glucocorticoids stimulate trh and c-fos/c-jun gene co-expression in cultured hypothalamic neurons

Ž .Brain Research 791 1998 56–62

Research report

Glucocorticoids stimulate TRH and c-fosrc-jun gene co-expression incultured hypothalamic neurons

Lu-Guang Luo, I.M.D. Jackson )

DiÕision of Endocrinology, Brown UniÕersity School of Medicine, Rhode Island Hospital, 593 Eddy Street, ProÕidence, RI 02903, USA

Accepted 23 December 1997

Abstract

To explore whether the protooncogenes, c-fosrc-jun, might be involved in regulating the effect of glucocorticoids on thyrotropin-re-leasing hormone in fetal rat diencephalic neurons, their localization and transcriptional activity were investigated using double-labeled insitu hybridization, Northern blot and nuclear run-on assays. The results showed that TRH mRNA was coexpressed with both c-jun andc-fos in the same neurons. Treatment with dexamethasone, a synthetic glucocorticoid, at 10y8 M, enhanced transcriptional activityresulting in an increase in both cell number and intensity of all three mRNAs. The existence of c-fosrc-jun in thyrotropin-releasinghormone neurons and the increased transcriptional activity following dexamethasone treatment suggests that these protooncogenes couldmediate the effect of glucocorticoids on thyrotropin-releasing hormone gene expression. q 1998 Elsevier Science B.V.

Keywords: Thyrotropin releasing hormone; c-fos; c-jun; Hypothalamic neuron; Glucocorticoid

1. Introduction

The hypothalamic tripeptide thyrotropin-releasing hor-Ž .mone TRH regulates the mammalian pituitary–thyroid

w xaxis 10 . Glucocorticoids appear to inhibit the pituitary–w xthyroid axis in man 1 through effects at a supra-pituitary

w xsite 1,12 . However, in the rat, both inhibitory and stimu-latory responses of the pituitary–thyroid axis have been

w xreported depending on time and dose factors 2,3,20 .Utilizing a culture system for fetal rat diencephalon devel-oped in this laboratory, we have shown that glucocorti-coids, added to the culture in the form of dexamethasoneŽ . y8DEX at 10 M, produce a significant stimulation ofpro-TRH peptide production, as well as mRNA levels asshown by in situ hybridization, and Northern blot withquantitative image analysis. This event is mediated by

w xtranscription as determined by nuclear run-on assay 16 .We have previously shown that the protooncogenes,

c-fosrc-jun are colocalized with TRH mRNA in anteriorw xpituitary cells 17 and may be involved in regulation of

w xthe tripeptide amide in this location 17 . Accordingly, we

) Corresponding author. Fax: q1-401-444-4921; E-mail:[email protected]

wondered whether c-fosrc-jun might also be involved inthe gene expression of TRH in hypothalamic neurons.There is now much evidence that the synthesis of secretoryproteins is dependent upon various nuclear transcription

Ž .factors encoded by an ‘immediate early gene’ IEG . AnIEG of especial importance in the CNS is the protoonco-gene c-fos, which encodes a protein that forms an het-erodimer with a member of the c-jun family through a

w xstructural motif called a leucine zipper 22 . This complexfunctions as a ‘third messenger’ that binds DNA at the

w xso-called AP-1 site 22,13 , which has been identified onw xthe TRH promoter 10 . Such findings are consistent with

reports of Fos-like immunoreactivity present within TRHŽ .neurons of the hypothalamic paraventricular nucleus PVN

w x13 . Further, there is evidence that the glucocorticoidŽ .receptor GR interacts with c-fosrc-jun products to either

w xactivate or inhibit gene expression 14 .In this study, we have investigated whether there is

colocalization of TRH and c-fosrc-jun in cultured hy-pothalamic neurons, and if the protooncogenes respondsimilarly to stimulation by glucocorticoids utilizing doublein situ hybridization, Northern blot and nuclear run-onmethods. The results show that TRH and c-fosrc-jun arecolocalized, and that they all increased following glucocor-ticoid exposure, suggesting that c-fosrc-jun may be in-

0006-8993r98r$19.00 q 1998 Elsevier Science B.V. All rights reserved.Ž .PII S0006-8993 97 01550-3

( )L.-G. Luo, I.M.D. JacksonrBrain Research 791 1998 56–62 57

volved in the glucocorticoid stimulation of TRH geneexpression in hypothalamic neurons.

2. Materials and methods

2.1. Primary neuronal cultures

The diencephalon was dissected from day 17 rat fe-tuses, dispersed enzymatically and plated in monolayer on100-mm cell culture plates for Northern blot analysis andnuclear run-on analysis, and on four-chamber glass LabTekcell culture slides for non-isotopic in situ hybridization.The culture plates and glass LabTek cell culture slides

Ž .were coated with poly-D-lysine 20 mgrml, Sigma . Theplating density was 106 cellsrml. The culture medium wasDMEMrL-15 supplemented with 10% fetal calf serumand 50 mM BrdU, a cell differentiating agent that was

w xpresent for the first four days of culture 4 . DEX was usedat 10y8 M in DMEM medium.

2.2. Double labeled in situ hybridization

Cells that had been maintained on LabTek slides for 7days in the presence or absence of 10y8 M DEX werewashed with PBS and fixed in 4% paraformaldehyde inPBS then dehydrated with ethanol. A Pst I–EcoRI frag-

Ž .ment 1241 bp of the preproTRH cDNA inserted into theexpression vector pSp65 in reverse orientation was used togenerate a digoxigenin guanosine 5X-tri-phosphate labeled,antisense, single-stranded RNA probe. c-jun cDNA plas-mid PUC18 was cut with the restriction enzymes HindIIIand BamHI, and c-fos cDNA plasmid pKSV10 cut withthe restriction enzymes NaeI and AÕaI. The digestionproducts were displayed on a 1% agarose gel. The 1625 bp

Žfragment, c-jun nucleotides 580–2205 of cDNA se-. Žquence and the 119 bp fragment, c-fos nucleotides 175–

.294 of cDNA sequence , were excised from the gel andŽ .purified with a Gene-Clean Kit Bio 101, CA . The frag-

ments were labeled with a biotin-dUTP by random oligo-nucleotide priming. For prehybridization, the slides wereincubated with 50% formamide and 10= Denhardt’s solu-tion at 428C for 2 h. Hybridization was performed with aTRH cRNA Dig-labeled probe, denatured c-fos or c-junbiotin-labeled probe each at 20 ngrwell in 50% for-mamide and 10= Denhardt’s solution containing 20 mgtRNA, 20 mg denatured sperm DNA incubated at 428C for16 h. After hybridization, the slides were washed with

Ž50% formamide in 2= SSC 0.3 M NaCl, 0.03 M sodium.citrate for 20 min, and then sequentially exposed to 1:800

Ž . Žstreptavidin–alkaline phosphatase AP -conjugate Boeh-.ringer Mannheim incubated for 30 min, anti-dig-fluo-

Ž .rescein Boehringer Mannheim 1:10 and streptavidin–Ž .texas-red Boehringer Mannheim 1:400 for 20 min all at

378C. The slides were observed under a microscope withfilter excitation at 494 nm, emission 523 nm for dig-

Ž .labeled-TRH green and 596 nm, emission 630 nm forŽ .biotin-labeled c-fos or c-jun red . Controls included slides

hybridized in the absence of either the Dig-labeled probeor the AP-conjugated antibody. Only background labelingwas observed when hybridization was conducted with thecorresponding sense probes. The cell mean intensity was

Žanalyzed by the Image Analysis System Biovision Image.Analysis, Perceptics TN without knowing the treatment

protocols. The mean intensity was calculated from themeasurement of more than 20 positive cells per image,taken from 6 areas chosen randomly from each well, 4wells from one slide and 24 wells as one group.

2.3. Northern blot analysis

Total RNA was extracted by the acid guanidiniumŽ .thiocyanate–phenol–chloroform extraction AGPC

w x Ž 7method 5 from hypothalamic neurons 10 cells per.point . RNA was quantitated spectrophotometrically by

Ž .absorbance at 260 nM the ratio of 260r280)1.8 andŽ .stored in 0.5% SDS at y708C. RNA samples 10 mg

were electrophoresed and transferred to a nylon membranew xusing established procedures 15 . After prehybridization

for 2 h, the membrane was hybridized with a 32 P-labeledantisense TRH cRNA probe. For c-fos and c-jun hybridiza-tion, the c-DNA fragment, described above was labeledwith 32 P using a random primer labeling method. Themembranes were hybridized respectively. Analysis of thehybridization signal was carried out with Pdi-Proteinq

Ž .DNA Imageware Systems NY .

2.4. Nuclear run-on analysis

Nuclear run-on analysis to determine the role of tran-w xscription was performed as described previously 18 . Hy-

Ž 7 .pothalamic neurons 10 cells per point were collectedŽinto ice-cold lysis buffer 10 mM Tris, 10 mM NaCl, 3.mM MgCl , 0.5% NP-40 and dounced on ice. Following2

Ž .centrifugation 1000=g, 5 min , the nuclear pellet wasŽresuspended in 200 ml of glycerol buffer 50 mM Tris,.40% Glycerol, 5 mM MgCl , 0.1 mM EDTA , snap-frozen,2

and stored in liquid nitrogen at y808C. The nuclei werethawed for transcription and mixed with 100 ml reaction

Žbuffer 10 mM Tris–HCl, pH 7.4, 5 mM MgCl , 300 mM2

KCl, 0.5 mM each of ATP, CTP, GTP, and 100 mCi ofŽ 32 .a- P UTP and reacted at 308C for 30 min. The reaction

Ž .mixture was then treated with DNase I 50 mgrml at308C for 8 min, deproteinized by digestion with proteinase

Ž .K 20 mgrml after addition of 10 mM Tris–HCl pH 7.4,10 mM EDTA, and 0.5% SDS, followed by phenol–chlo-roform extraction. RNA was rapidly precipitated from theaqueous phase with 2 M NH Ac and 2 volumes of ethanol.4

( )L.-G. Luo, I.M.D. JacksonrBrain Research 791 1998 56–6258

Ž . ŽFig. 1. a Double-labeled in situ hybridization for TRH A and D;. Ž .original green and c-fos B and E; original red which are colocalized in

Ž .the same hypothalamic neurons C and F; original yellow . A, B and C,Ž y8 .control; D, E and F, the effect of Dex 10 M treatment. The arrows

Ž .indicate neurons in which there are positive signals and colocalization. bŽ .Double-labeled in situ hybridization for TRH a and d; original green

Ž .and c-jun b and e; original red which are colocalized in the sameŽ .hypothalamic neurons c and f; original yellow . a, b and c, control; d, eŽ y8 .and f, the effect of Dex 10 M treatment. The arrows indicate neurons

in which there are positive signals and colocalization.

This step was repeated four times. Prior to hybridization,the 32 P-labeled RNA was heated at 1008C for 10 min andused to probe 5 mg of various cloned DNAs fixed to a

w xnylon matrix as described 24 . Plasmid clones used wereŽ .rat c-jun, c-fos cDNA, TRH cDNA 10 and pSp65 plasmid

Ž .Fig. 1 continued .

Ž .as negative control . Following hybridization for 40–60 h,w xthe filters were washed 24 , exposed to X-ray film for

various periods of time, and scanned as described above.

2.5. Statistical analysis

All the data are represented as means"S.E.M. andŽ .analyzed by analysis of variance ANOVA followed by

the Tukey–Kramer test unless otherwise indicated. Thelevel of significance was set at p-0.01. The correlation

Ž .coefficients r values for TRH and c-fosrc-jun from thedensitometric readings for each method—double-labeledin situ hybridization, Northern blot and nuclear run-on



analysis—were analyzed by the Cricket Graph linear fitcurve.

3. Results

3.1. Coexpression of c-fosrc-jun and TRH in hypothala-mic neurons

On in situ hybridization, the c-fosrc-jun proto-oncogenes were localized to the cultured hypothalamicneurons. Double-labeled in situ hybridization was thenused to determine the cellular relationship between c-


fosrc-jun and TRH. The results showed that the c-fosrc-jun positive cells were about 25% of the total hypothala-mic neurons, and that TRH colocalized with c-fosrc-jun in35% of the c-fosrc-jun expressing neurons. Almost allTRH positive neurons coexpressed c-fosrc-jun. Following

Ž y8 .exposure to DEX 10 M for 7 days both c-fosrc-junŽ .and TRH signals were similarly enhanced Fig. 1 . The

hybridization intensity of c-fosrc-jun and TRH stimulatedby DEX increased for TRH 133%, c-fos 148% and c-jun

Ž . Ž .121% ns40, p-0.01 Fig. 2 . The number of cellsexpressing c-fosrc-jun and TRH showed an approximate2.5-fold increase following glucocorticoid exposure.


Fig. 2. Percent change in TRH and c-fosrc-jun level in hypothalamicŽ y8 .neurons in response to Dex 10 M . ns60; ) p-0.01 vs. respective

control.

3.2. Northern blot

Total RNA isolated from hypothalamic neurons wasanalyzed by Northern blot with a- 32 P-labeled cRNA probe

Ž y8 .Fig. 3. Effect of Dex 10 M on TRH mRNA in hypothalamic neurons:Top Panel, Northern blot image; Bottom panel, densitometric analysis.) p-0.01; ns4.

Ž y8 .Fig. 4. Effect of Dex 10 M on TRH and c-fosrc-jun transcriptionalactivity in hypothalamic neurons, as determined by nuclear run-on analy-sis. Top panel, nuclear run-on image. Bottom panel, densitometric analy-sis. ) p-0.01, ns4.

w x y817 . After DEX 10 M treatment for 7 days, the positiveintensity of TRH mRNA enhanced 3-fold, c-fos 2.1-fold

Ž .and c-jun 4.2-fold ns4, p-0.01 . These results demon-strated that the response of c-fosrc-jun and TRH mRNA is

Ž .similar to the results from in situ hybridization Fig. 3 .

3.3. Nuclear run-on analysis

Following treatment with DEX 10y8 M for 7 days,TRH and c-fosrc-jun transcriptional activity as determinedby nuclear run-on analysis showed an increase of TRH

Ž7.7-fold, c-jun 5-fold and c-fos 3.4-fold vs. control ns3,. Ž .p-0.01 Fig. 4 .

The correlation coefficients of TRH and c-fosrc-jun areŽ . Žrs0.976 run-on analysis and rs0.957 in situ hy-. Ž .bridization both p-0.01 .

4. Discussion

We have previously shown that glucocorticoids stimu-late TRH gene expression in cultured hypothalamic neu-

w xrons by transcriptional activation 16 . However, the under-lying mechanism has been unclear. There is evidence thatthe effect of glucocorticoids at the cellular level may bemediated by the protooncogenes c-fos and c-junw x.8,17,19,23 . Accordingly, we speculate that c-fosrc-jun


may be involved in the process through which Dex en-hances TRH mRNA level in vitro.

To explore this issue, we determined the colocalizationof TRH and c-fosrc-jun expression in cultured hypothala-mic neurons by double labeling in situ hybridization with

Ž .two separate labeling techniques, a in vitro transcriptionŽ .labeling and b random labeling. These procedures used

digoxigenin and biotin, respectively, as labeling materialsas well as different developing colors that permitted clearseparation of each probe in the same sample. Our results

Ždemonstrated colocalization of TRH with c-fosrc-jun AP-.1 complex in hypothalamic neurons and a significant

correlation of c-fosrc-jun induction with changes in TRHŽmRNA activity after treatment with glucocorticoids p-

.0.01 .Ž .The involvement of multiple interacting cellular path-

ways in the regulation of c-fosrc-jun expression followingglucocorticoid stimulation of TRH likely reflects the pres-

Ž .ence of very strong steroid functional response domainsŽ .composed of steroid response elements SREs , as well as

w xelements for binding other transcriptional factors 21 .These domains, or steroid receptor units, are able to regu-late steroid action at the level of the DNA. Other studieshave demonstrated that a glucocorticoid response elementŽ .GRE promoter is recognized by both glucocorticoid re-

Ž .ceptor GR and AP-1 factors that are responsible for thew xpositive and negative regulation by glucocorticoids 8 .

w xBoth of these elements are present on the TRH gene 15 .It is also known that glucocorticoids interact with the AP-1

Ž .transcription heterodimer fosrjun or homodimerŽ .junrjun to either inhibit or activate, respectively, the

w xtranscription of the proliferin gene 8 . The level of the Fosoncoprotein accordingly determines whether the glucocor-

w xticoid induces or represses gene expression 9 .The studies reported herein indicate that TRH and

c-fosrc-jun show a similar response to DEX at bothŽ .mRNA in situ hybridization and gene transcription level

Ž .run-on analysis . The coefficient of correlation calcula-tions for both methods produced good fit curves. Thesimilar response of TRH and c-fosrc-jun to DEX exposurein the same hypothalamic neurons raises the possibility ofa functional relationship between these substances. Thereare two possible explanations for these findings. The firstis that DEX stimulates TRH by enhancing c-fosrc-jun.This could be investigated using a time course experimentto determine whether c-fosrc-jun increased prior to TRHin response to DEX treatment. In preliminary studies,

w xreported in abstract 11 , we found that while neither genewas expressed at 15 min, both were expressed at 1 h.Whether the 15–60-min time frame might show a sequen-tial response is unknown. Second, it is possible that theTRH gene and the c-fosrc-jun genes are expressed sepa-rately after DEX stimulation, and that the FosrJun pro-teins are responsible for prolonging the TRH gene expres-sion rather than initiating its activity. This would explainthe long-term response of the TRH gene to DEX. Prior

studies of hypothalamic cultures in this laboratory showedthat TRH and c-fosrc-jun gene expression exhibit a grad-ual increase over the time frame 1 h through 4 days after

w xDEX treatment 11 .Ž .The fact that the glucocorticoid receptor GR is ex-

w xpressed in hypothalamic TRH neurons 6,7 provides amorphological basis for glucocorticoids to directly regulateTRH gene expression in this location. Further, the pres-ence of a GRE and AP-1 regulatory site on the TRH

w xpromoter 15 provides a mechanism through which theprotooncogenes, c-fosrc-jun, could mediate the transcrip-tional activation of TRH gene expression in response toglucocorticoid treatment. However, further studies areneeded to establish such a relationship.

In conclusion, we have demonstrated the glucocorticoidinduction of the proto-oncogenes c-fos and c-jun in cul-tured hypothalamic neurons. The co-expression of TRHand c-fosrc-jun in the same neurons and the strong posi-tive correlation coefficient of TRH and c-fosrc-jun mR-NAs raise the possibility that c-fosrc-jun may mediate theeffect of glucocorticoids on TRH at this site by activatinggene transcriptional activity.

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glucocorticoids stimulate trh and c-fos/c-jun gene co-expression in cultured hypothalamic neurons

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