Cloning, Expression, and Localization of MNAR/PELP1 in Rodent Brain: Colocalization in Estrogen Receptor-- But Not in Gonadotropin-Releasing Hormone-P
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《内分泌学杂志》
Institute of Molecular Medicine and Genetics (M.M.K., M.H., C.W., L.M.D.S., K.M.D., V.B.M., D.W.B.)
Institute of Neuroscience (D.W.B.), School of Medicine, Medical College of Georgia, Augusta, Georgia 30912
Department of Genetics and Stanley S. Scott Cancer Center (R.K.V.), Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112
Abstract
MNAR/PELP1 is a recently identified scaffold protein in the human that modulates the nongenomic activity of estrogen receptors by facilitating linkage/cross talk with the Src/Erk activation cascade. We report herein the cloning of rat MNAR/PELP1 and provide new information concerning its distribution in the female rat brain and its degree of colocalization with estrogen receptor- (ER-) and GnRH. PCR-based cloning of MNAR/PELP1 from rat hypothalamus yielded a transcript of approximately 3.4 kb, which shows 86% homology to the published human MNAR/PELP1 sequence and retained all the key binding motifs (PXXP, LXXLL, and glutamic acid clusters) in its primary structure that are known to be critical for its interaction with Src and steroid receptors. RT-PCR revealed that the MNAR/PELP1 transcript is expressed in many regions of the brain, and immunohistochemistry studies showed intense MNAR/PELP1 immunoreactivity (MNAR/PELP1-ir) in areas such as the hypothalamus, cerebral cortex, hippocampus, amygdala, and cerebellum. MNAR/PELP1-ir principally localized in the nucleus, but some cytoplasmic and plasma membrane-associated staining was also observed. MNAR/PELP1-ir was also primarily neuronal, although some localization in glia cells was observed in select brain regions. Colocalization studies revealed that a majority of ER--positive cells in the brain colocalized MNAR/PELP1-ir. In contrast, MNAR/PELP1-ir rarely colocalized in GnRH neurons. In conclusion, the current study provides evidence that MNAR/PELP1 is expressed in key neural tissues of the rat brain that are known targets of steroid action, that its expression is primarily neuronal, and that MNAR/PELP1-ir is strongly colocalized in ER-, but not GnRH neurons in the rodent brain.
Introduction
THE STEROID HORMONE, 17-estradiol (E2) exerts multiple effects in the brain to regulate such key processes as reproduction, sexual behavior, neuroprotection, synaptic plasticity, and memory (1, 2, 3, 4, 5, 6, 7). E2 has generally been thought to exert its actions through the classical genomic mechanism of direct control of gene expression via interacting with estrogen receptor (ER)- and/or ER- (8, 9). However, within the past several years there has been growing evidence that E2 can also exert rapid nongenomic effects in a variety of tissues, including the brain, which could help mediate the multiple actions of E2 in the body (10, 11, 12, 13, 14). Along these lines, E2 has been shown to rapidly regulate activation of kinases, such as MAPKs, Akt, and protein kinase A (11, 15, 16, 17), as well as to rapidly modulate intracellular calcium mobilization (17). The regulation of MAPKs is of particular interest to the neural regulatory actions of E2, as ERK, a principal estrogen-regulated MAPK, has been implicated to play critical roles in the modulation of synaptic plasticity, memory and neuroprotection (18, 19). A key upstream regulator of ERK activation is the tyrosine kinase, Src, which regulates upstream ERK-regulatory signal kinases, Ras-Raf-MEK (20).
Until recently, the mechanism of how E2-ER signaling interacts with and regulates ERK activation was unclear. However, the recent cloning of a human protein termed MNAR/PELP1 (modulator of nongenomic activity of estrogen receptor/proline-glutamic acid-leucine-rich protein-1) has shed new light on the potential underlying mechanisms for E2 regulation of ERK signaling (21, 22). MNAR/PELP1 has been shown to act both as an estrogen receptor cofactor and as a scaffold protein (21, 22). As a scaffold protein, MNAR/PELP1 has been shown to contain LXXLL and PXXP motifs in its N terminal, which mediate MNAR/PELP1 binding to AP2 domains of ER- and the SH3 (Src Homology 3) domain of Src, respectively (21, 22, 23). This interaction of MNAR/PELP1 with ER- and Src appears functionally important as overexpression of MNAR/PELP1 in cells treated with E2 activates the Src/ERK signaling cascade and enhances ER transcriptional activity, effects blocked by inhibitors of Src and MAPK kinase (22). Furthermore, antisense oligonucleotides and small interfering RNA to MNAR/PELP1 have been shown to significantly attenuate E2-induced expression of target genes in MCF-7 cancer cells and endometrial cancer cells (22, 24). MNAR/PELP1 was shown to be expressed in a variety of tissues, with highest expression noted in brain, testes, ovaries, uterus, and thyroid (21, 22). The specific tissue localization, cell type-specific expression, and degree of colocalization with ER of MNAR/PELP1 in the brain have not been determined. Additionally, the colocalization of MNAR/PELP1 in GnRH neurons is unknown, and the gene has not yet been cloned in the rat, a step critical for further studies on structure/function of MNAR/PELP1 in this species. The present study was thus designed to address these key issues.
Materials and Methods
Animal models
All experiments were conducted in compliance with the National Institutes of Health guidelines for the care and use of experimental animals and were approved by the Institutional Animal Care and Use Committee at the Medical College of Georgia. Sprague Dawley female rats (2–4 months old) were purchased from Harlan (Indianapolis, IN). The animals were housed in individual cages and water and rat chow was provided ad libitum. Regular 4-d cycling animals were killed on proestrus (1500–1700 h) and processed for RT-PCR, immunohistochemistry or Western blot analysis as described below. Only animals that showed at least two consecutive 4-d cycles were included in the study. Cycle stages were determined by recording daily electrical impedance readings of the vaginal wall using a commercially available EC40 estrous cycle monitor (Fine Science Tools, Foster City, CA). The EC40 estrous cycle monitor measures the inherent electrical resistance of the vaginal wall, which fluctuates markedly across different phases of the estrous cycle (25). Vaginal lavage and microscopic determination of vaginal cytology was also used to confirm estrus cycle stage determinations. Additionally, regular cycling green fluorescent protein (GFP)-tagged GnRH transgenic mice (2–4 months old) (gift from Dr. Moenter, University of Virginia, Charlottesville, VA) were also killed at proestrus for GnRH and MNAR/PELP1 colocalization studies (26).
Cloning of MNAR/PELP1 from rat hypothalamus
The rat MNAR/PELP1 sequence was cloned from adult female rat hypothalamus by RT-PCR using primers direct against a computer-generated predicted rat MNAR sequence (XM_340831). mRNA was isolated with RNAqueous-4PCR isolation kit (Ambion, Austin, TX) and oligo-deoxythymidine primed immediately. The resulting template was used in PCR with the following sets of primers: forward primer, 5'-GAA GAT GGC GGC AGC CGT TCT TAG TG-3'; and reverse primer, 5'-CTA AGA GTC AGG CTC TGT AGC AGG TGG TGG C-3'. The PCR conditions were; 94 C, 2 min denaturation, followed by 30 cycles at 92 C for 45 sec, 67 C for 45 sec and 72 C for 2 min; and 4 min extension at 70 C. The resulting PCR product was gel-purified and cloned into a pT7-blue vector (Novagen, EMD Biosciences, Inc., Madison, WI) and individual clones were sequenced using the T7 primer. DNA sequence analysis revealed a 3393-bp MNAR/PELP1 sequence containing an open reading frame with a start and stop codon (accession no. AY 970831).
RT-PCR analysis
Total RNA from rat tissues was isolated and primed with oligo-deoxythymidine for the reverse transcriptase (RT) reaction. A negative control, which consisted of pooled total RNA run in the RT-PCR without RT added, was also included. A positive control was also used, which was the cloned rat MNAR/PELP1 plasmid. Five hundred nanograms of the RT reactions was used for PCR with the following primer set to identify MNAR/PELP1 transcription: forward primer, 5'-GAA GAT GGC GGC AGC CGT TCT TAG TG-3'; and reverse primer, 5'-CTA AGA GTC AGG CTC TGT AGC AGG TGG TGG C-3'. The hot-start PCR conditions [Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA) 1.5 mM MgCl] were 94 C for 2 min, followed by 30 cycles of 92 C for 1 min, 67 C for 1 min, and 72 C for 3 min. A total of 10 μl of the PCR product was electrophoresed and visualized with ethidium bromide, and photographed for documentation.
Neuronal cultures
Rat cortical neurons were cultured as described previously (27). Briefly, E18 rat cortices were dissected under a microscope in cold Hanks’ balanced salt solution. Under sterile conditions, tissue was minced and treated with 0.1% trypsin (Invitrogen) and 100 U/ml deoxyribonuclease (Promega, Madison, WI) for 30 min at 37 C in serum-free Neurobasal medium (Invitrogen). Neurobasal medium was supplemented with B27 serum-free supplement (Invitrogen), Penicillin-Streptomycin liquid (x1), 0.5 mM L-glutamine, and sodium pyruvate (1x). After trypsin digestion, tissue mechanically dissociated by trituration and then filtered through a 40 μm nylon cell strainer (Becton Dickson, Franklin Lakes, NJ). Cells were counted using the trypan blue assay, and plated in poly-D-lysine-coated four-well Biocoat plates (Becton Dickson) in Neurobasal medium. Cells were plated with a plating density of approximately 500,000/well.
Western blot analysis
For Western blot analysis, tissue was homogenized in RIPA buffer (1x PBS), 1% IGEPAL CA-630 (Sigma Chemicals, St. Louis, MO), 0.5% sodium deoxycholate, 0.1% SDS) and protease inhibitors using a polytron homogenizer. After homogenization, samples were centrifuged at 10,000 rpm for 10 min at 4 C. The supernatant fraction was the total cell lysate. Protein concentration in the lysate was determined using a total protein measurement kit from Sigma Chemicals. Protein samples were denatured in sample buffer containing -mercaptoethanol in 25 mM Tris-glycine buffer and separated on 7.5% sodium dodecyl sulfate-polyacrylamide gel after loading equal amount of protein in each lane. Separated proteins were transferred to Immobilon P membrane (Millipore, Billerica, MA) at 27 V for 15 h in 25 mM Tris-glycine buffer (pH 8.3), 10% methanol using a Transblot apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). After the transfer, the membranes were rinsed twice with T-TBS (20 mM Tris, 137 mM NaCl, 0.1% Tween 0) for 5 min each rinse and then incubated with 5% nonfat dry milk for 1 h at room temperature to block nonspecific/unbound surface. The membrane was incubated overnight with a well-characterized, commercially available rabbit polyclonal anti-MNAR/PELP1 antibody (1:2500; Bethyl Laboratories, Inc., Montgomery, TX). The membrane was then washed with T-TBS to remove unbound antibody, followed by incubation with secondary horseradish peroxidase-conjugated donkey antirabbit IgG (Transduction Laboratories, San Diego, CA) for 1 h at room temperature. The signal was detected using an ECL detection kit (Amersham, Buckinghamshire, UK) and the membranes were exposed to Kodak Biomax MR film (Eastman Kodak, Rochester, NY).
Immunohistochemistry
Perfusion and fixation.
Animals were deeply anesthetized with ketamine/xylazine and transcardially perfused with saline followed by fixation with 300–400 ml ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, at a flow rate of 20–25 ml/min. After fixation, brain samples were cut into 5-mm blocks and placed in the fixative overnight at 4 C followed by cryoprotection in 30% sucrose solution in 0.1 M PB, pH 7.4 at 4 C until the brains permeated. Tissue was frozen in OCT (optimum cutting temperature) compound under an atmosphere of nitrogen, and coronal sections (40-μm thickness) were cut on a cryostat microtome (Leica, Germany) through the entire brain and stored in a cryoprotection solution (FD Neurotechnology, Inc., Baltimore, MD) in stereological order for immunostaining. Identification of anatomical landmarks was based on stereotaxic coordinates reported previously (28).
Antibody/antiserum.
Two polyclonal antibodies were used to detect MNAR/PELP1 in rodent brain. The first was a commercially available polyclonal anti-MNAR/PELP1 purchased from Bethyl Laboratories, Inc. This antibody was produced against an epitope corresponding to C-terminal amino acids 1000–1050 of human MNAR/PELP1 and affinity purified by immobilizing the epitope on a solid support. The second MNAR/PELP1 antibody was an antiserum produced in rabbit against a 19-mer peptide encoding amino acids 558–576 (RDSLSPGQERPYSTVRTKV) of the human MNAR/PELP1 gene that has been extensively characterized by one of the authors (R.K.V.) (21). This antiserum detects MNAR/PELP1 from various tissues including brain (21). The regional distribution and density of ER--positive cells was determined using two different and well-characterized antibodies. The first one was a polyclonal antiserum purchased from Upstate Biotechnology (Lake Placid, NY), produced in rabbit against a peptide representing the last 15 amino acids of rat ER- (lot no. 26214) and has been used in earlier studies (29). The second antibody was a commercially available monoclonal antihuman ER- (catalog no. ab858) antibody purchased from Abcam Inc. (Cambridge, MA), produced in mouse against a full-length recombinant human ER-. Monoclonal antibodies specific for the glial marker, GFAP was purchased from Sigma, neuronal markers; NeuN and MAP-2, and synaptic markers PSD95 and synaptotagmin were purchased from Chemicon International Inc. (Temecula, CA). The specificity and reactivity of these antibodies have been well characterized.
Control experiments
The specificity of immunohistochemical labeling was confirmed on representative sections from proestrus female animals after blocking the antibody/antiserum with the corresponding peptide/protein immunogen or with the omission of the primary antibodies.
3,3'-Diaminobenzidine (DAB) staining method
For immunochemical analysis using the DAB staining method, 40-μm coronal sections from rat or mouse proestrous female brain, under free-floating conditions were incubated with 10% normal goat serum for 1 h at room temperature to block nonspecific surfaces. Sections were then incubated with primary antibody/antiserum at 1:1000 dilution for MNAR/PELP1 and 1:1000 for monoclonal ER- and 1:5000 for polyclonal ER- antibodies/antiserum for 36–48 h at 4 C. Afterward, sections were washed with PBS containing 0.04% Triton, followed by incubation with secondary goat antirabbit antibodies (Vector Laboratories, Inc., Burlingame, CA) at a dilution of 1:200 in PBS containing 0.04% Triton X-100 for 1 h at room temperature. Sections were then washed with PBS containing 0.04% Triton X-100, followed by incubation with ABC reagents for 1 h at room temperature in the same buffer. Tissue section were then washed and incubated with DAB reagent according to the manufacturer’s instructions (Vector Laboratories, Inc.). DAB staining results were also confirmed using silver-enhanced nanogold immunostaining for MNAR/PELP1, with a similar localization pattern and density observed in the brain for both procedures (data not shown).
Immunofluorescent staining
Free floating sections were incubated with 12% normal donkey serum for 2 h at room temperature in PBS containing 0.04% Triton X-100, followed by incubation with appropriate dilutions of the primary antibodies for 24–36 h at 4 C in the same buffer. The primary antibodies used were; monoclonal mouse-anti-ER- (1:1000; Abcam), anti-NeuN and anti-MAP2 (1:500; Chemicon), anti-GFAP (1:500; Sigma) and rabbit polyclonal anti-MNAR/PELP1 (1:500–1000; Bethyl Laboratories, Inc.). Sections were washed for 4 x 10 min at room temperature followed by incubation with Alexa-Fluor488 donkey antimouse IgG and Alexa Fluor594 donkey antirabbit IgG antibodies (1:300) for 1 h at room temperature. Sections were washed with PBS containing 0.04% Triton X-100 for 4 x 10 min, followed by 2 x 5 min with PBS and 2 x 1 min with water, and then mounted with water-based mounting medium containing antifading agents (Biomeda, Fischer Scientific, Pittsburgh, PA). For colocalization studies, a combination of one of the above monoclonal antibodies and MNAR/PELP1 antibody was used. To confirm nuclear staining, sections were incubated with 1 μM solution of either 4'-6-diamidino-2-phenylindole (DAPI) (Sigma) or TO-PRO-3 (Invitrogen) nuclear stains after appropriate dilution for 15 min at room temperature in the dark. A simultaneous examination of negative controls (after blocking the primary antibody with peptide antigen in five to six molar excess or after omitting the primary antibodies) confirmed the absence of nonspecific immunofluorescent staining, cross-immunostaining, or fluorescence bleedthrough.
For colocalization of MNAR/PELP1 with GnRH, both proestrous rats and proestrous GnRH-GFP-tagged transgenic mice were used for immunochemical analysis. Detection of MNAR/PELP1 in rats was accomplished using the rabbit polyclonal anti-MNAR/PELP1 antibody as described above, whereas GnRH detection was accomplished by using a monoclonal anti-GnRH antibody (Chemicon; 1:500). Immunohistochemistry on mice tissue sections was performed using either the Bethyl commercial MNAR/PELP1 antibody or a rabbit antiserum to MNAR/PELP1 characterized previously by one of the authors (R.K.V.) (21) (1:1000).
For in vitro double immunofluorescence staining, cultured cortical neurons were washed with Dulbecco’s phosphate buffer and fixed with 4% paraformaldehyde containing 2% glycerol at 37 C for 10 min followed by washing with PBS. Cultures were incubated with 0.1% Triton-X-100 in PBS for 10 min at room temperature and then with 10% normal donkey serum in PBS for 1 h. The cultures were then incubated with primary antibody in PBS for overnight or 24 h at 4 C. The primary antibodies used were as follows: mouse monoclonal anti-PSD95 (1:400), mouse monoclonal antisynaptotagmin (1:400), and rabbit polyclonal anti-MNAR/PELP1 (1:400; Bethyl Laboratories, Inc.). After washing with PBS, cells were incubated with fluorescent-labeled AlexaFluor488 donkey antimouse and AlexaFluor584 donkey antirabbit antibodies (1:200; Invitrogen) for 1 h at room temperature. Cell were then washed with PBS, followed by a brief wash with distilled water, and then mounted with aqueous mounting medium containing antifading agents.
Confocal microscopy and image analysis
Photomicrographs of the brain sections stained by the DAB method were captured on an Axiophot-2 visible/fluorescence microscope using an AxioVision4Ac software system (Carl Zeiss, Jena, Germany). Double fluorescence-labeled images were obtained through an inverted LSM-510 Meta confocal microscope either in XY or XYZ (Z-stacks) using a x40 oil immersion Neofluor objective (NA, 1.3). The Z-stacks (30–50 optical sections) were collected at 0.2- to 0.4-μm intervals, encompassing the entire cell bodies and cellular processes. Triple fluorescence-labeled images were captured through an LSM510 Meta confocal microscope (Carl Zeiss) attached to a Coherent (Mira 900 and Verdi-V10) two-photon laser-scanning system. The term MNAR/PELP1-ir (immunoreactivity) is used interchangeably to denote MNAR/PELP1 immunoreactivity and MNAR/PELP1 immunoreactive protein. For the immunohistochemical studies, at least six to 10 alternate sections per animal were analyzed and the experiments were repeated two to three times for verification of results.
Results
Cloning and expression of MNAR/PELP1 in rat brain
PCR-based cloning of MNAR/PELP1 from rat hypothalamus yielded a transcript of approximately 3.4 kb, which shows 86% homology to the published human MNAR/PELP1 sequence. The complete cDNA sequence has been deposited in GenBank (accession no. AY 970831). The primary protein structure consists of 1130 amino acids organized into several functionally conserved domains—such as glutamic acid-clusters, and LXXLL and PXXP motifs, the latter two having been implicated in mediating MNAR/PELP1 interaction with AF2 domains of steroid receptors and SH3 domains of proteins such as Src, respectively (Fig. 1, A and B). The glutamic acid-clusters in rat MNAR/PELP1 are located in the C terminus spanning the last approximately 280 amino acid residues, whereas the LXXLL motifs (n = 11) reside in N-terminal regions within the first 600 residues (Fig. 1B). The PXXP motifs (n = 17) are sequestered from one another, with the majority located at the C terminus encompassing the last 700 amino acids, and the remaining few motifs condensed at the N terminus within the first 70 amino acid residues (Fig. 1B). This organization of various motifs in the primary structure of rat MNAR/PELP1 is similar to that reported for other species including human (21, 22). RT-PCR using primers specific for rat MNAR/PELP1 revealed that the MNAR/PELP1 transcript is expressed in several rat brain regions, including hypothalamus, cerebral cortex, and hippocampus, and other organs such as ovary, uterus, muscle, and pituitary (Fig. 1C). A negative control (total pooled RNA used in the RT-PCR without addition of reverse transcriptase) yielded no product, demonstrating the lack of genomic DNA contamination. Additionally, a positive control (cloned rat MNAR/PELP1 plasmid) yielded a product of equivalent size to the products amplified from the various tissues.
Distribution of MNAR/PELP1 immunoreactivity in the rat CNS
Antibody specificity.
Figure 2A shows Western blot detection of MNAR/PELP1 protein from various brain regions using commercial anti-MNAR/PELP1 antibodies (Bethyl Laboratories, Inc.). Lysates from MCF-7 cell and human placental JAR cells (which have shown to have very high expression of MNAR/PELP1) were used as positive controls. A band corresponding to the size of human MNAR/PELP1 (170 kDa) was observed in all the brain tissues (hypothalamus, cortex and hippocampus) and positive controls. The specificity of the MNAR/PELP1 antibody was demonstrated by antigenic peptide blocking at 5 M excess for 3–4 h at room temperature or 24 h at 4 C, which abolished the reactivity of primary antibodies to MNAR/PELP1 protein in the brain tissues and greatly diminished MNAR/PELP1 antibody reactivity in the positive control (JAR cells) (Fig. 2B, upper panel). Similar results were observed in DAB immunohistochemistry studies on brain sections where antigenic peptide blocking of the primary antibodies led to complete abolition of MNAR/PELP1-specific immunostaining (compare Fig. 2, C and D). Antigenic preabsorption studies using the silver-enhanced nanogold procedure for MNAR/PELP1 in tissue sections also eliminated MNAR/PELP1 staining, and MNAR/PELP1 staining using the silver-enhanced nanogold procedure demonstrated a similar localization pattern as that observed using the DAB procedure (data not shown). A similar localization pattern for MNAR/PELP1-ir was also observed in the proestrous female mouse brain using the commercial (Bethyl Laboratories, Inc.) antibody (see supplemental Fig. 1 published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). The immunolocalization pattern for MNAR/PELP1-ir in rodent brain observed with the commercial antibody was confirmed using a second MNAR/PELP1 antiserum that has been extensively characterized by one of the authors (R.K.V.) previously (21) (data not shown). The commercial (Bethyl Laboratories, Inc.) antibody preparation was then used for mapping the MNAR/PELP1-ir in the entire brain of the young adult proestrous female rat as shown in Table 1. The results of the localization of MNAR/PELP1-ir throughout the brain are described in detail below.
Telencephalon
Olfactory region.
The olfactory bulb of the rodents has six well-defined cellular layers comprising; nerve cell layer, external plexiform layer, glomerular layer, mitral cell layer, internal plexiform and internal granule cell layers (Table 1). A dense population of MNAR/PELP1-ir cells was present throughout the internal granule cells and mitral cell layer. In the glomerular layer around the glomeruli and the anterior olfactory nucleus, only a scattered MNAR/PELP1-ir was observed. In the mitral cell layers, MNAR/PELP1-ir was located in both the small interneurons and large mitral cells (Fig. 2E). Similarly, a less scattered MNAR/PELP1-ir cell population was present in the medial, lateral, and dorsal subdivisions of the anterior olfactory nucleus. Sparse, MNAR/PELP1-ir was also present in several olfactory regions of the cerebral cortex, including the agranular insular cortex, the islands of Calleja, and layer III of the olfactory tubercle.
Cortex.
In the cerebral cortex, MNAR/PELP1-ir was localized throughout all the major cytoarchitectonic divisions of sensorimotor, association, visual, piriform and entorhinal cortex (Table 1). MNAR/PELP1-ir was primarily concentrated in cell nuclei (Fig. 2, F–H), with some staining localized in the cytoplasm and cell processes identified mainly in the large pyramidal neurons (Fig. 2F). Most labeled nuclei were round in shape and small to medium-sized (5–10 μm) in diameter. Occasionally, some MNAR/PELP1-ir cells were also found to have more irregularly, e.g. fusiform and star shaped nuclei suggesting their astrocytic origin (not shown). The majority of sensory, motor, and association areas of the rat cerebral cortex showed MNAR/PELP1-ir that was most prominent in layers-II-III and V-VI followed by layer IV. However, differences in the apparent density of MNAR/PELP1-ir cells distinguished the association from the sensorimotor areas. The association areas of the cerebral cortex including the medial prefrontal (Cg1–3), retrosplenial (RSG and RSA), and insular (AIV, AID, AIP) areas visually showed less density of MNAR/PELP1-ir cells compared with sensorimotor areas (e.g. the primary and secondary somatosensory, auditory, visual, and motor areas). Finally, in contrast to most cortical areas, MNAR/PELP1-ir cells in the three-layered piriform cortex were more concentrated in layer II than layer III (Fig. 2H).
Hippocampus.
In the hippocampus, a dense population of MNAR/PELP1-ir cells was observed in the CA1-CA3 pyramidal cell layers, dentate gyrus, the subiculum and parasubiculum (Fig. 2, I–L). Scattered density of MNAR/PELP1-ir cells also observed in the hilus, stratum oriens, stratum radiatum and alveus of the hippocampus (not shown). Interestingly, in these layers, MNAR/PELP1-ir cells predominantly showed characteristics morphological features of astroglial cells, which was confirmed by colocalization of MNAR/PELP1-ir with glial markers, as described later.
Septum.
Variable MNAR/PELP1-ir cells bodies were detected in the septum, being higher in the lateral septum with a slightly less density in the medial septum (Table 1). A moderate number of MNAR/PELP1-ir cells were also detected scattered in the septohippocampal nuclei, the vertical and horizontal limbs of the diagonal band of Broca, and the organum vasculosum of the lamina terminalis (Table 1).
Amygdala.
With respect to the amygdala, the density and intensity of MNAR/PELP1-ir cells was considerably higher in the dorsal portions of the medial amygdaloid nucleus (Fig. 2M) than the cortical, central, and basomedial subdivisions and amygdalohippocampal area (Table 1).
In the stria terminalis, the MNAR/PELP1-ir cells were more abundant in the dorsolateral subdivision compared with the ventral and intermediate subdivisions (Table 1). In the striatum; however, MNAR/PELP1-ir cells were more or less evenly distributed, whereas in caudate putamen they were more concentrated toward the ventral region (Table 1). Several MNAR/PELP1-ir cells with a characteristic morphology of astroglia were present in the internal and external subregions of globus pallidus and in the basal nucleus of Meynert.
Diencephalon
Thalamus.
In the thalamic areas of diencephalon, MNAR/PELP1-ir cells were scattered in all the subregions with variable numbers. Comparatively, high MNAR/PELP1-ir cell density was observed in the dorsal part of anterior medial thalamic nuclei, then the centrum medianum, paracentral and paraventricular thalamic and ventral anterior and ventrolateral sub-regions of the thalamus (Table 1).
Hypothalamus.
Among various regions of hypothalamus, a high number of intense MNAR/PELP1-ir cells was observed in the arcuate nucleus, ventromedial nucleus, suprachiasmatic nucleus, and premammillary nuclei. A moderate density of MNAR/PELP1-ir cells were also observed in the dorsomedial nucleus and the lateral hypothalamic areas (Table 1). In the anterior hypothalamus, MNAR/PELP1-ir cells were dense in the ventromedial nucleus and periventricular preoptic nuclei and in the rostral-caudal extent of the medial preoptic area. In the paraventricular nucleus, MNAR/PELP1-ir cells were present in all the subregions with the highest number of cells in the ventral part of the paraventricular nucleus (Table 1). No significant MNAR/PELP1-ir was detected in the supraoptic nucleus.
Mesencephalon
MNAR/PELP1-ir cells localized across all the subdivisions of the midbrain with a moderate density observed in dorsal and median raphe nuclei, periaqueductal gray and the substantia nigra (Table 1). Scattered MNAR/PELP1-labeled cell nuclei were also present in the dorsal part of the interpeduncular nucleus, central gray matter, dorsal and ventral nuclei of the lateral lemniscus, and the granular layer of the optic tectum. Additionally, slightly lower density, albeit strong MNAR/PELP1-ir cells were detected in the ventral tegmental area.
Metencephalon
In the cerebellum, an intense MNAR/PELP1-ir was localized in the Purkinje cells (not shown) of the outer molecular layer; whereas, in the internal molecular layer, the intensity of MNAR/PELP1-ir cells was considerably weaker (Table 1). No MNAR/PELP1-ir cells were detected in the posteriolateral or posteriodorsal fissures. Few small to medium size MNAR/PELP1-ir cells were present in the white matter. In the pons, strongly stained MNAR/PELP1-ir cells were present in the dorsal parabrachial nucleus, ventral parabrachial nucleus, the dorsal raphe nucleus, and the lateral supraolivary nucleus (Table 1).
Myelencephalon
In the medulla, a moderate number of intensely stained MNAR/PELP1-ir cells were scattered in most of the subdivisions of the solitary tract, raphe nuclei, spinal trigeminal nucleus and the lateral reticular nucleus. MNAR/PELP1-ir was present in both small interneurons and large pyramidal cells of the solitary tract and raphe nuclei. Scattered and variably stained MNAR/PELP1-ir cells were also present in the area postrema, spinal trigeminal nucleus, lateral reticular nucleus and the reticular formation (Table 1).
Spinal cord
A very low density of MNAR/PELP1-ir cells was observed in various subregions/layers of the spinal cord, including the layers II-III of the dorsal horn, layers VIII-IX of the ventral horn, and in the lamina X around the central canal. A small number of MNAR/PELP1-ir cells were also detected in the lateral cervical and lateral spinal nucleus and in the dorsal corticospinal tract (Table 1).
Table 1 summarizes the distribution of MNAR/PELP1-ir in the CNS, the intensity of the staining, and for the sake of comparison, the pattern of ER--ir staining. Figure 3 also provides a schematic representation of coronal sections depicting the distribution of MNAR/PELP1-ir in the rat brain.
MNAR/PELP1 localization in neurons and glia
We next examined the cell type (neuronal vs. glial) expressing MNAR/PELP1-ir protein by use of double/triple immunofluorescence staining and confocal microscopy. In the hippocampus, the majority (>95%) of MNAR/PELP1-ir cells in the molecular layers of dentate gyrus (DG) and pyramidal layers (CA1-CA3) showed colocalization with the neuronal-specific markers, NeuN (Fig. 4, upper left panel) or MAP-2 (lower left panel), suggesting a primarily neuronal origin of MNAR/PELP1. Interestingly, a strong colocalization of MNAR/PELP1-ir was also observed in astrocytes in the hilus of the dentate gyrus, oriens and stratum radiatum around the CA1 and CA3 pyramidal cell layers (Fig. 4, DG, CA1, and CA3; upper right panel). When quantified using confocal double label imaging, nearly 60–70% of the astrocytes showed colocalization with MNAR/PELP1-ir in the stratum radiatum and stratum oriens. However, in contrast, in the suprachiasmatic nucleus (SCN) and arcuate nucleus (ARC) of the hypothalamus, we observed that MNAR/PELP1-ir was mainly localized in the neurons, with a much lower number of astrocytes expressing MNAR/PELP1-ir. This was further confirmed by confocal two-photon microscopy of the triple fluorescent labeling of MNAR/PELP1 with the nuclear stain, DAPI and NeuN. As illustrated in Fig. 4 (ARC, lower right panels), a large number of MNAR/PELP1-ir nuclei (A; red) showed complete overlapping (D; white) with NeuN (B; green) and a nuclear stain DAPI (C; blue), indicating primarily a neuronal origin and nuclear localization of MNAR/PELP1-ir. However, it is also clear from Fig. 4 that not all the NeuN-positive neurons in the ARC show MNAR/PELP1-ir. With respect to the cerebral cortex, a majority of MNAR/PELP1-ir cells in various layers were found to colocalize NeuN (Fig. 4, SC in the upper left panel) and MAP-2 (PFC in the lower left panel). Additionally, in layer II-III and V-VI of the sensorimotor and association cortex, there were also a moderate number of astrocytes that colocalized MNAR/PELP1-ir (data not shown). Three-dimensional confocal microscopic analysis in the hippocampus using z-series stacking and three-dimensional reconstruction of z-stacks confirmed the colocalization of MNAR/PELP1-ir with the neuronal marker, NeuN and with the astrocyte marker [glial fibrillary acidic protein (GFAP)] (supplemental Fig. 2).
MNAR/PELP1 localization in ER--positive cells
We next investigated whether MNAR/PELP1-ir colocalized within ER--positive cells in regions where ER- is highly expressed. First, a systematic anatomical investigation of ER- immunoreactivity (ER--ir) was performed in the brain using polyclonal as well as monoclonal ER- antibody/antiserum preparations (Upstate and Abcam, respectively) as described in Materials and Methods. The ER--ir localization patterns obtained with these antibodies in the rat brain, were similar to those mentioned in other reports (29, 30, 31, 32, 33) and has been listed in Table 1 and illustrated in Fig. 5. As illustrated in Table 1, the localization patterns of MNAR/PELP1-ir and ER--ir, whereas similar in some regions, were not completely identical. Most notably, the density of ER--ir-positive cell bodies in the cerebral cortex and hippocampus was much lower than the density of MNAR/PELP1-ir cell bodies in these regions. In contrast, the overlap and density of MNAR/PELP1-ir and ER--ir-positive cells roughly corresponded with each other in the subregions of the hypothalamus, amygdala and bed nucleus of the stria terminalis (Table 1). Figure 5 shows photomicrographs of the areas of high density of ER--ir-positive cell bodies in the rat brain and the parallel pattern of MNAR/PELP1-ir localization in the same areas. As shown in Fig. 5 and Table 1, high density of ER--ir-positive cell bodies were observed in the septum, medial preoptic area, hypothalamus, amygdala, and bed nucleus of the stria terminalis. ER- immunostaining showed primarily nuclear localization; however, some cytoplasmic or membrane-associated ER--ir staining was also detected. To confirm that MNAR/PELP1-ir and ER--ir were indeed colocalized in cells in the above noted brain regions where significant overlap of staining pattern was observed, double-immunofluorescent colocalization studies for ER- and MNAR/PELP1 were performed. As illustrated in Fig. 6, double-immunofluorescent studies confirmed that many ER--ir-positive cells in the hypothalamus, amygdala and bed nucleus of the stria terminalis strongly colocalized with MNAR/PELP1-ir, with the highest degree (60–80%) of colocalization observed in the dorsal medial nucleus of amygdala, ventral medial nucleus of the hypothalamus, and arcuate nucleus of hypothalamus. Whereas ER--ir was lightly expressed in cortical and hippocampal regions, it is interesting to note that the few ER--ir cells observed in the cortex and hippocampus also colocalized MNAR/PELP1-ir (data not shown). Unfortunately, we were not able to ascertain MNAR/PELP1 colocalization with ER- due to unavailability of suitable primary monoclonal antibodies. Additional studies showed that MNAR/PELP1-ir is also expressed in embryonic rat cortical neurons in vitro (in both the cell body and dendritic processes), and that it colocalizes with both presynaptic (synaptotagmin) and postsynaptic markers (PSD-95) (supplemental Fig. 3).
Colocalization of MNAR/PELP1-ir in GnRH neurons
Because MNAR/PELP1 mRNA and immunoreactive protein was highly expressed in the hypothalamus, including the preoptic area and the organum vasculosum of the lamina terminalis regions of the hypothalamus where GnRH neuronal cell bodies are located, we next examined whether MNAR/PELP1-ir was colocalized in GnRH neurons in rats and GFP-tagged GnRH transgenic mice. As shown in Fig. 7, MNAR/PELP1-ir protein was rarely expressed in GnRH neurons of the rat or mouse, and there was also no overlap of MNAR/PELP1-ir fibers and GnRH neuronal fibers in the median eminence.
Discussion
Using a variety of methodological approaches, we have characterized the expression, cloning and localization of MNAR/PELP1 in the rodent brain. PCR-based cloning of MNAR/PELP1 from rat hypothalamus yielded a transcript of approximately 3.4 kb, which shows 86% homology to the published human MNAR/PELP1 sequence. The primary structure of rat MNAR/PELP1 protein consists of 1130 amino acids organized into various functionally conserved domains, similar to other species (21, 22). These conserved domains are comprised of various LXXLL, PXXP motifs and glutamic acid-clusters. The LXXLL motifs have been shown to interact with the AF2 domains of classical steroid receptors, whereas the PXXP domains serve as SH3 binding sites for Src and potentially other SH3 domain proteins (22). In vivo localization results using immunohistochemistry showed that MNAR/PELP1 is expressed with varying intensity throughout the rat brain, including a number of functionally important regions that are known targets of steroid actions. This appears to be one of the first detailed investigations of MNAR/PELP1 expression/localization in vivo, which we believe will open new directions for evaluating the functional roles of MNAR/PELP1 in the central nervous system. Our study revealed that MNAR/PELP1-ir is present throughout the telencephalon, diencephalon, mesencephalon, metencephalon, myelencephalon, and spinal cord in the rat. A similar immunohistochemical localization pattern for MNAR/PELP1 was observed in the mouse brain (supplemental Fig. 1) and monkey brain (Khan, M., and D. W. Brann, unpublished observation). In the rat olfactory bulb of telencephalon, a strong MNAR/PELP1-ir was located in the mitral and internal granule cell layers followed by glomerular layer. The cerebral cortex showed intense MNAR/PELP-ir present in all the major cytoarchitectonic divisions of the isocortex and allocortex. In the cortical regions with clear demarcation of cellular layers, a high density and intensity of MNAR/PELP1-ir cells was always detected in the layer II-III and V-VI followed by layer IV (Table 1). Strong MNAR/PELP1-ir was also observed in the piriform and entorhinal cortex. In the hippocampus, strong MNAR/PELP1-ir was noted in the pyramidal cell layers (CA1-CA3) and dentate gyrus, whereas in the hilus, stratum radiatum, stratum oriens, and alveus MNAR/PELP1-ir was significantly more scattered (Table 1), confined mainly in the astrocytes and glia. In the striatum, MNAR/PELP1-ir was higher in the lateral part than the dorsal region, and among various subregions of amygdala, MNAR/PELP1-ir was highest in the dorsal medial nucleus followed by cortical and others (Fig. 2M and Table 1). In the bed nucleus of stria terminalis, MNAR/PELP1-ir was highest both in the medial and lateral subdivisions and less scattered in ventral and intermediate subregions (Table 1). In the diencephalon, MNAR/PELP1-ir was densely localized throughout key hypothalamic nuclei, including ARC, SCN, organum vasculosum of the lamina terminalis, and VMH and all the subregions of the preoptic area (Fig. 2, N–P). In the thalamus, MNAR/PELP1-ir was present in all subdivisions, although with less density and intensity than the hypothalamus and cortex (Table 1). Lastly, variable density and intensity of MNAR/PELP1-ir cells was detected throughout the mesencephalon, metencephalon, myelencephalon and spinal cord (Table 1).
MNAR/PELP1 expression in the above brain regions, particularly the hypothalamic regions, hippocampus, amygdala, and cerebral cortex, may indicate an important role for MNAR/PELP1 in coordinating diverse estrogen actions, including such estrogen-regulated processes as reproductive function, circadian rhythms, feeding and sexual behavior, fear conditioning behavior, synaptic plasticity, neuroprotection and learning and memory (1, 2, 3, 4, 5, 6, 7, 10, 27, 33, 34, 35, 36, 37, 38, 39, 40). One also cannot exclude the possibility that MNAR/PELP1 expression in these and other brain regions could serve other functions, such as to mediate androgen and/or glucocorticoid actions, as MNAR/PELP1 has recently been shown to interact with androgen and glucocorticoid receptors, in addition to estrogen receptors (22). Interestingly, we detected a strong MNAR/PELP1-ir in the paraventricular nucleus of the hypothalamus, areas CA-1-CA-2 of the hippocampus, and in the thalamus (Table 1), sites where glucocorticoid receptors are densely located (41), which may explain localization of MNAR/PELP1 in these regions which have low expression of ER-. An important issue to consider is subcellular localization of MNAR/PELP1 and cell type of expression in the brain (e.g. neuron vs. glial expression). Regarding subcellular localization, the results of DAB, silver-enhanced nanogold and immunofluorescence staining studies suggest that MNAR/PELP1-ir localized primarily in the nuclei of cells in various brain regions, with a scattered staining observed in the cytoplasm/plasma membrane compartments. This pattern of subcellular localization of MNAR/PELP1 is consistent with the results of previous studies in MCF-7 breast cancer cells, which also showed a predominant nuclear localization with lighter cytoplasmic expression (21, 22). Cytoplasmic localization of MNAR/PELP1 would fit its proposed role in mediating nongenomic effects of estrogen through its ability to bind ER- and Src, leading to subsequent ERK activation. It is possible that MNAR/PELP1 is shuttled between the nuclear and cytoplasm compartments, although this remains to be demonstrated. The role of MNAR/PELP1 in the nucleus is less clear. MNAR/PELP1 has been shown to be a coactivator of ER and to be critical for its transcriptional effects (21, 22). MNAR/PELP1 can also interact with other factors, such as retinoblastoma, which is a nuclear protein implicated to regulate cell cycle and possibly act as a coactivator (42). Interaction with retinoblastoma has thus been proposed as an additional potential mechanism for MNAR/PELP1 enhancement of estrogen actions (42). Obviously, further studies are needed on the role/functions of MNAR/PELP1 in the nucleus.
Regarding cell type of expression, the results of our study suggest that MNAR/PELP1 is primarily expressed in neurons in most brain regions examined; however, the hippocampus, and to a lesser extent the cerebral cortex (data not shown), also showed significant colocalization of MNAR/PELP1 in GFAP-positive astrocytes. The localization of MNAR/PELP1 protein in hippocampal and cortical astrocytes is intriguing as there is evidence that in addition to neurons, astrocytes may also be targets for estrogen action (27, 43, 44, 45). Interestingly, in contrast to the hippocampus and cerebral cortex, the hypothalamus showed very little colocalization of MNAR/PELP1 protein in astrocytes. This finding demonstrates that regional differences exist in the coexpression of MNAR/PELP1 protein in astrocytes. The reason for such regional differences in colocalization of MNAR/PELP1 in astrocytes is unclear, as is the mechanism responsible for the phenomena. Nevertheless, as a whole, the studies suggest that MNAR/PELP1 could have roles in astrocytes as well as neurons.
Another important finding of the present study was that ER- and MNAR/PELP1 colocalized in many cells in the amygdala, several hypothalamic nuclei and various other regions. Approximately 60–80% of ER--positive cells in the dorsal medial region of amygdala, VMH, SCN, and ARC of the hypothalamus showed colocalization with MNAR/PELP1. As a scaffold protein, MNAR/PELP1 has been demonstrated to interact with SH3 domains of the tyrosine kinase, Src via N-terminal PXXP motifs and with ER- via LXXLL motifs (21, 22, 23). This interaction has been shown to be required for ERK signaling pathway activation by estrogen, and importantly, has been demonstrated to be critical for estrogen induction of target gene expression (21, 22). Thus, our observation of a high degree of colocalization of MNAR/PELP1 in ER--positive cells in various brain regions provides further support for a potential role for MNAR/PELP1 in E2/ER-mediated actions in the CNS. Additionally, MNAR/PELP1 has been shown to be a cofactor/coactivator for ER-induced transcription in breast cancer cells (21, 22), and MNAR/PELP1 could have similar cofactor functions in the brain. Finally, recent evidence suggests that E2 actions may also involve mediation by a G protein-coupled ER (GPR30) that is different from classical ER- and ER- (46, 47). Whether MNAR/PELP1 colocalizes/complexes with this G protein-coupled ER and mediates E2 actions via such interaction remains to be determined. Interestingly, in potential support of such a mechanism, recent work has shown that MNAR/PELP1 forms a complex with another steroid receptor, the androgen receptor, and the G protein G, and has been implicated in mediating the cross talk between androgen receptor signaling and G protein signaling (48). Further work is ongoing in our laboratory to address all of these questions.
In contrast to the high colocalization of MNAR/PELP1 and ER- that we observed in the rat brain, colocalization studies of MNAR/PELP1 and GnRH, the principal central regulatory signal for reproduction, showed that GnRH neurons rarely (<2%) colocalized MNAR/PELP1-ir in proestrous female rats. Additionally, a similar low degree of colocalization was observed for MNAR/PELP1-ir in GFP-tagged GnRH neurons in the proestrous female mouse. These studies demonstrate that MNAR/PELP1 is not significantly colocalized in GnRH neurons of the rat or mouse, at least on proestrus. The low degree of colocalization of MNAR/PELP1-ir in GnRH neurons is consistent with the reported lack of ER- colocalization in GnRH neurons (49, 50, 51, 52).
In conclusion, the rat MNAR/PELP1 gene has been cloned, revealing its high sequence and structural homology to the human MNAR/PELP1 gene. Furthermore, a detailed localization of MNAR/PELP1-ir in the rat female brain was conducted, with high intensity noted in the known targets of estrogen/steroid actions, including the hippocampus, cortex, hypothalamus, amygdala, and septum. MNAR/PELP1-ir was primarily observed in neurons of the rat brain, although some regions also showed significant colocalization of MNAR/PELP1 in astrocytes. MNAR/PELP1-ir was predominantly nuclear, but some cytoplasmic and plasma membrane-associated localization was also observed. In keeping with its proposed role as an ER interacting protein/coactivator, we observed that MNAR/PELP1-ir was localized in the majority of ER--positive cells in various regions of brain. In contrast, MNAR/PELP1-ir was not significantly colocalized in GnRH neurons in rats or mice, demonstrating that its colocalization varies depending upon cell type. In closing, the detailed characterization of MNAR/PELP1 in the brain provides a wealth of new information that may be useful for evaluating/elucidating the functional roles of MNAR/PELP1 in the CNS.
Footnotes
This research was supported by Research Grant (28965) from National Institute of Child Health and Human Development, National Institutes of Health.
First Published Online September 1, 2005
Abbreviations: ARC, Arcuate nucleus; DAB, 3,3'-diaminobenzidine; DAPI, 4'-6-diamidino-2-phenylindole; DG, dentate gyrus; E2, 17-estradiol; ER, estrogen receptor; GFAP, glial fibrillary acidic protein; ir, immunoreactivity; GFP, green fluorescent protein; RT, reverse transcriptase; SCN, suprachiasmatic nucleus; SH3, Src homology 3.
Accepted for publication August 25, 2005.
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Institute of Neuroscience (D.W.B.), School of Medicine, Medical College of Georgia, Augusta, Georgia 30912
Department of Genetics and Stanley S. Scott Cancer Center (R.K.V.), Louisiana State University Health Sciences Center, New Orleans, Louisiana 70112
Abstract
MNAR/PELP1 is a recently identified scaffold protein in the human that modulates the nongenomic activity of estrogen receptors by facilitating linkage/cross talk with the Src/Erk activation cascade. We report herein the cloning of rat MNAR/PELP1 and provide new information concerning its distribution in the female rat brain and its degree of colocalization with estrogen receptor- (ER-) and GnRH. PCR-based cloning of MNAR/PELP1 from rat hypothalamus yielded a transcript of approximately 3.4 kb, which shows 86% homology to the published human MNAR/PELP1 sequence and retained all the key binding motifs (PXXP, LXXLL, and glutamic acid clusters) in its primary structure that are known to be critical for its interaction with Src and steroid receptors. RT-PCR revealed that the MNAR/PELP1 transcript is expressed in many regions of the brain, and immunohistochemistry studies showed intense MNAR/PELP1 immunoreactivity (MNAR/PELP1-ir) in areas such as the hypothalamus, cerebral cortex, hippocampus, amygdala, and cerebellum. MNAR/PELP1-ir principally localized in the nucleus, but some cytoplasmic and plasma membrane-associated staining was also observed. MNAR/PELP1-ir was also primarily neuronal, although some localization in glia cells was observed in select brain regions. Colocalization studies revealed that a majority of ER--positive cells in the brain colocalized MNAR/PELP1-ir. In contrast, MNAR/PELP1-ir rarely colocalized in GnRH neurons. In conclusion, the current study provides evidence that MNAR/PELP1 is expressed in key neural tissues of the rat brain that are known targets of steroid action, that its expression is primarily neuronal, and that MNAR/PELP1-ir is strongly colocalized in ER-, but not GnRH neurons in the rodent brain.
Introduction
THE STEROID HORMONE, 17-estradiol (E2) exerts multiple effects in the brain to regulate such key processes as reproduction, sexual behavior, neuroprotection, synaptic plasticity, and memory (1, 2, 3, 4, 5, 6, 7). E2 has generally been thought to exert its actions through the classical genomic mechanism of direct control of gene expression via interacting with estrogen receptor (ER)- and/or ER- (8, 9). However, within the past several years there has been growing evidence that E2 can also exert rapid nongenomic effects in a variety of tissues, including the brain, which could help mediate the multiple actions of E2 in the body (10, 11, 12, 13, 14). Along these lines, E2 has been shown to rapidly regulate activation of kinases, such as MAPKs, Akt, and protein kinase A (11, 15, 16, 17), as well as to rapidly modulate intracellular calcium mobilization (17). The regulation of MAPKs is of particular interest to the neural regulatory actions of E2, as ERK, a principal estrogen-regulated MAPK, has been implicated to play critical roles in the modulation of synaptic plasticity, memory and neuroprotection (18, 19). A key upstream regulator of ERK activation is the tyrosine kinase, Src, which regulates upstream ERK-regulatory signal kinases, Ras-Raf-MEK (20).
Until recently, the mechanism of how E2-ER signaling interacts with and regulates ERK activation was unclear. However, the recent cloning of a human protein termed MNAR/PELP1 (modulator of nongenomic activity of estrogen receptor/proline-glutamic acid-leucine-rich protein-1) has shed new light on the potential underlying mechanisms for E2 regulation of ERK signaling (21, 22). MNAR/PELP1 has been shown to act both as an estrogen receptor cofactor and as a scaffold protein (21, 22). As a scaffold protein, MNAR/PELP1 has been shown to contain LXXLL and PXXP motifs in its N terminal, which mediate MNAR/PELP1 binding to AP2 domains of ER- and the SH3 (Src Homology 3) domain of Src, respectively (21, 22, 23). This interaction of MNAR/PELP1 with ER- and Src appears functionally important as overexpression of MNAR/PELP1 in cells treated with E2 activates the Src/ERK signaling cascade and enhances ER transcriptional activity, effects blocked by inhibitors of Src and MAPK kinase (22). Furthermore, antisense oligonucleotides and small interfering RNA to MNAR/PELP1 have been shown to significantly attenuate E2-induced expression of target genes in MCF-7 cancer cells and endometrial cancer cells (22, 24). MNAR/PELP1 was shown to be expressed in a variety of tissues, with highest expression noted in brain, testes, ovaries, uterus, and thyroid (21, 22). The specific tissue localization, cell type-specific expression, and degree of colocalization with ER of MNAR/PELP1 in the brain have not been determined. Additionally, the colocalization of MNAR/PELP1 in GnRH neurons is unknown, and the gene has not yet been cloned in the rat, a step critical for further studies on structure/function of MNAR/PELP1 in this species. The present study was thus designed to address these key issues.
Materials and Methods
Animal models
All experiments were conducted in compliance with the National Institutes of Health guidelines for the care and use of experimental animals and were approved by the Institutional Animal Care and Use Committee at the Medical College of Georgia. Sprague Dawley female rats (2–4 months old) were purchased from Harlan (Indianapolis, IN). The animals were housed in individual cages and water and rat chow was provided ad libitum. Regular 4-d cycling animals were killed on proestrus (1500–1700 h) and processed for RT-PCR, immunohistochemistry or Western blot analysis as described below. Only animals that showed at least two consecutive 4-d cycles were included in the study. Cycle stages were determined by recording daily electrical impedance readings of the vaginal wall using a commercially available EC40 estrous cycle monitor (Fine Science Tools, Foster City, CA). The EC40 estrous cycle monitor measures the inherent electrical resistance of the vaginal wall, which fluctuates markedly across different phases of the estrous cycle (25). Vaginal lavage and microscopic determination of vaginal cytology was also used to confirm estrus cycle stage determinations. Additionally, regular cycling green fluorescent protein (GFP)-tagged GnRH transgenic mice (2–4 months old) (gift from Dr. Moenter, University of Virginia, Charlottesville, VA) were also killed at proestrus for GnRH and MNAR/PELP1 colocalization studies (26).
Cloning of MNAR/PELP1 from rat hypothalamus
The rat MNAR/PELP1 sequence was cloned from adult female rat hypothalamus by RT-PCR using primers direct against a computer-generated predicted rat MNAR sequence (XM_340831). mRNA was isolated with RNAqueous-4PCR isolation kit (Ambion, Austin, TX) and oligo-deoxythymidine primed immediately. The resulting template was used in PCR with the following sets of primers: forward primer, 5'-GAA GAT GGC GGC AGC CGT TCT TAG TG-3'; and reverse primer, 5'-CTA AGA GTC AGG CTC TGT AGC AGG TGG TGG C-3'. The PCR conditions were; 94 C, 2 min denaturation, followed by 30 cycles at 92 C for 45 sec, 67 C for 45 sec and 72 C for 2 min; and 4 min extension at 70 C. The resulting PCR product was gel-purified and cloned into a pT7-blue vector (Novagen, EMD Biosciences, Inc., Madison, WI) and individual clones were sequenced using the T7 primer. DNA sequence analysis revealed a 3393-bp MNAR/PELP1 sequence containing an open reading frame with a start and stop codon (accession no. AY 970831).
RT-PCR analysis
Total RNA from rat tissues was isolated and primed with oligo-deoxythymidine for the reverse transcriptase (RT) reaction. A negative control, which consisted of pooled total RNA run in the RT-PCR without RT added, was also included. A positive control was also used, which was the cloned rat MNAR/PELP1 plasmid. Five hundred nanograms of the RT reactions was used for PCR with the following primer set to identify MNAR/PELP1 transcription: forward primer, 5'-GAA GAT GGC GGC AGC CGT TCT TAG TG-3'; and reverse primer, 5'-CTA AGA GTC AGG CTC TGT AGC AGG TGG TGG C-3'. The hot-start PCR conditions [Platinum Taq DNA polymerase (Invitrogen, Carlsbad, CA) 1.5 mM MgCl] were 94 C for 2 min, followed by 30 cycles of 92 C for 1 min, 67 C for 1 min, and 72 C for 3 min. A total of 10 μl of the PCR product was electrophoresed and visualized with ethidium bromide, and photographed for documentation.
Neuronal cultures
Rat cortical neurons were cultured as described previously (27). Briefly, E18 rat cortices were dissected under a microscope in cold Hanks’ balanced salt solution. Under sterile conditions, tissue was minced and treated with 0.1% trypsin (Invitrogen) and 100 U/ml deoxyribonuclease (Promega, Madison, WI) for 30 min at 37 C in serum-free Neurobasal medium (Invitrogen). Neurobasal medium was supplemented with B27 serum-free supplement (Invitrogen), Penicillin-Streptomycin liquid (x1), 0.5 mM L-glutamine, and sodium pyruvate (1x). After trypsin digestion, tissue mechanically dissociated by trituration and then filtered through a 40 μm nylon cell strainer (Becton Dickson, Franklin Lakes, NJ). Cells were counted using the trypan blue assay, and plated in poly-D-lysine-coated four-well Biocoat plates (Becton Dickson) in Neurobasal medium. Cells were plated with a plating density of approximately 500,000/well.
Western blot analysis
For Western blot analysis, tissue was homogenized in RIPA buffer (1x PBS), 1% IGEPAL CA-630 (Sigma Chemicals, St. Louis, MO), 0.5% sodium deoxycholate, 0.1% SDS) and protease inhibitors using a polytron homogenizer. After homogenization, samples were centrifuged at 10,000 rpm for 10 min at 4 C. The supernatant fraction was the total cell lysate. Protein concentration in the lysate was determined using a total protein measurement kit from Sigma Chemicals. Protein samples were denatured in sample buffer containing -mercaptoethanol in 25 mM Tris-glycine buffer and separated on 7.5% sodium dodecyl sulfate-polyacrylamide gel after loading equal amount of protein in each lane. Separated proteins were transferred to Immobilon P membrane (Millipore, Billerica, MA) at 27 V for 15 h in 25 mM Tris-glycine buffer (pH 8.3), 10% methanol using a Transblot apparatus (Bio-Rad Laboratories, Inc., Hercules, CA). After the transfer, the membranes were rinsed twice with T-TBS (20 mM Tris, 137 mM NaCl, 0.1% Tween 0) for 5 min each rinse and then incubated with 5% nonfat dry milk for 1 h at room temperature to block nonspecific/unbound surface. The membrane was incubated overnight with a well-characterized, commercially available rabbit polyclonal anti-MNAR/PELP1 antibody (1:2500; Bethyl Laboratories, Inc., Montgomery, TX). The membrane was then washed with T-TBS to remove unbound antibody, followed by incubation with secondary horseradish peroxidase-conjugated donkey antirabbit IgG (Transduction Laboratories, San Diego, CA) for 1 h at room temperature. The signal was detected using an ECL detection kit (Amersham, Buckinghamshire, UK) and the membranes were exposed to Kodak Biomax MR film (Eastman Kodak, Rochester, NY).
Immunohistochemistry
Perfusion and fixation.
Animals were deeply anesthetized with ketamine/xylazine and transcardially perfused with saline followed by fixation with 300–400 ml ice-cold 4% paraformaldehyde in 0.1 M phosphate buffer (PB), pH 7.4, at a flow rate of 20–25 ml/min. After fixation, brain samples were cut into 5-mm blocks and placed in the fixative overnight at 4 C followed by cryoprotection in 30% sucrose solution in 0.1 M PB, pH 7.4 at 4 C until the brains permeated. Tissue was frozen in OCT (optimum cutting temperature) compound under an atmosphere of nitrogen, and coronal sections (40-μm thickness) were cut on a cryostat microtome (Leica, Germany) through the entire brain and stored in a cryoprotection solution (FD Neurotechnology, Inc., Baltimore, MD) in stereological order for immunostaining. Identification of anatomical landmarks was based on stereotaxic coordinates reported previously (28).
Antibody/antiserum.
Two polyclonal antibodies were used to detect MNAR/PELP1 in rodent brain. The first was a commercially available polyclonal anti-MNAR/PELP1 purchased from Bethyl Laboratories, Inc. This antibody was produced against an epitope corresponding to C-terminal amino acids 1000–1050 of human MNAR/PELP1 and affinity purified by immobilizing the epitope on a solid support. The second MNAR/PELP1 antibody was an antiserum produced in rabbit against a 19-mer peptide encoding amino acids 558–576 (RDSLSPGQERPYSTVRTKV) of the human MNAR/PELP1 gene that has been extensively characterized by one of the authors (R.K.V.) (21). This antiserum detects MNAR/PELP1 from various tissues including brain (21). The regional distribution and density of ER--positive cells was determined using two different and well-characterized antibodies. The first one was a polyclonal antiserum purchased from Upstate Biotechnology (Lake Placid, NY), produced in rabbit against a peptide representing the last 15 amino acids of rat ER- (lot no. 26214) and has been used in earlier studies (29). The second antibody was a commercially available monoclonal antihuman ER- (catalog no. ab858) antibody purchased from Abcam Inc. (Cambridge, MA), produced in mouse against a full-length recombinant human ER-. Monoclonal antibodies specific for the glial marker, GFAP was purchased from Sigma, neuronal markers; NeuN and MAP-2, and synaptic markers PSD95 and synaptotagmin were purchased from Chemicon International Inc. (Temecula, CA). The specificity and reactivity of these antibodies have been well characterized.
Control experiments
The specificity of immunohistochemical labeling was confirmed on representative sections from proestrus female animals after blocking the antibody/antiserum with the corresponding peptide/protein immunogen or with the omission of the primary antibodies.
3,3'-Diaminobenzidine (DAB) staining method
For immunochemical analysis using the DAB staining method, 40-μm coronal sections from rat or mouse proestrous female brain, under free-floating conditions were incubated with 10% normal goat serum for 1 h at room temperature to block nonspecific surfaces. Sections were then incubated with primary antibody/antiserum at 1:1000 dilution for MNAR/PELP1 and 1:1000 for monoclonal ER- and 1:5000 for polyclonal ER- antibodies/antiserum for 36–48 h at 4 C. Afterward, sections were washed with PBS containing 0.04% Triton, followed by incubation with secondary goat antirabbit antibodies (Vector Laboratories, Inc., Burlingame, CA) at a dilution of 1:200 in PBS containing 0.04% Triton X-100 for 1 h at room temperature. Sections were then washed with PBS containing 0.04% Triton X-100, followed by incubation with ABC reagents for 1 h at room temperature in the same buffer. Tissue section were then washed and incubated with DAB reagent according to the manufacturer’s instructions (Vector Laboratories, Inc.). DAB staining results were also confirmed using silver-enhanced nanogold immunostaining for MNAR/PELP1, with a similar localization pattern and density observed in the brain for both procedures (data not shown).
Immunofluorescent staining
Free floating sections were incubated with 12% normal donkey serum for 2 h at room temperature in PBS containing 0.04% Triton X-100, followed by incubation with appropriate dilutions of the primary antibodies for 24–36 h at 4 C in the same buffer. The primary antibodies used were; monoclonal mouse-anti-ER- (1:1000; Abcam), anti-NeuN and anti-MAP2 (1:500; Chemicon), anti-GFAP (1:500; Sigma) and rabbit polyclonal anti-MNAR/PELP1 (1:500–1000; Bethyl Laboratories, Inc.). Sections were washed for 4 x 10 min at room temperature followed by incubation with Alexa-Fluor488 donkey antimouse IgG and Alexa Fluor594 donkey antirabbit IgG antibodies (1:300) for 1 h at room temperature. Sections were washed with PBS containing 0.04% Triton X-100 for 4 x 10 min, followed by 2 x 5 min with PBS and 2 x 1 min with water, and then mounted with water-based mounting medium containing antifading agents (Biomeda, Fischer Scientific, Pittsburgh, PA). For colocalization studies, a combination of one of the above monoclonal antibodies and MNAR/PELP1 antibody was used. To confirm nuclear staining, sections were incubated with 1 μM solution of either 4'-6-diamidino-2-phenylindole (DAPI) (Sigma) or TO-PRO-3 (Invitrogen) nuclear stains after appropriate dilution for 15 min at room temperature in the dark. A simultaneous examination of negative controls (after blocking the primary antibody with peptide antigen in five to six molar excess or after omitting the primary antibodies) confirmed the absence of nonspecific immunofluorescent staining, cross-immunostaining, or fluorescence bleedthrough.
For colocalization of MNAR/PELP1 with GnRH, both proestrous rats and proestrous GnRH-GFP-tagged transgenic mice were used for immunochemical analysis. Detection of MNAR/PELP1 in rats was accomplished using the rabbit polyclonal anti-MNAR/PELP1 antibody as described above, whereas GnRH detection was accomplished by using a monoclonal anti-GnRH antibody (Chemicon; 1:500). Immunohistochemistry on mice tissue sections was performed using either the Bethyl commercial MNAR/PELP1 antibody or a rabbit antiserum to MNAR/PELP1 characterized previously by one of the authors (R.K.V.) (21) (1:1000).
For in vitro double immunofluorescence staining, cultured cortical neurons were washed with Dulbecco’s phosphate buffer and fixed with 4% paraformaldehyde containing 2% glycerol at 37 C for 10 min followed by washing with PBS. Cultures were incubated with 0.1% Triton-X-100 in PBS for 10 min at room temperature and then with 10% normal donkey serum in PBS for 1 h. The cultures were then incubated with primary antibody in PBS for overnight or 24 h at 4 C. The primary antibodies used were as follows: mouse monoclonal anti-PSD95 (1:400), mouse monoclonal antisynaptotagmin (1:400), and rabbit polyclonal anti-MNAR/PELP1 (1:400; Bethyl Laboratories, Inc.). After washing with PBS, cells were incubated with fluorescent-labeled AlexaFluor488 donkey antimouse and AlexaFluor584 donkey antirabbit antibodies (1:200; Invitrogen) for 1 h at room temperature. Cell were then washed with PBS, followed by a brief wash with distilled water, and then mounted with aqueous mounting medium containing antifading agents.
Confocal microscopy and image analysis
Photomicrographs of the brain sections stained by the DAB method were captured on an Axiophot-2 visible/fluorescence microscope using an AxioVision4Ac software system (Carl Zeiss, Jena, Germany). Double fluorescence-labeled images were obtained through an inverted LSM-510 Meta confocal microscope either in XY or XYZ (Z-stacks) using a x40 oil immersion Neofluor objective (NA, 1.3). The Z-stacks (30–50 optical sections) were collected at 0.2- to 0.4-μm intervals, encompassing the entire cell bodies and cellular processes. Triple fluorescence-labeled images were captured through an LSM510 Meta confocal microscope (Carl Zeiss) attached to a Coherent (Mira 900 and Verdi-V10) two-photon laser-scanning system. The term MNAR/PELP1-ir (immunoreactivity) is used interchangeably to denote MNAR/PELP1 immunoreactivity and MNAR/PELP1 immunoreactive protein. For the immunohistochemical studies, at least six to 10 alternate sections per animal were analyzed and the experiments were repeated two to three times for verification of results.
Results
Cloning and expression of MNAR/PELP1 in rat brain
PCR-based cloning of MNAR/PELP1 from rat hypothalamus yielded a transcript of approximately 3.4 kb, which shows 86% homology to the published human MNAR/PELP1 sequence. The complete cDNA sequence has been deposited in GenBank (accession no. AY 970831). The primary protein structure consists of 1130 amino acids organized into several functionally conserved domains—such as glutamic acid-clusters, and LXXLL and PXXP motifs, the latter two having been implicated in mediating MNAR/PELP1 interaction with AF2 domains of steroid receptors and SH3 domains of proteins such as Src, respectively (Fig. 1, A and B). The glutamic acid-clusters in rat MNAR/PELP1 are located in the C terminus spanning the last approximately 280 amino acid residues, whereas the LXXLL motifs (n = 11) reside in N-terminal regions within the first 600 residues (Fig. 1B). The PXXP motifs (n = 17) are sequestered from one another, with the majority located at the C terminus encompassing the last 700 amino acids, and the remaining few motifs condensed at the N terminus within the first 70 amino acid residues (Fig. 1B). This organization of various motifs in the primary structure of rat MNAR/PELP1 is similar to that reported for other species including human (21, 22). RT-PCR using primers specific for rat MNAR/PELP1 revealed that the MNAR/PELP1 transcript is expressed in several rat brain regions, including hypothalamus, cerebral cortex, and hippocampus, and other organs such as ovary, uterus, muscle, and pituitary (Fig. 1C). A negative control (total pooled RNA used in the RT-PCR without addition of reverse transcriptase) yielded no product, demonstrating the lack of genomic DNA contamination. Additionally, a positive control (cloned rat MNAR/PELP1 plasmid) yielded a product of equivalent size to the products amplified from the various tissues.
Distribution of MNAR/PELP1 immunoreactivity in the rat CNS
Antibody specificity.
Figure 2A shows Western blot detection of MNAR/PELP1 protein from various brain regions using commercial anti-MNAR/PELP1 antibodies (Bethyl Laboratories, Inc.). Lysates from MCF-7 cell and human placental JAR cells (which have shown to have very high expression of MNAR/PELP1) were used as positive controls. A band corresponding to the size of human MNAR/PELP1 (170 kDa) was observed in all the brain tissues (hypothalamus, cortex and hippocampus) and positive controls. The specificity of the MNAR/PELP1 antibody was demonstrated by antigenic peptide blocking at 5 M excess for 3–4 h at room temperature or 24 h at 4 C, which abolished the reactivity of primary antibodies to MNAR/PELP1 protein in the brain tissues and greatly diminished MNAR/PELP1 antibody reactivity in the positive control (JAR cells) (Fig. 2B, upper panel). Similar results were observed in DAB immunohistochemistry studies on brain sections where antigenic peptide blocking of the primary antibodies led to complete abolition of MNAR/PELP1-specific immunostaining (compare Fig. 2, C and D). Antigenic preabsorption studies using the silver-enhanced nanogold procedure for MNAR/PELP1 in tissue sections also eliminated MNAR/PELP1 staining, and MNAR/PELP1 staining using the silver-enhanced nanogold procedure demonstrated a similar localization pattern as that observed using the DAB procedure (data not shown). A similar localization pattern for MNAR/PELP1-ir was also observed in the proestrous female mouse brain using the commercial (Bethyl Laboratories, Inc.) antibody (see supplemental Fig. 1 published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). The immunolocalization pattern for MNAR/PELP1-ir in rodent brain observed with the commercial antibody was confirmed using a second MNAR/PELP1 antiserum that has been extensively characterized by one of the authors (R.K.V.) previously (21) (data not shown). The commercial (Bethyl Laboratories, Inc.) antibody preparation was then used for mapping the MNAR/PELP1-ir in the entire brain of the young adult proestrous female rat as shown in Table 1. The results of the localization of MNAR/PELP1-ir throughout the brain are described in detail below.
Telencephalon
Olfactory region.
The olfactory bulb of the rodents has six well-defined cellular layers comprising; nerve cell layer, external plexiform layer, glomerular layer, mitral cell layer, internal plexiform and internal granule cell layers (Table 1). A dense population of MNAR/PELP1-ir cells was present throughout the internal granule cells and mitral cell layer. In the glomerular layer around the glomeruli and the anterior olfactory nucleus, only a scattered MNAR/PELP1-ir was observed. In the mitral cell layers, MNAR/PELP1-ir was located in both the small interneurons and large mitral cells (Fig. 2E). Similarly, a less scattered MNAR/PELP1-ir cell population was present in the medial, lateral, and dorsal subdivisions of the anterior olfactory nucleus. Sparse, MNAR/PELP1-ir was also present in several olfactory regions of the cerebral cortex, including the agranular insular cortex, the islands of Calleja, and layer III of the olfactory tubercle.
Cortex.
In the cerebral cortex, MNAR/PELP1-ir was localized throughout all the major cytoarchitectonic divisions of sensorimotor, association, visual, piriform and entorhinal cortex (Table 1). MNAR/PELP1-ir was primarily concentrated in cell nuclei (Fig. 2, F–H), with some staining localized in the cytoplasm and cell processes identified mainly in the large pyramidal neurons (Fig. 2F). Most labeled nuclei were round in shape and small to medium-sized (5–10 μm) in diameter. Occasionally, some MNAR/PELP1-ir cells were also found to have more irregularly, e.g. fusiform and star shaped nuclei suggesting their astrocytic origin (not shown). The majority of sensory, motor, and association areas of the rat cerebral cortex showed MNAR/PELP1-ir that was most prominent in layers-II-III and V-VI followed by layer IV. However, differences in the apparent density of MNAR/PELP1-ir cells distinguished the association from the sensorimotor areas. The association areas of the cerebral cortex including the medial prefrontal (Cg1–3), retrosplenial (RSG and RSA), and insular (AIV, AID, AIP) areas visually showed less density of MNAR/PELP1-ir cells compared with sensorimotor areas (e.g. the primary and secondary somatosensory, auditory, visual, and motor areas). Finally, in contrast to most cortical areas, MNAR/PELP1-ir cells in the three-layered piriform cortex were more concentrated in layer II than layer III (Fig. 2H).
Hippocampus.
In the hippocampus, a dense population of MNAR/PELP1-ir cells was observed in the CA1-CA3 pyramidal cell layers, dentate gyrus, the subiculum and parasubiculum (Fig. 2, I–L). Scattered density of MNAR/PELP1-ir cells also observed in the hilus, stratum oriens, stratum radiatum and alveus of the hippocampus (not shown). Interestingly, in these layers, MNAR/PELP1-ir cells predominantly showed characteristics morphological features of astroglial cells, which was confirmed by colocalization of MNAR/PELP1-ir with glial markers, as described later.
Septum.
Variable MNAR/PELP1-ir cells bodies were detected in the septum, being higher in the lateral septum with a slightly less density in the medial septum (Table 1). A moderate number of MNAR/PELP1-ir cells were also detected scattered in the septohippocampal nuclei, the vertical and horizontal limbs of the diagonal band of Broca, and the organum vasculosum of the lamina terminalis (Table 1).
Amygdala.
With respect to the amygdala, the density and intensity of MNAR/PELP1-ir cells was considerably higher in the dorsal portions of the medial amygdaloid nucleus (Fig. 2M) than the cortical, central, and basomedial subdivisions and amygdalohippocampal area (Table 1).
In the stria terminalis, the MNAR/PELP1-ir cells were more abundant in the dorsolateral subdivision compared with the ventral and intermediate subdivisions (Table 1). In the striatum; however, MNAR/PELP1-ir cells were more or less evenly distributed, whereas in caudate putamen they were more concentrated toward the ventral region (Table 1). Several MNAR/PELP1-ir cells with a characteristic morphology of astroglia were present in the internal and external subregions of globus pallidus and in the basal nucleus of Meynert.
Diencephalon
Thalamus.
In the thalamic areas of diencephalon, MNAR/PELP1-ir cells were scattered in all the subregions with variable numbers. Comparatively, high MNAR/PELP1-ir cell density was observed in the dorsal part of anterior medial thalamic nuclei, then the centrum medianum, paracentral and paraventricular thalamic and ventral anterior and ventrolateral sub-regions of the thalamus (Table 1).
Hypothalamus.
Among various regions of hypothalamus, a high number of intense MNAR/PELP1-ir cells was observed in the arcuate nucleus, ventromedial nucleus, suprachiasmatic nucleus, and premammillary nuclei. A moderate density of MNAR/PELP1-ir cells were also observed in the dorsomedial nucleus and the lateral hypothalamic areas (Table 1). In the anterior hypothalamus, MNAR/PELP1-ir cells were dense in the ventromedial nucleus and periventricular preoptic nuclei and in the rostral-caudal extent of the medial preoptic area. In the paraventricular nucleus, MNAR/PELP1-ir cells were present in all the subregions with the highest number of cells in the ventral part of the paraventricular nucleus (Table 1). No significant MNAR/PELP1-ir was detected in the supraoptic nucleus.
Mesencephalon
MNAR/PELP1-ir cells localized across all the subdivisions of the midbrain with a moderate density observed in dorsal and median raphe nuclei, periaqueductal gray and the substantia nigra (Table 1). Scattered MNAR/PELP1-labeled cell nuclei were also present in the dorsal part of the interpeduncular nucleus, central gray matter, dorsal and ventral nuclei of the lateral lemniscus, and the granular layer of the optic tectum. Additionally, slightly lower density, albeit strong MNAR/PELP1-ir cells were detected in the ventral tegmental area.
Metencephalon
In the cerebellum, an intense MNAR/PELP1-ir was localized in the Purkinje cells (not shown) of the outer molecular layer; whereas, in the internal molecular layer, the intensity of MNAR/PELP1-ir cells was considerably weaker (Table 1). No MNAR/PELP1-ir cells were detected in the posteriolateral or posteriodorsal fissures. Few small to medium size MNAR/PELP1-ir cells were present in the white matter. In the pons, strongly stained MNAR/PELP1-ir cells were present in the dorsal parabrachial nucleus, ventral parabrachial nucleus, the dorsal raphe nucleus, and the lateral supraolivary nucleus (Table 1).
Myelencephalon
In the medulla, a moderate number of intensely stained MNAR/PELP1-ir cells were scattered in most of the subdivisions of the solitary tract, raphe nuclei, spinal trigeminal nucleus and the lateral reticular nucleus. MNAR/PELP1-ir was present in both small interneurons and large pyramidal cells of the solitary tract and raphe nuclei. Scattered and variably stained MNAR/PELP1-ir cells were also present in the area postrema, spinal trigeminal nucleus, lateral reticular nucleus and the reticular formation (Table 1).
Spinal cord
A very low density of MNAR/PELP1-ir cells was observed in various subregions/layers of the spinal cord, including the layers II-III of the dorsal horn, layers VIII-IX of the ventral horn, and in the lamina X around the central canal. A small number of MNAR/PELP1-ir cells were also detected in the lateral cervical and lateral spinal nucleus and in the dorsal corticospinal tract (Table 1).
Table 1 summarizes the distribution of MNAR/PELP1-ir in the CNS, the intensity of the staining, and for the sake of comparison, the pattern of ER--ir staining. Figure 3 also provides a schematic representation of coronal sections depicting the distribution of MNAR/PELP1-ir in the rat brain.
MNAR/PELP1 localization in neurons and glia
We next examined the cell type (neuronal vs. glial) expressing MNAR/PELP1-ir protein by use of double/triple immunofluorescence staining and confocal microscopy. In the hippocampus, the majority (>95%) of MNAR/PELP1-ir cells in the molecular layers of dentate gyrus (DG) and pyramidal layers (CA1-CA3) showed colocalization with the neuronal-specific markers, NeuN (Fig. 4, upper left panel) or MAP-2 (lower left panel), suggesting a primarily neuronal origin of MNAR/PELP1. Interestingly, a strong colocalization of MNAR/PELP1-ir was also observed in astrocytes in the hilus of the dentate gyrus, oriens and stratum radiatum around the CA1 and CA3 pyramidal cell layers (Fig. 4, DG, CA1, and CA3; upper right panel). When quantified using confocal double label imaging, nearly 60–70% of the astrocytes showed colocalization with MNAR/PELP1-ir in the stratum radiatum and stratum oriens. However, in contrast, in the suprachiasmatic nucleus (SCN) and arcuate nucleus (ARC) of the hypothalamus, we observed that MNAR/PELP1-ir was mainly localized in the neurons, with a much lower number of astrocytes expressing MNAR/PELP1-ir. This was further confirmed by confocal two-photon microscopy of the triple fluorescent labeling of MNAR/PELP1 with the nuclear stain, DAPI and NeuN. As illustrated in Fig. 4 (ARC, lower right panels), a large number of MNAR/PELP1-ir nuclei (A; red) showed complete overlapping (D; white) with NeuN (B; green) and a nuclear stain DAPI (C; blue), indicating primarily a neuronal origin and nuclear localization of MNAR/PELP1-ir. However, it is also clear from Fig. 4 that not all the NeuN-positive neurons in the ARC show MNAR/PELP1-ir. With respect to the cerebral cortex, a majority of MNAR/PELP1-ir cells in various layers were found to colocalize NeuN (Fig. 4, SC in the upper left panel) and MAP-2 (PFC in the lower left panel). Additionally, in layer II-III and V-VI of the sensorimotor and association cortex, there were also a moderate number of astrocytes that colocalized MNAR/PELP1-ir (data not shown). Three-dimensional confocal microscopic analysis in the hippocampus using z-series stacking and three-dimensional reconstruction of z-stacks confirmed the colocalization of MNAR/PELP1-ir with the neuronal marker, NeuN and with the astrocyte marker [glial fibrillary acidic protein (GFAP)] (supplemental Fig. 2).
MNAR/PELP1 localization in ER--positive cells
We next investigated whether MNAR/PELP1-ir colocalized within ER--positive cells in regions where ER- is highly expressed. First, a systematic anatomical investigation of ER- immunoreactivity (ER--ir) was performed in the brain using polyclonal as well as monoclonal ER- antibody/antiserum preparations (Upstate and Abcam, respectively) as described in Materials and Methods. The ER--ir localization patterns obtained with these antibodies in the rat brain, were similar to those mentioned in other reports (29, 30, 31, 32, 33) and has been listed in Table 1 and illustrated in Fig. 5. As illustrated in Table 1, the localization patterns of MNAR/PELP1-ir and ER--ir, whereas similar in some regions, were not completely identical. Most notably, the density of ER--ir-positive cell bodies in the cerebral cortex and hippocampus was much lower than the density of MNAR/PELP1-ir cell bodies in these regions. In contrast, the overlap and density of MNAR/PELP1-ir and ER--ir-positive cells roughly corresponded with each other in the subregions of the hypothalamus, amygdala and bed nucleus of the stria terminalis (Table 1). Figure 5 shows photomicrographs of the areas of high density of ER--ir-positive cell bodies in the rat brain and the parallel pattern of MNAR/PELP1-ir localization in the same areas. As shown in Fig. 5 and Table 1, high density of ER--ir-positive cell bodies were observed in the septum, medial preoptic area, hypothalamus, amygdala, and bed nucleus of the stria terminalis. ER- immunostaining showed primarily nuclear localization; however, some cytoplasmic or membrane-associated ER--ir staining was also detected. To confirm that MNAR/PELP1-ir and ER--ir were indeed colocalized in cells in the above noted brain regions where significant overlap of staining pattern was observed, double-immunofluorescent colocalization studies for ER- and MNAR/PELP1 were performed. As illustrated in Fig. 6, double-immunofluorescent studies confirmed that many ER--ir-positive cells in the hypothalamus, amygdala and bed nucleus of the stria terminalis strongly colocalized with MNAR/PELP1-ir, with the highest degree (60–80%) of colocalization observed in the dorsal medial nucleus of amygdala, ventral medial nucleus of the hypothalamus, and arcuate nucleus of hypothalamus. Whereas ER--ir was lightly expressed in cortical and hippocampal regions, it is interesting to note that the few ER--ir cells observed in the cortex and hippocampus also colocalized MNAR/PELP1-ir (data not shown). Unfortunately, we were not able to ascertain MNAR/PELP1 colocalization with ER- due to unavailability of suitable primary monoclonal antibodies. Additional studies showed that MNAR/PELP1-ir is also expressed in embryonic rat cortical neurons in vitro (in both the cell body and dendritic processes), and that it colocalizes with both presynaptic (synaptotagmin) and postsynaptic markers (PSD-95) (supplemental Fig. 3).
Colocalization of MNAR/PELP1-ir in GnRH neurons
Because MNAR/PELP1 mRNA and immunoreactive protein was highly expressed in the hypothalamus, including the preoptic area and the organum vasculosum of the lamina terminalis regions of the hypothalamus where GnRH neuronal cell bodies are located, we next examined whether MNAR/PELP1-ir was colocalized in GnRH neurons in rats and GFP-tagged GnRH transgenic mice. As shown in Fig. 7, MNAR/PELP1-ir protein was rarely expressed in GnRH neurons of the rat or mouse, and there was also no overlap of MNAR/PELP1-ir fibers and GnRH neuronal fibers in the median eminence.
Discussion
Using a variety of methodological approaches, we have characterized the expression, cloning and localization of MNAR/PELP1 in the rodent brain. PCR-based cloning of MNAR/PELP1 from rat hypothalamus yielded a transcript of approximately 3.4 kb, which shows 86% homology to the published human MNAR/PELP1 sequence. The primary structure of rat MNAR/PELP1 protein consists of 1130 amino acids organized into various functionally conserved domains, similar to other species (21, 22). These conserved domains are comprised of various LXXLL, PXXP motifs and glutamic acid-clusters. The LXXLL motifs have been shown to interact with the AF2 domains of classical steroid receptors, whereas the PXXP domains serve as SH3 binding sites for Src and potentially other SH3 domain proteins (22). In vivo localization results using immunohistochemistry showed that MNAR/PELP1 is expressed with varying intensity throughout the rat brain, including a number of functionally important regions that are known targets of steroid actions. This appears to be one of the first detailed investigations of MNAR/PELP1 expression/localization in vivo, which we believe will open new directions for evaluating the functional roles of MNAR/PELP1 in the central nervous system. Our study revealed that MNAR/PELP1-ir is present throughout the telencephalon, diencephalon, mesencephalon, metencephalon, myelencephalon, and spinal cord in the rat. A similar immunohistochemical localization pattern for MNAR/PELP1 was observed in the mouse brain (supplemental Fig. 1) and monkey brain (Khan, M., and D. W. Brann, unpublished observation). In the rat olfactory bulb of telencephalon, a strong MNAR/PELP1-ir was located in the mitral and internal granule cell layers followed by glomerular layer. The cerebral cortex showed intense MNAR/PELP-ir present in all the major cytoarchitectonic divisions of the isocortex and allocortex. In the cortical regions with clear demarcation of cellular layers, a high density and intensity of MNAR/PELP1-ir cells was always detected in the layer II-III and V-VI followed by layer IV (Table 1). Strong MNAR/PELP1-ir was also observed in the piriform and entorhinal cortex. In the hippocampus, strong MNAR/PELP1-ir was noted in the pyramidal cell layers (CA1-CA3) and dentate gyrus, whereas in the hilus, stratum radiatum, stratum oriens, and alveus MNAR/PELP1-ir was significantly more scattered (Table 1), confined mainly in the astrocytes and glia. In the striatum, MNAR/PELP1-ir was higher in the lateral part than the dorsal region, and among various subregions of amygdala, MNAR/PELP1-ir was highest in the dorsal medial nucleus followed by cortical and others (Fig. 2M and Table 1). In the bed nucleus of stria terminalis, MNAR/PELP1-ir was highest both in the medial and lateral subdivisions and less scattered in ventral and intermediate subregions (Table 1). In the diencephalon, MNAR/PELP1-ir was densely localized throughout key hypothalamic nuclei, including ARC, SCN, organum vasculosum of the lamina terminalis, and VMH and all the subregions of the preoptic area (Fig. 2, N–P). In the thalamus, MNAR/PELP1-ir was present in all subdivisions, although with less density and intensity than the hypothalamus and cortex (Table 1). Lastly, variable density and intensity of MNAR/PELP1-ir cells was detected throughout the mesencephalon, metencephalon, myelencephalon and spinal cord (Table 1).
MNAR/PELP1 expression in the above brain regions, particularly the hypothalamic regions, hippocampus, amygdala, and cerebral cortex, may indicate an important role for MNAR/PELP1 in coordinating diverse estrogen actions, including such estrogen-regulated processes as reproductive function, circadian rhythms, feeding and sexual behavior, fear conditioning behavior, synaptic plasticity, neuroprotection and learning and memory (1, 2, 3, 4, 5, 6, 7, 10, 27, 33, 34, 35, 36, 37, 38, 39, 40). One also cannot exclude the possibility that MNAR/PELP1 expression in these and other brain regions could serve other functions, such as to mediate androgen and/or glucocorticoid actions, as MNAR/PELP1 has recently been shown to interact with androgen and glucocorticoid receptors, in addition to estrogen receptors (22). Interestingly, we detected a strong MNAR/PELP1-ir in the paraventricular nucleus of the hypothalamus, areas CA-1-CA-2 of the hippocampus, and in the thalamus (Table 1), sites where glucocorticoid receptors are densely located (41), which may explain localization of MNAR/PELP1 in these regions which have low expression of ER-. An important issue to consider is subcellular localization of MNAR/PELP1 and cell type of expression in the brain (e.g. neuron vs. glial expression). Regarding subcellular localization, the results of DAB, silver-enhanced nanogold and immunofluorescence staining studies suggest that MNAR/PELP1-ir localized primarily in the nuclei of cells in various brain regions, with a scattered staining observed in the cytoplasm/plasma membrane compartments. This pattern of subcellular localization of MNAR/PELP1 is consistent with the results of previous studies in MCF-7 breast cancer cells, which also showed a predominant nuclear localization with lighter cytoplasmic expression (21, 22). Cytoplasmic localization of MNAR/PELP1 would fit its proposed role in mediating nongenomic effects of estrogen through its ability to bind ER- and Src, leading to subsequent ERK activation. It is possible that MNAR/PELP1 is shuttled between the nuclear and cytoplasm compartments, although this remains to be demonstrated. The role of MNAR/PELP1 in the nucleus is less clear. MNAR/PELP1 has been shown to be a coactivator of ER and to be critical for its transcriptional effects (21, 22). MNAR/PELP1 can also interact with other factors, such as retinoblastoma, which is a nuclear protein implicated to regulate cell cycle and possibly act as a coactivator (42). Interaction with retinoblastoma has thus been proposed as an additional potential mechanism for MNAR/PELP1 enhancement of estrogen actions (42). Obviously, further studies are needed on the role/functions of MNAR/PELP1 in the nucleus.
Regarding cell type of expression, the results of our study suggest that MNAR/PELP1 is primarily expressed in neurons in most brain regions examined; however, the hippocampus, and to a lesser extent the cerebral cortex (data not shown), also showed significant colocalization of MNAR/PELP1 in GFAP-positive astrocytes. The localization of MNAR/PELP1 protein in hippocampal and cortical astrocytes is intriguing as there is evidence that in addition to neurons, astrocytes may also be targets for estrogen action (27, 43, 44, 45). Interestingly, in contrast to the hippocampus and cerebral cortex, the hypothalamus showed very little colocalization of MNAR/PELP1 protein in astrocytes. This finding demonstrates that regional differences exist in the coexpression of MNAR/PELP1 protein in astrocytes. The reason for such regional differences in colocalization of MNAR/PELP1 in astrocytes is unclear, as is the mechanism responsible for the phenomena. Nevertheless, as a whole, the studies suggest that MNAR/PELP1 could have roles in astrocytes as well as neurons.
Another important finding of the present study was that ER- and MNAR/PELP1 colocalized in many cells in the amygdala, several hypothalamic nuclei and various other regions. Approximately 60–80% of ER--positive cells in the dorsal medial region of amygdala, VMH, SCN, and ARC of the hypothalamus showed colocalization with MNAR/PELP1. As a scaffold protein, MNAR/PELP1 has been demonstrated to interact with SH3 domains of the tyrosine kinase, Src via N-terminal PXXP motifs and with ER- via LXXLL motifs (21, 22, 23). This interaction has been shown to be required for ERK signaling pathway activation by estrogen, and importantly, has been demonstrated to be critical for estrogen induction of target gene expression (21, 22). Thus, our observation of a high degree of colocalization of MNAR/PELP1 in ER--positive cells in various brain regions provides further support for a potential role for MNAR/PELP1 in E2/ER-mediated actions in the CNS. Additionally, MNAR/PELP1 has been shown to be a cofactor/coactivator for ER-induced transcription in breast cancer cells (21, 22), and MNAR/PELP1 could have similar cofactor functions in the brain. Finally, recent evidence suggests that E2 actions may also involve mediation by a G protein-coupled ER (GPR30) that is different from classical ER- and ER- (46, 47). Whether MNAR/PELP1 colocalizes/complexes with this G protein-coupled ER and mediates E2 actions via such interaction remains to be determined. Interestingly, in potential support of such a mechanism, recent work has shown that MNAR/PELP1 forms a complex with another steroid receptor, the androgen receptor, and the G protein G, and has been implicated in mediating the cross talk between androgen receptor signaling and G protein signaling (48). Further work is ongoing in our laboratory to address all of these questions.
In contrast to the high colocalization of MNAR/PELP1 and ER- that we observed in the rat brain, colocalization studies of MNAR/PELP1 and GnRH, the principal central regulatory signal for reproduction, showed that GnRH neurons rarely (<2%) colocalized MNAR/PELP1-ir in proestrous female rats. Additionally, a similar low degree of colocalization was observed for MNAR/PELP1-ir in GFP-tagged GnRH neurons in the proestrous female mouse. These studies demonstrate that MNAR/PELP1 is not significantly colocalized in GnRH neurons of the rat or mouse, at least on proestrus. The low degree of colocalization of MNAR/PELP1-ir in GnRH neurons is consistent with the reported lack of ER- colocalization in GnRH neurons (49, 50, 51, 52).
In conclusion, the rat MNAR/PELP1 gene has been cloned, revealing its high sequence and structural homology to the human MNAR/PELP1 gene. Furthermore, a detailed localization of MNAR/PELP1-ir in the rat female brain was conducted, with high intensity noted in the known targets of estrogen/steroid actions, including the hippocampus, cortex, hypothalamus, amygdala, and septum. MNAR/PELP1-ir was primarily observed in neurons of the rat brain, although some regions also showed significant colocalization of MNAR/PELP1 in astrocytes. MNAR/PELP1-ir was predominantly nuclear, but some cytoplasmic and plasma membrane-associated localization was also observed. In keeping with its proposed role as an ER interacting protein/coactivator, we observed that MNAR/PELP1-ir was localized in the majority of ER--positive cells in various regions of brain. In contrast, MNAR/PELP1-ir was not significantly colocalized in GnRH neurons in rats or mice, demonstrating that its colocalization varies depending upon cell type. In closing, the detailed characterization of MNAR/PELP1 in the brain provides a wealth of new information that may be useful for evaluating/elucidating the functional roles of MNAR/PELP1 in the CNS.
Footnotes
This research was supported by Research Grant (28965) from National Institute of Child Health and Human Development, National Institutes of Health.
First Published Online September 1, 2005
Abbreviations: ARC, Arcuate nucleus; DAB, 3,3'-diaminobenzidine; DAPI, 4'-6-diamidino-2-phenylindole; DG, dentate gyrus; E2, 17-estradiol; ER, estrogen receptor; GFAP, glial fibrillary acidic protein; ir, immunoreactivity; GFP, green fluorescent protein; RT, reverse transcriptase; SCN, suprachiasmatic nucleus; SH3, Src homology 3.
Accepted for publication August 25, 2005.
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