ARCHIVE23 OF BIOCHEMISTRY
AND BIOPHYSICS
Vol. 296, No. 1, July, pp. 198206,
Identification
1992
of Prolactin Receptors in Hepatic Nuclei
Arthur R. Buckley, *yl David W. Montg omery,?$lI Mary J. C. Hendrix,$ Charles F. Zukoski,$f and Charles W. Putnamt$lI *Department of Pharmacology, University of North Dakota School of Medicine, 501 North Columbia Road, Grand Forks, North Dakota 58203; Departments of ?Phurmacology, SSurgery, and $Anatomy and Cell Biology, University of Arizona College of Medicine, Tucson, Arizona 85724; and TResearch Service, Veterans Affairs Medical Center, Tucson, Arizona 85723
Received January
7,1992, and in revised form March 4,1992
Prolactin is a trophic hormone which may act directly at the hepatocyte nucleus. In this study, specific prolactin binding sites were sought in purified rat liver nuclei. Saturableandspecific,highaffinity128I-prolactinbinding sites were demonstrated to be on or within the nucleus. Prolactin binding was competitively inhibited by rat and ovine prolactins but not by rat growth hormone. Using immunogold electron microscopy, we detected prolactin receptors throughout the nucleus, in association with heterochromatin. Furthermore, endogenous immunoreactive prolactin was demonstrated to be within hepatic nuclei. We conclude that rat liver nuclei possess prolactin binding sites which likely participate in hormone-directed growth processes. c lee2 Academic PEW, I~C.
Evidence suggesting that prolactin may be a physiologically important hepatotrophic hormone has accumulated. Prolactin’s administration to rats elicits a growth response in liver characterized by expression of growthrelated genes (l), induction of ornithine decarboxylase (2, 3), stimulation of DNA synthesis (4, 5) and hypomethylation (6), and ultimately, increased hepatic mass (7). Notably, the hepatotrophic effects produced by prolactin are strikingly similar to the growth response in the liver following partial hepatectomy (g-lo), a procedure which rapidly elevates serum prolactin levels (9, 10). Prolactin appears to stimulate liver growth through activation of protein kinase C (10). Tumor promoting phorbol esters, which directly activate protein kinase C, mimic the hepatotrophic effects produced by prolactin treatment (11, 12). Moreover, prolactin administration cglses translocation of protein kinase C from the cytosol to the hepatic membrane, reflecting its activation (10). In uiuo 1 To whom correspondence
198
should be addressed.
and in primary hepatocyte cultures, prolactin-stimulated macromolecular synthesis is preceded by the generation of diacylglycerol, the physiologic activator of protein kinase C (1, 13). Other studies have shown that, in the nuclear compartment as well, prolactin is coupled to protein kinase C. The addition of prolactin to purified rat liver nuclei markedly enhanced protein kinase C activity, an effect again mimicked by phorbol esters (14). Furthermore, a monoclonal antibody to the rat liver prolactin receptor prevents the activation of nuclear protein kinase C by prolactin, suggesting that hepatocyte nuclei contain prolactin receptors (14, 15). In this report, we demonstrate for the first time that prolactin interacts with a specific, high affinity, immunoreactive binding site in rat hepatic nuclei. In addition, evidence is presented that normal rat liver nuclei contain endogenous prolactin which may bind to this site. These observations suggest that prolactin exerts at least a portion of its trophic actions directly at the nucleus. MATERIALS
AND
METHODS
Nuclear preparations. Hepatic nuclei were prepared from the livers of male Sprague-Dawley rats (100-140 g, Hilltop Lab Animals, Scottdale, PA) essentially as previously described (14) except that detergents were omitted in all of the buffers. Briefly, livers were perfused with 0.9% NaCl via portal vein cannulation, then minced, and washed in homogenization buffer (Tris-HCl, pH 7.4, 3 mM MgClz, 0.32 M sucrose, 2 mM ethylene glycol his@-aminoethyl ether) N,N’-tetraacetic acid (EGTA),’ 0.1 mM spermine, and 50 rg/ml of leupeptin. The tissue was homogenized using a glass Dounce homogenizer, filtered, and diluted to 0.2 M sucrose. Homogenization buffer was layered beneath the homogenate and the sample was centrifuged at 750g for 10 min at 4°C. The pellet was re-
2 Abbreviations used: EGTA, ethylene glycol bis(@-aminoethyl ether) N,N’-tetraacetic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; NRS, normal rabbit serum; FITC, fluorescein isothiocynate; FACS, fluorescence-activated cell sorting; EGF, epidermal growth factor.
0003-9&x31/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
NUCLEAR
PROLACTIN
199
RECEPTORS 6
120 looso
-
60
-
40
-
20 -
0
2
Time
4
of
6
Incubation
0
6
IO
Eomd
Nous)
20
hnol/5x
30
1 O6 Nuclei)
FIG. 1. Time course for ‘261-prolactin binding to rat liver nuclei. Nuclei (5 X 106) were incubated in quadruplicate with ‘BI-prolactin (0.1 nM) in the presence and absence of unlabeled hormone. Specific binding was determined as the difference between total (I’?-prolactin only) and nonspecific (‘261-prolactin plus 1 pg/tube unlabeled rat prolactin) binding. The experiment was repeated three times with similar results.
FIG. 3. Scatchard plot generated from an “‘1-prolactin competition experiment. Purified hepatic nuclei (1 X 106) were incubated in quadruplicate with ‘251-prolactin in the presence and absence of increasing unlabeled hormone (0.1-1000 nM) for 4 h at 37°C. A representative binding experiment is presented. Mean values: Kd = 0.42 f 0.07 nM; B mu = 29.2 ? 3.0 fmol/5 X lo6 nuclei (mean f SE, n = 6).
suspended in homogenization buffer, diluted, and again washed. The crude nuclear fraction was resuspended in a Tris-HCl buffer, pH 7.4, containing 2.4 M sucrose, 1.0 mM MgC&, 2.0 mM EGTA, 0.1 mM spermine, and 25 pg/ml leupeptin and centrifuged at 50,OOOgfor 60 min. The nuclei were washed and resuspended in 10 mM Tris-HCl (pH 7.4), 25% glycerol, and 1 mM MgClz . Purity of the nuclear preparations Verificution of nuclear purity. was assessed utilizing both morphological and biochemical criteria. For the morphological assessment, nuclei were fixed in 1% glutaraldehyde and embedded in LR white resin (Polysciences, Warrington, PA), and 60- to 80-nm sections were mounted on 200-mesh Ni grids. Subsequently,
the grids were stained with uranyl acetate and lead citrate prior to examination with a JEOL 100 CX transmission electron microscope, operating at 80 kV. Nuclear preparations were seen to be free of any visible cellular contamination (Figs. 5-8). Purity of the nuclei was also verified by biochemical means. Five different biochemical markers-three enzymes specific for plasma membrane (6-nucleotidase, phosphodiesterase I, and Na’,K+-ATPase) (16-18) and two for microsomes (NADPH-cytochrome c reductase and 2-acetylaminofluorene deacetylase) (19, 20)-were examined. Of the three plasma membrane markers (Table I), only 5’-nucleotidase activity was detectable in the nuclear preparations. If extrapolated to the entire liver, the nuclear level of 5’-nucleotidase represents less than 0.1% of total enzyme activity. Similar results were obtained when nuclear preparations were examined for the presence of microsomal enzyme markers.
‘6 20Yi +
Prolactin
3
60
.-E 2
40
WA)
FIG. 2. Saturation curve for ‘251-prolactin binding to rat liver nuclei. Nuclei (5 X 106) were incubated with increasing concentrations of lz51prolactin in the presence of 1 pg/tube unlabeled rat prolactin for 4 h at 37’C. Specific hormone binding was determined as the difference between total (1251-prolactin only) and nonspecific (‘251-prolactin plus 1 fig/tube unlabeled prolactin) binding. This experiment was repeated three times with similar results.
b
a
10
Competing
1
Ligand
0.1
Concentration
()lg/tube)
FIG. 4. Specificity of ‘%I-prolactin binding in rat liver nuclei. Purified liver nuclei (5 X 10”) were incubated in quadruplicate with “‘1-prolactin (0.1 nM) in the presence of competing ligands at the concentrations indicated for 4 h at 37°C. Data are presented as the mean -t SE of five separate experiments. CTL, ‘251-prolactin only.
BUCKLEY
FIG. 5. Electron microscopy of hepatic nuclei. Ultrathin conjugated anti-mouse IgG. Magnification, X19,000.
ET AL.
sections of purified
Radio&and binding studies. Binding experiments were performed with rat prolactin (rPRL-I-4, NIDDK), iodinated using a solid phase oxidizing reagent (Iodogen, Pierce, Rockford, IL). Radiolabeled prolactin (specific activity, approximately 50 &i/fig) eluted as a single peak from a Sephedex G-50 column and appeared as a single 24,000-Da band following SDS-PAGE and autoradiography. Radiolabeled prolactin retained full bioactivity, as assessed in the Nb2 lymphoma cell bioassay (21). Nuclear binding experiments were performed under conditions previously reported by us to be optimal for prolactin stimulation of nuclear protein kinase C (14). Purified rat liver nuclei were incubated with iz61-prolactin (150,000 cpm) in the presence and absence of increasing concentrations of unlabelled hormone (rat prolactin, B-6, ovine prolactin, O-19; rat growth hormone, GH-B-12; NIDDK) for 4 h at 37°C in a 10 mM sodium phosphate (pH 8.0) buffer containing 4 mM MgCIZ, 0.15 M NaCl, 0.1 mM spermine, 0.1% bovine serum albumin, and 25 pg/ ml leupeptin. Bound ‘261-prolactin was separated from free ligand by centrifugation at 12,000g for 3 min through a layer of 25% glycerol in binding buffer. The tubes were immediately frozen at -8O”C, the pellets was determined in a y counter. were cut out, and bound ‘%I-prolactin
nuclei were incubated
with PBS followed
by colloidal
gold-
Under these conditions, total and nonspecific binding were approximately 25 and 8%, respectively, of added iodinated prolactin. Immunogold electron microscopy. Sections (60-80 nm) of demonstrably pure nuclei mounted on ZOO-mesh Ni grids were initially incubated with 1% goat serum followed by monoclonal antibodies to the rat hepatic prolactin receptor (generously provided by Dr. Paul Kelly, INSERM, Paris, France), an irrelevant isotypic control monoclonal antibody (human myoglobin, IgGJ, or phosphate-buffered saline (PBS). Grids were incubated with antibody concentrations ranging from lo-’ to lo-’ M for 18 h at 4°C. Following extensive washing and a second blocking with 1% goat serum, the grids were incubated with goat antimouse IgG coupled to colloidal gold (20 nm, Polysciences). Subsequently, the grids were washed and analyzed by transmission electron microscopy. Fkwrescence-activated cell sorting (FAGS) of hpatk nuclei. To determine whether nuclei contain endogenous prolactin, purified hepatic nuclei were first exposed to goat serum (30 min, 4°C) and then incubated for 60 min at 4°C with a highly specific rabbit anti-rat prolactin antibody (&, dilution, Arnel, Cherokee Station, NY) or normal rabbit serum
NUCLEAR
PROLACTIN
RECEPTORS
201
FIG. 6. Electron microscopy of hepatic nuclei. Ultrathin sections of purified nuclei were incubated with an isotype control monoclonal antibody (anti-human myoglobin, IgG,) followed by colloidal gold-conjugated anti-mouse IgG. Magnification, ~19,000.
(NRS). The nuclei were subsequently washed and treated with an affinity-purified, FITC-conjugated goat anti-rabbit IgG (Fab’)r. Washed nuclei were then subjected to FACS (Becton-Dickinson, Braintree, MA), using an argon laser with excitation at 488 nm for FITC. RESULTS
Initially, the time course of ‘251-prolactin binding to rat liver nuclei was ascertained. Specific prolactin binding increased with time to 4 h, after which it remained stable through 6 h (Fig. 1). Since binding equilibrium was reached within 4 h of incubation, this interval was employed for all subsequent radioligand binding experiments. Total and nonspecific binding was found to increase at all radiolabel concentrations when purified nuclei were incubated with increasing concentrations of ‘251-prolactin.
In contrast, specific 1251-prolactin binding increased to 18-35 fmol/5 X lo6 nuclei, where it plateaued despite increasing radioligand concentrations (Fig. 2). Thus, specific binding of ‘251-prolactin to rat liver nuclei appears to be saturable. To determine the affinity as well as the binding capacity of the nuclear prolactin binding site, purified liver nuclei were incubated with 1251-prolactin(0.1 nM) in the presence or absence of increasing concentrations of unlabeled hormone. Figure 3 shows a Scatchard plot generated from one such displacement experiment. Analysis of these data reveals a high affinity (Kd = 0.42 -I 0.07 nM) nuclear 1251prolactin binding site with a capacity (B,,) of 29.2 k 3.0 fmol/5 X lo6 nuclei (n = 6). The affinity of the nuclear prolactin binding site correlates well with those reported
202
BUCKLEY
ET AL.
FIG. 7. Electron microscopy of hepatic nuclei. Ultrathin sections of purified nuclei were incubated with U6 (IgGI), a monoclonal the rat hepatic prolactin receptor, followed by colloidal gold-conjugated anti-mouse IgG. Magnification, X19,000.
for both the liver membrane prolactin receptor (22, 23) and the nuclear growth hormone binding protein (24). As mentioned above, a nuclear growth hormone receptor has been identified in liver (24). Since prolactins and growth hormones exhibit significant amino acid sequence homology (they are thought to be derived from a common ancestral gene (25)), it was important to determine the specificity for prolactin of the lz51-prolactin binding site. In inhibition experiments (Fig. 4) rat and ovine prolactins successfully compete with ‘251-prolactin for binding sites in nuclei; rat growth hormone did not, even at high concentrations. These data demonstrate that the [‘251]-prolactin binding site of hepatic nuclei is specific for lactogens and does not recognize a homologous but nonlactogenic hormone. Thus, this binding site appears to be specific for prolactin.
antibody
to
Immunogold electron microscopy was employed to visualize the nuclear prolactin receptor. Verifiably pure hepatic nuclei mounted on Ni grids were incubated with IgG1-subtype monoclonal antibodies specific for the rat hepatic prolactin receptor with antigenic domains either distinct from (U6) or directed to (Tl) the hormone binding site (26), a human myoglobin monoclonal antibody as an isotype control, or PBS alone. The results achieved by transmission electron microscopy are presented in Figures 5-8. The gold-conjugated secondary antibody was nonreactive when added to nuclei previously exposed to either PBS (Fig. 5) or the isotype control antibody (Fig. 6). In contrast, when nuclei were first incubated with anti-prolactin receptor antibodies followed by the immunogold-conjugated secondary antibody, both anti-
NUCLEAR
PROLACTIN
RECEPTORS
FIG. 8. Electron microscopy of hepatic nuclei. Ultrathin sections of purified nuclei were incubated with Tl (IgG1), a monoclonal the rat hepatic prolactin receptor, followed by colloidal gold-conjugated anti-mouse IgG. Magnification, X19,000.
receptor antibodies recognized the nuclear receptor (Figs. 7 and 8). Immunoreactive prolactin receptors are seen throughout the nuclei (arrows). Notably, immunoreactive prolactin receptors appear to be associated with heterochromatin, the most active sites for gene transcription (27). Prolactin is rapidly internalized by receptor-mediated endocytosis in several tissues, including liver (28). In pituitary mammotrophs, for example, lz51-prolactin has been shown to accumulate in and around the nucleus (29). Therefore, if prolactin exerts its effects via binding to a nuclear site, endogenous prolactin should be demonstrable in the nucleus. To test this hypothesis, purified liver nuclei were incubated with either a highly purified anti-rat prolactin antibody or NRS and then with FITC-conjugated,
203
antibody
to
affinity-purified goat anti-rabbit IgG (Fab’)z. The nuclei were subjected to FACS analysis and plotted as FITC fluorescent intensity versus size (forward light scatter). When normal hepatic nuclei were incubated with NRS as a control (Fig. 9a), two separate populations of low fluorescent intensity nuclei, differing in size, were detected. In contrast, the contour plot (Fig. 9b) obtained when the nuclei were first incubated with a highly specific antibody to rat prolactin (prior to the addition of the FITC-conjugated secondary antibody and FACS analysis) again showed two populations of nuclei. Now, however, both were of much higher fluorescent intensity than the controls. Hepatic nuclei therefore contain endogenous immunoreactive rat prolactin, presumably bound to the nuclear receptor.
204
BUCKLEY
ET AL. 64.
b .:: c
48
- 1;;;:;;;
p
.
. ...
,, .,. E F 8
32,..
8
4 ti 16.
.. ,..... B
.
16
FORWFiD LIGHT SCATTER
F-D
32
48
64
LIGHT SCATTER
FIG. 9.
Endogenous rat prolactin is present in purified liver nuclei. Purified liver nuclei were first incubated with either normal rabbit serum (a) or rabbit anti-rat prolactin antibody (b) prior to exposure to a FITC-conjugated goat anti-rabbit IgG (Fab’), and FACS analysis. These experiments were repeated three times with similar results. Total events at or above 1 were 19,181 (a) and 13,493 (b).
DISCUSSION
This report describes the first demonstration, to our knowledge, of a specific nuclear receptor for prolactin. Employing radioligand binding techniques, we found that rat hepatic nuclei possess high affinity binding specific for prolactin. Furthermore, this nuclear binding of prolactin was both time-dependent and saturable. Using monoclonal antibodies directed to the rat hepatic prolactin receptor, immunoreactive receptors were demonstrable within the nucleus but were not associated with the nuclear membrane. Significantly, the prolactin receptors appeared (by transmission electron microscopy) to be located within the condensed chromatin material. Since one of the earliest cellular effects provoked by exposure to prolactin is enhanced transcription (1) and since heterochromatin is the most active site for this process, it is possible that the nuclear prolactin receptor is directly coupled to expression of specific mRNAs, such as that for the protooncogene c-myc (1). Finally, we demonstrated immunoreactive prolactin within normal hepatocyte nuclei. The significance of the two populations, differing in size, found by FACS analysis is not clear. However, it is noteworthy that endogenous immunoreactive prolactin, possibly bound to a nuclear receptor, is present in purified nuclei prepared from normal animals. Historically, reports suggesting the existence of nuclear receptors for polypeptide hormones and growth factors
have been greeted with skepticism (30). Contamination of nuclei by nonnuclear cellular constituents could account for our observations. However, we have rigorously examined all of our nuclear preparations to ensure that they were always of the utmost purity. By both morphological and biochemical criteria, the nuclei employed in these studies were free of contamination by membrane or other cellular constituents (Table I and Figs. 5-8). While contamination of the nuclei by “invisible” receptors cannot be entirely excluded, it is notable that hepatic nuclei possess a relative abundance of prolactin receptors (about 3600/nucleus), specifically associated with heterochromatin, a hydrophilic nuclear constituent. Thus, it seems quite unlikely that contaminating “invisible” receptors could account for these results. A final criticism leveled against many nuclei isolation procedures is the use of detergents, such as Triton X-100 (30), which could potentially solubilize membrane receptors and cause their translocation onto hydrophobic nuclear regions. It is important to note that the nuclei utilized in all of the experiments presented were prepared in the absence of detergent. Thus, the data in toto demonstrate a true prolactin receptor present within the nucleus. Recently, a specific receptor for epidermal growth factor (EGF) in hepatocyte nuclei has been reported (31). EGF, like prolactin, stimulates hepatocyte replication (32-34), participates in the liver regeneration stimulated by partial hepatectomy (35, 36), and has been linked to activation
205
NUCLEAR PROLACTIN RECEPTORS TABLE I Characterization of Rat Hepatic Nuclei
Total protein % recovery
(mg)
DNA bd % recovery 5’-Nucleotidase specific activity Relative enrichment”
(nmol/min/mg
Phosphodiesterase I specific activity Relative enrichment”
protein)
(pmol/min/mg
protein)
Na+,K+-ATPasespecificactivity (pmol/min/mg protein) Relative
enrichment”
2-AAF-deacetylase specific activity Relative enrichment” NADPH-cytochrome min/mg protein) Relative enrichment”
(pmol/min/mg
c reductase specific activity
Note. N.D., not determined. a Activity/mg protein each fraction
protein)
Nuclei
Cytosol
Particulate
50.6 1.4
1295.3 34.9
124.9 3.4
6.9 24.6
N.D.
0.3 0.04
3712 100 28.0 100
17.7 2.5
7.0 1.0
Nondetectable
Nondetectable
41.3 3.9
10.5 1
Nondetectable -
Nondetectable -
1630 2.3
710 1.0
11.5 4.1
2.8 1.0
84.3 4.0
21.0 1.0
Nondetectable -
11.7 4.2
(nmol/ Nondetectable -
% activity/mg
0.4 0.06
N.D.
Homogenate
protein
4.6 0.22
in homogenate.
of protein kinase C within the hepatocyte nucleus (14). The demonstration that hepatocyte nuclei contain receptors for both prolactin and EGF and that both are coupled to protein kinase C suggests a mechanism by which these polypeptides may regulate the early transcriptional events requisite for cell replication. Prolactin is rapidly internalized by hormonally responsive cells (20-22, 28, 29, 37). Importantly, prolactin is required within the lymphocyte nucleus in order to modulate interleukin-2-stimulated proliferation (38, 39). Moreover, an intracellular role for proteolytically processed prolactin has been hypothesized (40). Taken together, the demonstration of endogenous immunoreactive prolactin within the nucleus, and of a nuclear binding site for this hormone, provides a mechanism by which previously reported nuclear effects of prolactin may occur (14,15). Further studies are required to fully address the biochemical and molecular consequences of prolactin binding to its nuclear receptor. ACKNOWLEDGMENTS We thank Dr. David W. Hein for his expertise and assistance with the determination of 2-acetylaminotluorene deacetylase. This work was supported by USPHS Grants DK40519 (D.W.M.), CA54984 (M.J.C.H.), and DK42279 (C.W.P.); by the Arizona Disease Control Research Commission (D.W.M., C.W.P.); and by Department of Veterans Affairs Merit Review Grants (D.W.M., C.W.P., C.F.Z.), the PMA Foundation, and the NSF ASEND Program (A.R.B.).
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AND BIOPHYSICS
Vol. 296, No. 1, July, pp. 198206,
Identification
1992
of Prolactin Receptors in Hepatic Nuclei
Arthur R. Buckley, *yl David W. Montg omery,?$lI Mary J. C. Hendrix,$ Charles F. Zukoski,$f and Charles W. Putnamt$lI *Department of Pharmacology, University of North Dakota School of Medicine, 501 North Columbia Road, Grand Forks, North Dakota 58203; Departments of ?Phurmacology, SSurgery, and $Anatomy and Cell Biology, University of Arizona College of Medicine, Tucson, Arizona 85724; and TResearch Service, Veterans Affairs Medical Center, Tucson, Arizona 85723
Received January
7,1992, and in revised form March 4,1992
Prolactin is a trophic hormone which may act directly at the hepatocyte nucleus. In this study, specific prolactin binding sites were sought in purified rat liver nuclei. Saturableandspecific,highaffinity128I-prolactinbinding sites were demonstrated to be on or within the nucleus. Prolactin binding was competitively inhibited by rat and ovine prolactins but not by rat growth hormone. Using immunogold electron microscopy, we detected prolactin receptors throughout the nucleus, in association with heterochromatin. Furthermore, endogenous immunoreactive prolactin was demonstrated to be within hepatic nuclei. We conclude that rat liver nuclei possess prolactin binding sites which likely participate in hormone-directed growth processes. c lee2 Academic PEW, I~C.
Evidence suggesting that prolactin may be a physiologically important hepatotrophic hormone has accumulated. Prolactin’s administration to rats elicits a growth response in liver characterized by expression of growthrelated genes (l), induction of ornithine decarboxylase (2, 3), stimulation of DNA synthesis (4, 5) and hypomethylation (6), and ultimately, increased hepatic mass (7). Notably, the hepatotrophic effects produced by prolactin are strikingly similar to the growth response in the liver following partial hepatectomy (g-lo), a procedure which rapidly elevates serum prolactin levels (9, 10). Prolactin appears to stimulate liver growth through activation of protein kinase C (10). Tumor promoting phorbol esters, which directly activate protein kinase C, mimic the hepatotrophic effects produced by prolactin treatment (11, 12). Moreover, prolactin administration cglses translocation of protein kinase C from the cytosol to the hepatic membrane, reflecting its activation (10). In uiuo 1 To whom correspondence
198
should be addressed.
and in primary hepatocyte cultures, prolactin-stimulated macromolecular synthesis is preceded by the generation of diacylglycerol, the physiologic activator of protein kinase C (1, 13). Other studies have shown that, in the nuclear compartment as well, prolactin is coupled to protein kinase C. The addition of prolactin to purified rat liver nuclei markedly enhanced protein kinase C activity, an effect again mimicked by phorbol esters (14). Furthermore, a monoclonal antibody to the rat liver prolactin receptor prevents the activation of nuclear protein kinase C by prolactin, suggesting that hepatocyte nuclei contain prolactin receptors (14, 15). In this report, we demonstrate for the first time that prolactin interacts with a specific, high affinity, immunoreactive binding site in rat hepatic nuclei. In addition, evidence is presented that normal rat liver nuclei contain endogenous prolactin which may bind to this site. These observations suggest that prolactin exerts at least a portion of its trophic actions directly at the nucleus. MATERIALS
AND
METHODS
Nuclear preparations. Hepatic nuclei were prepared from the livers of male Sprague-Dawley rats (100-140 g, Hilltop Lab Animals, Scottdale, PA) essentially as previously described (14) except that detergents were omitted in all of the buffers. Briefly, livers were perfused with 0.9% NaCl via portal vein cannulation, then minced, and washed in homogenization buffer (Tris-HCl, pH 7.4, 3 mM MgClz, 0.32 M sucrose, 2 mM ethylene glycol his@-aminoethyl ether) N,N’-tetraacetic acid (EGTA),’ 0.1 mM spermine, and 50 rg/ml of leupeptin. The tissue was homogenized using a glass Dounce homogenizer, filtered, and diluted to 0.2 M sucrose. Homogenization buffer was layered beneath the homogenate and the sample was centrifuged at 750g for 10 min at 4°C. The pellet was re-
2 Abbreviations used: EGTA, ethylene glycol bis(@-aminoethyl ether) N,N’-tetraacetic acid; SDS, sodium dodecyl sulfate; PAGE, polyacrylamide gel electrophoresis; PBS, phosphate-buffered saline; NRS, normal rabbit serum; FITC, fluorescein isothiocynate; FACS, fluorescence-activated cell sorting; EGF, epidermal growth factor.
0003-9&x31/92 $5.00 Copyright 0 1992 by Academic Press, Inc. All rights of reproduction in any form reserved.
NUCLEAR
PROLACTIN
199
RECEPTORS 6
120 looso
-
60
-
40
-
20 -
0
2
Time
4
of
6
Incubation
0
6
IO
Eomd
Nous)
20
hnol/5x
30
1 O6 Nuclei)
FIG. 1. Time course for ‘261-prolactin binding to rat liver nuclei. Nuclei (5 X 106) were incubated in quadruplicate with ‘BI-prolactin (0.1 nM) in the presence and absence of unlabeled hormone. Specific binding was determined as the difference between total (I’?-prolactin only) and nonspecific (‘261-prolactin plus 1 pg/tube unlabeled rat prolactin) binding. The experiment was repeated three times with similar results.
FIG. 3. Scatchard plot generated from an “‘1-prolactin competition experiment. Purified hepatic nuclei (1 X 106) were incubated in quadruplicate with ‘251-prolactin in the presence and absence of increasing unlabeled hormone (0.1-1000 nM) for 4 h at 37°C. A representative binding experiment is presented. Mean values: Kd = 0.42 f 0.07 nM; B mu = 29.2 ? 3.0 fmol/5 X lo6 nuclei (mean f SE, n = 6).
suspended in homogenization buffer, diluted, and again washed. The crude nuclear fraction was resuspended in a Tris-HCl buffer, pH 7.4, containing 2.4 M sucrose, 1.0 mM MgC&, 2.0 mM EGTA, 0.1 mM spermine, and 25 pg/ml leupeptin and centrifuged at 50,OOOgfor 60 min. The nuclei were washed and resuspended in 10 mM Tris-HCl (pH 7.4), 25% glycerol, and 1 mM MgClz . Purity of the nuclear preparations Verificution of nuclear purity. was assessed utilizing both morphological and biochemical criteria. For the morphological assessment, nuclei were fixed in 1% glutaraldehyde and embedded in LR white resin (Polysciences, Warrington, PA), and 60- to 80-nm sections were mounted on 200-mesh Ni grids. Subsequently,
the grids were stained with uranyl acetate and lead citrate prior to examination with a JEOL 100 CX transmission electron microscope, operating at 80 kV. Nuclear preparations were seen to be free of any visible cellular contamination (Figs. 5-8). Purity of the nuclei was also verified by biochemical means. Five different biochemical markers-three enzymes specific for plasma membrane (6-nucleotidase, phosphodiesterase I, and Na’,K+-ATPase) (16-18) and two for microsomes (NADPH-cytochrome c reductase and 2-acetylaminofluorene deacetylase) (19, 20)-were examined. Of the three plasma membrane markers (Table I), only 5’-nucleotidase activity was detectable in the nuclear preparations. If extrapolated to the entire liver, the nuclear level of 5’-nucleotidase represents less than 0.1% of total enzyme activity. Similar results were obtained when nuclear preparations were examined for the presence of microsomal enzyme markers.
‘6 20Yi +
Prolactin
3
60
.-E 2
40
WA)
FIG. 2. Saturation curve for ‘251-prolactin binding to rat liver nuclei. Nuclei (5 X 106) were incubated with increasing concentrations of lz51prolactin in the presence of 1 pg/tube unlabeled rat prolactin for 4 h at 37’C. Specific hormone binding was determined as the difference between total (1251-prolactin only) and nonspecific (‘251-prolactin plus 1 fig/tube unlabeled prolactin) binding. This experiment was repeated three times with similar results.
b
a
10
Competing
1
Ligand
0.1
Concentration
()lg/tube)
FIG. 4. Specificity of ‘%I-prolactin binding in rat liver nuclei. Purified liver nuclei (5 X 10”) were incubated in quadruplicate with “‘1-prolactin (0.1 nM) in the presence of competing ligands at the concentrations indicated for 4 h at 37°C. Data are presented as the mean -t SE of five separate experiments. CTL, ‘251-prolactin only.
BUCKLEY
FIG. 5. Electron microscopy of hepatic nuclei. Ultrathin conjugated anti-mouse IgG. Magnification, X19,000.
ET AL.
sections of purified
Radio&and binding studies. Binding experiments were performed with rat prolactin (rPRL-I-4, NIDDK), iodinated using a solid phase oxidizing reagent (Iodogen, Pierce, Rockford, IL). Radiolabeled prolactin (specific activity, approximately 50 &i/fig) eluted as a single peak from a Sephedex G-50 column and appeared as a single 24,000-Da band following SDS-PAGE and autoradiography. Radiolabeled prolactin retained full bioactivity, as assessed in the Nb2 lymphoma cell bioassay (21). Nuclear binding experiments were performed under conditions previously reported by us to be optimal for prolactin stimulation of nuclear protein kinase C (14). Purified rat liver nuclei were incubated with iz61-prolactin (150,000 cpm) in the presence and absence of increasing concentrations of unlabelled hormone (rat prolactin, B-6, ovine prolactin, O-19; rat growth hormone, GH-B-12; NIDDK) for 4 h at 37°C in a 10 mM sodium phosphate (pH 8.0) buffer containing 4 mM MgCIZ, 0.15 M NaCl, 0.1 mM spermine, 0.1% bovine serum albumin, and 25 pg/ ml leupeptin. Bound ‘261-prolactin was separated from free ligand by centrifugation at 12,000g for 3 min through a layer of 25% glycerol in binding buffer. The tubes were immediately frozen at -8O”C, the pellets was determined in a y counter. were cut out, and bound ‘%I-prolactin
nuclei were incubated
with PBS followed
by colloidal
gold-
Under these conditions, total and nonspecific binding were approximately 25 and 8%, respectively, of added iodinated prolactin. Immunogold electron microscopy. Sections (60-80 nm) of demonstrably pure nuclei mounted on ZOO-mesh Ni grids were initially incubated with 1% goat serum followed by monoclonal antibodies to the rat hepatic prolactin receptor (generously provided by Dr. Paul Kelly, INSERM, Paris, France), an irrelevant isotypic control monoclonal antibody (human myoglobin, IgGJ, or phosphate-buffered saline (PBS). Grids were incubated with antibody concentrations ranging from lo-’ to lo-’ M for 18 h at 4°C. Following extensive washing and a second blocking with 1% goat serum, the grids were incubated with goat antimouse IgG coupled to colloidal gold (20 nm, Polysciences). Subsequently, the grids were washed and analyzed by transmission electron microscopy. Fkwrescence-activated cell sorting (FAGS) of hpatk nuclei. To determine whether nuclei contain endogenous prolactin, purified hepatic nuclei were first exposed to goat serum (30 min, 4°C) and then incubated for 60 min at 4°C with a highly specific rabbit anti-rat prolactin antibody (&, dilution, Arnel, Cherokee Station, NY) or normal rabbit serum
NUCLEAR
PROLACTIN
RECEPTORS
201
FIG. 6. Electron microscopy of hepatic nuclei. Ultrathin sections of purified nuclei were incubated with an isotype control monoclonal antibody (anti-human myoglobin, IgG,) followed by colloidal gold-conjugated anti-mouse IgG. Magnification, ~19,000.
(NRS). The nuclei were subsequently washed and treated with an affinity-purified, FITC-conjugated goat anti-rabbit IgG (Fab’)r. Washed nuclei were then subjected to FACS (Becton-Dickinson, Braintree, MA), using an argon laser with excitation at 488 nm for FITC. RESULTS
Initially, the time course of ‘251-prolactin binding to rat liver nuclei was ascertained. Specific prolactin binding increased with time to 4 h, after which it remained stable through 6 h (Fig. 1). Since binding equilibrium was reached within 4 h of incubation, this interval was employed for all subsequent radioligand binding experiments. Total and nonspecific binding was found to increase at all radiolabel concentrations when purified nuclei were incubated with increasing concentrations of ‘251-prolactin.
In contrast, specific 1251-prolactin binding increased to 18-35 fmol/5 X lo6 nuclei, where it plateaued despite increasing radioligand concentrations (Fig. 2). Thus, specific binding of ‘251-prolactin to rat liver nuclei appears to be saturable. To determine the affinity as well as the binding capacity of the nuclear prolactin binding site, purified liver nuclei were incubated with 1251-prolactin(0.1 nM) in the presence or absence of increasing concentrations of unlabeled hormone. Figure 3 shows a Scatchard plot generated from one such displacement experiment. Analysis of these data reveals a high affinity (Kd = 0.42 -I 0.07 nM) nuclear 1251prolactin binding site with a capacity (B,,) of 29.2 k 3.0 fmol/5 X lo6 nuclei (n = 6). The affinity of the nuclear prolactin binding site correlates well with those reported
202
BUCKLEY
ET AL.
FIG. 7. Electron microscopy of hepatic nuclei. Ultrathin sections of purified nuclei were incubated with U6 (IgGI), a monoclonal the rat hepatic prolactin receptor, followed by colloidal gold-conjugated anti-mouse IgG. Magnification, X19,000.
for both the liver membrane prolactin receptor (22, 23) and the nuclear growth hormone binding protein (24). As mentioned above, a nuclear growth hormone receptor has been identified in liver (24). Since prolactins and growth hormones exhibit significant amino acid sequence homology (they are thought to be derived from a common ancestral gene (25)), it was important to determine the specificity for prolactin of the lz51-prolactin binding site. In inhibition experiments (Fig. 4) rat and ovine prolactins successfully compete with ‘251-prolactin for binding sites in nuclei; rat growth hormone did not, even at high concentrations. These data demonstrate that the [‘251]-prolactin binding site of hepatic nuclei is specific for lactogens and does not recognize a homologous but nonlactogenic hormone. Thus, this binding site appears to be specific for prolactin.
antibody
to
Immunogold electron microscopy was employed to visualize the nuclear prolactin receptor. Verifiably pure hepatic nuclei mounted on Ni grids were incubated with IgG1-subtype monoclonal antibodies specific for the rat hepatic prolactin receptor with antigenic domains either distinct from (U6) or directed to (Tl) the hormone binding site (26), a human myoglobin monoclonal antibody as an isotype control, or PBS alone. The results achieved by transmission electron microscopy are presented in Figures 5-8. The gold-conjugated secondary antibody was nonreactive when added to nuclei previously exposed to either PBS (Fig. 5) or the isotype control antibody (Fig. 6). In contrast, when nuclei were first incubated with anti-prolactin receptor antibodies followed by the immunogold-conjugated secondary antibody, both anti-
NUCLEAR
PROLACTIN
RECEPTORS
FIG. 8. Electron microscopy of hepatic nuclei. Ultrathin sections of purified nuclei were incubated with Tl (IgG1), a monoclonal the rat hepatic prolactin receptor, followed by colloidal gold-conjugated anti-mouse IgG. Magnification, X19,000.
receptor antibodies recognized the nuclear receptor (Figs. 7 and 8). Immunoreactive prolactin receptors are seen throughout the nuclei (arrows). Notably, immunoreactive prolactin receptors appear to be associated with heterochromatin, the most active sites for gene transcription (27). Prolactin is rapidly internalized by receptor-mediated endocytosis in several tissues, including liver (28). In pituitary mammotrophs, for example, lz51-prolactin has been shown to accumulate in and around the nucleus (29). Therefore, if prolactin exerts its effects via binding to a nuclear site, endogenous prolactin should be demonstrable in the nucleus. To test this hypothesis, purified liver nuclei were incubated with either a highly purified anti-rat prolactin antibody or NRS and then with FITC-conjugated,
203
antibody
to
affinity-purified goat anti-rabbit IgG (Fab’)z. The nuclei were subjected to FACS analysis and plotted as FITC fluorescent intensity versus size (forward light scatter). When normal hepatic nuclei were incubated with NRS as a control (Fig. 9a), two separate populations of low fluorescent intensity nuclei, differing in size, were detected. In contrast, the contour plot (Fig. 9b) obtained when the nuclei were first incubated with a highly specific antibody to rat prolactin (prior to the addition of the FITC-conjugated secondary antibody and FACS analysis) again showed two populations of nuclei. Now, however, both were of much higher fluorescent intensity than the controls. Hepatic nuclei therefore contain endogenous immunoreactive rat prolactin, presumably bound to the nuclear receptor.
204
BUCKLEY
ET AL. 64.
b .:: c
48
- 1;;;:;;;
p
.
. ...
,, .,. E F 8
32,..
8
4 ti 16.
.. ,..... B
.
16
FORWFiD LIGHT SCATTER
F-D
32
48
64
LIGHT SCATTER
FIG. 9.
Endogenous rat prolactin is present in purified liver nuclei. Purified liver nuclei were first incubated with either normal rabbit serum (a) or rabbit anti-rat prolactin antibody (b) prior to exposure to a FITC-conjugated goat anti-rabbit IgG (Fab’), and FACS analysis. These experiments were repeated three times with similar results. Total events at or above 1 were 19,181 (a) and 13,493 (b).
DISCUSSION
This report describes the first demonstration, to our knowledge, of a specific nuclear receptor for prolactin. Employing radioligand binding techniques, we found that rat hepatic nuclei possess high affinity binding specific for prolactin. Furthermore, this nuclear binding of prolactin was both time-dependent and saturable. Using monoclonal antibodies directed to the rat hepatic prolactin receptor, immunoreactive receptors were demonstrable within the nucleus but were not associated with the nuclear membrane. Significantly, the prolactin receptors appeared (by transmission electron microscopy) to be located within the condensed chromatin material. Since one of the earliest cellular effects provoked by exposure to prolactin is enhanced transcription (1) and since heterochromatin is the most active site for this process, it is possible that the nuclear prolactin receptor is directly coupled to expression of specific mRNAs, such as that for the protooncogene c-myc (1). Finally, we demonstrated immunoreactive prolactin within normal hepatocyte nuclei. The significance of the two populations, differing in size, found by FACS analysis is not clear. However, it is noteworthy that endogenous immunoreactive prolactin, possibly bound to a nuclear receptor, is present in purified nuclei prepared from normal animals. Historically, reports suggesting the existence of nuclear receptors for polypeptide hormones and growth factors
have been greeted with skepticism (30). Contamination of nuclei by nonnuclear cellular constituents could account for our observations. However, we have rigorously examined all of our nuclear preparations to ensure that they were always of the utmost purity. By both morphological and biochemical criteria, the nuclei employed in these studies were free of contamination by membrane or other cellular constituents (Table I and Figs. 5-8). While contamination of the nuclei by “invisible” receptors cannot be entirely excluded, it is notable that hepatic nuclei possess a relative abundance of prolactin receptors (about 3600/nucleus), specifically associated with heterochromatin, a hydrophilic nuclear constituent. Thus, it seems quite unlikely that contaminating “invisible” receptors could account for these results. A final criticism leveled against many nuclei isolation procedures is the use of detergents, such as Triton X-100 (30), which could potentially solubilize membrane receptors and cause their translocation onto hydrophobic nuclear regions. It is important to note that the nuclei utilized in all of the experiments presented were prepared in the absence of detergent. Thus, the data in toto demonstrate a true prolactin receptor present within the nucleus. Recently, a specific receptor for epidermal growth factor (EGF) in hepatocyte nuclei has been reported (31). EGF, like prolactin, stimulates hepatocyte replication (32-34), participates in the liver regeneration stimulated by partial hepatectomy (35, 36), and has been linked to activation
205
NUCLEAR PROLACTIN RECEPTORS TABLE I Characterization of Rat Hepatic Nuclei
Total protein % recovery
(mg)
DNA bd % recovery 5’-Nucleotidase specific activity Relative enrichment”
(nmol/min/mg
Phosphodiesterase I specific activity Relative enrichment”
protein)
(pmol/min/mg
protein)
Na+,K+-ATPasespecificactivity (pmol/min/mg protein) Relative
enrichment”
2-AAF-deacetylase specific activity Relative enrichment” NADPH-cytochrome min/mg protein) Relative enrichment”
(pmol/min/mg
c reductase specific activity
Note. N.D., not determined. a Activity/mg protein each fraction
protein)
Nuclei
Cytosol
Particulate
50.6 1.4
1295.3 34.9
124.9 3.4
6.9 24.6
N.D.
0.3 0.04
3712 100 28.0 100
17.7 2.5
7.0 1.0
Nondetectable
Nondetectable
41.3 3.9
10.5 1
Nondetectable -
Nondetectable -
1630 2.3
710 1.0
11.5 4.1
2.8 1.0
84.3 4.0
21.0 1.0
Nondetectable -
11.7 4.2
(nmol/ Nondetectable -
% activity/mg
0.4 0.06
N.D.
Homogenate
protein
4.6 0.22
in homogenate.
of protein kinase C within the hepatocyte nucleus (14). The demonstration that hepatocyte nuclei contain receptors for both prolactin and EGF and that both are coupled to protein kinase C suggests a mechanism by which these polypeptides may regulate the early transcriptional events requisite for cell replication. Prolactin is rapidly internalized by hormonally responsive cells (20-22, 28, 29, 37). Importantly, prolactin is required within the lymphocyte nucleus in order to modulate interleukin-2-stimulated proliferation (38, 39). Moreover, an intracellular role for proteolytically processed prolactin has been hypothesized (40). Taken together, the demonstration of endogenous immunoreactive prolactin within the nucleus, and of a nuclear binding site for this hormone, provides a mechanism by which previously reported nuclear effects of prolactin may occur (14,15). Further studies are required to fully address the biochemical and molecular consequences of prolactin binding to its nuclear receptor. ACKNOWLEDGMENTS We thank Dr. David W. Hein for his expertise and assistance with the determination of 2-acetylaminotluorene deacetylase. This work was supported by USPHS Grants DK40519 (D.W.M.), CA54984 (M.J.C.H.), and DK42279 (C.W.P.); by the Arizona Disease Control Research Commission (D.W.M., C.W.P.); and by Department of Veterans Affairs Merit Review Grants (D.W.M., C.W.P., C.F.Z.), the PMA Foundation, and the NSF ASEND Program (A.R.B.).
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