 12 h dark)
and allowed free access to normal rat chow. Control rats were allowed free access to tap water and adrenalectomized rats to normal saline. Adrenalectomized rats were used l-2 weeks post-surgery. The adrenalectomized rats were divided into three treatment groups with four rats per group. The control group received 200 ~1 of 0.005% ethanol/PBS vehicle intraperitoneally, a second group received 1 mg/lOO g body weight corticosterone and a third group was injected with 1 pg/lOO g body weight aldosterone. Rats were sacrificed 1 h after steroid administration. All animals were killed with an overdose of sodium pentobarbital intraperitoneally, perfused intracardially with PBS for 5 min followed by 3% phosphate-buffered paraformaldehyde for 8 min. Salivary glands and segments of distal colon were dissected out and postfixed in 3% paraformaldehyde for 2 h followed by cryoprotection in 30% sucrose. The tissues were then frozen on dry ice and stored at -70°C until sectioned. Pieces of kidney and liver were also taken from intact rats after perfusion fixation, postfixed for 6 h and processed for paraplast embedding as previously described (Krozowski et al., 1989). Sections 30 pm thick were cut on a cryostat, collected onto chromalum-subbed slides and stored with desiccant at -70°C until use. 7 pm thick sections of the paraplast embedded kidney and liver were also cut and collected onto gelatin-coated slides. Imtnunocytochemistry (i) SaliLlary glands and distal colon. Sections were dried in a vacuum chamber, rinsed in PBS for 10 min and permeabilized with 0.2% Triton X-100 for 10 min. Non-specific staining was blocked by incubating the sections in 0.1% PBS/bovine serum albumin (BSA) plus 0.02% normal goat serum for 20 min. Sections were then rinsed in PBS for 10 min and incubated with a 1: 1000 dilution of either MINREC2 or MINREC4 antiserum for 48 h at 4°C. Adjacent control sections were incubated with either normal rabbit serum, MINREC2 antiserum (1: 1000) preabsorbed with 10 pmol of MINREC2 peptide (Krozowski et al., 19891, MINREC4 antiserum
(1: 1000) preabsorbed with 10 pmol of GTMR4 fusion protein or GST protein, or a 1: 500 dilution of GST antiserum. All preabsorption steps were carried out at 4°C for 24 h. (ii) Kidney and lkler. Sections were deparaffinized with two 3 min changes of xylene, immersed for 1 min each in lOO%, 95% and 70% ethanol and hydrated in PBS. Endogenous peroxidase was reduced by immersing the sections in 0.3% hydrogen peroxide in methanol for 15 min and then rinsing in PBS for 10 min. Sections were then processed for immunocytochemical localization of type I receptor using MINREC2 and MINREC4 antisera, or type II (glucocorticoid) receptor using the BUGR2 monoclonal antibody (Eisen et al., 1985). Kidney and liver sections were incubated with the same dilutions of MINREC antisera and preabsorption controls as used for the salivary glands and colon. BUGR2 antibody was used at a dilution of 1: 500. A control section for the monoclonal antibody study was incubated with non-immune P3 AgX-653 myeloma cell supernatant. Sections were processed for immunostaining using the Vectastain ABC immunoperoxidase (goat anti-rabbit and horse anti-mouse) kits for MINREC antisera and BUGR2 respectively. The reaction product was visualised using 3,3’-diaminobenzidine (DAB) tetrahydrochloride or nickel-DAB as chromogen. The reaction was stopped by immersion of the sections in PBS, the sections dehydrated in 70%, 95% and 100% ethanol, defatted in Histoclear (National Diagnostics), and coverslipped using Permount (Fisher Scientific).
Microscopy Sections were viewed with the aid of a Nikon Optiphot microscope and photographs taken with a Nikon FX35A camera and Technical Pan (Kodak) film. Slides of salivary glands and distal colon were coded by one investigator and analysed blindly by two others in order to determine (i) cell types exhibiting MINREC immunostaining and (ii) intracellular distribution of MINREC immunostaining.
24 602670734
1
ICIO]
351 m
964 E
hMR MINREC 4
516 672 @6?
MINREC 2
Fig, 1. Schematic representation of the human type 1 receptor showing locations of the immunogens used to raise the MINREC4 and MINREC2 polyclonal antibodies. Numbering refers to the location of amino acids according to Arriza et al. (1987). Lettering designates domains of the receptor according to Gronemeyer et al. (1987): A/B is the N-terminal domain, some of which is highly immunogenic, C is the DNA binding domain, D is the linker region and E represents the hormone binding domain. The GTMR4 fusion protein used as an immunogen to generate the MINREC4 antibody consists of glutathione S-transferase (GST) linked to the N-terminus of the 167 amino acid peptide.
Results Early studies on steroid receptors showed that the N-terminus of these proteins contained a highly immunogenic domain (for a review see Gustafsson et al., 1987). Fig. 1 shows the location of the MINREC4 immunogen used in the present study in relation to MINREC2 epitopes. A hydrophilicity profile of the receptor protein (Hopp and Woods, 1981) showed seven regions of high antigenicity within a 167 amino acid fragment, including a peptide with the highest predicted antigenicity of the A/B domain. The MINREC4 immunogen of the human receptor is 88% identical with the corresponding region of the rat protein (Pate1 et al., 1989). The expression of foreign proteins in E. cofi frequently results in degradation or insolubility of the protein of interest. Often both these problems can be overcome by fusing mammalian peptides to proteins that are stable in bacterial cells. In the present study we have fused 167 amino acids of the human type I receptor with the carboxy terminus of glutathione S-transferase (EC 2.5.1.18), an enzyme cloned from the parasite Schistosoma juponicum (Smith and Johnson, 1988). Fig. 2A shows a Coomassie stained SDSpolyac~lamide gel of the GST and GTMR4 protein preparations after affinity purification. GST displayed a molecuIar weight of 28 kDa while GTMR4 showed the largest molecular weight species at 46 kDa. The 18 kDa difference be-
tween the molecular weights of GST and GTMR4 proteins is consistent with the addition of 167 amino acids of the type I receptor to GST. The GTMR4 protein showed variable degrees of degradation between preparations but the 46 kDa species was aIways present. Western blot analysis using the anti-GST antiserum (Fig. 2B) showed that CJST and GTMR4 were indeed related proteins. Furthermore, the major proteins migrating at 30-40 kDa in preparations of fusion protein also bound GST antiserum consistent with the existence of shorter forms of GTMR4. However, attempts to cIeave the 167 amino acid fragment from the GTMR4 fusion protein with thrombin were unsuccessful. In other experiments when the C-terminal half of the rat mineralocorticoid receptor was fused to the GST protein the fusion protein proved highly unstable and was not used in further studies (results not shown). When three rabbits were immunized with GST protein and then with GTMR4 one rabbit produced an antiserum (MINREC4) which reacted selectively with the receptor portion of the GTMR4 fusion protein. Fig. 2C shows the results obtained when GST and GTMR4 proteins were reacted with the MINREC4 antiserum in the presence or absence of competing antigens. In the absence of competitor MINREC4 bound both GST and GTMR4. Preincubating the antiserum in the presence of 10 pg GST abolished all binding to filter bound GST while binding to GTMR4 was only slightly reduced. Preincubation of the antiserum in the presence of both 10 pug GST and 10 pg GTMR4 abolished binding to both proteins. These results were not simply due to titration with a higher concentration of GST protein since the addition of up to SO /*g of GST did not eliminate all MINREC4 binding to GTMR4 (Fig. 2L)). These results show that MINREC4 binds selectively to both GST and the type I receptor fragment of GTMR4. In order to confirm that the MINREC4 antiserum would be suitable for the immunohistological detection of the type I receptor we performed immunostaining on sections of rat kidney and liver using MINREC4 and BUGR2, the latter monoclonal antiserum was generated against the closely related type II adrenocorticoid receptor (Eisen et al., 1985). In the kidney, the prototypic
25
LANE
1
2
LANE
1
2
C. GST+ GTMR4 I, 10
pg GST
10
pg GTMR4
D. GST+ GTMR4--+ 20
50
pg GST
Fig. 2. Production of the GTMR4 fusion protein and characterization of the MINREC4 antiserum. A: Coomassie stained SDS gel showing the GST and GTMR4 proteins after single step purification on glutathione agarose beads. Lane 1 contains GST, the GTMR4 preparation was loaded in lane 2. 10 pg of protein was loaded in each lane. B: Western blot analysis of GST and GTMR4 proteins using the anti-GST antiserum. 1 pg of protein was loaded in each lane. Lane 1 contains GST protein, the GTMR4 preparation was run in lane 2. C: Binding of MINREC4 antiserum to GST and GTMR4 proteins in the presence and absence of immunogen. GST (0.1 kg) or GTMR4 (0.1 Fg1 were dotted onto nitrocellulose filters in duplicate and the membranes probed with MINREC4 antiserum in the absence of competitor or after preincubation of the antiserum with either 10 Fg GST or 10 Fg GST plus 10 pg GTMR4. D: Titration of the MINREC4 antiserum with increasing amounts of GST protein.
mineralocorticoid target tissue, predominantly nuclear staining was observed in epithelial cells of the distal convoluted tubule and collecting tubule (Fig. 3A). This staining could be abolished by preincubation of antiserum with the GTMR4 fusion protein (results not shown). Furthermore, the tubular staining pattern obtained using MINREC4 in the absence of competing antigen was identical to that obtained in our previous study using the MINREC2 antiserum Krozowski et al., 1989). In the liver, where high concentrations of the type II receptor and glutathione S-transferase
are known to be present (Eisen et aI., 1985; Johnson et al., 19901, MINREC4 staining was clearly absent (Fig. 3Bl. Liver sections stained for type II receptor with BUGR2 showed predominantly nuclear staining (Fig. 3C). When MINREC4 antiserum was used to stain sections of rat parotid gland the results shown in Fig. 4 were obtained. In the parotid gland of intact rats intense immunoreactivity was observed in ductal epithelial cells, but not in glandular acinar cells (Fig. 4A). The specificity of ductal staining was confirmed when immunoreactivity
26 Fig. 3. Type I and type II receptor-like immunoreactivity in kidney and liver. A: MINREC4 staining of rat kidney. Type I receptor-like immunoreactivity was predominantly observed in nuclei of distal tubular epithelial cells (arrows). B: MINREC4 staining of rat liver. Note the absence of staining in hepatocytes. C: BUGRZ staining of rat liver. Type II receptor-like immunoreactivity appeared as intense, predominantly nuclear staining in hepatocytes. Scale bar: SO pm. Magnification: x 700.
B
’
* j
-*
1 XL ~ ‘I $
‘1;,a _ *is* i _
_
$8> =;f. --
_,L x ” ‘~.,*.*#_.p & yy” a,> ‘,,Z” : ; ,, and crypts of Lieberkuhn of intact rat colon by preincubation of the antiserum with GTMR4 fusion protein. Note the persistence of non-specific signal in the muscle layer. C: MINREC4 staining of a transverse section of crypts of Lieberkuhn in an intact rat. Solid arrow: cell with nuclear immunostaining. Open arrow: cell showing extension of immunostaining into apical (luminal) portion. Goblet cells are devoid of staining. Scale bar: 200 pm (A, B), 50 pm (Cl. Magnification: X 150 (A, B), X560 (Cl.
cytoplasmic with no nuclear labeling. MINREC4 immunoreactivity was also reduced in the submandibular glands of adrenalectomized rats, although not to the same extent as in the parotid; however, hormone replacement restored immunostaining to intact levels in the submandibular ducts (results not shown). When MINREC2 antiserum was used on sections of parotid gland
from intact, adrenaIectomized or adrenalectomized plus hormone treated rats a higher background immunoreactivity was obtained but the pattern of staining was essentially the same. A parallel study was performed on the distal colon from the same rats using both the MINREC2 and MINREC4 antisera. Surface columnar cells of the mucosa of the distal colon showed
29
Fig. 6. Effect of ligand on MINREC4 staining in distal colonic mucosa. A: Reduction in MINREC4 immunostaining in surface columnar cells 1 week post-adrenalectomy. B: Restoration of MINREC4 immunostaining in colonic mucosa of 1 week adrenalectomized rat by administration of corticosterone 1 h before sacrifice. Identical results were obtained when aldosterone was used instead of corticosterone. Scale bar: 300 pm. Magnification: X 90.
intense immunoreactivity when stained with the MINREC4 antiserum (Fig. 5A) and this staining could be abolished by preabsorption of the antiserum with the GTMR4 antigen (Fig. 5B). Some cells in the crypts of Lieberkuhn showed apical as well as nuclear immunoreactivity, while goblet cells were distinctly devoid of staining (Fig. 50 Immunostaining in the surface columnar cells and crypts of Lieberkuhn could be abolished by adrenalectomy (Fig. 6A) while the administration of corticosterone or aldosterone to adrenally ablated rats 1 h before sacrifice restored a considerable amount of staining (Fig. 6B). In the colon MINREC2 and MINREC4 antisera produced identical patterns of immunoreactivity. Discussion
The parotid gland and the colon represent two sodium transporting tissues which modulate total body sodium by regulating the amount of salt resorbed from saliva and colonic fluids. Type I receptors have been shown to be aldosterone selective in vivo in both tissues (Sheppard and
Funder, 19871, with the concentration in the colon being amongst the highest of all tissues studied (Schulman et al., 1986). We have examined the distribution of the type I receptor in these tissues using an antiserum raised against a 167 amino acid N-terminal portion of the protein and verified the results using antibodies raised against a 16 amino acid synthetic peptide derived from the linker region of the receptor. The specificity of the MINREC4 immunostaining on tissue sections was confirmed by a number of criteria. Firstly, MINREC2 and MINREC4 antisera showed identical staining patterns when used to immunostain salivary gland or colonic tissues. Secondly, MINREC4 antibodies could be competed for by antigen, could be diluted out and did not bind rat glutathione S-transferase or the type II receptor, structurally the most similar to the type I receptor (Arriza et al., 1987). Thirdly, MINREC4 staining was present in distal and collecting duct epithelial cells of the kidney, in agreement with previous reports (Arriza et al., 1988; Krozowski et al., 1989; Lombes et al., 1990).
30
Fourthly, MINREC4 immunostaining was modulated by aldosterone and corticosterone, the cognate ligands of the type I receptor. In addition others have recently used MINREC4 to detect a highly specific, approximately 100 kDa band on Western blots of partially purified human kidney and ileum preparations, consistent with the detection of type I receptor (Fukushima et ai., 1992). In the brain MINREC4 has been used to map the distribution of type I sites during development; an excellent correlation was observed between the localization of immunoreactivity and previous reports of corticosteroid binding (Lawson et al.. 1991). In the present study immunostaining of tissue sections showed that the distribution of the type I receptor was highly cell specific and appeared to be concentrated in those cells with secretory or absorptive functions. Epithelial cells of the ductal elements of the salivary glands and the surface epithelial and absorptive ceils of the coIonic crypts showed intense specific type I receptor-like immunorea~tivity. Neither the glandular exocrine ceils of the parotid and submandibular glands nor goblet cells of the colonic crypts contained type I receptor. In parotid ducts staining was often concentrated in the apical region of cells. We have previously observed apical staining in the cortica1 collecting ducts of the kidney when using the MINREC2 antiserum (Krozowski et al., 1989). It is tempting to speculate that apical staining represents some non-genomic effect of the type I receptor such as binding to luminai ionic channels or is associated with induction of a second messenger system as proposed for the glucocorticoid receptor in rat hepatoma cells (Simons et al., 1989). Since adrenalectomy abolished and corticosteronc restored immunostaining, in both the salivary gland and colon when using either antiserum, it appears that antibody recognition is contingent on the presence of ligand. Ligand binding to type I receptors may unmask immunogenic epitopes by altering receptor conformation, or it could reduce the interaction between unliganded receptor and other intracellular proteins which may be masking these epitopes. Ligand binding has been shown to dissociate the hsp90
protein from the glucocorticoid receptor (Denis et al., 1988). In another study antibodies directed at the DNA binding domain of the progesterone receptor were found to bind to transformed, but not untransformed receptor (Smith et al., 1988). It is also possible that the ligand alters type I receptor conformation indirectly by stimulating phosphoryIation of the hormone binding domain as has been shown in studies with the estrogen receptor (Auricchio et al., 1987). Though the epitopes recognised by the MINREC2 and MINREC4 antisera are widely spaced on the type I receptor, and it is possible that different mechanisms facilitate recognition of the epitopes after hormone binding, the staining parallelism between the two antibodies suggests a common mechanism of epitope revelation such as the dissociation of a protein. Furthermore, recent studies from our laboratory have demonstrated cell specific, ligand mediated induction and abolition of type I and type II immunoreactivity in the rat central nervous system, consistent with the involvement of trans-acting factors in the modulation of immunostaining (Ahima et al., 1991; Ahima and I-Iarlan, 1991). The present study confirms previous observations (Krozowski et al., 1989; Lombes et al., 1990) that the intracellular distribution of the type I receptor is both cytoplasmic and nuclear in the presence of hormone, resembling the distribution of the type II glucocorticoid receptor (Gustafsson et al., 19871, but contrasting with the exclusive nuclear vocalization of the estrogen (King and Green, 1984; Welshons et al., 1984), progesterone (Perrot-Applanat et al., 1985) and androgen receptors (Chang et al., 1989). A comparison of the nuclear localization mechanism of the glucocorticoid and estrogen receptors has shown that nuclear localization is differentially regulated in these two receptors. In contrast to the glucocorticoid receptor, the estrogen receptor lacked a second nuclear localization domain within the hormone binding region. It has been proposed that the second domain possesses a nuclear translocation inactivation function which is erased after hormone activation of the receptor (Picard et al., 1990). The cytoplasmic localization of the type I receptor suggests that it too may possess a translocation inactivation function.
31
While MINREC4 staining was clearly localized in the apical region and nucleus of the parotid of intact rats, in adrenalectomized rats injected with corticosterone or aldosterone parotid staining was diffuse over most of the cell. In the submandibular gland and colon, however, corticosterone or aldosterone replacement resulted in a staining pattern which was not different from that of intact rats. These results suggest that different mechanisms may be involved in eliciting the hormonal response in the parotid; in particular the failure of parotid MINREC4 staining to reach pre-adrenalectomy levels 1 h after administration of hormone to adrenalectomized rats may simply reflect an incomplete revelation of epitopes during this period or it may be related to the turnover or synthesis of new receptor. Further work needs to be done in the salivary glands and colon in order to understand the kinetics of receptor turnover following hormonal withdrawal. It appears that both the kinetics and conformation of the type I receptor may be modulated in a tissue specific and hormone dependent manner. Even though the parotid gland and colon are aldosterone selective in vivo, we have shown that corticosterone can modulate type I receptor-like immunoreactivity in these tissues. It would appear that, under the conditions used in the present study, the level of intracellular llp-hydroxysteroid dehydrogenase activity is insufficient to keep corticosterone from binding to the type I receptor. The availability of the MINREC4 antibody should permit mapping of the anatomical distribution of the type I glucocorticoid receptor in peripheral tissues and the brain where the role of corticosteroids in learning, memory and stress as well as cardiovascular regulation is well known. Both MINREC antibodies appear to recognize ligand dependent conformations of the receptor and an examination of the immunoreactivity of different hormone-receptor complexes may lead to a better understanding of the mechanism of corticosteroid action. Our results suggest that there may be tissue specific differences in type I receptor activation and that the MINREC antibodies could be used to further explore this hypothesis.
Acknowledgement
The constant support of Professor J.W. Funder throughout these studies is gratefully acknowledged. References Ahima, R.S. and Harlan, R.E. (1991) Endocrinology 129, 226-236. Ahima, R., Krozowski, Z. and Harlan, R. (1991) J. Comp. Nemo]. 313, 1-17. Arriza, J.L., Weinberger, C., Cerelli, G., Glaser, T.M., Handelin, B.L., Housman, D.E. and Evans, R.M. (1987) Science 231, 268-275. Arriza, J., Simerly, R., Swanson, L. and Evans, R.M. (1988) Neuron 1, 887-900. Auricchio, F., Migliaccio, A., Di Domenico, M. and Nola, E. (1987) EMBO J. 6, 2923-2929. Beaumont, K. and Fanestil, D.D. (1983) Endocrinology 113, 2043-2049. Chang, C., Whelan, C.T., Popovich, T.C., Kokontis, J. and Liao, S. (1989) Endocrinology 123, 1097-1099. Denis, M., Poellinger, L., Wikstom, A.C. and Gustafsson, J.A. (1988) Nature 333, 686-687. Edwards, C.R.W., Stewart, P.M., Burt, D., Brett, L., McIntyre, M.A.. Sutanto, W.S., DeKloet, E.R. and Monder, C. (1988) Lancet ii, 986-988. Eisen, L.P., Reichman, M.E., Thompson, E.B., Gametchu, B., Harrison, R.W. and Eisen, H.J. (198.5) J. Biol. Chem. 260, 11805-11810. Fukushima, K., Sasano, H., Nagura, H., Sasaki, I., Matsuo, S. and Krozowski, Z. (1992) Ann. Surg. (submitted). Funder, J.W., Pearce, P.T., Smith, R. and Smith, I. (1988) Science 242, 583-585. Gronemeyer, H., Turcotte, B., Quirin-Stricker, C., Bocquel, M.T., Meyer, M.E., Krozowski, Z., Jeltsch, J.M., Lerouge, T., Garnier, J.M. and Chambon, P. (1987) EMBO 6, 3985-3994. Gustafsson, J.A., Carlstedt-Duke, J., Poellinger, L., Okret, S., Wilkstrom, A.C., Bronnegard, M., Gillner, M., Dong, Y., Fuxe, K., Cintra, A., Harfstrand, A. and Agnati, L. (1987) Endocr. Rev. 8, 185-234. Hopp, T.P. and Woods, K.R. (1981) Proc. Natl. Acad. Sci. USA 78, 3824-3828. Johnson, J.A., Neal, T.L., Collins, J.H. and Siegel, F.L. (1990) Biochem. J. 270, 483-489. King, W.J. and Green, G.L. (1984) Nature 307, 745-746. Krozowski, Z. (1989) Mol. Cell. Endocrinol. 63, 15-22. Krozowski, Z.S. and Funder, J.W. (1983) Proc. Natl. Acad. Sci. USA 80, 6056-6060. Krozowski, Z.S., Rundle, S.E., Wallace, C., Castell, M.J., Shen, J.H., Dowling, J., Funder, J.W. and Smith, A.Z. (1989) Endocrinology 12.5, 192-198. Krozowski, Z.S., Stuchbery, S., White, P., Monder, C. and Funder, J.W. (1990) Endocrinology 127, 3009-3013.
32 Lawson, A., Ahima, R., Krozowski, Z.S. and Harlan, R. (1991) Dev. Brain Res. 62, 69-79. Lombes, M., Farman, N., Oblin, M.E., Baulieu, E.E., Bonvalet, J.P., Erlanger, B.F. and Gasc. J.M. (1990) Proc. Nat]. Acad. Sci. USA 87, 1086-188. Patel, P.D., Sherman, T.G., Goldman, D.J. and Watson, S.J. 11989) Mol. Endocrinol. 3, 1877-1885. Perrot-Appianat, M., Logeat, F., Groyer-Picard, M.T. and Milgrom, E. (1985) Endocrinology 116, 3473-1489. Rundle, S.E., Funder, J.W., Lakshmi, V. and Monder, C. (1989) Endocrinology 125, 1700-1704. Schulman, G., Miller-Diener, A., Lihvack, G. and Bastl, C.P. (1986) J. Biol. Chem. 261, 12102-12106.
Sheppard, K. and Funder, J.W. (1987) Am. J. Physiol. 253, E467-E471. Simons, S.S., Mercier, L.. Miller, N.R., Miller, P.A., Oshima, H., Sistare, F.D., Thompson, E.B., Wasner. G. and Yen, P.M. (1989) Cancer Res. 49, 2244s-2252s. Smith, D.B. and Johnson, K.S. (1988) Gene 67, 31-40. Smith, D.F., Lubahn, D.B., McCormick, D.L., Wilson, E.M. and Toft, D.O. (1988) Endocrinology 122, 2816-2825. Welshons, W., Lieberman, M.E. and Gorski, J. (1984) Nature 307. 747-749.
MOLCEL
21
85 (1992) 21-32 Ireland, Ltd. 0303-7207/92/$05.00
02738
Type I corticosteroid receptor-like immunoreactivity in the rat salivary glands and distal colon: modulation by corticosteroids Zygmunt Krozowski a, Kelli Wendell a, Rexford Ahima b and Richard Harlan b aPrince Henry’s Institute of Medical Research, Prince Henry’s Hospital, Melbourne, Vie. 3205, Australia and ’ Department of Anatomy, Tulane Unicersity Medical Center, New Orleans, LA 70112, USA (Received
Key words: Receptor,
type I; Mineralocorticoid
20 August
receptor;
1991; accepted
Colon rat; Parotid
2 January
gland;
1992)
Fusion
protein;
Antibody
Summary A 167 amino acid fragment of the N-terminal domain of the human type I corticosteroid (mineralocorticoid) receptor was fused to the glutathione S-transferase gene using the Gex expression plasmid and the fusion protein used to raise the monospecific polyclonal antibody, MINREC4. Immunostaining experiments showed that MINREC4 specifically bound type I receptor in the distal tubule of the kidney, the ductal elements of the salivary glands and the epithelium of the distal colon in the rat. Adrenalectomy abolished staining in the parotid and colon, and reduced immunoreactivity in the submandibular gland. Administration of corticosterone or aldosterone resulted in partial restoration of immunostaining in the parotid, and a complete restoration of staining to intact levels in the submandibular gland and colon. These results suggest that adrenocorticoid binding to the type I receptor may result in tissue specific conformational changes in the binding protein and that the MINREC4 antibody may be used to study these effects.
Introduction The type I adrenocorticoid (mineralocorticoid) receptor displays equivalent affinity for corticosterone and aldosterone (Beaumont and Fanestil, 1983; Krozowski and Funder, 1983). It is thought that the presence of lip-hydroxysteroid dehydrogenase in target cells serves to reduce the high level of glucocorticoid and allows aldosterone access to the type I receptor in mineralocorticoid selective target tissues (Edwards et al., 1988; Funder et al., 1988). However, other evidence sug-
Correspondence to: Dr. Z. Krozowski, Baker Institute of Medical Research, P.O. Box 348, Prahran, Vie. 3181, Australia. Fax 61-3-521.1362.
gests that the specificity conferring mechanism may be far more complex (Rundle et al., 1989; Krozowski et al., 1990). The availability of a panel of monospecific antibodies to the type I receptor may aid the resolution of this conundrum. We have previously raised a polyclonal antibody to the type I receptor by immunizing with a synthetic peptide corresponding to a region between the DNA and hormone binding domains of the human mineralocorticoid receptor (Krozowski et al., 1989). The overall pattern of staining obtained in that study has recently been confirmed by use of an auto-anti-idiotypic antibody which recognizes the unbound form of the receptor (Lombes et al., 1990). However, the subcellular distribution of staining between the two studies appeared to differ, possibly reflecting the de-
22
tection of bound and unbound forms of the receptor. These observations suggest that different antisera may be useful in discriminating between various conformations of the receptor and may aid the elucidation of the specificity conferring mechanism in mineralocorticoid selective target tissues. In our continuing efforts to raise a battery of polyclonal antibodies to the type I receptor we have constructed a fusion protein comprising part of the N-terminus of the receptor fused to glutathione S-transferase. The full-length fusion protein can be expressed in Escherichia coli and can be purified by a single step on a glutathione affinity column. Antibodies raised against the fusion protein were used to map type I receptor distribution in various tissues in the presence and absence of corticosteroids. The results obtained with this antiserum were then compared with those obtained using the previously described MINREC2 antibody. Materials
and methods
Production of bacterial proteins A 497 base pair BamHI (1273 bp) to EcoRI (1770 bp) fragment of the human type I adrenocorticoid receptor cDNA (Arriza et al., 19871, corresponding to G1y-&er5i9, was cloned into the multiple cloning site of the pGEX2 expression vector (Smith and Johnson, 1988) to produce the pGTMR500 recombinant plasmid. Either pGEX2 or pGTMR500 was transfected into Escherichia coli. JM109 cells, an overnight culture of the cells diluted 1: 10 in culture medium and grown for 1 h at 37°C before induction of protein synthesis with 0.1 mM isopropylthiolgalatoside. After a further 3 h of growth the cells were pelleted and resuspended in phosphate-buffered saline (PBS) containing 1% Triton X-100 and 0.1 mM phenylmethylsulfonyl fluoride (PMSF). E. coli were lysed by freezing at -20°C overnight and thawing at room temperature. Centrifugation of the lysate at 10,000 X g for 5 min yielded a clear supernatant which was mixed at room temperature for 30 min with preswollen agarose beads. The beads were pelleted at 500 X g for 10 s and washed 3 times with PBS containing 0.1% Triton X-100. Protein was eluted from the affinity
beads by competition with free glutathione in 50 mM Tris pH 8.0 containing 5 mM reduced glutathione. The fusion protein derived from pGTMR500 was designated GTMR4 while the parent plasmid pGEX2 produced glutathione Stransferase (GST). Bacterial proteins were freeze-dried (Speed-Vat) and stored at - 20°C. Immunization and screening protocol Three New Zealand White rabbits were injected subcutaneously along the spine with 100 pg GST protein in complete adjuvant and were boosted 4 weeks later with 500 pg GST in complete adjuvant. Rabbits were bled every 2-3 weeks. Serum was separated by incubating the blood at 37°C for 1 h, storing overnight at 4°C and then spinning down the red blood cells. AntiGST antibodies appeared 2 weeks after the booster injection of GST, were of maximum titre by 7 weeks and then showed a decline in antibody titre by week 10. All rabbits were then injected with 5 pg GTMR4 in complete adjuvant and boosted 4 weeks later with 50 pg GTMR4 in complete adjuvant. The appearance of anti-GST and anti-GTMR4 antibodies was monitored by dot blot analysis of GST and GTMR4 proteins using a 1: 1000 dilution of antiserum preincubated for 1 h at room temperature in the presence or absence of 10 pg GST or 10 pg GST plus 10 pg GTMR4. GST (0.1 pg) and GTMR4 (0.1 pg> were spotted onto nitrocellulose filters in 1 ~1 PBS and the filters processed as described for Western blot analysis. Rabbit C showed high levels of anti-GTMR4 antibodies and was bled out 1 month after injection of the booster dose of GTMR4; the antiserum thus obtained was designated MINREC4. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot analysis was performed as previously described (Krozowski, 1989) except that gel electrophoresis was performed using a 10% polyactylamide gel and Western blot analysis was performed using a 1: 200 dilution of anti-GST antiserum. Animals Adult male Sprague-Dawley rats were used throughout the study. Rats were kept under standard lighting conditions (12 h light and 12 h dark)
and allowed free access to normal rat chow. Control rats were allowed free access to tap water and adrenalectomized rats to normal saline. Adrenalectomized rats were used l-2 weeks post-surgery. The adrenalectomized rats were divided into three treatment groups with four rats per group. The control group received 200 ~1 of 0.005% ethanol/PBS vehicle intraperitoneally, a second group received 1 mg/lOO g body weight corticosterone and a third group was injected with 1 pg/lOO g body weight aldosterone. Rats were sacrificed 1 h after steroid administration. All animals were killed with an overdose of sodium pentobarbital intraperitoneally, perfused intracardially with PBS for 5 min followed by 3% phosphate-buffered paraformaldehyde for 8 min. Salivary glands and segments of distal colon were dissected out and postfixed in 3% paraformaldehyde for 2 h followed by cryoprotection in 30% sucrose. The tissues were then frozen on dry ice and stored at -70°C until sectioned. Pieces of kidney and liver were also taken from intact rats after perfusion fixation, postfixed for 6 h and processed for paraplast embedding as previously described (Krozowski et al., 1989). Sections 30 pm thick were cut on a cryostat, collected onto chromalum-subbed slides and stored with desiccant at -70°C until use. 7 pm thick sections of the paraplast embedded kidney and liver were also cut and collected onto gelatin-coated slides. Imtnunocytochemistry (i) SaliLlary glands and distal colon. Sections were dried in a vacuum chamber, rinsed in PBS for 10 min and permeabilized with 0.2% Triton X-100 for 10 min. Non-specific staining was blocked by incubating the sections in 0.1% PBS/bovine serum albumin (BSA) plus 0.02% normal goat serum for 20 min. Sections were then rinsed in PBS for 10 min and incubated with a 1: 1000 dilution of either MINREC2 or MINREC4 antiserum for 48 h at 4°C. Adjacent control sections were incubated with either normal rabbit serum, MINREC2 antiserum (1: 1000) preabsorbed with 10 pmol of MINREC2 peptide (Krozowski et al., 19891, MINREC4 antiserum
(1: 1000) preabsorbed with 10 pmol of GTMR4 fusion protein or GST protein, or a 1: 500 dilution of GST antiserum. All preabsorption steps were carried out at 4°C for 24 h. (ii) Kidney and lkler. Sections were deparaffinized with two 3 min changes of xylene, immersed for 1 min each in lOO%, 95% and 70% ethanol and hydrated in PBS. Endogenous peroxidase was reduced by immersing the sections in 0.3% hydrogen peroxide in methanol for 15 min and then rinsing in PBS for 10 min. Sections were then processed for immunocytochemical localization of type I receptor using MINREC2 and MINREC4 antisera, or type II (glucocorticoid) receptor using the BUGR2 monoclonal antibody (Eisen et al., 1985). Kidney and liver sections were incubated with the same dilutions of MINREC antisera and preabsorption controls as used for the salivary glands and colon. BUGR2 antibody was used at a dilution of 1: 500. A control section for the monoclonal antibody study was incubated with non-immune P3 AgX-653 myeloma cell supernatant. Sections were processed for immunostaining using the Vectastain ABC immunoperoxidase (goat anti-rabbit and horse anti-mouse) kits for MINREC antisera and BUGR2 respectively. The reaction product was visualised using 3,3’-diaminobenzidine (DAB) tetrahydrochloride or nickel-DAB as chromogen. The reaction was stopped by immersion of the sections in PBS, the sections dehydrated in 70%, 95% and 100% ethanol, defatted in Histoclear (National Diagnostics), and coverslipped using Permount (Fisher Scientific).
Microscopy Sections were viewed with the aid of a Nikon Optiphot microscope and photographs taken with a Nikon FX35A camera and Technical Pan (Kodak) film. Slides of salivary glands and distal colon were coded by one investigator and analysed blindly by two others in order to determine (i) cell types exhibiting MINREC immunostaining and (ii) intracellular distribution of MINREC immunostaining.
24 602670734
1
ICIO]
351 m
964 E
hMR MINREC 4
516 672 @6?
MINREC 2
Fig, 1. Schematic representation of the human type 1 receptor showing locations of the immunogens used to raise the MINREC4 and MINREC2 polyclonal antibodies. Numbering refers to the location of amino acids according to Arriza et al. (1987). Lettering designates domains of the receptor according to Gronemeyer et al. (1987): A/B is the N-terminal domain, some of which is highly immunogenic, C is the DNA binding domain, D is the linker region and E represents the hormone binding domain. The GTMR4 fusion protein used as an immunogen to generate the MINREC4 antibody consists of glutathione S-transferase (GST) linked to the N-terminus of the 167 amino acid peptide.
Results Early studies on steroid receptors showed that the N-terminus of these proteins contained a highly immunogenic domain (for a review see Gustafsson et al., 1987). Fig. 1 shows the location of the MINREC4 immunogen used in the present study in relation to MINREC2 epitopes. A hydrophilicity profile of the receptor protein (Hopp and Woods, 1981) showed seven regions of high antigenicity within a 167 amino acid fragment, including a peptide with the highest predicted antigenicity of the A/B domain. The MINREC4 immunogen of the human receptor is 88% identical with the corresponding region of the rat protein (Pate1 et al., 1989). The expression of foreign proteins in E. cofi frequently results in degradation or insolubility of the protein of interest. Often both these problems can be overcome by fusing mammalian peptides to proteins that are stable in bacterial cells. In the present study we have fused 167 amino acids of the human type I receptor with the carboxy terminus of glutathione S-transferase (EC 2.5.1.18), an enzyme cloned from the parasite Schistosoma juponicum (Smith and Johnson, 1988). Fig. 2A shows a Coomassie stained SDSpolyac~lamide gel of the GST and GTMR4 protein preparations after affinity purification. GST displayed a molecuIar weight of 28 kDa while GTMR4 showed the largest molecular weight species at 46 kDa. The 18 kDa difference be-
tween the molecular weights of GST and GTMR4 proteins is consistent with the addition of 167 amino acids of the type I receptor to GST. The GTMR4 protein showed variable degrees of degradation between preparations but the 46 kDa species was aIways present. Western blot analysis using the anti-GST antiserum (Fig. 2B) showed that CJST and GTMR4 were indeed related proteins. Furthermore, the major proteins migrating at 30-40 kDa in preparations of fusion protein also bound GST antiserum consistent with the existence of shorter forms of GTMR4. However, attempts to cIeave the 167 amino acid fragment from the GTMR4 fusion protein with thrombin were unsuccessful. In other experiments when the C-terminal half of the rat mineralocorticoid receptor was fused to the GST protein the fusion protein proved highly unstable and was not used in further studies (results not shown). When three rabbits were immunized with GST protein and then with GTMR4 one rabbit produced an antiserum (MINREC4) which reacted selectively with the receptor portion of the GTMR4 fusion protein. Fig. 2C shows the results obtained when GST and GTMR4 proteins were reacted with the MINREC4 antiserum in the presence or absence of competing antigens. In the absence of competitor MINREC4 bound both GST and GTMR4. Preincubating the antiserum in the presence of 10 pg GST abolished all binding to filter bound GST while binding to GTMR4 was only slightly reduced. Preincubation of the antiserum in the presence of both 10 pug GST and 10 pg GTMR4 abolished binding to both proteins. These results were not simply due to titration with a higher concentration of GST protein since the addition of up to SO /*g of GST did not eliminate all MINREC4 binding to GTMR4 (Fig. 2L)). These results show that MINREC4 binds selectively to both GST and the type I receptor fragment of GTMR4. In order to confirm that the MINREC4 antiserum would be suitable for the immunohistological detection of the type I receptor we performed immunostaining on sections of rat kidney and liver using MINREC4 and BUGR2, the latter monoclonal antiserum was generated against the closely related type II adrenocorticoid receptor (Eisen et al., 1985). In the kidney, the prototypic
25
LANE
1
2
LANE
1
2
C. GST+ GTMR4 I, 10
pg GST
10
pg GTMR4
D. GST+ GTMR4--+ 20
50
pg GST
Fig. 2. Production of the GTMR4 fusion protein and characterization of the MINREC4 antiserum. A: Coomassie stained SDS gel showing the GST and GTMR4 proteins after single step purification on glutathione agarose beads. Lane 1 contains GST, the GTMR4 preparation was loaded in lane 2. 10 pg of protein was loaded in each lane. B: Western blot analysis of GST and GTMR4 proteins using the anti-GST antiserum. 1 pg of protein was loaded in each lane. Lane 1 contains GST protein, the GTMR4 preparation was run in lane 2. C: Binding of MINREC4 antiserum to GST and GTMR4 proteins in the presence and absence of immunogen. GST (0.1 kg) or GTMR4 (0.1 Fg1 were dotted onto nitrocellulose filters in duplicate and the membranes probed with MINREC4 antiserum in the absence of competitor or after preincubation of the antiserum with either 10 Fg GST or 10 Fg GST plus 10 pg GTMR4. D: Titration of the MINREC4 antiserum with increasing amounts of GST protein.
mineralocorticoid target tissue, predominantly nuclear staining was observed in epithelial cells of the distal convoluted tubule and collecting tubule (Fig. 3A). This staining could be abolished by preincubation of antiserum with the GTMR4 fusion protein (results not shown). Furthermore, the tubular staining pattern obtained using MINREC4 in the absence of competing antigen was identical to that obtained in our previous study using the MINREC2 antiserum Krozowski et al., 1989). In the liver, where high concentrations of the type II receptor and glutathione S-transferase
are known to be present (Eisen et aI., 1985; Johnson et al., 19901, MINREC4 staining was clearly absent (Fig. 3Bl. Liver sections stained for type II receptor with BUGR2 showed predominantly nuclear staining (Fig. 3C). When MINREC4 antiserum was used to stain sections of rat parotid gland the results shown in Fig. 4 were obtained. In the parotid gland of intact rats intense immunoreactivity was observed in ductal epithelial cells, but not in glandular acinar cells (Fig. 4A). The specificity of ductal staining was confirmed when immunoreactivity
26 Fig. 3. Type I and type II receptor-like immunoreactivity in kidney and liver. A: MINREC4 staining of rat kidney. Type I receptor-like immunoreactivity was predominantly observed in nuclei of distal tubular epithelial cells (arrows). B: MINREC4 staining of rat liver. Note the absence of staining in hepatocytes. C: BUGRZ staining of rat liver. Type II receptor-like immunoreactivity appeared as intense, predominantly nuclear staining in hepatocytes. Scale bar: SO pm. Magnification: x 700.
B
’
* j
-*
1 XL ~ ‘I $
‘1;,a _ *is* i _
_
$8> =;f. --
_,L x ” ‘~.,*.*#_.p & yy” a,> ‘,,Z” : ; ,, and crypts of Lieberkuhn of intact rat colon by preincubation of the antiserum with GTMR4 fusion protein. Note the persistence of non-specific signal in the muscle layer. C: MINREC4 staining of a transverse section of crypts of Lieberkuhn in an intact rat. Solid arrow: cell with nuclear immunostaining. Open arrow: cell showing extension of immunostaining into apical (luminal) portion. Goblet cells are devoid of staining. Scale bar: 200 pm (A, B), 50 pm (Cl. Magnification: X 150 (A, B), X560 (Cl.
cytoplasmic with no nuclear labeling. MINREC4 immunoreactivity was also reduced in the submandibular glands of adrenalectomized rats, although not to the same extent as in the parotid; however, hormone replacement restored immunostaining to intact levels in the submandibular ducts (results not shown). When MINREC2 antiserum was used on sections of parotid gland
from intact, adrenaIectomized or adrenalectomized plus hormone treated rats a higher background immunoreactivity was obtained but the pattern of staining was essentially the same. A parallel study was performed on the distal colon from the same rats using both the MINREC2 and MINREC4 antisera. Surface columnar cells of the mucosa of the distal colon showed
29
Fig. 6. Effect of ligand on MINREC4 staining in distal colonic mucosa. A: Reduction in MINREC4 immunostaining in surface columnar cells 1 week post-adrenalectomy. B: Restoration of MINREC4 immunostaining in colonic mucosa of 1 week adrenalectomized rat by administration of corticosterone 1 h before sacrifice. Identical results were obtained when aldosterone was used instead of corticosterone. Scale bar: 300 pm. Magnification: X 90.
intense immunoreactivity when stained with the MINREC4 antiserum (Fig. 5A) and this staining could be abolished by preabsorption of the antiserum with the GTMR4 antigen (Fig. 5B). Some cells in the crypts of Lieberkuhn showed apical as well as nuclear immunoreactivity, while goblet cells were distinctly devoid of staining (Fig. 50 Immunostaining in the surface columnar cells and crypts of Lieberkuhn could be abolished by adrenalectomy (Fig. 6A) while the administration of corticosterone or aldosterone to adrenally ablated rats 1 h before sacrifice restored a considerable amount of staining (Fig. 6B). In the colon MINREC2 and MINREC4 antisera produced identical patterns of immunoreactivity. Discussion
The parotid gland and the colon represent two sodium transporting tissues which modulate total body sodium by regulating the amount of salt resorbed from saliva and colonic fluids. Type I receptors have been shown to be aldosterone selective in vivo in both tissues (Sheppard and
Funder, 19871, with the concentration in the colon being amongst the highest of all tissues studied (Schulman et al., 1986). We have examined the distribution of the type I receptor in these tissues using an antiserum raised against a 167 amino acid N-terminal portion of the protein and verified the results using antibodies raised against a 16 amino acid synthetic peptide derived from the linker region of the receptor. The specificity of the MINREC4 immunostaining on tissue sections was confirmed by a number of criteria. Firstly, MINREC2 and MINREC4 antisera showed identical staining patterns when used to immunostain salivary gland or colonic tissues. Secondly, MINREC4 antibodies could be competed for by antigen, could be diluted out and did not bind rat glutathione S-transferase or the type II receptor, structurally the most similar to the type I receptor (Arriza et al., 1987). Thirdly, MINREC4 staining was present in distal and collecting duct epithelial cells of the kidney, in agreement with previous reports (Arriza et al., 1988; Krozowski et al., 1989; Lombes et al., 1990).
30
Fourthly, MINREC4 immunostaining was modulated by aldosterone and corticosterone, the cognate ligands of the type I receptor. In addition others have recently used MINREC4 to detect a highly specific, approximately 100 kDa band on Western blots of partially purified human kidney and ileum preparations, consistent with the detection of type I receptor (Fukushima et ai., 1992). In the brain MINREC4 has been used to map the distribution of type I sites during development; an excellent correlation was observed between the localization of immunoreactivity and previous reports of corticosteroid binding (Lawson et al.. 1991). In the present study immunostaining of tissue sections showed that the distribution of the type I receptor was highly cell specific and appeared to be concentrated in those cells with secretory or absorptive functions. Epithelial cells of the ductal elements of the salivary glands and the surface epithelial and absorptive ceils of the coIonic crypts showed intense specific type I receptor-like immunorea~tivity. Neither the glandular exocrine ceils of the parotid and submandibular glands nor goblet cells of the colonic crypts contained type I receptor. In parotid ducts staining was often concentrated in the apical region of cells. We have previously observed apical staining in the cortica1 collecting ducts of the kidney when using the MINREC2 antiserum (Krozowski et al., 1989). It is tempting to speculate that apical staining represents some non-genomic effect of the type I receptor such as binding to luminai ionic channels or is associated with induction of a second messenger system as proposed for the glucocorticoid receptor in rat hepatoma cells (Simons et al., 1989). Since adrenalectomy abolished and corticosteronc restored immunostaining, in both the salivary gland and colon when using either antiserum, it appears that antibody recognition is contingent on the presence of ligand. Ligand binding to type I receptors may unmask immunogenic epitopes by altering receptor conformation, or it could reduce the interaction between unliganded receptor and other intracellular proteins which may be masking these epitopes. Ligand binding has been shown to dissociate the hsp90
protein from the glucocorticoid receptor (Denis et al., 1988). In another study antibodies directed at the DNA binding domain of the progesterone receptor were found to bind to transformed, but not untransformed receptor (Smith et al., 1988). It is also possible that the ligand alters type I receptor conformation indirectly by stimulating phosphoryIation of the hormone binding domain as has been shown in studies with the estrogen receptor (Auricchio et al., 1987). Though the epitopes recognised by the MINREC2 and MINREC4 antisera are widely spaced on the type I receptor, and it is possible that different mechanisms facilitate recognition of the epitopes after hormone binding, the staining parallelism between the two antibodies suggests a common mechanism of epitope revelation such as the dissociation of a protein. Furthermore, recent studies from our laboratory have demonstrated cell specific, ligand mediated induction and abolition of type I and type II immunoreactivity in the rat central nervous system, consistent with the involvement of trans-acting factors in the modulation of immunostaining (Ahima et al., 1991; Ahima and I-Iarlan, 1991). The present study confirms previous observations (Krozowski et al., 1989; Lombes et al., 1990) that the intracellular distribution of the type I receptor is both cytoplasmic and nuclear in the presence of hormone, resembling the distribution of the type II glucocorticoid receptor (Gustafsson et al., 19871, but contrasting with the exclusive nuclear vocalization of the estrogen (King and Green, 1984; Welshons et al., 1984), progesterone (Perrot-Applanat et al., 1985) and androgen receptors (Chang et al., 1989). A comparison of the nuclear localization mechanism of the glucocorticoid and estrogen receptors has shown that nuclear localization is differentially regulated in these two receptors. In contrast to the glucocorticoid receptor, the estrogen receptor lacked a second nuclear localization domain within the hormone binding region. It has been proposed that the second domain possesses a nuclear translocation inactivation function which is erased after hormone activation of the receptor (Picard et al., 1990). The cytoplasmic localization of the type I receptor suggests that it too may possess a translocation inactivation function.
31
While MINREC4 staining was clearly localized in the apical region and nucleus of the parotid of intact rats, in adrenalectomized rats injected with corticosterone or aldosterone parotid staining was diffuse over most of the cell. In the submandibular gland and colon, however, corticosterone or aldosterone replacement resulted in a staining pattern which was not different from that of intact rats. These results suggest that different mechanisms may be involved in eliciting the hormonal response in the parotid; in particular the failure of parotid MINREC4 staining to reach pre-adrenalectomy levels 1 h after administration of hormone to adrenalectomized rats may simply reflect an incomplete revelation of epitopes during this period or it may be related to the turnover or synthesis of new receptor. Further work needs to be done in the salivary glands and colon in order to understand the kinetics of receptor turnover following hormonal withdrawal. It appears that both the kinetics and conformation of the type I receptor may be modulated in a tissue specific and hormone dependent manner. Even though the parotid gland and colon are aldosterone selective in vivo, we have shown that corticosterone can modulate type I receptor-like immunoreactivity in these tissues. It would appear that, under the conditions used in the present study, the level of intracellular llp-hydroxysteroid dehydrogenase activity is insufficient to keep corticosterone from binding to the type I receptor. The availability of the MINREC4 antibody should permit mapping of the anatomical distribution of the type I glucocorticoid receptor in peripheral tissues and the brain where the role of corticosteroids in learning, memory and stress as well as cardiovascular regulation is well known. Both MINREC antibodies appear to recognize ligand dependent conformations of the receptor and an examination of the immunoreactivity of different hormone-receptor complexes may lead to a better understanding of the mechanism of corticosteroid action. Our results suggest that there may be tissue specific differences in type I receptor activation and that the MINREC antibodies could be used to further explore this hypothesis.
Acknowledgement
The constant support of Professor J.W. Funder throughout these studies is gratefully acknowledged. References Ahima, R.S. and Harlan, R.E. (1991) Endocrinology 129, 226-236. Ahima, R., Krozowski, Z. and Harlan, R. (1991) J. Comp. Nemo]. 313, 1-17. Arriza, J.L., Weinberger, C., Cerelli, G., Glaser, T.M., Handelin, B.L., Housman, D.E. and Evans, R.M. (1987) Science 231, 268-275. Arriza, J., Simerly, R., Swanson, L. and Evans, R.M. (1988) Neuron 1, 887-900. Auricchio, F., Migliaccio, A., Di Domenico, M. and Nola, E. (1987) EMBO J. 6, 2923-2929. Beaumont, K. and Fanestil, D.D. (1983) Endocrinology 113, 2043-2049. Chang, C., Whelan, C.T., Popovich, T.C., Kokontis, J. and Liao, S. (1989) Endocrinology 123, 1097-1099. Denis, M., Poellinger, L., Wikstom, A.C. and Gustafsson, J.A. (1988) Nature 333, 686-687. Edwards, C.R.W., Stewart, P.M., Burt, D., Brett, L., McIntyre, M.A.. Sutanto, W.S., DeKloet, E.R. and Monder, C. (1988) Lancet ii, 986-988. Eisen, L.P., Reichman, M.E., Thompson, E.B., Gametchu, B., Harrison, R.W. and Eisen, H.J. (198.5) J. Biol. Chem. 260, 11805-11810. Fukushima, K., Sasano, H., Nagura, H., Sasaki, I., Matsuo, S. and Krozowski, Z. (1992) Ann. Surg. (submitted). Funder, J.W., Pearce, P.T., Smith, R. and Smith, I. (1988) Science 242, 583-585. Gronemeyer, H., Turcotte, B., Quirin-Stricker, C., Bocquel, M.T., Meyer, M.E., Krozowski, Z., Jeltsch, J.M., Lerouge, T., Garnier, J.M. and Chambon, P. (1987) EMBO 6, 3985-3994. Gustafsson, J.A., Carlstedt-Duke, J., Poellinger, L., Okret, S., Wilkstrom, A.C., Bronnegard, M., Gillner, M., Dong, Y., Fuxe, K., Cintra, A., Harfstrand, A. and Agnati, L. (1987) Endocr. Rev. 8, 185-234. Hopp, T.P. and Woods, K.R. (1981) Proc. Natl. Acad. Sci. USA 78, 3824-3828. Johnson, J.A., Neal, T.L., Collins, J.H. and Siegel, F.L. (1990) Biochem. J. 270, 483-489. King, W.J. and Green, G.L. (1984) Nature 307, 745-746. Krozowski, Z. (1989) Mol. Cell. Endocrinol. 63, 15-22. Krozowski, Z.S. and Funder, J.W. (1983) Proc. Natl. Acad. Sci. USA 80, 6056-6060. Krozowski, Z.S., Rundle, S.E., Wallace, C., Castell, M.J., Shen, J.H., Dowling, J., Funder, J.W. and Smith, A.Z. (1989) Endocrinology 12.5, 192-198. Krozowski, Z.S., Stuchbery, S., White, P., Monder, C. and Funder, J.W. (1990) Endocrinology 127, 3009-3013.
32 Lawson, A., Ahima, R., Krozowski, Z.S. and Harlan, R. (1991) Dev. Brain Res. 62, 69-79. Lombes, M., Farman, N., Oblin, M.E., Baulieu, E.E., Bonvalet, J.P., Erlanger, B.F. and Gasc. J.M. (1990) Proc. Nat]. Acad. Sci. USA 87, 1086-188. Patel, P.D., Sherman, T.G., Goldman, D.J. and Watson, S.J. 11989) Mol. Endocrinol. 3, 1877-1885. Perrot-Appianat, M., Logeat, F., Groyer-Picard, M.T. and Milgrom, E. (1985) Endocrinology 116, 3473-1489. Rundle, S.E., Funder, J.W., Lakshmi, V. and Monder, C. (1989) Endocrinology 125, 1700-1704. Schulman, G., Miller-Diener, A., Lihvack, G. and Bastl, C.P. (1986) J. Biol. Chem. 261, 12102-12106.
Sheppard, K. and Funder, J.W. (1987) Am. J. Physiol. 253, E467-E471. Simons, S.S., Mercier, L.. Miller, N.R., Miller, P.A., Oshima, H., Sistare, F.D., Thompson, E.B., Wasner. G. and Yen, P.M. (1989) Cancer Res. 49, 2244s-2252s. Smith, D.B. and Johnson, K.S. (1988) Gene 67, 31-40. Smith, D.F., Lubahn, D.B., McCormick, D.L., Wilson, E.M. and Toft, D.O. (1988) Endocrinology 122, 2816-2825. Welshons, W., Lieberman, M.E. and Gorski, J. (1984) Nature 307. 747-749.
