Caveolin-1 Regulates Genomic Action of the Glucocorticoid Receptorin Neural Stem Cells

Melanie E. Peffer,a,b Uma R. Chandran,c Soumya Luthra,c Daniela Volonte,b Ferruccio Galbiati,b Michael J. Garabedian,e

A. Paula Monaghan,d Donald B. DeFrancoa,b

Program in Integrative Molecular Biology,a Department of Pharmacology and Chemical Biology,b Department of Biomedical Informatics,c and Department ofNeurobiology,d University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania, USA; Department of Microbiology, New York University School of Medicine, NewYork, New York, USAe

While glucocorticoids (GCs) are used clinically to treat many conditions, their neonatal and prenatal usage is increasingly con-troversial due to reports of delayed adverse outcomes, especially their effects on brain development. Such alterations may reflectthe impact of GCs on neural progenitor/stem cell (NPSC) function. We previously demonstrated that the lipid raft proteincaveolin-1 (Cav-1) was required for rapid GC signaling in embryonic mouse NPSCs operating through plasma membrane-bound glucocorticoid receptors (GRs). We show here that genomic GR signaling in NPSCs requires Cav-1. Loss of Cav-1 impactsthe transcriptional response of many GR target genes (e.g., the serum- and glucocorticoid-regulated kinase 1 gene) that are likelyto mediate the antiproliferative effects of GCs. Microarray analysis of wild-type C57 or Cav-1-deficient NPSCs identified approx-imately 100 genes that are differentially regulated by GC treatment. These changes in hormone responsiveness in Cav-1 knock-out NPSCs are associated with the loss of GC-regulated phosphorylation of GR at serine 211 but not at serine 226. Chromatinrecruitment of total GR to regulatory regions of target genes such as Fkbp-5, RhoJ, and Sgk-1, as well as p211-GR recruitment toSgk-1, are compromised in Cav-1 knockout NPSCs. Cav-1 is therefore a multifunctional regulator of GR in NPSCs influencingboth rapid and genomic action of the receptor to impact cell proliferation.

Glucocorticoid (GC) hormones are essential for multiple post-natal processes, including immune responses, glucose metab-

olism, blood pressure regulation, and central nervous systemfunction (1, 2). Given their potent anti-inflammatory actions,GCs are widely used therapeutically in children and adults to treata variety conditions and diseases such as asthma, allergies, rheu-matoid arthritis, chronic inflammation, and induction treatmentfor acute leukemias (2).

Dexamethasone (Dex) is one of the synthetic glucocorticoidsrecommended by the American College of Obstetricians and Gy-necologists for antenatal therapy (betamethasone being the other)to limit adverse respiratory and vascular events in premature ba-bies (3). Antenatal GC therapy is also used to reduce virilization ofthe external genitalia for female infants with congenital adrenalhyperplasia. However, this therapy remains highly controversialgiven the early gestational age required for clinical benefit (4).

Despite their efficacy in the treatment of many diseases, thediverse action of GCs in various tissues and cell types results inmany side effects (2). Although the use of synthetic prenatal GCsto promote lung maturation in preterm infants is undisputed,clinical and preclinical studies have revealed the potential for pro-longed or delayed side effects linked to antenatal or neonatal GCusage (5–7). In several species, including humans, these studiesindicate that exogenous treatment with GCs during fetal or neo-natal development or fetal exposure to high levels of endogenousGCs produced as a result of maternal stress can be associated withneurological, cognitive, and affective disorders that emerge laterin childhood (7, 8). Although the mechanism(s) responsible forthese affects are not well established, they may involve alterationsin neural progenitor/stem cell (NPSC) proliferation and/or differ-entiation, thereby disrupting the development of neuronal cir-cuits essential for higher-order cognitive or behavioral function.

Studies examining GC effects on NPSC function in a variety of

experimental systems uncovered a number of affected pathwaysand signaling molecules. For example, in rat embryonic neuralstem cells, GCs decreased proliferation by enhanced degradationof the cell cycle regulator cyclin D1 (9). In the mouse cerebellum,neonatal treatment with Dex increased apoptosis of the neuralprogenitor cells within the extra granule cell layer and decreasedoverall numbers of internal granule layer neurons (10). GC effectson the differentiation of NPSCs are varied and conflicting; re-duced differentiation of glial cells by GCs was reported in mesen-cephalic NPSCs and adult rat hippocampal progenitor cells, whileGCs increased the differentiation of glia in human NPSC cultures(11–13).

GC responses are mediated primarily by the glucocorticoidreceptor (GR), a member of the nuclear receptor superfamily oftranscription factors (1, 14), through the actions of two separatebut interrelated mechanisms. In classical or genomic signaling,binding of ligand results in GR translocation to the nucleus, whereit can either activate or repress the transcription of target genes(15). In contrast, rapid or nonclassical signaling involves a plasmamembrane-associated GR that is capable of activating kinases,such as mitogen-activated protein kinase (MAPK). These kinasescan then influence GR transcriptional response through direct

Received 23 August 2013 Returned for modification 12 September 2013Accepted 19 March 2014

Published ahead of print 28 April 2014

Address correspondence to Donald B. DeFranco, dod1@pitt.edu.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/MCB.01121-13.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/MCB.01121-13

July 2014 Volume 34 Number 14 Molecular and Cellular Biology p. 2611–2623 mcb.asm.org 2611

modification of the receptor or its associated transcriptional co-regulators (16). Although both rapid and genomic GR signalingoperates in NPSCs (17), the degree of interaction between the twopathways and how they intersect to influence GC effects on NPSCbiology are unknown.

Caveolin-1 (Cav-1) is a major protein subunit of specializedregions of the plasma membrane known as caveolae. Caveolae canact as signaling organizers, and previous work from our laboratoryestablished a role for Cav-1 in a rapid GC signaling pathway thattriggers MAPK activation in embryonic mouse NPSC cultures(17). One of the consequences of Cav-1-dependent activationof MAPK by GCs is an inhibition of gap junction intercellularcommunication (GJIC) between coupled NPSCs (17). Studieswith other steroid hormone receptors, such as the androgenand estrogen receptor, have revealed mechanisms of cross talkbetween genomic and rapid actions of the receptors (reviewedin reference 18).

Given the role of Cav-1 in mediating the rapid response of GR(16, 17), we set out to examine whether Cav-1 could also partici-pate in classical genomic GR signaling and impact the antiprolif-erative response of NPSCs to Dex. Here we show that loss of theDex-mediated antiproliferative response in NPSCs derived fromCav-1 knockout (KO) mice is associated with the alterations inDex-induced expression of an established negative regulator ofthe cell cycle in NPSCs, the serum- and GC-induced kinase 1 gene,Sgk-1 (19). We also revealed more global effects of Cav-1 deletionon the GR transcriptome and uncovered a mechanism for Cav-1-mediated cross talk between rapid and genomic GR signaling thatimpacts site-specific GR phosphorylation and chromatin recruit-ment of GR.

MATERIALS AND METHODSMouse NPSC cultures. NPSCs were derived from embryonic (E14.5) ce-rebral cortex of wild-type C57BL/6 (C57) or Cav-1 knockout (KO) miceand grown as three-dimensional neurosphere cultures (17). Cells werepassaged every 7 days, and experiments were performed at passage 3 un-less indicated otherwise.

NPSC BrdU assays. Passage 1 neurospheres were cultured for 3 daysafter passaging before replenishment with fresh epidermal growth factorand fibroblast growth factor 1. Approximately 6 h later, cells were treatedwith 100 nM Dex (Sigma Chemicals, St. Louis, MO) or vehicle (ethanol[EtOH]). Then, 10 �M bromodeoxyuridine (BrdU; Sigma Chemicals,catalog no. B-9285) was added for 1 h after 23 h of Dex treatment. Neu-rospheres were then dissociated into single cells and attached to poly-D-lysine-treated coverslips prior to fixation with 4% paraformaldehyde.Fixed cells were processed for immunocytochemistry according to stan-dard methods. A rat anti-BrdU antibody (Abcam; catalog no. ab6326) wasused at 1:500 and anti-rat antibody conjugated to Alexa Flour 488 (Invit-rogen; catalog no. A21208) at 1:1,000. Images were captured using aNikon Eclipse E400 microscope and a Photometrics Cool Snap E52 cam-era. For SGK-1 inhibitor experiments, GSK650394 (Tocris Bioscience)was used at a concentration of 50 nM and added coincident with Dex.

Microarrays. Total RNA was extracted by using a Qiagen RNeasy kit(catalog no. 74104) from cell pellets comprising biologically distinct neu-rosphere cultures from C57 or Cav-1 KO embryos treated with 100 nMDex or vehicle for 4 h (n � 5/treatment condition). Total RNA of thehighest quality and integrity was subjected to further processing afterpurification, as defined by a 260/280 absorption ratio of �1.8 using spec-trophotometry on the NanoDrop 1000 (NanoDrop, Wilmington, DE)and an RNA integrity number of �8.0 determined via electrophoreticanalysis on a Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA).Each of the samples met these standards, and in vitro transcription wasperformed using the MessageAmp Premier Enhanced assay protocol

(Ambion, Inc., Austin, TX) starting with 500 ng of purified total RNA.Confirmation of cRNA diversity was obtained using the Bioanalyzer 2100to generate an electrophoretogram for each in vitro transcription reactionregarding sample yield, integrity, and size diversity against a UniversalHuman Reference RNA (Stratagene, La Jolla, CA). A 15-�g portion ofpurified, amplified, biotin-labeled cRNA was fragmented and hybridizedonto Affymetrix Mouse Genome 430A 2.0 arrays (13,687 genes, 22,690probes; Affymetrix Corp., Santa Clara, CA) for 18 h. Washing, staining,and scanning of arrays was performed on an Affymetrix Fluidics Station450 and Scanner 3000 immediately after completion of hybridization.

Bioinformatic analysis. The data were normalized and summarizedusing the robust multichip average (RMA) method (20). For the genesrepresented by multiple probe sets, the probe set with the highest inter-quartile ratio (a descriptive statistic used to summarize the extent of thespread of the data) was selected to represent the gene. An additional fil-tering step was performed to remove the genes that were flagged as absentacross all 20 samples by the MAS5 normalization method. This reducesthe data set to 9,916 genes that were used for further statistical analysis.

Statistical tests were performed using BRB-ArrayTools developed byRichard Simon and the BRB-ArrayTools Development Team (21). A Stu-dent t test was used to identify genes significantly different in the followingcomparisons: Cav-1 KO Dex versus vehicle, C57 Dex versus vehicle, andCav-1 KO versus C57 vehicle. To find genes differentially regulated by Dextreatment of Cav-1 KO versus C57 NPSCs but not by the KO effect alone,a one-way analysis of variance (ANOVA) was performed. Genes that werefound significantly different (false discovery rate, 10%) in the pairwisecomparisons were further analyzed for canonical pathways, networks andbiological functions using the Ingenuity pathway analysis (IPA) software(Ingenuity Systems, Redwood City, CA). Hierarchical clustering figuresand Venn diagrams were produced in R-Bioconductor.

Meta-analysis was performed in NextBio Research (Cupertino, CA), aweb-based data search and analysis engine, to compare the GC signaturein our studies to 12 other individual publicly available data sets selectedusing relevant keywords. Differentially expressed genes from each of thedata sets were combined to create the master list of GC-regulated genes.This list was then further filtered to identify genes in a subset of studies, forexample, genes differentially expressed only in neuronal cells. The datasets used are shown in Table 1.

qRT-PCR. For microarray validations, RNAs treated with Dex or eth-anol for 4 h were isolated from biologically independent samples of eachgenotype using Macherey-Nagel Nucleospin RNA II kit. cDNA synthesiswas performed using iScript Select cDNA synthesis kit (Bio-Rad; catalogno. 170-8897). Quantitative real-time PCRs (qRT-PCRs) were performedon a Stratagene Mx3000P and used iTaq Universal SYBR green Supermix(Bio-Rad; catalog no. 172-5121) and primers, with efficiencies calculatedto be �80%.

Phospho-GR Westerns. Neurospheres from C57 or Cav-1 KO micewere treated with 100 nM Dex for 1 h. After treatment, cells were lysed inradioimmunoprecipitation assay buffer (10 mM Tris-Cl [pH 8.0], 1 mMEDTA, 0.5 mM EGTA, 140 mM NaCl, 1% Triton X-100, 0.1% sodiumdeoxycholate, 0.1% sodium dodecyl sulfate [SDS]) supplemented withHALT protease and phosphatase inhibitors (Thermo, catalog no.1861281) at 1:1,000. Lysate was subjected to immunoprecipitation usingthe BuGR2 mouse monoclonal antibody or incubated with nonimmuneIgG using standard laboratory conditions. Immunoprecipitated materialwas run on SDS–7.5% PAGE gel and transferred to a polyvinylidene di-fluoride membrane (Immobilon-P, catalog no. IPVH00010; Millipore).Western blot analysis was performed using the p211-GR antibody (29)and an anti-rabbit horseradish peroxidase-conjugated secondary anti-body (Bio-Rad, catalog no. 170-6515) using a chemiluminescence detec-tion system (Advansta Western Bright ECL; catalog no. K-12045-D50).

Nuclear fractionation. Subcellular fractionation was performed ac-cording to guidelines provided in the NE-PER kit (Thermo; catalog no.78833) with lysates from C57 or Cav-1 KO neurospheres treated with Dexor ethanol for 4 h. Fractions were then separated on SDS–10% PAGE gels,

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and Western blotting was performed to detect total GR using the M20rabbit polyclonal antibody (Santa Cruz; catalog no. sc-1004) and Cav-1(BD Biosciences; catalog no. 610059). The purity of fractions was assessedusing GAPDH (glyceraldehyde-3-phosphate dehydrogenase) as a markerfor cytoplasmic (Santa Cruz; catalog no. sc-32233) and Lamin-B1 fornuclear proteins (Abcam; catalog no. ab16048).

ChIP. NPSCs isolated from C57 or Cav-1 KO mice were grown asdescribed above and treated with Dex or vehicle for 1.5 h. Cells were thenfixed in 1% formaldehyde for 10 min at room temperature. After fixation,0.125 M glycine was added for 5 min to quench the cross-linking reaction.Cells were washed with phosphate-buffered saline twice and frozen at �80until immunoprecipitation. Prior to sonication, cell pellets were thawedon ice and lysed in chromatin immunoprecipitation (ChIP) lysis buffer(50 mM HEPES, 1 mM EDTA, 140 mM NaCl, 10% glycerol, 0.5% NP-40,0.25% Triton X-100) with freshly added HALT protease and phosphataseinhibitor cocktail (Thermo-Fisher). Cell lysate was centrifuged at 5,000rpm for 5 min at 4°C, and crude nuclear pellets were collected and washedonce with ChIP wash buffer (10 mM Tris-Cl, 1 mM EDTA, 200 mM NaCl)with freshly added HALT protease and phosphatase inhibitor cocktail.The pellet was then resuspended in ChIP sonication buffer (10 mM Tris-Cl, 1 mM EDTA, 0.5 mM EGTA, 0.5% N-lauroylsarcosine) with freshlyadded HALT protease and phosphatase inhibitor cocktail and chromatinsonicated using a Bioruptor into fragments of approximately 500 to 1,000bp. Immunoprecipitation of GR was performed with 8 �g of a GR anti-body cocktail (2 �g each of M20 [Santa Cruz], P20 [Santa Cruz], H300[Santa Cruz] and BuGR2), 8 �g of phosphoserine-211 (ab55189), or nor-mal rabbit IgG (Santa Cruz; catalog no. sc-2027) overnight at 4°C. A 1:1mix of anti-rabbit and anti-mouse antibodies cross-linked to Dynabeads(Life Technologies) were used according to the manufacturer’s instruc-tions. After incubation, the beads were washed with low-salt immunecomplex buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mMTris-Cl [pH 8.1], 150 mM NaCl), high-salt immune complex buffer (0.1%SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl [pH 8.1], 500 mMNaCl), LiCl immune complex buffer (0.25 M LiCl, 1% NP-40, 1% deoxy-cholate, 1 mM EDTA, 10 mM Tris-Cl [pH 8.1]), and finally 1� Tris-EDTA. Cross-links were then removed via an overnight proteinase K di-gestion (Ambion; catalog no. AM2546), and DNA was purified using astandard phenol-chloroform extraction protocol. Purified DNA was thenanalyzed using qRT-PCR. Primers were designed based on GR ChIP-Seqdata published previously (30–33), and the coordinates used were as fol-lows: Fkbp-5, chromosome 17, 28556870 to 28557847; RhoJ, chromosome12, 76404969 to 76405723; and Sgk-1, chromosome 10, 21683041 to21683190.

Microarray data accession number. The CEL files have been submit-ted to GEO under accession number GSE49804.

RESULTSThe antiproliferative effects of Dex in NPSCs require Cav-1. Thecontribution of rapid GR signaling to the antiproliferative effectsof GCs exerted on a variety of tissue and cell types remains unre-solved (9, 11, 16). Since rapid signaling effects of GCs on MAPKactivation and GJIC are lost upon Cav-1 deletion in embryonicNPSCs (17), we tested whether the antiproliferative effects of GCswere altered in Cav-1 KO NPSCs. As shown in Fig. 1, Dex did notsignificantly impact proliferation of NPSCs in Cav-1 KO neuro-spheres, which contrasts with C57 NPSCs, where proliferation wasreduced by 10%. Furthermore, basal proliferation of embryonicNPSCs was not affected by Cav-1 deletion (Fig. 1). Therefore, inembryonic NPSC cultures, Cav-1 was an essential component of aGR signaling pathway that operated to limit proliferation.

Cav-1 deletion abolishes GR induction of Sgk-1, a gene re-quired for the antiproliferative effect of Dex in NPSCs. We pre-viously established that inhibition of GJIC by the rapid action ofGR was associated with an inhibition of S-phase progression incultured NPSCs (17). Our observation that transient pharmaco-logic inhibition of GJIC is also sufficient to reduce NPSC prolifer-ation (17) was recently confirmed in cultured embryonic stemcell-derived neural progenitors and in embryonic neural progen-itors isolated from the cerebral cortex (34). Recent studies in hu-man hippocampal neural progenitor cells indicate that the anti-proliferative properties of GCs are dependent upon at least onegenomic GR target gene, Sgk-1 (19). Consistent with these find-ings, Sgk-1 is also required for the antiproliferative effect of GCs inour NPSC cultures, since treatment with an SGK-1 inhibitor(GSK650394) blunts the growth-inhibitory affect of Dex (Fig. 2Aand B). Sgk-1 was a GR target gene in C57 but not in Cav-1 KONPSCs, as revealed by qRT-PCR analysis following a 4-h Dextreatment (Fig. 2C). Thus, the role of Cav-1 in GC-mediated ef-fects on the cell cycle may extend beyond its impact on rapid GRsignaling and influence genomic action of the receptor.

Analysis of Cav-1 effects on the GR transcriptome in NPSCs.To examine Cav-1 regulation of genome-wide GR transcriptionalresponses, C57 and Cav-1 KO NPSCs were subjected to microar-ray expression analysis. Compared to C57 NPSCs, Cav-1 KO cellsdemonstrate differential basal expression of 186 genes (false dis-covery rate � 0.1) with 44 genes exhibiting higher expression in

TABLE 1 Data sets utilized for NextBio analysis

Set IDno. Cell type Hormone Concn (�M)

Duration oftreatment (h)

Source orreference

1 E14.5 cerebral cortex from Cav-1 KO mice Dex 0.1 4 This study2 E14.5 cerebral cortex from C57 (wild type) mice Dex 0.1 4 This study3 AtT-20 mouse pituitary Dex 0.1 4 224 Multipotent human fetal hippocampal progenitor cell line (HPC03A/07) Cortisol 0.1 12 235 3134 mouse mammary adenocarcinoma Dex 0.1 4 226 C2C12 mouse myotubes Dex 1 24 247 C2C12 mouse myotubes Dex 1 6 248 Multipotent human fetal hippocampal progenitor cell line (HPC03A/07) Cortisol 100 12 239 E14.5 cerebral cortex from rat Corticosterone 1 28 2510 Mouse oligodendrocyte precursor cell line (Oli-Neu) Dex 1 10 2611 Mouse oligodendrocyte precursor cell line (Oli-Neu) Dex 1 24 2612 Mouse 3T3-L1 preadipocytes Dex 1 2 2713 Mouse 3T3-L1 preadipocytes Dex 1 36 2714 Mouse C3H10T1/2 pluripotent stem cell line Dex 1 1.5 28

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Cav-1 KO cells and 20 genes lower expression by at least 1.5-fold(Table S1). The transcripts with lowest expression in the Cav-1KO versus C57 cells were Cav-1, as expected, and the ribosomalprotein S9 (Rps9) gene, whose expression had previously beenshown to be highly dependent upon Cav-1 (35). When Dextreated Cav-1 KO and C57 NPSCs were compared for gene expres-sion, 568 genes (excluding Cav-1 and Rps9) were differentiallyDex responsive (false discovery rate � 0.1) (see Table S1 in thesupplemental material). In general, Dex-responsive genes wereregulated in the same direction in both C57 and Cav-1 KO NPSCsas shown in a heat map (Fig. 3A). However, individual genes doexhibit differences in Dex responsiveness between the two geno-types.

A number of genes that were induced by Dex at least 2.5-foldand exhibited robust differences in Dex-regulated expression be-tween C57 and Cav-1 KO NPSCs (see Table S1 in the supplemen-

tal material) were validated by qRT-PCR using independentNPSC cultures (Fig. 3B to I). For this analysis, we examined bothwell-established GR target genes (e.g., Fkbp5) and genes with lim-ited reports of GC responsiveness (e.g., Gcnt2). Specifically, Dexinduction of Fkbp5, Gcnt2, RhoJ, Pcolce2, Mal, Adm, and Arl4dmRNA expression was significantly reduced in Cav-1 KO versusC57 NPSCs (Fig. 3). We also observed that Cxcr4 did not attainappropriate level of repression in response to Dex treatment inCav-1 KO NPSCs (Fig. 3). Therefore, Cav-1 effects on GR actionare not limited to rapid signaling (17) but also extend globally togenomic responses.

IPA was used to examine components of molecular networksand pathways defined by Dex-regulated genes in C57 and Cav-1KO NPSCs. As shown in Fig. S1C and D in the supplementalmaterial, the most significant functional networks defined byDex-responsive genes in C57 NPSCs were distinct from networks

C57 EtOH

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FIG 1 Antiproliferative effects of Dex in NPSCs require Cav-1. (A) C57 or Cav-1 KO NPSCs were treated with Dex or vehicle (ethanol [EtOH]) for 24 h and pulsed withBrdU during the last hour of treatment. Immunocytochemistry was performed to detect BrdU-positive nuclei (pink). Nuclei were visualized by DAPI staining (blue). (B)Three independent coverslips per biological replicate were counted to ascertain the percentage of cells that passed through S-phase (i.e., BrdU-positive nuclei). Error barsrepresent the standard error of the mean (SEM; n � three biological replicates). P � 0.001 (one-way ANOVA with Tukey’s posttest).

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formed in Cav-1 KO NPSCs. For example, in C57 NPSCs, cellcycle genes comprise the top two highly rated networks; 9 of thenext 10 highly rated gene networks included molecules involved indevelopment, particularly in the nervous system (see Fig. S1C inthe supplemental material). Figure S1A in the supplemental ma-terial shows the most highly rated cell cycle network in Dex-treated C57 NPSCs. Included in this network are genes such as

Pcolce2 (Fig. 3D), which was validated as a GR target by qRT-PCRof independent samples. In addition, the top canonical pathwayswithin this cell cycle network include a number of nuclear recep-tors: retinoid acid receptor, GR, peroxisome proliferation-activat-ing receptor alpha/retinoid X receptor alpha (RXR�), estrogenreceptor, and thyroid hormone receptor/RXR�.

In contrast to C57 Dex-regulated networks, Cav-1 KO NPSCs

EtOH + D

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FIG 2 Antiproliferative effects of Dex in NPSCs require SGK-1. (A) C57 or Cav-1 KO cells were treated with Dex and/or the SGK-1 inhibitor, GSK650394, andthe appropriate vehicle (ethanol [EtOH] or dimethyl sulfoxide [DMSO]) for 24 h and pulsed with BrdU during the last hour of treatment. Immunocytochemistrywas performed to detect BrdU-positive nuclei (pink). Nuclei were visualized by DAPI staining (blue). (B) Three independent coverslips per biological replicatewere counted to ascertain the percentage of cells that passed through S phase (i.e., BrdU-positive nuclei). Error bars represent the SEM (n � 3 biologicalreplicates). P � 0.05 (one-way ANOVA with Tukey’s posttest). (C) qRT-PCR analysis of Sgk-1 mRNA indicates that significant induction of Sgk-1 occurs aftera 4-h Dex treatment in C57 but not Cav-1 KO NPSCs (Student’s t test, P � 0.01).

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demonstrated only one highly rated network involved in nervoussystem development (see Fig. S1D in the supplemental material).Although GR signaling pathways were part of nervous system de-velopment networks in both Dex-treated C57 and Cav-1 KONPSCs, the genes in the two networks were different (see Fig. S2Aand B in the supplemental material). In addition, the only cellcycle network (sixth highest) in Dex-regulated Cav-1 KO NPSCswas unrelated to the highest-rated cell cycle network in Dex-treated C57 NPSCs (see Fig. S1A and B).

The analysis described above used Dex-regulated genes fromC57 and Cav-1 KO NPSCs, including those that were similarlyresponsive to hormone in the two genotypes. To examine differ-ences in Dex responsiveness between the two genotypes, IPA wasperformed using genes determined to be significantly different byANOVA with regard to the Dex response. This analysis showedthat approximately half (i.e., 13) of the 25 highly rated networksformed by differentially Dex-responsive genes were involved insome aspect of organ or tissue development (see Fig. S3C in thesupplemental material). In fact, the most highly rated networksfunction in nervous system development (see Fig. S3A and B in thesupplemental material), protein ubiquitylation (see Fig. S3C), andaxon guidance or cellular/extracellular matrix interactions (seeFig. S3C). GC effects on neuronal migration and ubiquitin-pro-teasome mediated degradation have previously been observed inneural stem cells (9, 36) and may be influenced by Cav-1. Tosummarize, in addition to identifying many novel primary GCresponsive genes in NPSCs, our microarray data implicated Cav-1as a modulator of both rapid signaling and genomic action of GR.

To identify Dex-regulated genes that are unique to neural stemcells, the NextBio data mining tools (see Materials and Methods)were used to compare the GR-regulated transcriptome in NPSCsto others from cell and tissue sources as diverse as the 3134 mousemammary adenocarcinoma cell line (22) and a mouse oligoden-drocyte progenitor cell line (26) (Table 1). Specifically, thisanalysis included 14 separate studies: a union of differentially ex-pressed genes from each of these studies generated 5,000 GC-regulated genes (see Table S2 in the supplemental material). Acomparison of the GC-regulated gene list in our NPSC cultures(Fig. 4A, set 3) with a subset of these studies that used closelyrelated cell types (i.e., rat neural [25] and oligodendrocyte [26]progenitors; Fig. 4A, set 2) revealed 176 common genes (Fig. 4A).However, of the 5,000 GC-regulated genes from 14 data sets,only six were uniquely GC regulated in our mouse NPSC cul-tures: Gjb6, Gbx2, Card10, Plcl2, Smc5, and Rnf157. Three ofthese genes (Gjb6, Gbx2, and Plcl2) were validated as Dex reg-ulated in qRT-PCR analysis of independent samples (Fig. 4B toD). Therefore, the GR transcriptome in three-dimensional cul-tures of mouse NPSCs only included a limited number ofunique target genes.

Cav-1 influences site-specific GR phosphorylation. To deter-mine a mechanism for Cav-1 effects on GR transcriptional activ-ity, we first sought to determine whether Cav-1 was found in the

nucleus of NPSCs. In ovarian carcinoma cell lines, Cav-1 regulatescell cycle regulatory gene expression through direct DNA binding(37). Cav-1 also affects transcription in lung epithelial Beas-2Bcells by directly binding the transcription factor nuclear erythroid2 p45-related factor 2 (Nrf2) (38). Our previous work (17) re-vealed an interaction between Cav-1 and GR in NPSCs. However,Cav-1 was undetectable in nuclear fractions prepared from un-treated or Dex-treated NPSCs (Fig. 5A). Dex-dependent nucleartranslocation of GR occurred in both genotypes (Fig. 5A). Fur-thermore, GR protein and mRNA levels were unaltered in Cav-1KO neurospheres (Fig. 5D). Therefore, Cav-1 may indirectly af-fect GR transcriptional response via regulation of a cytoplasmicsignaling pathway that ultimately impacts GR bound at specificgene targets.

Site-specific GR phosphorylation influences its transcriptionalregulatory activity. In fact, different phosphorylation patternswithin the amino-terminal activation function-1 domain can dic-tate which target genes will be bound by GR (29). Both MAPKs,targets of rapid GR signaling and cyclin-dependent kinase 2(CDK2), phosphorylate GR and influence its transcriptional ac-tivity (39). Therefore, we tested the impact of Cav-1 deletion onMAPK- and CDK2-dependent phosphorylation of GR at serine-211 (S211) and serine-226 (S226), respectively. As shown in Fig. 6,phosphorylation at S211, a Dex-responsive site, was undetectablein Cav-1 KO neurospheres. Since hormone-dependent phosphor-ylation at S211 is associated with transcriptionally active GR, theloss of phosphorylation at S211 may explain diminished Dex re-sponsiveness of select genes in Cav-1 KO NPSCs (e.g., Fig. 3). GRphosphorylation at S226 is unaltered in Cav-1 KO NPSCs. There-fore, Cav-1 does not alter global GR phosphorylation.

Cav-1 influences recruitment of GR to chromatin of targetgenes. We next sought to determine whether any of the genesdifferentially regulated in C57 and Cav-1 KO NPSCs (Fig. 3) weredirect targets of GR and whether recruitment of GR to gene regu-latory regions was altered in Cav-1 KO cells. Directed ChIP assayswere therefore used to reveal GR recruitment to select target genesaffected by Cav-1 KO, using previously reported GR binding sitesin other cell lines (30–33). As shown in Fig. 6, three genes validatedas differentially expressed in C57 versus Cav-1 KO NPSCs havesignificantly decreased chromatin recruitment of GR in responseto Dex treatment in Cav-1 KO NPSCs (Fig. 6B to D). Since GRphosphorylation at Ser211 is undetectable in Cav-1 KO cells (Fig.6A), we did not expect to detect any recruitment of pSer211-GR toGC-regulated genes. Nonetheless, this we confirmed by ChIP as-says with the pSer211-GR antibody, as shown in Fig. 6E, whichalso demonstrated diminished recruitment of pSer211-GR to theSgk-1 promoter in Cav-1 KO NPSCs. Therefore, reduced tran-scriptional responses to Dex in Cav-1 KO NPSCs can be mediatedby diminished recruitment of select GR phosphoisoforms to tar-get gene regulatory sites.

FIG 3 A subset of genes are differentially regulated by GR in C57 versus Cav-1 KO NPSCs. (A) Hierarchical gene clustering of 20 NPSC cultures usingDex-responsive genes in C57 and Cav-1 KO NPSCs (Student t test; false discovery rate � 0.1) using Pearson dissimilarity as the distance measure and the averagelinkage method for linkage analysis. The data are represented using a z-score normalized before plotting the heat map. For each gene (row), the z-score wascalculated by subtracting the expression value by mean expression across all samples (centering) and dividing by the standard deviation (scaling). (B-I) C57 orCav-1 KO NPSCs from tissues independent of those used for microarray analysis were treated for 4 h with 100 nM Dex and mRNA expression of indicated genesanalyzed using qRT-PCR. Although all genes shown are significantly induced in response to Dex, activation is attenuated in the Cav-1 KO cells. Error barsrepresent the SEM (n � 6). *, P � 0.05; **, P � 0.01 (Student’s t test).

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DISCUSSION

The nonclassical rapid activation of nuclear receptors residing atthe plasma membrane mobilizes various cytoplasmic signalingpathways that can either directly alter cellular physiology (16, 17)or indirectly modulate transcriptional responses (40). Such crosstalk between rapid nuclear receptor signaling and classicalgenomic action can utilize nuclear receptors at target genes orindependent transcription factors. In this report, we demon-strated that cross talk between GR rapid and genomic pathways inembryonic NPSCs utilized Cav-1, a lipid raft protein. Cav-1 didnot act as a classical coregulator of GR on genomic targets butrather altered the phosphorylation of GR on at least one site,S211. Phosphorylation of S211 in U2OS cells influences the GCtranscriptional program (29) and may likewise impact targetgenes regulated by GR in NPSCs. In fact, alterations of the GRtranscriptome in NPSCs upon Cav-1 deletion define uniquenetworks that potentially alter GC regulation of the cell cycle(see model in Fig. 7).

The most prominent pathways defined by the GR transcrip-tome in C57 NPSCs regulated cell cycle progression and contain anumber of genes that could contribute to the antiproliferative

effects of GCs (9, 16, 17). Notably, one validated GR target gene,Sgk-1, is required for the antiproliferative effects of GC in embry-onic mouse NPSCs (the present study) and a human hippocampalprogenitor cell line (19) but did not reach significance as GC reg-ulated in our microarray analysis or that reported in hippocampalprogenitors (see Table S2 in the supplemental material) (23). Thehigh stringency applied to microarray data sets, while reducing thenumber of false positives, may miss bona fide GR targets that playan important role in the biological effects of GCs. The inability ofGR to induce Sgk-1 expression in Cav-1 KO NPSCs may underliein part the loss of an antiproliferative response to Dex. Impor-tantly, a number of cell cycle-regulated genes were also found to bedifferentially responsive to Dex in C57 versus Cav-1 KO NPSCs,so there are likely to be multiple GR targets that participate in thecomplex regulation of proliferation in NPSCs, which are influ-enced by Cav-1. Since the GR transcriptome in C57 and Cav-1 KOembryonic NPSC cultures also defines a number of pathways im-plicated in neuronal development, future studies may reveal a rolefor Cav-1 operating through either rapid signaling (17) or crosstalk with genomic GR pathways to influence antenatal effects ofGCs on neurodevelopment.

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FIG 4 NextBio analysis reveals a subset of genes regulated by Dex in embryonic mouse NPSC cultures. (A) Venn diagram comparing the GC-regulated gene listsin the 14 studies used to perform meta-analysis in NextBio. The genes contained in set 1 are from study ID numbers 3 to 8 and 12 to 14 (Table 1) and derived fromcell types most distant from our mouse embryonic NPSCs (set 3). The genes contained in set 2 are from study ID numbers 9 to 11 (Table 1) and derived from celltypes closely related to our mouse embryonic NPSCs (i.e., rat neural progenitors or mouse oligodendrocyte progenitor cells). (B to D) C57 KO NPSCs fromtissues independent of those used in the microarray were treated for 4 h with 100 nM Dex and induction of Gjb6 (B), Plcl2 (C), and Gbx2 (D) mRNA analyzedusing qRT-PCR. Error bars represent the SEM (n � 6). *, P � 0.05; ***, P � 0.001 (Student’s t test).

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Our study supports a role for Cav-1 in mediating the genomicaction of GR in addition to its role in rapid GR signaling. NPSCsnull for Cav-1 contain a subset of genes that are differentiallyregulated by GR in response to Dex. Of the subset that we vali-dated, Fkbp5 is a known target of GR involved in negative regula-tion of GR and the stress response. Improper activity of Fkbp5 cancontribute to neuropsychiatric diseases such as posttraumaticstress disorder (41). Alterations in the regulation of Fkbp5 mayexplain why adult Cav-1 KO animals tend to exhibit more anxiousbehaviors than their wild-type counterparts (42).

Gjb6, Gbx2, and Plcl2 were novel validated GR target genes inNPSC cultures but their role in regulating neural stem or progen-itor cell function has yet to be determined. Gjb6 encodes connexin30, a gap junction protein expressed primarily in astrocytes butnot until postnatal day 10 in mice (43). Therefore, Dex inductionof Gjb6 in our neurosphere cultures may be reflective of GR action

in astrocyte progenitor cells. Gbx2 is a homeobox transcriptionalfactor that has been shown to regulate various aspects of neuralstem cell differentiation (44) and may contribute to the reportedeffects of GCs on differentiation of distinct neuroprogenitors (11–13). No role for phospholipase C-related protein (Plcl2) in neuro-development or GC action has been reported, although Plcl2 wasidentified in exome sequencing analysis as one of 40 genes withprotein coding sequence variations in schizophrenia patients (45).

Of the 25 highly rated networks identified by our analysis ofgenes differentially responsive to Dex in C57 versus Cav-1 KONPSCs, approximately half were related to organ or tissue devel-opment—the top two from our data set involving nervous systemdevelopment. Notably, one of these pathways involves proteinubiquitinylation. Multiple studies have revealed an impact of GCson the protein degradation of NPSCs. For example, GC treatmentin rat embryonic neural stem cells results in decreased prolifera-tion by enhancing ubiquitin-mediated degradation of the cell cy-cle regulator cyclin D1 (9). In embryonic rat cells, Dex treatmentincreases the expression of a deubiquitinating enzyme, Usp8/Ubpy that can indirectly cause increased degradation of theBRUCE/Apollo inhibitor of apoptosis protein. As a result of in-creased BRUCE degradation, there is a consequent decrease in cellproliferation (36). Interestingly, Cav-1 interacts with and regu-lates the polyubiquitinylation of active Rac1 (46). Rac1 is a mem-ber of the Rho-like GTPases and also involved in actin polymer-ization and protrusion and cell migration.

The second most highly regulated pathway that we uncoveredduring our network analysis involved axon guidance. Interest-ingly, RhoJ and Arl4D were validated as genes differentially regu-lated between C57 and Cav-1 KO and are both implicated in actin-mediated cell migration (47, 48). Furthermore, Cxcr4 was alsovalidated as a differentially regulated gene and is implicated indopaminergic cell migration (49). GC effects on neural stem cellmigration are well documented (25). Therefore, GR signaling me-diated by Cav-1 could have a critical impact on the biology anddevelopment of NPSCs by regulating the degradation of cell cycleregulators or migration of differentiated cells derived from NPSCsto their final position in the cortex.

Although Cav-1 was not found in the nucleus of NPSCs, itinfluenced site-specific GR phosphorylation, which in turn couldimpact the GR transcriptome. Specifically, phosphorylation atS211 but not S226 was affected in Cav-1 KO NPSCs. Therefore,the GC response of genes that require pSer211-GR to reach peakactivation may be altered in Cav-1 KO NPSCs. For example,pSer211-GR is recruited to promoters of genes such as Gilz andTat to activate their transcription (29). Since GR phosphorylatedat S211 is associated with activated gene transcription, alterationsin pSer211-GR are consistent with the attenuated activation ofselect GR target genes in Cav-1 KO NPSCs. The results from ChIPassays on select GR target genes provide additional mechanisticinsights regarding the role of Cav-1 in GC-regulated transcriptionand show reduced recruitment of GR (and pSer211-GR) to selectregulatory regions of genes differentially responsive to Dex in C57versus Cav-1 KO NPSCs. Since GR is recruited to multiple bindingsites of its target genes, a more comprehensive analysis of the GRcistrome in C57 versus Cav-1 KO NPSCs using ChIP-seq will berequired to uncover other mechanistic features of site-specificCav-1-dependent GR recruitment. Since the majority of Dex-reg-ulated genes in NPSCs were unaffected by Cav-1 KO, GR chroma-tin recruitment is not globally regulated by Cav-1. Finally, other

FIG 5 Cav-1 protein is not detectable in the nucleus, nor does the loss of Cav-1affect GR protein or mRNA expression. (A) Cytoplasmic and nuclear fractionsprepared from C57 and Cav-1 KO NPSCs treated for 1 h with 100 nM Dex orEtOH vehicle were subjected to Western blot analysis to detect GR, Cav-1, andmarkers for cytoplasmic (GAPDH) or nuclear (lamin B1) proteins. Cav-1 wasnot detected in nuclear fractions from C57 NPSCs lysates and in either cyto-plasmic or nuclear fractions from Cav-1 KO cells. The blot shown is represen-tative of three independent experiments. (B) qRT-PCR analysis indicated nodifference in GR mRNA expression between C57 and Cav-1 KO cells (n � 5).(C and D) Western blot analysis also indicated no difference in GR protein(relative to GAPDH) expression between C57 and Cav-1 KO cells treated with100 nM Dex or EtOH vehicle for 4 h (n � 4). Error bars represent the SEM.

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future experiments directed toward identifying genes occupied bydistinct GR phosphoisoforms (e.g., pSer211-GR) in NPSCs couldidentify specific GC-regulated gene networks that are influencedby cross talk with cytoplasmic signaling pathways. In fact, altera-

tions in GR phosphorylation in specific adult rat brain regions aregenerated in response to specific stresses and may impact selectgenes that modulate behavioral responses to stressful states (50).

Phosphorylation of GR at S211 and S226 is mediated by two

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FIG 6 GR phosphorylation at S211 but not S226 is altered in Cav-1 KO NPSCs. (A) Whole-cell lysates from C57 and Cav-1 KO NPSCs treated for 1 h with 100nM Dex or EtOH vehicle were subjected to immunoprecipitation with the BuGR-2 mouse monoclonal antibody against GR or nonimmune mouse IgG and thensubjected to Western blot analysis to detect total GR or phospho-S211 or phospho-S226 GR isoforms. Asterisks show the nonspecific band detected in all lanesfollowing pulldown with nonimmune IgG. The identity of the higher-molecular-weight band in the input lane (star) is unknown, but it was not detected inanti-GR antibody immunoprecipitates. The Dex-inducible phospho-S211 isoform, detectable in C57 but not Cav-1 KO NPSC lysates, is indicated by the arrows.GR phosphorylation at S226 is similar in C57 and Cav-1 KO lysates. The blot is representative of three biologically independent experiments. ChIP experimentsusing total GR (B to D) or phospho-S211 (E) antibodies indicates attenuated recruitment of GR to target genes in response to a 1.5-h treatment with 100 nM Dex.DNA was analyzed by using qRT-PCR, and final values are shown relative to total and IgG-negative controls before comparing enrichment in Dex versus EtOH(n � 3 biological replicates for C57 and n � 5 for Cav-1 KO cells). Error bars represent the SEM. *, P � 0.05; **, P � 0.01 (Student’s t test).

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different kinases—CDK2 at S211 and MAPK at S226. Others havesuggested that Cav-1 is a tumor suppressor gene due to its role inmitogenic signaling (51). Ablation of Cav-1 in metastatic lungcancer cell lines results in proliferation arrest and decreased ex-pression of cyclin D1 and CDK4 (51). Therefore, Cav-1 loss mayindependently or in conjunction with rapid GR signaling impactCDK2 and consequently genomic action of GR (Fig. 7).

In summary, Cav-1 is a multifunctional regulator of GR actionin NPSCs. Cav-1 influences both GR-dependent rapid changes inintercellular communication through gap junctions (17), which isrequired for the establishment of cerebral cortical architecture(34) and the GR transcriptome, including genes responsible forregulating NPSC proliferation. The wide variety of GR gene net-works affected by Cav-1 also suggests that this novel regulator ofreceptor action could impact the ultimate fate and laminar posi-tion of cells derived from embryonic cerebral cortical NPSCs ex-posed prematurely to GCs during fetal development.

ACKNOWLEDGMENTS

We thank Teresa Liu, Marcia Lewis, Christin Sculli, and Janie Zhang fortechnical assistance in these studies. In addition, we thank Bill LaFram-boise and Selma Witchel for helpful critiques and suggestions.

This study was supported by Public Health Service grants DK078394from the National Institute of Diabetes and Digestive and Kidney Diseases(D.B.D.), MH086651 from the National Institute of Mental Health(M.J.G.), AG030636 from the National Institute on Aging (F.G.), and aT32 training grant T32GM008424 from the National Institute of Gen-eral Medical Sciences (M.E.P.). In addition, support was received fromthe American Heart Association (13GRNT16560012 to F.G. and12SDG8800012 to D.V.). This project used the University of PittsburghCancer Institute’s Cancer Informatics and Cancer Biomarkers FacilityCores, which are supported in part by Public Health Service awardP30CA047904.

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