Cross-talk of vitamin D and glucocorticoids in hippocampal cells

Darja Obradovic,* Hinrich Gronemeyer,� Beat Lutz� and Theo Rein*

*Max Planck Institute of Psychiatry, Munich, Germany

�Institut de Genetique et de Biologie Moleculaire et Cellulaire, Illkirch, Strasbourg, France

�Department of Physiological Chemistry, Johannes Gutenberg University, Mainz, Germany

Abstract

There is growing evidence for a role of vitamin D3 signalling in

the brain. In this study, we investigated the influence of vita-

min D3, in combination with glucocorticoids, on differentiation

of the hippocampal progenitor line HIB5, as well as survival of

rat primary hippocampal cells. In HIB5, pre-treatment with

dexamethasone (Dex) alone inhibited neurite outgrowth and

abolished activation of the mitogen-activated protein kinase

(MAPK) pathway during platelet-derived growth factor

(PDGF)-induced differentiation, consistent with previous find-

ings. Interestingly, pre-treating HIB5 with vitamin D3 signifi-

cantly reduced these effects of Dex and, in addition, lowered

the transactivational function of the glucocorticoid receptor

(GR) in transient reporter gene assays. A further impact of

vitamin D3 on glucocorticoid effects was observed in a rat

primary hippocampal culture known to be particularly sensitive

to prolonged GR activation. In this model, Dex induced con-

siderable cell death after 72 h of exposure in vitro. However,

24 h of pre-treatment with low doses of vitamin D3 substan-

tially reduced the degree of Dex-induced apoptosis in primary

hippocampal cells. Taken together, our experiments demon-

strate a cross-talk between vitamin D3 and glucocorticoids in

two hippocampal models, a feature that may have important

implications in disorders with dysregulated glucocorticoid

signalling, including major depression.

Keywords: brain, glucocorticoid, hippocampus, MAPK,

phosphorylation, vitamin D.

J. Neurochem. (2006) 96, 500–509.

Vitamin D3 is mostly associated with its role in regulating thebody levels of calcium and phosphorus, and the mineralizationof bone (Bouillon et al. 2003; Holick 2003, 2004). The termvitamin D3 actually refers to a group of sterol-derivatives thatincludes the precursors 7-dehydrocholesterol (previtamin D3),cholecalciferol (major circulating form of vitamin D3) and thefinal bioactive metabolite calcitriol (1,25(OH)2 vitamin D3

which was used in this study and which is further referred tosimply as ‘vitamin D’) (Haussler et al. 1998; Jones et al.1998). Until recently, kidneywas considered to be the only siteof the production of calcitriol (DeLuca 1975; Zehnder andHewison 1999). However, recent findings indicate that theenzymatic machinery involved in vitamin D synthesis is alsopresent at several extra-renal sites in humans, including areassuch as hypothalamus, cerebellum, substantia nigra and retinalneurones (Zehnder et al. 2001; Eyles et al. 2005), suggestingthat vitamin D may have autocrine and/or paracrine propertiesin the human brain.

The genomic effects of vitamin D are mediated by thevitamin D receptor (VDR) (McDonnell et al. 1987; Normanet al. 2002; Carlberg 2003; Holick 2003). VDR expression inthe brain was first demonstrated in autoradiographic hormonebinding studies more than 20 years ago (Stumpf et al. 1982).

Subsequent studies have confirmed the presence of VDR inboth neuronal and glial cells of the limbic system, choroidplexus and several other brain regions (Walters et al. 1992;Baas et al. 2000; Langub et al. 2001), further indicating apotential role for vitamin D as a neuroactive hormone.

VDR-deficient mice were shown to have motor impair-ments as well as increased anxiety and grooming (Kalueffet al. 2004; Burne et al. 2005), indicating that VDR may beinvolved in physiological processes in the brain. In humans,vitamin D signalling has been linked to the incidence of

Received August 24, 2005; revised manuscript received September 27,2005; accepted September 27, 2005.Address correspondence and reprint requests to Darja Obradovic,

Max Planck Institute of Psychiatry, Kraepelinstr. 2–10, 80804 Munich,Germany. E-mail: darja@mpipsykl.mpg.deAbbreviations used: AP-1, activation protein-1; CRE, CREB response

element; CREB, cAMP response element binding protein; Dex, dexa-methasone; DMEM, Dulbecco’s modified Eagle’s medium; FBS, fetalbovine serum; GR, glucocorticoid receptor; GRE, glucocorticoidresponse element; HA, haemaglutinin; MAPK, mitogen-activated proteinkinase; MMTV, mouse mammary tumour virus; NGF, nerve growthfactor; PBS, phosphate-buffered saline; PDGF, platelet-derived growthfactor; SDS, sodium dodecyl sulfate; SV40, simian virus 40; TBS, Tris-buffered saline; VDR, vitamin D receptor.

Journal of Neurochemistry, 2006, 96, 500–509 doi:10.1111/j.1471-4159.2005.03579.x

� 2005 The Authors500 Journal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 500–509

various brain tumours, as well as to brain detoxificationpathways, growth factor synthesis (Neveu et al. 1994; Hahnet al. 1997; Garcion et al. 2002), multiple sclerosis andschizophrenia (McGrath 1999; Hayes 2000). A recent studydemonstrated that oral intake of 4000 IU vitamin D per daygreatly improved the sense of wellbeing and eased depres-sion in a large group of patients (Vieth et al. 2004),supporting previous studies which suggested that raisingvitamin D blood levels may be useful as an alternative and/orsupplementation in treating various affective disorders(Stumpf and Privette 1989; Gloth et al. 1999).

Furthermore, the offspring of vitamin D-deficient motherswas reported to have profoundly altered brain morphology atbirth (Eyles et al. 2003), and vitamin D was demonstrated tobe a modulator of key molecular events in the brain related togrowth factor signalling, cellular proliferation and differen-tiation (Garcion et al. 2002).

The strong differentiation-inducing and anti-proliferativeeffects of vitamin D in various tissues are well established(Lin and White 2003; Bikle 2004; van Driel et al. 2004), butalthough VDR has been localized to the brain areas oftenaffected by neurodegenerative diseases, such as hippocam-pus, prefrontal cortex and cerebellum (Walbert et al. 2001;Eyles et al. 2005), little is known about vitamin D signallingin these brain tissues.

The hippocampus is known to express glucocorticoidreceptors (GRs) and to be strongly affected by the circulatinglevels of corticosteroids. Given the well established role ofglucocorticoids in several major pathological conditions(Sousa and Almeida 2002 and references therein), theexperiments in this study focused mainly on the capabilityof vitamin D to modulate such glucocorticoid-related actionsin cells of hippocampal origin.

Experimental procedures

Chemicals

All media, media supplements and sera were from Invitrogen

(Karlsruhe, Germany). Vitamin D was purchased from Biomol

(Hamburg, Germany) [1,25 (OH)2 dihydroxyvitamin D3], luciferin

from Roche (Penzberg, Germany), platelet-derived growth factor

(PDGF) from Biochrom (Berlin, Germany) (A/B chain), and all

other hormones/chemicals, unless otherwise mentioned, from Sigma

(Munich, Germany).

Plasmids

pcHA-VDR was cloned by the author as previously described

(Puccetti et al. 2002). AP-1-Luc reporter was kindly provided by

Marcelo P. Pereda (Munich, Germany), and pRShGR and pMMTV-

Luc by R. M. Evans (La Jolla, CA, USA).

Cell culture and differentiation

HIB5 cells (Renfranz et al. 1991) were maintained in Dulbecco’s

modified Eagle’s medium (DMEM), supplemented with 10% fetal

bovine serum (FBS), under a humidifying atmosphere containing

5% CO2, at 32�C. Cells were split after reaching 80–90%

confluency. To induce differentiation in HIB5, cells were first

transferred to 39�C for 24 h, to inactivate the T-antigen, in a

chemically-defined medium consisting of a 1 : 1 mixture of

DMEM/F12 enriched with N2 growth supplements. The differ-

entiation inducer PDGF was then added at 30 ng/mL, and cells

were treated for the indicated times (24 h before inspection of

morphological changes or 6 h prior to measuring luciferase

activity).

Neurite outgrowth assay

HIB5 cells were cultured and differentiated in the presence of

hormones as described above; their morphology was examined

with a phase-contrast microscope, and photographs were taken

from at least 30 randomly chosen in each experiment, using the

Olympus BX-60 microscope linked to image-processing software

(Maryland, USA). Any neurone that sprouted at least one neurite

twice as long as the diameter of its cell body was regarded as a

neurone with outgrown neurites, and we determined the number of

such neurones in each plate. Values are presented as mean

percentage ± SEM.

Transfections, reporter assays

Transfections were performed using the lipofectamine 2000 reagent

according to the manufacturer’s instructions. Prior to the experi-

ment, 105 cells/well were plated in 24-well plates; transfection

was carried out 14–24 h later, after cells had attached. When co-

transfecting HA-VDR together with GR, 125 ng of each DNA

construct were used, and the total amount of DNA was adjusted

with ‘empty’ pcDNA vector. At 24 h following the transfection, the

medium was changed to 1% charcoal-stripped fetal bovine serum

(FBS) medium, and cells were treated with the specific GR ligand

dexamethasone (Dex) at a final concentration of 10)7 M (commonly

used in transfection assays, sufficient to provide maximum receptor

activation without having side effects on cell viability). After 24 h,

cells were lysed (with 75 mM Tris-HCl, 10 mM MgCl2, 10 mM

Triton-X 100, 2 mM ATP), briefly sonicated, and luciferase activity

measured. Aliquots (50 lL) of each supernatant were transferred to

a 96-well plate; 150 lL of 33 mM KHPO4, 1.7 mM ATP, 3.3 mM

MgCl2 and 13 mM luciferin (Roche, Germany) were automatically

added to each sample (Luminat LB 96, Wallac, Germany), and light

emission was measured for 10 s. Raw luciferase values varied

according to cell number and transfection efficiency. To correct for

this and to allow comparison of results from independent

experiments, values were normalized to their corresponding b-galactivity. The latter was determined after incubating cell extracts with

BAB buffer (60 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl,

1 mM MgCl2, 50 mM b-mercaptoethanol) and O-nitrophenyl-D-galactopyranoside (Sigma) for 30 min and measuring light absorp-

tion at 405 nm. In the transfection experiments where mouse

mammary tumour virus (MMTV)- or activation protein-1 (AP-1)-

Luc activation by endogenous receptors was determined, 500 ng of

the corresponding plasmid was transfected together with b-gal. Thenext day, hormones were added for the indicated times and further

processed as described above (in the case of AP-1, cells were

transferred to 39�C for 24 h and treated for 6 h with 30 ng/mL of

PDGF prior to lysis).

Novel vitamin D actions in the hippocampus 501

� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 500–509

Mitogen-activated protein kinase (MAPK) phosphorylation/

immunodetection

At 24 h following the 39�C temperature shift, hormones (Dex, vitamin

D or a combination of both) were added to cultures for an additional

24 h. Then, MAPK stimulator PDGF (30 ng/mL) was administered

for the times indicated as previously described (Son et al. 2001), andcells were harvested with ice-cold RIPA–PBS buffer [1 · PBS, 0.1%

sodium dodecyl sulfate (SDS), 1%NP40, 0.5% sodiumdeoxycholate]

supplemented with protease/phosphatase inhibitors (Roche,

Germany) and lysed for 30 min at 4�C. Cell lysates were cleared by

centrifugation at 14 000 g for 15 min, and the protein concentrations

were measured (Bio-Rad assay, Munich, Germany). A 20 lg aliquotof total protein was separated under reducing and denaturing

conditions by 10% SDS–polyacrylamide gel electrophoresis (SDS–

PAGE) and electro-transferred onto polyvinylidene difluoride mem-

branes (Millipore, Bedford, MA, USA). Non-specific binding sites

were blocked with 5% non-fat milk in TBS-T (Tris-buffered saline

containing 0.1% Tween-20). For phospho-ERK1/2 determination, a

phospho-p44/42 MAPK (Thr202/Tyr204) antibody (Cell Signalling,

Beverly,MA, USA; 1 : 2000 in TBS-T/5% non-fat milk) was used. In

order to quantify the signals of p42/p44, membranes were stripped

(62.5 mM Tris-HCl, pH 8.0, 100 mM b-mercaptoethanol, 2% SDS)

and re-probed with total ERK1/2 antibody (Cell Signalling; 1 : 2000

in TBS-T/5% non-fat milk). Membranes were incubated with the

corresponding secondary antibody conjugated to horseradish peroxi-

dase (Dako, Hamburg, Germany), and proteins were visualized by

enhanced chemiluminescence (Amersham Biosciences, Freiburg,

Germany). The values obtained in each experiment were normalized

to the vehicle-treated controls, and amounts of phosphorylatedMAPK

were determined relative to total MAPK expression by optical density

analysis using the Bio-Rad imaging system. Data are given as mean ±SEM for at least three independent experiments. For other immuno-

assays, cell lysates were processed accordingly and the following

antibodies used: anti-HA (1 : 2000 TBST/5% non-fat milk; Roche);

anti-hGR, anti-hVDR (1 : 2000 TBST/5% non-fat milk, Santa Cruz

Biotechnology, Santa Cruz, CA, USA).

Primary cell culture

Cultures were prepared from Wistar rats aged 4 days (Charles River,

Sulzfeld, Germany), following a previously described protocol

(Almeida et al. 2000). Briefly, hippocampal slices were digested

using the Papain Dissociation System from Worthington Biochem-

icals (Lakewood, NJ, USA), and the dissociated cells plated on poly

D-lysine-coated glass coverslips at a density of 400 cells/mm2.

Cultures were maintained in Neurobasal A medium to which 2% B27

supplement, 1 mMGlutamaxI (Invitrogen, Eggenstein, Germany) and

0.1 mg/mL kanamycin were added. Culture medium was half-

renewed every 3 days and experiments started 4–6 days after plating.

Cell death assay

Cell death was examined in 4% paraformaldehyde-fixed cells by

Hoechst 33342 staining. Apoptotic cells were characterized by

characteristic nuclear fragmentation; only those nuclei showing

evidence of DNA fragmentation without plasma membrane damage

were taken to be apoptotic cells. Fixed cells were incubated with the

dye (1 : 1000) for 15 min before examination under fluorescence.

Apoptotic cells as a percentage of total cells were quantified in at

least five, randomly-chosen microscopic fields (0.072 mm2, magni-

fication of 400 · ) across the long axis of the coverslips on which

primary cells were grown (randomly-chosen fields of a 24-well plate

in the case of HIB5).

Statistics

All data are presented as mean ± SEM. Numerical data were

subjected to statistical analysis (ANOVA, followed by post hoc test).

Significant differences were accepted when p £ 0.05.

Results

Vitamin D acts against glucocorticoid-induced inhibition

of morphological differentiation in hippocampal cells

In view of recent reports on VDR expression and vitamin Daction in the brain with respect to the impact of corticoster-oids on various processes occurring in hippocampus, thisstudy aimed at investigating the potential effects of vitaminD on those cellular phenomena. For this purpose, we firstused the progenitor cell line, HIB5, derived from embryonicrat hippocampus (day 16) and immortalized with a tempera-ture-sensitive variant of simian virus 40 (SV40) (Renfranzet al. 1991). HIB5 are known to functionally differentiate ina region-specific manner, when implanted into developing ratbrain in vivo (Renfranz et al. 1991) as well as in vitro, byshifting the temperature from 32�C to 39�C and using adefined medium containing PDGF. Under these conditions,cells stop proliferating and develop neurite-like extensions(Son et al. 2001; Kim et al. 2002).

Here, HIB5 cells were first treated at 39�C for 24 h eitherwith the synthetic glucocorticoid dexamethasone, or withvitamin D, or with a combination of both, followed by theaddition of differentiation-inducer PDGF for a further 24 h.The majority of cells in the control cultures extended neuritesof various lengths (Figs 1ai, ii); the same was observed incells pre-treated with vitamin D alone (Fig. 1aiii), whereastreatment with Dex inhibited morphological changes ofHIB5 cells and allowed further proliferation, as expected(Fig. 1aiv). In contrast, administration of 100 nM vitamin Din addition to Dex prevented, to a significant degree, theinhibitory effects of Dex, thus facilitating morphologicalchanges in HIB5 (Fig. 1av). It should be noted that treatingHIB5 cells with either vitamin D or Dex under proliferatingconditions (32�C) did not have any influence on cellmorphology or viability (not shown).

Next, the above cultures were examined for viability(trypan blue) and incidence of apoptosis (Hoechst staining).As depicted in Figs 1(b) and (c), vitamin D treatment did notsignificantly affect either when applied alone, but it signi-ficantly decreased the number of viable cells (Fig. 1b) andincreased appoptosis of the Dex-treated differentiating cul-tures (Fig. 1c).

To define the effect of vitamin D on HIB5 morphologicaldifferentiation more precisely, neurite outgrowth was

502 D. Obradovic et al.

� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 500–509

measured and quantified as described (see Experimentalprocedures). As depicted in Fig. 1(d), addition of PDGF at39�C induced differentiation in HIB5 control cells, howeverin HIB5 that were cultured in the presence of 10)7 M vitaminD, a marginal and only statistically insignificant increase ofneurite outgrowth relative to the control was observed. HIB5cultured with Dex showed very few morphological changesin their cell bodies, remaining rounded as previouslydescribed (Son et al. 2001). However, when differentiatingHIB5 cells were supplemented with vitamin D in addition toDex, the number of cells that sprouted neurites twice as longas the diameter of their cell body increased 2.5 fold

compared with Dex alone (13.5 ± 2.04% vs.5.38 ± 0.72 inthe Dex group), further indicating that application of vitaminD does not on its own significantly impact on neuriteoutgrowth, but can act against the inhibition by gluco-corticoids in the HIB5 model of hippocampal celldifferentiation.

Vitamin D and glucocorticoids: cross-talk on p42/p44

MAPK phosphorylation during early differentiation

The differentiating process in HIB5 cells involves severaldistinct signal transduction cascades (Sung et al. 2001; Kimet al. 2002). In particular, p42/p44 MAPK activation was

(a)

(b) (c) (d)

(i) (ii)

(iii) (iv) (v)

Fig. 1 Vitamin D reverses the inhibitory action of dexamethasone on

morphological differentiation of hippocampal cells. (a) Differentiating

HIB5 cells were pre-treated with vehicle (1, 2), vitamin D (10)7 M; 3),

dexamethasone (10)7 M; 4), or a combination of both (5) for 24 h and

then treated with the differentiation agent, PDGF (2–5). After 24 h,

cultures were examined under the microscope; representative photo-

graphs are shown (200-fold magnifications). (b, c) Cells from (1)–(5)

were Trypan blue (b) or Hoechst (c) stained to determine the per-

centage of viable and apoptotic cells, respectively; results represent

the mean ± SEM of three independent experiments. (d) Vitamin D

significantly increases the length of neurites in Dex-treated HIB5

cultures. Phase-contrast images from cells in (1)–(5) were taken, and

numbers of neurite-bearing cells were counted from 10, randomly-

chosen fields (treatments performed in triplicate); *p < 0.05.

Novel vitamin D actions in the hippocampus 503

� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 500–509

reported to be indispensable at early differentiation stages,and glucocorticoids were proposed to inhibit the differenti-ation of HIB5 cells, largely by blocking the p42/p44 MAPKpathway (Son et al. 2001). In order to obtain further insight

into the counteracting effects of vitamin D on the glucocor-ticoid-mediated inhibition of HIB5 morphological differen-tiation observed previously, we analysed MAPK activation inHIB5 cells following 24 h hormone pre-treatment with Dex,

(c)

(a)

(d)

(b)

Fig. 2 Vitamin D and glucocorticoids: cross-talk on p42/p44 MAPK

phosphorylation during early differentiation steps. (a) Succession of

treatments. (b) Following the temperature shift, HIB5 were treated with

different hormones (vitamin D 10)7 M, dexamethasone 10)7 M, or a

combination of both) for 24 h and then exposed to PDGF (30 ng/mL)

for the indicated times. Lysates were taken at different time points

following the PDGF treatment (15 min to 3 h) and phospho-MAPK

immunoreactivity was compared with the vehicle-treated controls (set

to 100). Bar graphs represent mean ± SEM of three independent

experiments; *p < 0.05, ***p < 0.001 compared with the respective

vehicle-treatment. (c) Representative blots from lysates at 15 and

30 min following the PDGF treatment are shown. (d) Schematic

summary of experiments in (b) and (c) displaying the kinetics of vita-

min D influence on phospho-MAPK levels.

504 D. Obradovic et al.

� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 500–509

vitamin D or both (summarized in Fig. 2a). As PDGF wasshown to influence MAPK phosphorylation most in the earlyphases of HIB5 cell differentiation (Son et al. 2001), celllysates were collected 15 min and 3 h after addition ofPDGF. p42/p44 MAPK phosphorylation levels were deter-mined by western blotting, and MAPK activation in hormonepre-treated cells was compared with vehicle-treated samples(Fig. 2b).

As illustrated in Figs 2(b–d), cells that were pre-treatedwith 10)7 M vitamin D showed significantly more activationof p42/p44 MAPK than vehicle-treated controls as early as15 min following PDGF treatment. Vitamin D pre-exposureresulted in a maximal p42 MAPK immunoreactivity increaseof approximately 60% at 30 min, with levels returning to thecontrol after 1–3 h (Figs 2b and c). In contrast, exposingcells to Dex (10)7 M) prior to PDGF administration dimin-ished p42 MAPK activation as expected (Figs 2b and c).However, this inhibition was significantly reversed bysimultaneous pre-treatment with vitamin D (Fig. 2b). In allcases, changes in p44 MAPK activation paralleled thoseobserved for p42 MAPK (Fig. 2b, inset).

In summary, we demonstrated that vitamin D leads toincreased levels of MAPK activation in this model ofhippocampal differentiation, both when administered aloneand when ceasing the phosphorylation block induced by Dex(schematically summarized in Fig. 2d).

Vitamin D down-regulates GR-mediated reporter gene

transactivation

In order to test the possibility that the antagonizing effects onthe glucocorticoid-mediated differentiation block could bedue to a direct or indirect cross-talk between the VDR and GRsignal transduction pathways, the transactivation potential ofGR was investigated after simultaneous VDR activation inreporter gene assays. HIB5 cells were transiently transfectedwith either VDR or GR, or both, and GR-induced transacti-vation was measured as luciferase activity originating froma co-transfected MMTV-Luc reporter that carries theGR-responsive elements upstream of the luciferase-encodinggene. In agreement with the presence of endogenous GR andVDR in HIB5 cells, as confirmed by western blots (data notshown), Dex activated the MMTV-Luc reporter about 20-fold(Fig. 3a: lane A). This induction was significantly reducedwhen co-administering vitamin D (lane B), most likely due tothe action of endogenous VDR. In cells transiently overex-pressing GR, MMTV-Luc activity was strongly enhancedsubsequent to a 24 h Dex treatment, and addition of vitamin Dreduced this activity only marginally (lanes E and F), inkeeping with the low level of endogenous VDR relative to thevery high levels of exogenous GR. Further supporting thehypothesis of a VDR–GR cross-talk that is expected to dependon the relative expression levels of the two receptors, thisinhibitory effect was dramatically increased when VDRwas overexpressed in addition to GR (the MMTV-Luc

transactivation decreased by more than 50% after addition ofvitamin D; compare lanes G and H). Additional experiments,performed to address the dose–response of vitamin D onendogenous receptors, suggested that the effects of vitamin Don GR-mediated transactivation are concentration-dependent(Fig. 3b). A similar, albeit less pronounced, inhibitory effect ofVDR on GR-transactivation (endogenous receptors) wasobserved with another GR-responsive reporter, GRE-Luc(not shown).

Since the actions of GR on the transcriptome also includetransrepression of certain signalling pathways, such as thoseresulting in the activation of AP-1-dependent promoters(Unal et al. 2004), we were curious to assess the effect ofVDR on GR-mediated AP-1 repression in HIB5 cells.Notably, suppression of AP-1-Luc reporter activity byactivation of endogenous GR could not be reversed bysimultaneous vitamin D incubation, indicating that theobserved cross-talk between VDR and GR signal transduc-tion pathways may affect only the transactivation functionsof GR and not its cross-talk with AP1 (Fig. 3c).

Vitamin D protects against glucocorticoid-induced cell

death in primary hippocampal cell culture

Having revealed that vitamin D counteracts GR-mediatedeffects in HIB5 cells (commonly used as a hippocampaldifferentiation model), it was important to investigate itseffects in a more physiological system. To do this, we usedprimary hippocampal cultures (rat, postnatal day 4), alsoknown to be vulnerable to glucocorticoids. For reasons thatare not yet completely understood, prolonged GR activationby its specific agonist Dex induces a considerable amountof cell death following exposure in vitro (Lu et al. 2003).Here, we pre-treated the hippocampal cells with vitamin D(10)8 and 10)9 M, concentrations close to physiologicallevels) for 24 h before Dex was applied for an additional72 h (in the continued presence of vitamin D), after whichcultures were analysed for nuclear fragmentation and theincidence of apoptosis (Hoechst staining). Vitamin Dtreatment alone had a small, but not significant effect oncellular viability (Fig. 4; white bars). However, in hippo-campal cultures exposed for 72 h to Dex, apoptosis wassignificantly increased (Fig. 4; black bars). Notably, cellspre-treated with vitamin D prior to Dex administrationexhibited markedly lower levels of GR-mediated cell death,indicating that vitamin D also counteracts at least certainaspects of GR-mediated signal transduction in rat primaryhippocampal cultures. In order to examine which populationof cells may benefit most from the vitamin D treatment,various immunocytochemical stainings were performed.However, no significant differences in numbers of neuronalor glial cells were observed across the different treatments(not shown).

In summary, these observations are in agreement withpreviously published reports of the neuroprotective actions of

Novel vitamin D actions in the hippocampus 505

� 2005 The AuthorsJournal Compilation � 2005 International Society for Neurochemistry, J. Neurochem. (2006) 96, 500–509

vitamin D (Brewer et al. 2001; Lin et al. 2003), andrepresent the first evidence of its effects in a model ofDex-mediated cell death in rat primary hippocampal cultures.

Discussion

The data presented here suggest that vitamin D counteractssome of the known GR-mediated actions such as glucocor-ticoid-induced inhibition of morphological differentiation inhippocampal HIB5 cells, thus expanding our knowledge ofthe role of vitamin D in differentiation gathered previously

Fig. 3 Vitamin D down-regulates GR-mediated transactivation. (a)

HIB5 cells were transfected with a reporter plasmid pMMTV, together

with either an hHA-VDR-expressing vector, hGR-expressing vector, or

a combination of both. The total amounts of transfected DNA were

kept constant and controls were filled up with ‘empty’ pcDNA plasmid

where necessary. At 12 h following the transfection, cells were treated

with ligands as indicated, and the reporter gene activity measured

after another 24 h. Luciferase values were normalized to b-Gal activity

and results are given as the ‘induction-fold’ (ratio of b-Gal-normalized

luciferase values in the presence vs. absence of Dex). Bars represent

mean ± SEM of at least three independent experiments performed in

triplicate; asterisks indicate p-values £ 0.05. (b) Effect of vitamin D on

the transactivation of MMTV-Luc by endogenous GR, VDR. Experi-

ments were performed as in (a) (without co-transfecting VDR and GR).

Different concentrations of vitamin D were added as indicated, and

activation of the reporter by endogenous receptors measured after a

further 24 h. (c) Effect of vitamin D on GR-mediated AP-1 transre-

pression. Cells were transfected with an AP-1-dependent luciferase

reporter together with the b-Gal control plasmid. Dex and vitamin D

were given the next day as indicated for 24 h, and AP-1 stimulated by

PDGF for another 6 h. Data represent the mean ± SEM of at least

three independent experiments; the values of AP-1-dependent tran-

scription in the absence of hormone was set to 100.

0

100

AP-1-Luc

Rel

ativ

e Lu

c ac

tivity

++

++

--

--

Dex 10-7

VitD10-7

Fol

d in

duct

ion

0

50

100

control VDR GR GR+VDR

MMTV-Luc

*

*

***

Dex 10-7

VitD10-7+ ++++++ +

+ +++- - --

A B C D E F G H

0

10

20MMTV-Luc

+ +++Dex 10-7

VitD10-9

VitD10-8

VitD10-7

+ + ++ -- - + - -- +- - - + -- -- + - - +

Fol

d in

duct

ion

(a)

(b)

(c)

0

50

100

150

200

Apo

ptot

ic c

ells

(%

of c

ontr

ol)

Controllevel

- Dex+ Dex

*

Dex 10-6

Vit D 10-9

Vit D 10-8

-+-

+--

++-

--+

+-+

Fig. 4 Vitamin D protects against glucocorticoid-induced cell death in

primary hippocampal cells. Exposure of primary hippocampal cells

(rat, mixed culture, postnatal day 4) to vitamin D for 24 h before and

during application of Dex resulted in a significant reduction in Dex-

induced apoptosis, as measured by Hoechst staining. An average of

1000 cells was sampled on each coverslip, and the results represent

average values ± SEM from 6–10 coverslips per treatment; asterix

indicates p < 0.05.

506 D. Obradovic et al.

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mostly from studies of non-neuronal cells (Holick 2003 andreferences therein). Since the MAPK pathway was previ-ously demonstrated to have a prominent role in early HIB5neuronal differentiation, we followed its activation status anddiscovered that pre-exposure of immature progenitors tovitamin D partially overcomes the Dex-induced block ofMAPK phosphorylation. As maturation of a progenitor cellto a neurone is a complex process that depends oninterrelated actions of numerous pathways, the possibilitythat vitamin D affects some of them, irrespective of its cross-talk with glucocorticoids, cannot be ruled out. However,cAMP response element binding protein (CREB), JunN-terminal kinase (JNK) and p38 showed no significantactivation following vitamin D pre-treatment (not shown).Moreover, our results suggest that the GR-VDR cross-talk isfunctionally distinct from that observed between GR andAP-1, which is still not fully understood at the molecularlevel but already pharmacologically exploited to generate anovel type of anti-inflammatory drugs.

To our knowledge, this is the first time that vitaminD-induced phosphorylation of p42/p44 MAPK has beendemonstrated in a hippocampal progenitor cell line. Studieson the potential of vitamin D to modulate p42/p44 MAPKactivity have so far been performed in non-neuronal cells,and both stimulatory and inhibitory effects of vitamin D havebeen found (Ravid et al. 2002; Rossi et al. 2004; Vertinoet al. 2005). This suggests, together with our findings in aneuronal system, that the nature of the cross-talk between theVDR and MAPK pathways is cell type-specific (Narayananet al. 2004). In conclusion, our work demonstrates activatingeffects of vitamin D on the MAPK pathway in the HIB5model of hippocampal neuronal differentiation, pointing toanother important signalling pathway in the brain that can besubject to modulation by vitamin D.

Interestingly, our analysis of VDR-GR cross-talk byin vitro transcription assays suggested that ligand-activatedVDR decreases cognate reporter activity of GR, with theeffect seemingly confined to the transactivational function ofGR as transrepression by GR was not affected.

In spite of evidence from the literature on down-regulationof some TATA-less and SP-1-rich promoters (like the one ofVDR) by glucocorticoids in rodent hippocampus (Meijer2002), there is to our knowledge no study describing theeffects of VDR on GR-driven transactivation. Although thismechanism remains to be elucidated in molecular detail, thefollowing mechanisms are prime candidates for subsequentscrutiny. First, VDR and GR may compete for commonco-factors. However, it should be noted that the compositionof co-factors may vary in a cell-specific manner, thusproviding an extensive variability in gene regulation intissues rich in cell types like the hippocampus. Second, holo-VDR and holo-GR may directly interact, resulting inimpaired transactivation potential of (one of) the transacti-vation functions. In this respect, our co-immunoprecipitation

experiments failed to find supportive evidence for directbinding in either the presence or absence of their respectiveligands (not shown). However, these data neither exclude thepossibility of a very weak but still functional interaction, norof an indirect interaction that is mediated by a yet to beidentified third factor or complex.

A similar transactivation-differentiating behaviour wasreported for the co-chaperone BAG1 (Schneikert et al.1999). Moreover, effects similar to the cross-talk reportedhere were observed in the case of vitamin D and Dex onG-protein-dependent signalling in osteoblast-like cells (Hom-me et al. 2003), as well as counteracting effects of vitamin Dand glucocorticoids on nerve growth factor (NGF) produc-tion in an astroglial cell line (Hahn et al. 1997).

In our experiments, the antagonizing effect of vitamin Don certain glucocorticoid-mediated actions was also apparentin another cellular model (primary hippocampal cultures,known to undergo increased apoptosis upon prolonged GRactivation), which was significantly protected against celldeath by addition of vitamin D. Thus, vitamin D acts alsoagainst Dex-induced cell death, in addition to its protectivefunction against excitotoxic insults in brain cells (Hahn et al.1997; Brewer et al. 2001; Wang et al. 2001; Lin et al. 2003).

Given the involvement of high amounts of circulatingcorticosteroids in the pathogenesis of several mental disor-ders, including cognitive impairments and major depression(Holsboer 2000), a possible role of vitamin D in alleviatingdetrimental GR-dependent processes in the brain may be putforward. Further elucidation of the molecular mechanismsunderlying the GR-VDR cross-talk will hopefully contributeto novel approaches towards the prevention and/or treatmentof such disorders.

Acknowledgements

This project was supported by a NARSAD Young Investigator

Award to DO. We thank members of the Lutz laboratory and

Drs Carlberg, McGrath, Kato, Mangelsdorf, Eyles and Klocker for

comments and/or suggestions at various stages of this work or

manuscript preparation.

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