THE HYPOTHALAMUS is a key central nervous system

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1 /08/$15.00/0 Endocrinology 149(12): Printed in U.S.A. Copyright 2008 by The Endocrine Society doi: /en Genes Associated with Membrane-Initiated Signaling of Estrogen and Energy Homeostasis T. A. Roepke, C. Xue, M. A. Bosch, T. S. Scanlan, M. J. Kelly, and O. K. Rønnekleiv Departments of Physiology and Pharmacology (T.A.R., C.X., M.A.B., T.S.S., M.J.K., O.K.R.), and Anesthesiology and Perioperative Medicine (O.K.R.), and Division of Neuroscience (M.A.B., O.K.R.), Oregon National Primate Research Center, Oregon Health & Science University, Portland, Oregon During the reproductive cycle, fluctuations in circulating estrogens affect multiple homeostatic systems controlled by hypothalamic neurons. Two of these neuronal populations are arcuate proopiomelanocortin and neuropeptide Y neurons, which control energy homeostasis and feeding. Estradiol modulates these neurons either through the classical estrogen receptors (ERs) to control gene transcription or through a G protein-coupled receptor (mer) activating multiple signaling pathways. To differentiate between these two divergent ER-mediated mechanisms and their effects on homeostasis, female guinea pigs were ovariectomized and treated systemically with vehicle, estradiol benzoate (EB) or STX, a selective mer agonist, for 4 wk, starting 7 d after ovariectomy. Individual body weights were measured after each injection day for 28 d, at which time the animals were euthanized, and the arcuate nucleus was microdissected. As predicted, the THE HYPOTHALAMUS is a key central nervous system center for controlling many homeostatic processes, including reproduction, stress responses, fluid balance, temperature, and appetite. However, the cellular mechanisms and hypothalamic circuits involved in the control of energy homeostasis are only partially understood. Estradiol is known to affect many different types of neurons involved in the control of energy homeostasis, including the hypothalamic arcuate neurons, proopiomelanocortin (POMC), and neuropeptide Y (NPY), through multiple receptor-mediated mechanisms (1 4). Estradiol is known to attenuate weight First Published Online August 28, 2008 Abbreviations: AKAP11, A-kinase anchoring protein 11; CaM-1, calmodulin 1; CaMKII, calcium/calmodulin-dependent kinase II subunit; Ca v 3.1, T-type calcium channel 3.1 subunit; Cy3, cyanine 3; Cy5, cyanine 5; EB, estradiol benzoate; ER, estrogen receptor; ERE, estrogen response element; FC, fold change; FDR, false discovery rate; GABA, -amino-butyric acid; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GEC-1, -amino-butyric acid-a receptor associated protein-like 1; GLR, glycine receptor -subunit; KCNQ5, M-current potassium channel subunit 5; KO, knockout; mer, putative G protein-coupled ER; NPY, neuropeptide Y; pcreb, phosphorylated camp response elementbinding protein; PI3K, phosphatidylinositol 3-kinase; PI3K p85, phosphatidylinositol 3-kinase, p85 subunit; PLC, phospholipase C; PKC, protein kinase C; PITP, phosphatidylinositol transfer protein ; PKA 1, protein kinase A 1 subunit; POMC, proopiomelanocortin; qpcr, quantitative real-time PCR; SDS, sodium dodecyl sulfate; SSC, standard saline solution; SSH, suppression subtractive hybridization; SUR1, sulfonylurea receptor 1; TH, tyrosine hydroxylase; VMH, ventromedial nucleus of the hypothalamus. Endocrinology is published monthly by The Endocrine Society ( the foremost professional society serving the endocrine community. body weight gain was significantly lower for EB-treated females after d 5 and for STX-treated females after d 12 compared with vehicle-treated females. Total arcuate RNA was extracted from all groups, but only the vehicle and STXtreated samples were prepared for gene microarray analysis using a custom guinea pig gene microarray. In the arcuate nucleus, 241 identified genes were significantly regulated by STX, several of which were confirmed by quantitative realtime PCR and compared with EB-treated groups. The lower weight gain of EB-treated and STX-treated females suggests that estradiol controls energy homeostasis through both ER and mer-mediated mechanisms. Genes regulated by STX indicate that not only does it control neuronal excitability but also alters gene transcription via signal transduction cascades initiated from mer activation. (Endocrinology 149: , 2008) gain after ovariectomy in multiple rodent models primarily through classical estrogen receptor (ER) -mediated mechanisms and potentially through a novel G protein-coupled receptor (mer) (3, 5). Furthermore, physiological levels of estradiol can rapidly attenuate food intake within hours of third ventricular intracerebroventricular administration (6). Previously, we have identified a putative mer that is G q coupled to a phospholipase C (PLC)-protein kinase C (PKC) C-PKA pathway (see Fig. 5) (2). Through this receptor, estradiol reduces the efficacy of the -amino-butyric acid (GABA) B receptor agonist baclofen to activate G proteincoupled inwardly rectifying K channels in hypothalamic neurons (3). The inhibition of the G protein-coupled inwardly rectifying potassium channel activity will depolarize POMC neurons and increase neuronal activity potentially adding another mechanism for estradiol to control POMCassociated functions. Although the mer gene or proteins has not been identified, the putative mer has been functionally identified in at least three types of arcuate neurons, including POMC, dopamine, and GABA neurons. The mer was initially characterized in female guinea pigs but has also been functionally examined in ER, ER, and ER knockout (KO) mice (3). To elucidate this estradiol-induced signaling pathway, a selective agonist for the mer was discovered that has no binding capacity to classical ERs (7). This compound, called STX, is more potent than estradiol in attenuating the activation of G protein-coupled inwardly rectifying K channels by GABA B receptors in POMC neurons (2, 3). Because STX can increase the neuronal excitability of POMC neurons, we hypothesized that the putative mer has a role in hypo- 6113

2 6114 Endocrinology, December 2008, 149(12): Roepke et al. Estradiol Regulates Gene Regulation via a Membrane Receptor thalamic functions controlled by POMC neurons such as energy homeostasis. Indeed, we have previously reported that chronic, systemic injections of STX lowered post-ovariectomy body weight gain in guinea pigs similar to estradiol treatment (3). In the present study, we repeated the previous whole animal studies using a higher dose of the mer selective ligand, STX, to demonstrate a dose effect of the compound on body weight gain. Furthermore, we hypothesized that STX like estradiol acting through a mer can also activate signaling cascades that could modulate gene transcription. To measure STX-induced gene regulation in the arcuate nucleus, we used a brainspecific cdna library, produced by suppression subtractive hybridization (SSH), of estradiol-regulated genes from the guinea pig (8) coupled with cdna gene microarray production. We have previously published a description of the genes in the cdna library (9) and the initial microarray results of estradiol-treated hypothalamic tissue (8). Presently, we found that STX altered gene transcription in the arcuate nucleus, including genes that are relevant to neuronal excitability, signaling pathways, and energy homeostasis. Animals Materials and Methods Our chosen model for the human reproductive cycle is the female guinea pig because the guinea pig has a long ovulatory cycle (16 18 d), and the regulation of reproductive functions by estrogens is similar to that of primates (10 13), with a suppression of LH secretion 24 h after estradiol treatment (negative feedback), followed by the LH surge at 42 h after estradiol treatment (positive feedback) (14). All animal procedures described in this study are in accordance with institutional guidelines based on National Institutes of Health standards, and were performed with institutional Animal Care and Use Committee approval at the Oregon Health & Science University. Female multicolor guinea pigs ( g; Elm Hill Breeding Labs, Chelmsford, MA) were used in these experiments. The guinea pigs were maintained under constant temperature (22 C), and the lights were on between 0630 and 2030 h. Animals were housed in social groups with food and water provided ad libitum. Measurement of the effects of estradiol and STX on post-ovariectomy body weight gain Adult female guinea pigs weighing approximately 400 g were ovariectomized under ketamine-xylazine anesthesia (33 and 6 mg/kg, respectively, sc) and allowed to recover for 1 wk before initiation of injection treatments. The animals were injected three times per week (Monday, Wednesday, and Friday) for 4 wk with propylene glycol (vehicle) (n 5), estradiol benzoate (EB) (8 g/kg; n 5), or STX (6 mg/kg; n 7). The body weight was recorded before each injection. The dosage of EB used in this experiment produced serum estradiol levels well below the preovulatory surge of estradiol. Using RIA, the average level of estradiol from the EB-treated females was pg/ml, with the vehicle-treated serum estradiol levels less than 10 pg/ml, which is near or below the detection limit of the assay (Oregon National Primate Research Center Hormone Assay Core, Beaverton, OR). Previously, we have reported the biochemical recipe for the production of a nonsteroidal diphenylacrylamide compound, STX, which does not bind to ER or ER (2, 7). The STX was produced in the laboratory of T.S.S. with a purity of more than 95%. A two-way ANOVA (repeated measures) followed by a Newman-Keuls post hoc test was used to determine statistical significance for body weight. Tissue dissection Vehicle-, STX-, and EB-treated ovariectomized females were decapitated after sedation with ketamine (33 mg/kg, sc) 24 h after the final treatment injection. Hypothalamic nuclei were microdissected for RNA extraction and microarray analysis using our customized guinea pig microarray gene chip created from a cdna library of estradiol-regulated, brain-specific genes (9). The basal hypothalamus was cut using a brain slicer (EM Corp., Chestnut Hill, MA) into 1-mm thick coronal rostral and caudal blocks corresponding to certain figures in the Bleier (15) guinea pig atlas of the hypothalamus (Figs and 23 26). The tissue blocks were placed in RNAlater (Ambion, Inc., Austin, TX), and the rostral and caudal parts of the arcuate nucleus were dissected from the rostral and caudal blocks, respectively, using a dissecting microscope. Dissected tissue was stored at 80 C. Total RNA was extracted from the combined nuclei (rostral and caudal arcuate) using Ambion RNAqueous Micro Kits according to the manufacturer s protocol, and tested for RNA quantity using the NanoDrop ND-100 spectrophotometer (NanoDrop Technologies, Wilmington, DE). RNA quality was assessed by applying 200 ng of each RNA sample to a channel of an Agilent Nanochip and analyzed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA). Microarray chip production and printing Two microliters of bacterial culture containing the cloned DNA fragments were used for amplification. Amplifications were performed using DynaZyme EXT (New England Biolabs, Ipswich, MA) with universal primers. Reactions were run in PTC-225 thermocyclers (Bio-Rad Laboratories, Inc., Hercules, CA) for 30 cycles. Reaction products were separated from unincorporated nucleotides using MultiScreen PCR plates (Millipore Corp., Billerica, MA) and verified by visualization by agarose gel electrophoresis. Reaction products were reduced to dryness, then resuspended in 30 l printing solution [50% dimethylsulfoxide/te (10 mm Tris, 10 mm EDTA)] and stored for use in 96-well microtiter plates at 20 C. Printing plates were prepared using 3 l of each reaction product in 384-well microtiter plates (Whatman, Maidstone, Kent, UK). Arrays were printed onto UltraGAPS slides (Corning, Corning, NY) on a PixSys 5500XL microarray printer (Genomic Solutions, Ann Arbor, MI) using CMP3 printing pins (TeleChem, Sunnyvale, CA). Arrays were printed in duplicate on each slide. After printing, slides were baked at 80 C for 3.5 h and stored in a desiccator until use. RNA amplification, labeling, and cdna hybridization Amplified RNA was produced from total RNA with a single round of amplification using MessageAmp kits (Ambion). Labeled target was then made from 2.5 g amplified RNA using the SuperScript Indirect cdna labeling kit (Invitrogen Corp., Carlsbad, CA) with either singleuse cyanine-5 (Cy5) or cyanine-3 (Cy3) dye packs (GE Healthcare, Chalfont St. Giles, Buckinghamshire, UK). Before hybridization, slides were blocked for 1 h in 2% BSA (Fisher Scientific, Waltham, MA). Hybridizations were done with two slides per sample pair, arranged facing each other with the barcode labels functioning as the slide separators. Cy5- labeled sample and Cy3-labeled control were combined in 5 standard saline solution (SSC)/0.1% sodium dodecyl sulfate (SDS) and hybridized at 65 C overnight in InSlideOut slide hybridization chambers (Boekel, Feasterville, PA). After hybridizations, slide sandwiches were placed in 0.5 SSC/0.1% SDS and separated, then washed sequentially in 0.5 SSC/0.1% SDS, 0.06 SSC/0.1% SDS, and 0.06 SSC. Slides were then spun dry and placed in dark slide boxes until scanned. Microarray scanning and analysis Slides were scanned on a ScanArray 4000XL microarray scanner (PerkinElmer, Waltham, MA) using the proprietary ScanArray software. Scans were done at 5- m resolution. Images were stored as 16-bit tiff files. Images were quantified using ImaGene image analysis software (BioDiscovery, El Segundo, CA). Preprocessing for quality control was done using custom R scripts based on BioConductor 1 (16). Microarray data preprocessing and normalization We computed the background-adjusted signal intensities by subtracting the mean local background intensity from the mean signal intensity. Negatively adjusted signals were flagged and removed from subsequent analysis. The data were filtered using a threshold value

3 Roepke et al. Estradiol Regulates Gene Regulation via a Membrane Receptor Endocrinology, December 2008, 149(12): based on the 95th percentile of blank spots. For each gene there were four technical replicates. Any genes with expression values below this threshold for any of the four technical replicates were considered absent or not expressed, and were not included for further analysis. Data were transformed into a ratio by dividing the signal of the Cy3 channel by Cy5 channel. A ratio of one is considered normal expression, and a ratio of more than one or less than one is considered overexpression and underexpression, respectively. In general, when the distribution of the ratios is positively skewed, the ratios are Log 2 transformed. Normalization was performed using the within-slide, intensity-dependent normalization method of Yang et al. (17). The normalized log ratio is the main measurement used to analyze the data set. To address the issue of outlier detection, any gene with an absolute value of studentized residual 2 or Cook s distance 2 for any of the four technical replicates was marked as an outlier and filtered out. Discovery of differentially expressed genes One-way ANOVA was used to determine whether the STX treatment group is significantly expressed or not, and whether genes from STX treatment are significantly different compared with vehicle. The false discovery rate (FDR) (18) adjusted P value had been used to identify the differentially expressed genes. The FDR of a set of predictions is the expected percentage of false predictions in the set of predictions. For example, if the algorithm returns 100 genes with a FDR of 0.3, then we expect 70% of them to be correct. A fold-change (FC) cutoff of more than 1.25 (FDR P 0.05) was used as an initial criterion for the selection of STX-regulated genes. All these genes were sorted into functional categories. Quantitative real-time PCR (qpcr) To confirm the microarray results of selected genes known to be regulated by estradiol, we designed guinea pig-specific primers using areas of high homology between multiple species by aligning cdna sequences from our guinea pig cdna library (8) or known guinea pig sequences with sequences from human and rodents. All primers were designed to span introns and synthesized by Invitrogen using Clone Manager 5 software (Scientific & Educational Software, Cary, NC). See Table 1 for a listing of all the primer sets used for qpcr. Because of the loss of one of the EB-treated RNA samples leaving us with a number of four EB-treated samples, we decided to also compare selected STXregulated genes with another set of long-term EB-treated arcuate RNA samples (n 6) from a previous body weight experiment (3). The vehicle for this earlier set of RNA samples was sesame oil, therefore, the oil samples (n 7) from that experiment were used as the control for the EB-treated qpcr analysis. Both EB-treated groups were analyzed by qpcr for some of the selected genes. In preparation for qpcr, the remaining total RNA from the microdissected arcuate nucleus samples was deoxyribonuclease I treated (DNAfree; Ambion) at 37 C for 30 min to minimize any genomic DNA contamination. cdna was synthesized from 200 ng total RNA using 50 U murine leukemia virus reverse transcriptase (Applied Biosystems, Foster City, CA), 4 l5 buffer, 25 mm MgCl 2,10mm deoxynucleotide triphosphate, 100 ng random hexamer primers (Promega Corp., Madison, WI), 40 U/ l Rnasin (Promega), and 100 mm dithiothreitol in diethylpyrocarbonate-treated water (Ambion) in a total volume of 20 l. Reverse transcription was conducted using the following protocol: 60 TABLE 1. Primer sequences used for qpcr Name Product length Primer efficiency Primer sequence Base pair no. Accession no. KCNQ5 225 bp 100 CTC ACC CCA CCA CTT AAA NM_ CTC TCG GCT CTT CTT ATC TG SUR1 145 bp 91 TCG CCG GGA CAG AAG ATT GG AF GGG TGT GCA GTG GCA GTT TG GEC bp 100 GCG CGA GTC TTC ATC AAG GG AF TGA GGT CAG AGG GCA CAA GG ER CTG CGC AGT GTG CAA TGA C DQ TCA CAG GAC CAG ACC CCA TAA ER AGA ACC GGC GGA AAA GCT DQ CAT TCC TAC TCC ATA GCA CTT TCG Ca v bp 96 CTG GAG CTG GAG ATG AAG AC DQ AGT GGG CTG CCT TGT ATG CaM bp 92 TCT GCA CAT CAT CCT GAG NM_ CAT GCA GCT TGA CGA TTG AKAP bp 94 GTC CAA TGA TGG CAA GTC AG NM_ CGT GCA ATC CAT CTA CGC AG PKA bp 95 GAG CAG CCA CTG TCA AAG C NM_ CGT TCC CAC TTG TCC AGA G PI3K p bp 93 GCC AGA CCT CAT TCA GCT AAG NM_ GTT GCT GCT GCC AAC ATT C GLR 246 bp 98 CAT GCC CTT TGG ACT TGA C NM_ TCC TCA GGG TGA AGA TGA C CaMKII 101 bp 93 GAA GAC ACC AAA GTG CGG AAA C BC TCG CAC ATC TTC GTG TAG GAC PITP 114 bp 93 GTTG TGC CTA TAA GCT GGT GAC BC AGC TGG CGA TGG AAG TTT G TH 223 bp 93 TCC ACG TTA TAC TGG TTC AC DQ TTG CAT CAC TGA AGC TCT C GAPDH 212 bp 100 CAT CCA CTG GTG CTG CCA U51572 GTC CTC GGT GTA GCC CAA G NPY CCC GAG ACA CTG ATT TCA GAT CT NM_ Probe CCC ATC ACC ACA TAG AAG GAT CTT AAA GCA CAG AAA ATG TCC CCA GAA CTA GGC POMC CGC TGG TGA CGC TGT TCA S Probe CGC CTC ACT GGC CCT TCT AAC GCC ATC GTC AAG A Actin AAG CCC AGA GCA AAA GAG GTA TC AF Probe TAA CGA TGC CGT GCT CAA TG TGACCCTGAAATACC The sense primer is listed first with the antisense primer below.

4 6116 Endocrinology, December 2008, 149(12): Roepke et al. Estradiol Regulates Gene Regulation via a Membrane Receptor min at 42 C, 5 min at 95 C, and 5 min at 4 C. The cdna was diluted to 1:20 with Nuclease-free water (Ambion) for a final cdna concentration of 0.5 ng/ l and stored at 20 C. Basal hypothalamic test tissue RNA was used for positive and negative controls (no reverse transcriptase) and processed simultaneously with the experimental samples. For qpcr, 4 l cdna template (an equivalent of 2 ng total RNA) was amplified using PowerSyber Green master mix (Applied Biosystems) on an ABI 7500 Fast Real-time PCR instrument. Standard curves for each primer pair were prepared using serial dilutions of basal hypothalamus cdna in triplicate to determine the efficiency E 10( 1/m ) 1, m slope of each primer pair. All efficiencies expressed as percent efficiency were approximately equal (one doubling per cycle, %; Table 1), therefore, the relative mrna expression data were analyzed using the CT (cycle threshold) method (19, 20). The amplification protocol for all the genes was as follows: 95 C for 10 min (initial denaturing), followed by 45 cycles of amplification at 94 C for 15 sec (denaturing); 60 C for 30 sec (annealing); and completed with a dissociation step for melting point analysis with 35 cycles of 95 C for 15 sec, C (in increments of 1 C) for 1 min, and 95 C for 15 sec. However, primers for M-current potassium channel subunit 5 (KCNQ5) and protein kinase A 1 subunit (PKA 1) were optimized with an annealing temperature of 57 C and 62 C for 30 sec, respectively. For NPY and POMC, TaqMan primers and probes were used with -actin as the reference gene. Positive and negative controls were added to each amplification run, including a water blank. Quantification values were generated only from samples showing a single product at the expected melting point. Final relative quantitation was done using the comparative CT method (19, 20) using a calibrator of pooled, diluted cdnas from each oil-treated arcuate sample (50 l/sample). The data are reported as relative mrna expression. To determine the CT for each transcript, the threshold was consistently set at the lowest point of the exponential curve where the slope of the curve was the steepest and above the baseline of the first 15 cycles. The CT method normalizes the CT from each sample for each target gene by subtracting the CT of the reference gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or -actin, which is unresponsive to estradiol-treatment ( CT). The CT values were calculated using the pooled oil-cdna calibrator CT: CT (CT target gene CT reference gene) CT of calibrator. The relative linear quantity of target molecules was calculated using the formula 2 CT. Therefore, all transcription data are expressed as an n-fold difference relative to the calibrator. The n-fold difference was averaged for each treatment and analyzed statistically using a two-tailed Student s t test (P 0.05). Results Body weight gain is attenuated in ovariectomized females by EB and STX We have previously shown that systemic treatment with STX (2 mg/kg) in guinea pigs will lower the body weight gain after ovariectomy similar to systemic treatment with EB (3). We repeated the experiments with more animals and with a higher dose (6 mg/kg) of STX (Fig. 1). The present study not only reproduced the same effect on post-ovariectomy body weight gain but even showed a significantly greater reduction in body weight gain than in the former STX study. Therefore, when both studies were analyzed for percent body weight gain at the end of each experiment normalized to body weight at ovariectomy, the lower dose of STX has significantly higher weight gain ( %) compared with the higher dose used in the present study ( %; P 0.05). To confirm further the reproducibility of the former study, we also measured uterine weight and found that STX did not have a significant effect on uterine weight (STX: g vs. vehicle: ), whereas EB did significantly increase uterine weight by nearly 10-fold (EB: ). FIG. 1. STX and estradiol significantly reduced body weight gain in ovariectomized female guinea pigs. Estradiol significantly reduced body weight gain by the fifth day after first injection. STX significantly reduced body weight gain compared with vehicle by d 12 (*, P 0.05; **, P 0.01). The body weights of STX-treated females were also significantly different from EB-treated females (#, P 0.05; ##, P 0.01). Data points represent the mean SEM of five, five, and seven animals per group for vehicle (circles), EB (triangles), and STX (squares) treatments, respectively. Comparisons between groups were made using a two-way ANOVA (repeated measures) with post hoc Newman-Keuls paired analysis. STX regulates gene expression in the arcuate nucleus Pairwise comparisons of the mean difference between the vehicle- and STX-treated samples (n 5 and 7, respectively) for each gene determined that 241 identified genes were significantly different ( 1.25 FC; P 0.05) between the treatments. We used this FC and P value cutoff because these genes were previously identified as estradiol-regulated during the suppressive subtractive hybridization to produce the cdna library. Moreover, we found that several of our known estradiol-regulated genes, including tyrosine hydroxylase (TH), GABA-A receptor associated protein-like 1 (GEC-1), and NPY, were regulated by STX according to this criterion. Of these 241 genes, 80 genes were up-regulated, and 161 genes were down-regulated (Fig. 2A). On the microarray gene chip, over 400 cdna transcripts were not identified by Basic Local Alignment Search Tool searching, and of these unidentified transcripts, 196 were significantly regulated by STX. Thus, a combined total of 437 transcripts in the cdna library were regulated by STX according to the microarray. Of the identified genes, 10.7% of them were functionally categorized to be involved in metabolism and mitochondrial function, and 9.4% were channels or other membrane proteins (Fig. 2B). Signaling molecules made up 11.7% of the total, whereas 12.7% of the genes were involved in protein trafficking and catabolism. Genes involved in transcription and translation made up 16.8% of the total with genes involved in cell communication or cell cycle and growth made up 4.6 and 14.1%, respectively. Another cutoff commonly used in microarray analysis is the statistical P values produced during the ANOVA comparison between the treatments. In our study the genes more than 1.25 FC were divided into four groups of P value ranges. Of the 241 genes, 34 had a P value between 0.05 and

5 Roepke et al. Estradiol Regulates Gene Regulation via a Membrane Receptor Endocrinology, December 2008, 149(12): FIG. 2.A,STX(gray) significantly (FDR P 0.05; FC 1.25) regulates a number of genes compared with vehicle in the arcuate. Up are up-regulated genes, and Down are down-regulated genes. B, The percent distribution of the STX-regulated genes from the arcuate total RNA samples analyzed by microarray and divided into eight functional categories: Translation & Transcription; Channels & and Membrane Proteins; Signaling Molecules; Cell Cycle & Growth; Metabolism and Mitochondria; Protein Trafficking & Catabolism; Cell Communication; and Unknown. The cdna microarray chip was created from a brain-specific, estrogen-regulated guinea pig cdna library produced using SSH. FIG. 3. A representation of the percentage of the STX-regulated genes within each of the four P value ranges. All 241 genes with a FC more than 1.25 were divided into four categories based on their associated P value for the FDR. These categories are: P 0.05 but more than 0.01; P 0.01 but more than 0.001; P but more than ; and P , and 95 had a P value between 0.01 and (Fig. 3). Another 58 genes have a P value less than but above , with the remaining 54 less than A P value cutoff of P 0.01 for eliminating false positives is commonly used in the analysis of microarray data (21 23). However, we chose a modification of the FDR to estimate the percentage of observed total positives expected to be false. With our microarray of 954 genes, a P 0.05 would result in 48 false positives ( ). The FDR would be 48/241 (the number of genes with a FC 1.25) or This FDR indicates that 20% of the 241 genes should be positive by chance alone and that any single positive has a 20% chance of being a false positive (24). Because the genes are from an estradiol-regulated cdna library and many of them are known to be involved in homeostatic and neuroendocrine functions, we used the P 0.05 cutoff and present selected genes from each P value range in Tables 2 5. Confirmation of selected genes by qpcr The genes listed in Tables 2 5 demonstrate the wide variety of genes that are regulated by systemic STX in the arcuate nucleus. These identified genes have a multitude of functions in neurons but collectively suggest that estradiol through the mer may affect a wide variety of cellular mechanisms through the control of gene transcription. However, because we are primarily interested in the signaling pathways involved in energy homeostasis, we selected nine genes isolated during the cdna library production that were regulated by STX plus eight genes added to the microarray as controls to be analyzed by qpcr. These 17 genes include receptors, cation channel subunits, calcium and phosphatidylinositol signaling molecules, and genes involved in the control of energy homeostasis and feeding behavior. Estradiol has previously regulated a number of these genes in the arcuate nucleus (9, 25 27). Of these 17 genes, 16 of them had significantly different expression levels in STX-treated females compared with vehicle-treated females, with ER the only gene that did not reach significance in the microarray according to our criteria but was significantly up-regulated by STX treatment using qpcr (Fig. 4A). In Table 2 (P 0.05 to 0.01), we confirmed STX-induced expression differences by qpcr for five of the seven genes in this table (Fig. 4, A and C). In Table 3 (P 0.01 to P 0.001), we examined four genes with qpcr and confirmed directional regulation in only one gene T-type calcium channel 3.1 (Ca v 3.1). Another gene, phosphatidylinositol 3-kinase (PI3K), p85 subunit (PI3K p85 ), was suppressed by STX according tomicroarray analysis but was significantly up-regulated by STX using qpcr. This confir-

6 6118 Endocrinology, December 2008, 149(12): Roepke et al. Estradiol Regulates Gene Regulation via a Membrane Receptor TABLE 2. A list of genes regulated by long-term STX treatment (0.01 P 0.05) Accession no. Gene name Function FC SE P value AF SUR1 a Channels NM_ Adenylate cyclase-inhibiting G protein I1 G proteins AB Na /K -ATPase -1 Membrane transport NM_ Double cortin and CaM kinase-like 1 PKs NM_ p21/cdc42/rac1-activated kinase AY /rac-interacting citron kinase AF GEC-1 a Protein trafficking DQ Glucose-regulated protein GRP DQ ER a Receptors NM_ AKAP11 a Signal transduction AF Dopamine receptor interacting protein NR_ Inositol polyphosphate-5-phosphatase F NM_ Phosphatidylinositol-specific PLC, X domain containing 3 NM_ NPY a Synaptic transmission S POMC a DQ TH a a Transcript tested with qpcr. mation of gene regulation in the opposite direction occurred with two more genes, and in all three instances, gene expression was suppressed based on the microarray but enhanced using qpcr. The other two genes were this occurred are presented in Table 4 (P to P ) along with three other genes that were analyzed with qpcr. In this P value category, three of the five genes were regulated by STX treatment in the arcuate nucleus. Comparison to estradiol treatment using qpcr For comparison to chronic STX treatment, we also analyzed the expression of the 17 selected genes in the arcuate nucleus from long-term EB- and vehicle-treated females (an independent set of experiments in which body weight was significantly reduced by EB) using qpcr (Fig. 4, B and D). Among the control genes that are known to be regulated by estradiol, EB up-regulated Ca v 3.1 ( 1 G) ( vs , vehicle; all genes were normalized to vehicle expression, i.e. 1.00), ER ( ), POMC ( ), and TH ( ), while down-regulating ER ( ). STX up-regulated Ca v 3.1 ( ), ER ( ), and TH ( ), and also up-regulated PI3K p85 ( ), which EB did not affect. Unlike EB, STX did not have an effect on POMC relative mrna expression but did significantly decrease NPY expression in the arcuate nucleus ( ). Among the genes isolated during the SSH, chronic EB treatment up-regulated calmodulin 1 (CaM-1) ( ), GEC-1 ( ), and KCNQ5 ( ), while down-regulating A-kinase anchoring protein 11 (AKAP11) ( ). Conversely, STX up-regulated AKAP11 ( ), calcium/calmodulin-dependent kinase II subunit (CaMKII ) ( ), GEC-1 ( ), glycine receptor -subunit (GLR ) ( ), and phosphatidylinositol transfer protein (PITP ) ( ). Although the microarray indicated a 2-fold increase in the expression of -actin, a commonly used qpcr reference gene, neither EB- nor STX-treatment changed the relative expression of -actin using qpcr and GAPDH as the reference gene. To compare between the two sets of EB-treated samples long-term treated EB samples from the earlier study (n 6) or the current study (n 4), we analyzed gene expression TABLE 3. A list of genes regulated by long-term STX treatment (0.001 P 0.01) Accession no. Gene name Function FC SE P value XM_ Contactin 1 Cell adhesion XM_ Cyclin I Cell cycle NM_ NCK-associated protein 1 Cell proliferation DQ Ca v 3.1 a Channels AF Guanine nucleotide exchange factor G proteins NM_ Ras-related C3 botulinum toxin substrate BC F-box and leucine-rich repeat protein 5 Protein catabolism BC F-box protein NM_ Ubiquitin-like NM_ PKA 1 a PKs NM_ CaM-1 a NM_ Cyclin-dependent kinase M29550 Calcineurin A1 Protein phosphatase AF Syntaxin-interacting protein Protein trafficking AJ GABA(B)-R1 Receptors NM_ PI3K p85 a Signal transduction NM_ Phosphatidic acid phosphatase type 2B NM_ Glutamate decarboxylase 67 Synaptic transmission a Transcript tested with qpcr.

7 Roepke et al. Estradiol Regulates Gene Regulation via a Membrane Receptor Endocrinology, December 2008, 149(12): TABLE 4. A list of genes regulated by long-term STX treatment ( P 0.001) Accession no. Gene name Function FC SE P value NM_ KCNQ5 a Channels NM_ Maxi K NM_ Voltage-dependent anion channel AF Actin a Cytoskeleton NM_ G protein, -polypeptide 2-like 1 G proteins NM_ GTPase activating Rap/RanGAP domain-like NM_ G, 1 activating activity polypeptide NM_ RAB10, member RAS oncogene family AF GTPase activating protein BC CaMKII a PKs NM_ Centaurin, 1 Receptors NM_ GLR a NM_ Progesterone receptor membrane component BC PITP a Signal transduction NM_ PLC-like a Transcript tested with qpcr. of 11 of the 17 selected genes using the current EB-treated samples (n 4) and found similar levels of relative mrna expression in nine of the 11 genes. In the current EBtreated group (n 4), AKAP11 and ER mrna expression was suppressed ( ** and *, respectively; *, P 0.05; **, P 0.01 compared with oil-treated samples). Also in this group, EB treatment increased the relative mrna expression of CaM-1 ( **) Ca v 3.1 ( *), ER ( *), and KCNQ5 ( *). Similar to the earlier experiment, EB treatment did not have an effect on NPY, PKA 1, and PI3K p85 expression. However, in the current (n 4) samples, EB treatment did not affect GEC-1 expression but did increase sulfonylurea receptor 1 (SUR1) expression ( **). It should be noted that in the current group with a lower sample number, the variation was greater compared with the n 6 group, but the majority of genes were similarly affected by EB. Discussion In this study we examined the effects of STX, a selective agonist for the putative mer, on body weight gain in ovariectomized guinea pigs. We then determined whether systemic STX regulates gene transcription in the arcuate nucleus using microarray analysis in conjunction with qpcr in the same group of animals. The potential signaling pathways leading to gene expression are illustrated in Fig. 5. We have shown that a unique gene regulation signature occurs in the arcuate nucleus in the STX-treated females. The genes regulated have a variety of functions in the cell, including signal transduction, transcription, translation, protein trafficking, and other vital aspects of neuronal activity and survival. Because microarray analysis is a crude indicator of individual gene expression, relevant genes should be verified by other techniques of mrna analysis such as qpcr. Of the 16 selected genes that had significant differences in expression between the vehicle and STX after microarray analysis, 10 of these selected genes were regulated by STX using qpcr. Therefore, the confirmation of the microarray results suggests that our novel array is useful in obtaining data for a global picture of gene regulation in the context of STX treatment and an adequate indicator of the genes regulated by STX. The role of a mer and membrane-initiated gene transcription in energy homeostasis The central, hypothalamic effects of estradiol on energy homeostasis are primarily thought to function through ER mediated mechanisms. This hypothesis is partially due to TABLE 5. A list of genes regulated by long-term STX treatment (P ) Accession no. Gene name Function FC SE P value AY Cytochrome c oxidase subunit VII Electron transport BC Ubiquinol-cytochrome c reductase core protein II NM_ Ca 2 -dependent secretion activator Exocytosis BT RAB, RAS oncogene family-like 2B G proteins NM_ Longevity assurance gene homolog 1 Lipid biosynthesis NM_ Glutamate transporter A2 Membrane transport X Phosphatidylethanolamine binding protein Metabolism NM_ Ubiquitin specific peptidase 15 Protein catabolism NM_ Protein phosphatase 3, catalytic subunit, Protein phosphatase BC Protein phosphatase 2, regulatory subunit A, NM_ ADP-ribosylation factor 1 Protein trafficking BC01083 Ribophorin I NM_ IGF binding protein, acid labile Signal transduction NM_ MAPK 8 interacting protein AF Phospholipase D AY Migration-inducing protein 6 Stress response BC Amyloid (A4) precursor-like protein 1 Synaptic transmission

8 6120 Endocrinology, December 2008, 149(12): Roepke et al. Estradiol Regulates Gene Regulation via a Membrane Receptor FIG. 4. qpcr confirms regulation of multiple genes by STX in the arcuate nucleus. The relative mrna expression for selected control genes (A and B) and selected genes of interest (C and D) from microdissected arcuate tissue from vehicle-treated propylene glycol (n 5) or oil (n 7); black bars females and STX- (n 7) or EB-treated (n 6) females (gray bars). The expression values were calculated using the CT method where the calibrator was the average CT of the vehicle-treated samples. Bar graphs represent the mean SEM. Statistics: two-tailed Student s t test (*, P 0.05; **, P 0.01; ***, P compared with vehicle). early observations of an obesity phenotype in ERKO mice. The ERKO mice exhibited an obesity phenotype, whereas the ERKO mice did not (28, 29). ER is also considered important for the attenuation of post-ovariectomy body weight gain by estradiol in mice because estradiol did not have a significant effect in ERKO mice on weight gain (5). An important nucleus in the hypothalamic control of energy homeostasis besides the arcuate nucleus is the ventromedial nucleus of the hypothalamus (VMH). Estradiol has many actions in this nucleus, and, in fact, the suppression of ER by RNA interference in the VMH of both mice and rats produced a phenotype similar to metabolic syndrome with an increase in body weight, food intake, and reduced metabolism (30). There are synaptic interactions between the neurons of the VMH and arcuate POMC neurons, and VMH neurons do express the melanocortin-4 receptors (31, 32). Because these neurons are also activated by STX and estradiol increasing their neuronal excitability, this suggests that estradiol may have more than one pathway to effect VMHmediated control of energy homeostasis. The effects of estradiol in the transgenic mouse model are also thought to be due to the loss of estradiol-mediated actions at extrahypothalamic sites in the brain such as the nucleus tractus solitarius. The nucleus tractus solitarius controls feeding behaviors and is prominently involved in the effects of the gut peptide, cholecystokinin, on the control of food intake, which is potentiated by estradiol (5, 33, 34). In peripheral tissues, another study suggested a role for ER in adipose tissue accumulation based on an increase in weight gain and fat accumulation during estradiol treatment in ERKO mice (29). These findings collectively suggest that ER is the prominent nuclear receptor involved in the estrogenic effects on the neural control of energy homeostasis, although ER -mediated signaling may have a part in the periphery. Another possible pathway for estradiol to control certain aspects of energy homeostasis is through the activation of the

9 Roepke et al. Estradiol Regulates Gene Regulation via a Membrane Receptor Endocrinology, December 2008, 149(12): FIG. 5. A, Classical ER membrane-associated and nuclear-associated signaling. B, Signaling pathways for the novel membrane ER (mer). 1) Estrogen can activate the classical ERmediated (ER / ) pathways leading to changes in mrna transcription of relevant genes via ERE. 2) Both ERs are regulated by STX, and may be a mechanism for the interaction of classical ER and mer signaling. 3) Estrogen can bind to membrane-associated classical ER leading to the activation of signal transduction pathways to control gene transcription and cellular functions. The putative mer is a G protein-coupled receptor (GPCR) linked to G q/11 protein that activates PLC-PKC-PKA pathway may control gene transcription via pcreb- CRE. The activation of PKC can also lead to changes in gene transcription via the phosphorylation of other signaling proteins (MAPK/ERK). 4) The hydrolysis of phosphatidylinositol (4,5)-bisphosphate (PIP 2 ) into 1,4,5-trisphosphate (IP3) may also initiate calciumdependent signaling molecules (CaM/CaMK) to control transcription. Several of these genes are associated with cation and anion channels (Ca v 3.1, GLR) or proteins that impinge on channel functions (AKAP). 5) Changes in neuronal excitability either by channel expression or channel modulation may also control gene expression. A few of the genes analyzed by qpcr are involved in calcium signaling (CaMK II) and protein trafficking (GEC1). putative mer in arcuate neurons. Our previous data and the current data suggest a role for the mer (via STX-induced activation) in the effects of estradiol on energy homeostasis. At this time, we are not certain what part of energy homeostasis (metabolism, feeding behavior, etc.) is directly affected by STX (mer activation). Because estradiol and STX are known to activate POMC neurons, we can hypothesize that part of the STX-mediated effects involves those functions that are controlled by POMC activation and gene expression, i.e. feeding behaviors (35). These effects on POMC neuronal excitability occur before the activation of the melanocortin 3/4 receptors in other hypothalamic nuclei that control energy homeostasis. Indeed, injections of a melanocortin 3/4 receptor antagonist in the lateral ventricle, which blocked the downstream effects of melanocortin signaling, ablated any estrogenic effects on food intake in ovariectomized rats (36). In recent years, substantial evidence indicates that estradiol can activate a host of rapid signaling cascades to induce gene transcription (membrane-initiated steroid signaling) (37 40). Through membrane-associated ER, estradiol activates multiple signaling pathways, including PI3K, PLC, and PKA/phosphorylated camp response element-binding protein (pcreb). In fact, estrogenic activation of pcreb can induce transcription of genes for important neurotransmitters such as dopamine, enkephalin, dynorphin, and neurotensin (41, 42), and regulate CRH gene expression through ER - and/or ER -CRE pathways (43). Although there is little to no direct evidence suggesting that membrane-initiated signaling via classical ER controls energy homeostasis, the rapid signaling initiated by the putative mer (PLC-PKC-PKA) has now been shown to generate nascent transcription potentially through multiple signaling pathways or even due to an increase in neuronal excitability (44, 45) (Fig. 5). Interestingly, STX up-regulated the mrna expression of both types of ERs in the arcuate nucleus. Although both ER and ER are expressed in the guinea pig arcuate, ER expression is 4-fold higher than ER (9), and estradiol-induced down-regulation of ER has been previously found to be an estrogen response element (ERE)-dependent transcriptional event (46). In our study, long-term EB treatment also down-regulated ER ; however, in STX-treated females, ER is up-regulated, which is not surprising because ER down-regulation is ERE dependent. Expression of the other nuclear ER, ER, is increased by both EB and STX treatment. An increase in ER expression in the hypothalamus of females during long-term estradiol treatment has not been previously reported. In the pituitary, estradiol treatment suppressed ER expression after ovariectomy in rats again through an ER -dependent mechanism (47). In peripheral cells, PKC- and Ras-mediated pathways regulate the expression of ER / genes (48, 49), and potentially, similar mechanisms may be involved in the STXinduced increase in classical ER gene expression. The upregulation of ER / by STX in the arcuate nucleus is a potential mechanism whereby the activity of the mer and the classical ERs intersect to control arcuate neuronal functions. We found that chronic treatment with estradiol increased POMC mrna expression, whereas STX decreased NPY expression according to qpcr. These two types of arcuate neurons (POMC and NPY) are relevant to the estrogenic effects on energy homeostasis (2 4, 50, 51). POMC neurons decrease food intake (anorectic), whereas NPY neurons increase food intake (orexigenic) (35). Previous studies have shown that estradiol regulates the expression of both of these

10 6122 Endocrinology, December 2008, 149(12): Roepke et al. Estradiol Regulates Gene Regulation via a Membrane Receptor genes depending upon the treatment paradigm and experimental model (1, 4, 27, 50 55). In our study, estradiol did not affect NPY expression, whereas STX suppressed NPY expression. Potentially, STX, the mer selective ligand, exerts some of its effects by suppressing NPY while not directly effecting POMC mrna expression. The suppression of NPY or the increase in POMC expression would both lead to a similar physiological effect: a decrease in food intake. The changes in POMC or NPY mrna expression may be a response to changes in caloric intake, and not directly from STX or EB treatments. However, in the mouse the increase in POMC gene expression by estradiol treatment occurs within 12 h (4), which is before there is a measurable effect on body weight in the guinea pigs under the current experimental design. Other genes regulated by the long-term treatments by EB and/or STX are also regulated by 24-h treatment, including AKAP11, Ca v 3.1, CaM-1, KCNQ5, etc. (9, 26), which suggests that some of the actions of STX on gene expression are directly on arcuate neurons. Although we cannot exclude the long-term impact of ovariectomy on NPY expression as reported by Clegg et al. (56), further studies are needed to elucidate central and peripheral effects of STX treatment on homeostatic functions. Regardless of the different responses to EB or STX treatment, it is apparent that the effects of STX are mediated, in part, by direct effects on POMC and NPY neurons in the arcuate nucleus. Another arcuate neuronal cell type is dopaminergic neurons that are marked by the expression of TH, the ratelimiting enzyme in the catecholamine synthesis pathway. Our data suggest that long-term estradiol treatment upregulates TH expression in the arcuate nucleus partially through activation of the mer, which has been functionally identified in arcuate dopamine neurons (2). The mer when activated by STX initiates a PLC-PKC signaling pathway that activates PKA. The activation of PKA will initiate pcreb gene regulation, which has already been suggested as a transcriptional pathway for estradiol-induced regulation of TH mrna expression (41, 57). Previous studies have shown that estradiol treatment will increase the expression of both TH mrna and protein in other dopaminergic neurons (58, 59). Although dopamine-deficient mice exhibit a lack of feeding through the loss of motivation that is controlled by the midbrain region (60), it is unknown at this time if the activation of dopamine neurons in the arcuate nucleus by estradiol is a part of estradiol s control of energy homeostasis. The regulation of channels and signaling molecules Estradiol induces gene expression of a Ca 2 channel (Ca v 3.1) subunit in the arcuate nucleus after 24 h treatment (26) and after long-term estradiol treatment. STX also significantly up-regulates the Ca v 3.1 in the arcuate nucleus after long-term treatment. Because the mer is found in POMC neurons, one can hypothesize that the up-regulation after STX treatment is occurring in these neurons. The estradiolinduced increase in Ca v 3.1 expression has previously been demonstrated to increase the peak T-type Ca 2 current by 2-fold in arcuate neurons (26), which includes POMC neurons. The increase in the T-type Ca 2 current would augment burst firing, and cause increases in neurotransmitter and secretory protein ( -MSH) release. STX, but not estradiol, altered the expression of another type of channel subunit in the arcuate nucleus, the GLR, which is the structural subunit for this ligand-gated Cl channel. Glycine is a major inhibitory neurotransmitter and initiates a postsynaptic increase in chloride conductance through the glycine receptor (61). The inhibitory nature of the glycine receptor may have a role in rapid leptin signaling in the arcuate nucleus because the 1 subunit of the glycine receptor associates with leptin receptors in rat brainstem and may do so in the arcuate nucleus where the effects of leptin on energy homeostasis is centered (62). Therefore, any alteration in the expression of the glycine receptor by the mer may be relevant to the anorectic effects of leptin that occurs through hypothalamic neurons. There are two signaling pathways that STX controls through modulation of gene expression. The first such pathway is the PI3K pathway. STX up-regulates the p85 subunit of PI3K and PITP, whereas estradiol did not significantly regulate either of these genes. Estradiol has recently upregulated PI3K p85 in the dorsomedial portion of the ventromedial hypothalamic nuclei (adjacent to the arcuate nucleus) after 24 h treatment but did not regulate p85 expression in the arcuate nucleus (25). The other PI3K-associated gene, PITP, transports lipids (phosphatidylinositols) from their site of synthesis (endoplasmic reticulum) to the cellular membrane where they are the preferred substrates for the lipid kinases (PI3K, PI4K, etc.) (63). Not only is PITP activity required for PI3K, this protein is also necessary for PLC-mediated signaling (64). Because PI3K- and PLC-mediated signaling is implicated in the membrane-mediated effects of estradiol and other factors that control energy homeostasis (leptin, insulin, etc.), any changes in activity or expression of the transfer proteins may be another indirect mechanism for estradiol to potentiate the effects of these peripheral signals on energy homeostasis. Another signaling pathway affected transcriptionally by STX is the calcium signaling pathways specifically calmodulin-dependent kinase, CaM kinase II. CaMK II is a modulator of ion channels (Ca 2,K,Na ) (65) and is required for the Ca 2 -sensitive production of long-term potentiation in neurons from the hypothalamus (66) and the hippocampus, where leptin enhances long-term potentiation through the Ca 2 -independent activation of CaMK II (67). STX-induced gene expression of CaMK II is one more indirect mechanism for estradiol to potentiate the effects of peripheral signals and neuronal functions necessary for the control of energy homeostasis. Furthermore, calmodulin is up-regulated by estradiol both at 24 h (9) and after long-term treatment, as presently illustrated, and an increase in calmodulin expression would also affect the activity of the CaM kinase II. The regulation of trafficking and scaffold protein genes In our study we show that long-term EB and STX treatment increased the expression of the GABA A receptor trafficking gene, gec1, in the arcuate nucleus, confirming and expanding previous findings that estradiol increases gec1