pH is an intracellular effector controlling differentiation of oligodendrocyte precursors in culture...

pH Is an Intracellular Effector ControllingDifferentiation of OligodendrocytePrecursors in Culture Via Activation of theERK1/2 Pathway

Frederic Bernard,1,2,3 Peter Vanhoutte,2,3 Amar Bennasroune,1 Gerard Labour-dette,1 Martine Perraut,1 Dominique Aunis,1 and Stephane Gaillard1*1Inserm U 575, Physiopathologie du Systeme Nerveux, IFR des Neurosciences, Strasbourg, France2CNRS UMR 7102, Paris, France3Universite Pierre et Marie Curie - Paris VI, Paris, France

We reported previously that onset of oligodendrocyteprecursor cell (OPC) differentiation is accompanied byan increase in intracellular pH (pHi). We show that OPCdifferentiation is dependent primarily on a permissivepHi value. The highest differentiation levels were ob-served for pHi values around 7.15 and inhibition of differ-entiation was observed at slightly more acidic or alkalinevalues. Clamping the pHi of OPCs at 7.15 caused a tran-sient activation of ERK1/2 that was not observed atmore acidic or alkaline values. Furthermore, inhibition ofERK activation with the UO126 compound totally pre-vented OPC differentiation in response to pHi shift.These results indicate that pHi, acting through theERK1/2 pathway, is a key determinant for oligodendro-cyte differentiation. We also show that this pHi pathwayis involved in the process of retinoic acid-induced OPCdifferentiation. VVC 2006 Wiley-Liss, Inc.

Key words: oligodendrocyte; pH; signaling; differentiation;ERK; retinoic acid

The control of cell growth is a complex interplay ofmultiple extracellular and intracellular signals that governthe balance between cell cycle progression and commit-ment of terminal differentiation. Changes in intracellularpH (pHi) have been shown to be one of these signalsplaying a critical role in the control of cellular prolifera-tion (Grinstein et al., 1989; Shrode et al., 1997b) and dif-ferentiation (Alvarez et al., 1989; Hazav et al., 1989;McAdams et al., 1998). We have shown recently that anintracellular alkalinization occurred at the onset of the‘‘spontaneous’’ differentiation of oligodendrocyte precur-sor cells (OPCs) in primary culture (Boussouf et al., 1997;Boussouf and Gaillard, 2000). This observation suggestedthat, as for some other cell types, intracellular pH couldbe one of the intracellular signals, acting as a second mes-senger, involved in the control of oligodendrocyte differ-entiation. This hypothesis is further supported by the de-monstration that retinoic acid (RA), a potent inducer ofOPC differentiation (Barres et al., 1994; Tokumoto et al.,

1999, 2001; Billon et al., 2001), has been shown to exertits differentiating effect on other cell types through an in-tracellular alkalinization mediated by an increase of bothexpression level and activity of Naþ/Hþ exchanger. Inthis way, an increased Naþ/Hþ exchanger function hasbeen well documented for human leukemic HL-60 cells(Ladoux et al., 1987; Rao et al., 1992) and for murineP19 embryonal carcinoma cells (Dyck and Fliegel, 1995;Wang et al., 1997), a cell line that can be induced to dif-ferentiate into myelinating oligodendrocytes on RA treat-ment (Staines et al., 1996).

We investigated the potential role of intracellular pHas a key regulator in the process of ‘‘spontaneous’’ as wellas of RA-induced OPC differentiation. We showed, byexperimental manipulations of pHi, that the level of OPCdifferentiation was primarily dependent on intracellularpH. The maximum level of differentiation was found forintracellular pH value around 7.15 and inhibition of differ-entiation was observed at more acidic or alkaline values.We also showed that a pHi experimentally clamped to7.15 triggered a specific transient activation of the MAPkinase of the ERK1/2 subtype. Furthermore, we establishedthat a pharmacologic inhibition of the ERK1/2 pathwaytotally blocked the pHi-induced OPC differentiation. Theseresults show that intracellular pH changes are related cau-sally to the differentiation of OPC via the activation of theERK1/2 MAP kinase signaling pathway.

Our results also show the key role of pHi in RA-induced OPC differentiation. RA increased OPC pHi byabout 0.25 U and this intracellular alkalinization was asso-ciated with an increase in the Naþ/Hþ exchanger activ-ity. We further showed that RA-induced OPC differ-entiation was abolished completely by preventing the

*Correspondence to: Stephane Gaillard Inserm U 575, Centre de Neuro-

chimie, 5, rue Blaise Pascal, 67084 Strasbourg Cedex France.

E-mail: [email protected]

Received 22 May 2006; Revised 7 July 2006; Accepted 14 July 2006

Published online 18 September 2006 in Wiley InterScience (www.

interscience.wiley.com). DOI: 10.1002/jnr.21051

Journal of Neuroscience Research 84:1392–1401 (2006)

' 2006 Wiley-Liss, Inc.

alkalinization that follows RA treatment. Conversely,mimicking this alkalinization by experimental manipula-tion of pHi, in the absence of RA, also stimulated OPCdifferentiation to the same extent as RA.

MATERIALS AND METHODS

Materials

Dulbecco’s modified Eagle’s medium (DMEM) was pur-chased from Gibco (Grand Island, NY). 20,70-Bis(carboxyethyl)-carboxyfluorescein tetraacetoxymethyl ester (BCECF AM) waspurchased from Molecular Probes Europe (Leiden, Holland).Monoclonal anti-galactocerebroside and A2B5 antibodies werefrom Boehringer-Mannheim Biochemicals (Indianapolis, IN).Western blot experiments were carried out using the followingprimary antibodies: polyclonal anti-phospho-(Thr202-Tyr204)-ERK1/2 (diluted to 1:5,000) or anti-phospho-(Thr183-Tyr185)-JNK (diluted to 1:750) from Cell Signaling (San QuentinYvelines, France) and a monoclonal anti-alpha-tubulin (cloneDM1A) (diluted to 1/5,000) purchased from Sigma (SaintQuentin Fallavier, France). Horseradish peroxidase-conjugatedsecondary antibodies anti-rabbit (diluted to 1:5,000) and anti-mouse (diluted to 1/10,000) were from Amersham PharmaciaBiotech. Proteins were detected using the ECL kit (AmershamPharmacia Biotech, Orsay, France) according to the manufac-turer’s instructions. Biosynthetic human insulin was from EliLilly & Co. (Indianapolis, IN). All-trans-retinoic acid, nigericin,and other reagents were from Sigma.

Cell Culture

Primary culture of rat cerebellar oligodendrocytes and intra-cellular pH measurements were carried out as described previously(Boussouf et al., 1997). Briefly, oligodendrocytes were mechani-cally dissociated from newborn rat cerebella and maintained inDMEM supplemented with sodium bicarbonate (25 mM), insulin(0.5 lM), gentamycin (0.05 mg/ml), and 10% decomplementedhorse serum. Cells were plated at a density of 1 × 105 cells per35 mm culture dish and were incubated at 378C in a water satu-rated incubator equilibrated with 95% air-5% CO2. Two daysafter plating, the cells were rinsed and exposed to a serum-free,chemically defined medium (CDM) (Gaillard and Bossu, 1995)composed of DMEM supplemented with bovine serum albumin(100 lg/ml), human transferrin (100 lg/ml), progesterone (20 nM),insulin (0.5 lM), putrescine (100 lM), sodium selenite (30 nM),penicillin (50 UI/ml), and streptomycin (50 lg/ml). The effectsof retinoic acid and intracellular pH changes on OPC differen-tiation were analyzed after three days in defined medium. Simi-lar time point has been used in many studies to analyze the stim-ulation of OPC differentiation by various factors such as retinoicacid or thyroid hormone (Tokumoto et al., 1999) and norepi-nephrine (Ghiani et al., 1999a). Cells were fixed and immuno-labeled with anti-galactocerebroside (Gal-C) antibody to identifydifferentiated oligodendrocytes, as described previously (Bernardet al., 2001). Gal-C is a specific marker for differentiated oligo-dendrocytes (Raff et al., 1978; Ranscht et al., 1982) and hasbeen used widely to quantify stimulation of OPC differentiationby retinoic acid (Billon et al., 2001; Tokumoto et al., 2001),allowing an efficient comparison of our results with already pub-lished data. Fluorescent Gal-C positive cells were counted in

randomly selected fields observed with a ×40 objective and thetotal number of cells in the same field was determined underphase contrast optic. A minimum of 30 fields were analyzed foreach experimental series.

For morphologic analyses, primary anti-Gal-C and A2B5antibodies were revealed with peroxidase-conjugated secondaryantibodies as described previously (Boussouf and Gaillard, 2000).The complex morphology of oligodendroglial cells was quanti-fied by calculating the fractal dimension of individual living orfixed cells with the box-counting method as described previ-ously (Bernard et al., 2001). We have shown that changes infractal dimension during oligodendrocyte differentiation followthe well known pattern of markers expression by these cells andthat A2B5, O4, and Gal-C-expressing cells were identified con-fidently from their respective fractal dimension values (Bernardet al., 2001).

To analyze MAP kinase activation on cell lysates, purifiedsecondary cultures of OPCs from brain of newborn rat wereprepared as described previously (Besnard et al., 1989). Thistype of culture was used for obtention of cell lysates because itcontains exclusively oligodendroglial cells whereas the primarycell culture described above contains about 10% of contaminat-ing astrocytes (see Results). Furthermore, the use of cortical vs.cerebellar OPCs allows to collect much more material. Briefly,whole brain hemispheres were dissected in phosphate bufferedsaline and transferred to culture medium composed of DMEMsupplemented with penicillin (50 U/ml), streptomycin (50 lg/ml), and calf serum (10%). The dissociation was carried out bysieving the tissue through a 82 lm mesh nylon sieve in the cul-ture medium. The cell suspension was dispensed in 100 mm di-ameter plastic tissue culture dishes coated with poly-L-lysine.The cultures were incubated at 378C in a water saturated incu-bator equilibrated with 95% air-5% CO2. Culture medium waschanged 4 days after seeding and twice a week thereafter. After10–12 days oligodendrocyte precursor cells were detachedselectively by gentle syringing the culture medium on the celllayer. Dislodged cells were then submitted to three successivedifferential attachment over a 48-hr period in non-coated plas-tic culture dishes to allow adhesion of remaining astrocytes andmicroglia. The non- and loosely adherent cells were subcul-tured in 100 mm plastic culture dishes coated with poly-L-lysine in the chemically defined medium described above. In theabsence of additional mitogen these subcultures give rise to analmost homogeneous cell population containing >90% Gal-Cpositive cells after 10 days (Besnard et al., 1989). To maintainthese cells at the OPC stage and to prevent premature differentia-tion before processing, PDGFAA (10 ng/ml) and bFGF (10 ng/ml)were added to the culture medium (Besnard et al., 1989; Bogleret al., 1990).

Intracellular pH Analysis

For intracellular pH measurements, the cells were loadedby incubation in bicarbonate-buffered solution containing 5 lMof the permeant BCECF-AM for 60 min at 378C. The plasticculture dishes were then rinsed three times with physiologic so-lution and directly placed on the thermostated stage of the in-verted microscope equipped for epi-fluorescence. The ratio ofthe fluorescent signals measured at 530 nm in response to an al-ternating excitation at 450 and 490 nm was calibrated at the end

Intracellular pH Controls OPC Differentiation 1393

Journal of Neuroscience Research DOI 10.1002/jnr

of each experiment using nigericin (3 lM) in high externalpotassium (130 mM) allowing pHi to be clamped to variousexternal pH values. The nigericin-containing solutions wereintroduced in the plastic culture dishes through specific syringesand tubings, distinct from the lines used for introducing the phys-iologic solutions. The lines used for calibration solutions were rinsedwith ethanol after each experiment. These operating procedureswere used to eliminate the effects of trace levels of nigericin dur-ing the subsequent intracellular pH measurements (Bevenseeet al., 1999). All experiments were carried out at 378C. Intracel-lular pH recovery rates (pH units × min�1) were calculatedwithin time intervals of 20–30 sec, from individual cells after anacid load imposed by the NH4þ prepulse technique. The rates ofHþ efflux (mmol × l�1

× min�1) were calculated as the productof the recovery rate times the buffering power (mmol × l�1

×pH�1), calculated on the same cell, from the amplitude of theintracellular alkalinization observed after the external applicationof NH4Cl according to Roos and Boron (1981).

Cell Lysis andWestern Blot Analysis of ERK1/2 and JNK

After treatments, culture medium was removed quicklyand dishes were placed on dry ice. Cells were then placed onice and ice cold lysis buffer was added for 5 min (10 mM Tris-HCl, 50 mM NaCl, 1% Triton X-100, 30 mM sodium pyro-phosphate, 50 mM NaF, 5 lM ZnCl2, 100 mM Na3VO4, 0.5 mMDTT, 100 nM okadaic acid, 2.5 lg/ml aprotinin, 2.5 lg/mlpepstatin, 0.5 mM PMSF, 0.5 mM benzamidine, 2.5 lg/mlleupeptin). Lysates were then vortexed for 1 min and insolublematerial was removed by centrifugation (13,000 × g; 20 min;48C). A volume of cell lysate corresponding to 20 lg of pro-teins was boiled for 5 min and analyzed by SDS-PAGE on 10%gels as already described (Vanhoutte et al., 1999).

Results are given as means 6 SEM. Significance wastested using Student’s two-tailed t-test. P values of <0.01 weretaken as statistically significant.

RESULTS

Characteristics of Cell Cultures

After 1 day in CDM, the population of cerebellarcells is composed exclusively of glial cells. The numerous

neuronal cells (mainly granular neurons) that were presentin the mixed cells died during the 2 days after plating,these cells requiring serum and high external potassiumconcentration (25 mM) to survive (Balazs et al., 1988).The remaining glial cells were composed mainly ofA2B5þ/O4� oligodendrocyte precursors that averaged85% of the total number of cells (Fig. 1A). A few numberof A2B5þ / O4þ pro-oligodendrocytes was observed,representing 5% of the total cells. The difference in cellmorphology between these two distinct developmentalstages was clearly quantified by calculating their fractaldimension as reported previously (Bernard et al., 2001).A2B5þ/O4� OPCs displaying fractal dimensions rangingfrom 1.053–1.233 whereas A2B5þ/O4þ pro-oligoden-drocytes were characterized by fractal dimensions >1.233(Bernard et al., 2001). No Gal-Cþ cells were observed atthis stage. The remaining cells (about 10% of the totalcells) were contaminating A2B5�/GFAPþ Type 1 astro-cytes.

Analysis of oligodendrocyte differentiation in variousconditions was carried out after 3 days in CDM. Whencultured in control conditions (external pH 7.40) the pop-ulation of cells was composed of 23% of Gal-Cþ cells char-acterized by fractal dimensions ranging from 1.351–1.574and 67% of A2B5þ/Gal-C� OPCs (Fig. 1B). During the 3days in CDM, about 30% of the A2B5þ/Gal-C� cellsshowed a single division and 10% showed two successivedivisions, shown by time-lapse video microscopic observa-tions (not illustrated). After 8 days in culture (6 days inCDM) >80% of cells were Gal-Cþ/MBPþ many cells dis-playing a thin membrane-like sheet emanating from thedistal processes. Even at this later stage, a few percent (5–7%) of A2B5 positive, Gal-C negative progenitors werestill observed.

The population of purified secondary cultures, usedto analyze MAP kinase activation, was exclusively com-posed of A2B5þ/Gal-C� progenitors as long as growthfactors (PDGF and bFGF) were present. The morphologyof these cells was typical of OPCs, with few and poorlybranched processes and characterized by fractal dimension<1.050.

Fig. 1. Immunocytochemical characterization of primary cell culture.A: After 1 day in CDM, oligodendrocyte precursor cells immunola-beled with A2B5 antibody were characterized by few (2–4) and poorlybranched processes. An immature, A2B5 negative cell (arrow head),identified by a fractal dimension close to 1 is characteristic of the vimþ,PSA N-CAMþ, A2B5� pre-progenitors already described (Grinspanand Franceschini, 1995). A cell with more complex morphology is seen

in the upper right. This cell, identified by a fractal dimension of 1.264,corresponds to the population of A2B5þ, O4þ, Gal-C� pro-oligoden-drocytes previously described (Bernard et al., 2001). B: after 3 days inCDM, more differentiated oligodendrocytes, immunolabeled with anti-Gal-C antibody, appeared as round cell bodies emitting a profuse net-work of radial processes. Undifferentiated progenitors (not stained withanti-Gal-C antibody) are still present at this stage (arrows).

1394 Bernard et al.


Effects of pHi Changes on OPC Differentiation

We analyzed the pattern of OPC differentiation oncells cultured for 3 days in various conditions of intracel-lular pH clamp. Experimental manipulations of intracellu-lar pH were achieved by adjusting the pH of the culturemedium to given values. As shown in Figure 2, a directlinear relationship was found between external pH (pHe)and pHi indicating a substantial pHi tracking of pHe forOPCs, as already observed for mature oligodendrocytes(Boussouf et al., 1997). pHi was also experimentallyshifted by adding weak acid (propionic acid) or base (tri-methylamine) to the culture medium at constant pHe

(7.40). As shown in Figure 3, independently of the methodused to change pHi, the level of OPC differentiation wasfound to be primarily dependent on intracellular pH. Thehighest level of differentiation was found around pHi 7.15and a strong decline was observed for slightly more acidicor alkaline values.

Effects of pHi Changes on ERK1/2and JNK Activity

We next investigate the potential signaling pathway(s)acting downstream from intracellular pH changes. It hasbeen reported previously that ERK1/2 signaling pathwaywas involved in the differentiation of oligodendrocyte CG-4cell line (McNulty et al., 2001). Furthermore, the re-quirement of ERK1/2 pathway for neurotrophin-induced oligodendrocyte differentiation has been reportedrecently (Hu et al., 2004). We investigated whether theactivation of ERK1/2 (i.e., phosphorylation of ERK1/2)

was modulated by intracellular pH changes. Levels ofERK1/2 activation were analyzed by Western blot usingan antibody raised against the dually phosphorylated formof ERK1/2 (Vanhoutte et al., 1999). Using this method,we pointed out that clamping intracellular pH of OPCs to6.70, 7.15, and 7.35 resulted in a transient increase ofphosphorylated ERK compared to control cells (pHi 6.88)(Fig. 4A). This activation was detectable as early as 5 minand was not observed after 15 or 30 min. Interestingly,ERK phosphorylation was about three times stronger atpHi 7.15 for which we measured the highest level of OPCdifferentiation when compared to more acidic (6.70) ormore alkaline (7.35) values for which we measured thelowest levels of differentiation. On the other hand, it hasbeen reported previously that experimental cytosolic alka-linization rapidly increases c-Jun N-terminal kinase/stress-activated protein kinase (JNK/ SAPK) activity in theU937 cell line (Shrode et al., 1997a). We investigatedwhether the activity of JNK could be modified by intracel-lular pH changes in OPCs. Unlike ERK, activation ofJNK was not induced at any time tested (Fig. 4B). Theseresults show that intracellular pH can modulate with somespecificity one of the major intracellular signaling pathwaysinvolved in cell growth and differentiation. To determinethe role of ERK1/2 activation in pHi-dependent OPCdifferentiation, we pharmacologically blocked ERK1/2activation observed at pHi 7.15 by using the MEK1/2 (theupstream kinase of ERK1/2) inhibitor UO126 (10 lM).As shown in Figure 4A (upper right), adding UO126 inthese conditions blocked totally ERK1/2 activation at pHi

7.15. Differentiation of OPCs was then measured after

Fig. 2. Dependence of intracellular pH on external pH. In CO2/bicarbonate containing medium, external pH (pHe) was varied under5% CO2 by adjusting the NaHCO3 concentration (filled circles) withrespect to the control (open square: pH 7.40, NaHCO3 25 mM). Therelationship between pHi and pHe was best fitted by a linear regressionwith the following equation: pHi ¼ 1.7125 þ 0.7 pHe. At constantexternal pH (7.40), pHi was increased by adding 5 mM trimethyl-amine (down triangle) and decreased by adding 10 mM propionicacid (up triangle) to the culture medium. Each symbol represents themean 6 SEM of at least 17 cells recorded.

Fig. 3. Dependence of OPC differentiation level on intracellular pH.Intracellular pH was changed either by adjusting external pH (opencircles) or by adding trimethylamine (down triangle) or propionic acid(up triangle) at the external pH of 7.40, according to Figure 2.



3 days in the presence or not of UO126, the mediumbeing renewed every 12 hr. The percentage of differenti-ated oligodendrocytes (Gal-C positive cells) at pHi 7.15was 45.70% 6 2.16% and fell to 18.88% 6 0.51%, P <0.01, in the presence of UO126 (Fig. 5). After 3 days inthe presence of UO126, no significant change in total cellnumber was observed but all the cells were characterizedby a less complex morphology. The numerous processesobserved in control condition (Fig. 1B) were reduced in

number and length on exposure to the MEK inhibitorUO126. A similar observation has been reported recentlyon exposure of oligodendrocytes to the other MEK inhibi-tor PD098059 for several days (Baron et al., 2000). Ourresults further show that ERK1/2 plays a key role in trig-gering OPC differentiation in response to pHi shift.Although no phospho-ERK signal could be detected byWestern blot in OPCs in control conditions (pHe 7.40,pHi 6.88) (Fig. 4A) the activated form of the kinase is pres-ent most likely at a low basal level in these conditions. Wetested the effect of UO126 on OPC differentiation at pHi

6.88 and we found that blockade of ERK also inhibitedOPC differentiation, (30.8% 6 0.42% vs. 22.12% 60.38% in the presence of UO126, P < 0.01) (Fig. 5). Thisrate of differentiation have to be compared to the oneobtained at pHi 7.15 þ UO126, 18.88% 6 0.51%. Fromthese results it seems that whatever the pHi values, around20% of OPC are found differentiated when ERK1/2 isblocked.

Mechanisms of RA-Induced IntracellularAlkalinization

We investigated whether extracellular cues couldtrigger this pHi-controlled pathway to control OPC differ-entiation. We studied the effects of retinoic acid, wellknown for its ability to induce differentiation in oligoden-drocyte (Barres et al., 1994; Tokumoto et al., 1999, 2001;Billon et al., 2001) and pHi changes through activation ofNaþ/Hþ exchange in some other cell types (Ladoux et al.,1987; Rao et al., 1992; Dyck and Fliegel, 1995; Wanget al., 1997). We tested first whether intracellular pH wasmodified in OPCs by addition of RA. Intracellular pHwas monitored on single OPC bathed in physiologic CO2/bicarbonate-buffered saline (external pH 7.40). Addition of1 lM retinoic acid elicited an increase in intracellularpH. The elevated pHi was maintained as long as RA was

Fig. 5. Inhibition of pH-dependent OPC differentiation by inhibitionof ERK 1/2 activation. Differentiation of OPCs was measured after3 days in the presence (filled bar) or not of UO126 (UO) (10 lM).Addition of UO126 decrease significantly the level of differentiated(Gal-C positive) cells (*P < 0.01) but independently of the pHi, abasal level of differentiated oligodendrocytes is still observed.

Fig. 4. ERK, but not JNK, activity is modulated by changes in intra-cellular pH in OPC. Western blot analysis of ERK (A) and JNK (B)phosphorylation were carried out from OPCs incubated for the timesindicated, in experimental conditions leading to clamp intracellular pHto 6.70, 7.15, or 7.35 as described in the text. Alpha-tubulin stainingswere carried out on the same membrane as the one used for eitherphospho-ERK or phospho-JNK and served as a loading control with

regard to the amount of protein loaded in each lane of these gels. Allexperiments were carried out in duplicates. Because no phospho-JNKsignal could be detected in OPCs in any of the experimental condi-tions, we added a positive control (labeled ‘‘neuron’’) that was a lysateobtained from striatal neurons, a cerebral structure where the basallevel of JNK phosphorylation is high and detectable by western blot(Vanhoutte et al., 1999).

1396 Bernard et al.


present (Fig. 6A). After 3 days of incubation in the pres-ence of 1 lM RA, OPC pHi was significantly higher(7.14 6 0.01; n ¼ 166) than in its absence (6.88 6 0.01;n ¼ 80), P < 0.01.

Two distinct alkalinizing mechanisms have beendescribed in OPCs: an electroneutral, CO2 independent,Naþ/Hþ exchange and an electrogenic Naþ-HCO3

� co-transport, the latter being active only in the presence ofCO2/bicarbonate (Boussouf and Gaillard, 2000). A seriesof experiments was carried out in bicarbonate-free solu-tion (HEPES buffer) to determine whether HCO�

3 con-tributes to the RA-induced intracellular alkalinization. Inthese experimental conditions, retinoic acid still elicitedan increase in pHi (Fig. 6B). This intracellular alkaliniza-tion was abolished completely when all external sodiumwas removed or by adding 1.5 mM amiloride, a blockerof Naþ/Hþ exchanger (not illustrated), suggesting thatretinoic acid-induced elevation of OPC pHi is due toincreased Naþ/Hþ exchanger activity. This was furthershown by comparing the acid efflux rates mediated byNaþ/Hþ exchanger in control and RA-treated cells, inbicarbonate/CO2-free solutions. For these experiments,intracellular pH recoveries were analyzed after acute acidloads induced by ammonium prepulse technique (Boronand De Weer, 1976). Figure 6B shows a record of a rep-resentative experiment on a single OPC. It is apparentthat the cell recovered from the acid load at a muchgreater rate in the presence of retinoic acid compared tothe control. Hþ efflux rates were plotted against the cor-responding pHi values (Fig. 6C) showing the well knowndependency of Naþ/Hþ exchange activity on intracellu-lar pH. The initial proton efflux rate was approximatelythree times greater in the presence of RA. Figure 6C alsoshows that in the presence of retinoic acid, the relation-ship between proton efflux rate, and pHi is shifted towardmore alkaline values. The intercept of the linear regres-sion with the abscissa is at pHi 6.76 for unstimulatedOPC and at 7.17 after treatment with RA. These datashow that retinoic acid activates Naþ/Hþ exchanger byincreasing its intracellular pH sensitivity (Grinstein andRothstein, 1986).

pHi Dependency of RA-Induced OPCDifferentiation

We investigated whether the RA-induced intracellu-lar alkalinization is a downstream effector of OPC differ-entiation. The effect of RA on OPC differentiation wastherefore tested on cells with intracellular pH experimen-tally shifted to more acidic or alkaline values compared tocontrol cells. As shown Figure 7A, incubation of OPCswith 1 lM RA in a culture medium buffered at pH 7.4(control) triggers a significant increase in pHi (6.88 to7.14). When the same experiment is done with a pHe buf-fered at 7.00, RA treatment still elicits a significant increasein pHi (from 6.62 6 0.09 [n ¼ 166] to 6.84 6 0.08 [n ¼73]; see Fig. 7A). Culturing OPCs in a medium bufferedat pHe of 7.75 mimics the intracellular alkalinizationobserved in the presence of RA at pHe 7.40 (pHi 7.14 6

Fig. 6. Intracellular alkalinization induced by retinoic acid. A: Originalrecording carried out on a single OPC perfused with CO2/bicarbonatesaline, representative of >50 similar experiments. Addition of retinoicacid (1 lM) at the time indicated by arrow induced pHi to increase to anew steady state level. In all experiments carried out, the alkalinizationwas observed after a lag time ranging from 2–10 min. B: Original re-cording carried out on a single OPC in the absence of CO2/bicarbon-ate, ammonium chloride prepulse was used to induce intracellular acidi-fication. The following recovery was observed before and after additionof RA (arrow) on the same cell. Recordings were carried out after 1 or2 days in CDM on OPCs similar to those illustrated in Figure 1A, char-acterized by a fractal dimension lower than 1.233 (Bernard et al., 2001).C: Analysis of the Hþ efflux rates during the pHi recoveries before(open circles) and after (filled circles) RA addition illustrated in (B).The relationship between Hþ efflux rate and pHi is shifted toward amore alkaline value. This recording and the corresponding analysis arerepresentative of six similar experiments.



0.03; see Fig. 7A). Moreover, in this condition, additionof RA induced a significant increase in pHi from 7.14 60.03 to 7.236 0.02, P < 0.01 (Fig. 7A). As shown above,the differentiation rate of OPCs is dependent on pHi values.In the set of experiments shown in Figure 7B, we showthat, whatever the presence or absence of RA, the highestrate of differentiation is observed at pHi 7.15 (Fig. 7B).Shifting pHi from this value (more acidic or more alkaline)greatly decreased differentiation. Furthermore, we showthat clamping pHi value at 7.15 triggers OPC differentia-tion as efficiently as RA treatment does in control condi-tions (pHe 7.40). As illustrated, an increase of pHi from7.14 to 7.23 on RA incubation results in a decrease of dif-ferentiation from 41.80% 6 1.70 to 32.28% 6 1.05 (n ¼

420), P < 0.01 (Fig. 7B). These data establish that a pHi of7.15 is the optimal value to trigger OPC differentiation.

DISCUSSION

This study shows for the first time that an intracellu-lar pH change is able by itself to trigger the differentiationof OPCs in vitro. This is illustrated in Figure 3, whichshows clearly the existence of an optimal pHi value, setaround 7.15, for OPC differentiation. A similar biphasicrelationship, but shifted toward more acidic values hasbeen reported to describe the pHi dependence of astro-cyte proliferation via the modulation of Kþ channels ac-tivity (Pappas et al., 1994). These authors showed that thehighest level of astrocyte proliferation was observed atpHi around 6.70, a value for which we observed the lowestrate of OPC differentiation. Conversely, astrocyte prolifer-ation was sharply reduced at a pHi value of 7.15 for whichwe observed the highest rate of OPC differentiation. Boththe study of Pappas et al. (1994) and the present studystrongly suggest the existence of at least two distinct pHi-sensitive processes involved in the development of theseglial cells. At least one that promotes cellular proliferationaround pHi 6.70, coincident with reduced differentiationlevel, and at least one that promotes cellular differentia-tion around pHi 7.15, coincident with reduced prolifera-tion level.

Although stimulation of differentiation and increasein intracellular pH have been correlated in some othercells (Ladoux et al., 1987; Rao et al., 1992; Dyck andFliegel, 1995; Wang et al., 1997), this is the first evidencethat intracellular pH changes are sufficient by themselvesto control the level of cellular differentiation in vitro. Wehave used two distinct methods to induce experimentalchanges in intracellular pH: first, by adjusting external pHto different values and second, by adding weak acid orbase at constant external pH. The similarity of the resultsobtained with both strategies strengthens the conclusionthat the level of OPC differentiation is controlled byintracellular pH and not by external pH.

To cast some light on the intracellular mechanismsmediating the effect of pHi on OPC differentiation, weinvestigated the possible involvement of signaling kinases.We found that intracellular pH changes, which are suffi-cient to modify the level of OPC differentiation, also tran-siently modulate the ERK 1/2 activity. MAP kinase activa-tion has been already observed after either intracellularacidification (Souza et al., 2002; Sarosi et al., 2005; Zhenget al., 2005) or alkalinization (Shrode et al., 1997a; Mukhinet al., 2004). These observations suggest that cells could re-spond to any cytosolic pH shift by activating a commonsignalling pathway. This could explain the slight stimulationof ERK 1/2 also observed at pH 6.70 in the present study(Fig. 4A). The pattern of ERK1/2 phosphorylation as afunction of OPC pHi, illustrated in Figure 4A, parallels themodulation of OPC differentiation (Fig. 3). The maxi-mum level of ERK phosphorylation was found for pHi

7.15 for which we also observed the maximum level ofOPC differentiation. It is well known that both the

Fig. 7. Quantitation of RA-induced pHi shifts and differentiation. A:Whatever the resting pHi of OPC, addition of 1 lM RA to the cul-ture medium induced an intracellular alkalinization. pHi was measuredat least on 73 cells. B: Illustrates the effect of RA-induced pHi changeson OPC differentiation. Intracellular pH and quantitation of Gal-Cpositive cells were carried out after 3 days in CDM supplemented(filled circles) or not (open circles) with 1 lM RA. A pHi dependenceof differentiation could be observed and these results match well withthe bell-shaped relationship illustrated in Figure 3.

1398 Bernard et al.


strength and duration of ERK signalling can regulate vari-ous cell fate decisions (Werlen et al., 2003; Murphy et al.,2004) and a fundamental difference in threshold levels ofERK activation after NGF vs. EGF has been describedrecently in PC12 cells (Ho et al., 2005). Our results show-ing that ERK was actually much more activated at the pHcorresponding to the highest level of differentiation com-pared to more acidic or alkaline values represent anotherexample of the implication of ERK activation in this sig-nalling pathway. Moreover, we showed the inhibition ofpH-dependent OPC differentiation when ERK was blocked(Fig. 5). This indicates that ERK1/2 activation mediatesthe triggering of pHi-driven OPC differentiation. We ob-served that whatever the pHi values, around 20% of theOPCs were still differentiated when ERK1/2 was blocked(Fig. 5). These data showed that even if pHi is the majorsignal controlling the differentiation, this process could bealso mediated, at least in control conditions, in a ERK1/2and pHi -independent manner.

The absence of a dependence of JNK activation onintracellular pH indicates some specificity in the pHi targetsand strengthens the idea that the control of OPC differen-tiation by pH is specifically mediated by ERK. This obser-vation is consistent with the demonstration that intracellularalkalinization per se from 6.60 to 7.10 (in the absence ofNaþ/Hþ exchanger NHE1 activity) caused a significant2-fold increase in ERK1/2 phosphorylation in vascularsmooth muscle cells, although the increase in ERK phos-phorylation was much more pronounced when NHE1 ac-tivity was restored (Mukhin et al., 2004). In addition, It hasbeen reported previously that retinoic acid and 1, 25-dihy-droxyvitamin D3 that induce intracellular alkalinization anddifferentiation of HL 60 cells (Hazav et al., 1989; Ladouxet al., 1987) also activate the ERK1/2 signaling pathway inthese cells (Yen et al., 1998; Marcinkowska, 2001).

Interestingly, ERK1/2 (and mostly ERK2) have alsobeen shown to be activated in OPCs after PDGF or bFGFactivation that stimulate OPC proliferation (Bhat andZhang, 1996). This involvement of ERK1/2 in the prolif-eration of OPCs has been confirmed by other authors. In amore recent study, however, it was shown that inhibitionof ERK1/2 blocked the OPC differentiation when OPCsare not treated with growth factors (Baron et al., 2000).These authors concluded that ERK1/2 activity is essentialfor the progression of late progenitors (that we have in ourcultures) to the Gal-Cþ oligodendrocytes stage of the line-age. This conclusion is consistent with our results.

Both proliferation and differentiation are regulatedby ERK1/2 activation. This duality is explained by differ-ences in the kinetic of MAPK activation (Marshall, 1995).It is thus tempting to speculate that a transient activation ofERK, as those observed in the present study may specifi-cally triggers OPC differentiation rather than proliferation.

Stimulation of cellular differentiation by retinoicacid has been reported for many cell types of the nervoussystem (McCaffery et al., 2003). The near doubling ofdifferentiated rat oligodendrocytes in the presence of1 lM RA, reported in the present study, is very similar tothe results already published for mouse optic nerve OPCs

(Billon et al., 2001). In both studies differentiated oligo-dendrocytes were quantified by staining the cells for gal-actocerebroside that is a specific marker of such cells (Raffet al., 1978; Ranscht et al., 1982). Our results also showthat an increase in the Naþ/Hþ exchanger activity is re-sponsible for the intracellular alkalinization induced byretinoic acid. The presence of RA produces an alkalineshift in the pHi dependence of proton extrusion rate inOPCs. This observation is consistent with the effects ofRA already described in HL 60 cells (Ladoux et al., 1987)and in P19 embryonic carcinoma cells (Dyck and Fliegel,1995). Here, we found that a three-fold increase in themaximal proton efflux rate was induced by RA (Fig. 6C).This value is identical to the values also reported in thetwo studies aforementioned.

Increased Naþ/Hþ exchange activity, leading to sub-sequent intracellular alkalinization has been suggested to bethe intracellular signal necessary for RA-induced differen-tiation (Ladoux et al., 1987; Rao et al., 1992; Wang et al.,1997). Our results show the existence of an optimal pHi

value (around 7.15) for OPC differentiation rather than theobligation of an intracellular alkalinization. Indeed, whenOPCs were cultivated in experimental conditions leadingto an alkaline shift in their resting pHi (7.15), addition ofretinoic acid still elicited an intracellular alkalinization (from7.15 to 7.23) that resulted not in an increase but in a reduc-tion of the level of OPC differentiation (Fig. 7B).

The intracellular mechanisms controlling the exit ofOPCs from the cell cycle and the initiation of their differ-entiation are not elucidated and most likely, multiple intra-cellular signaling molecules are implicated in these pro-cesses. The cellular mechanisms of OPC differentiationdescribed in the present study are in agreement with thepH-dependence of cellular development described in otherstudies. It has been shown that the cell cycle progression offibroblasts was modulated by slight changes in intracellularpH, with a more efficient timing of G2/M entry and tran-sition requiring a pHi higher than 7.30 (Putney and Barber,2003). Putney and Barber (2003) also showed that this pHcontrol involves modulations of the cyclin B1 expressionand of the Cdc2 and Wee1 kinase activities. A p53-de-pendent accumulation of the CdKi p27/Kip1 has beenshown to be an essential part of the effector component thatcontrols OPC differentiation by retinoic acid (Tokumotoet al., 2001). Interestingly, a strong modulation of p53-de-pendent induction of p27/Kip1 by pH has been describedin human glioblastoma cells (Ohtsubo et al., 1997). Thussimilar pH-dependent modulation of the CdK/CdKi path-ways, acting in parallel or downstream of ERK1/2 activa-tion, could also be potentially responsible for the pH-de-pendence of OPC differentiation. Gallo et al. (1996) haveshown the fundamental role of functional Kþ channels inthis pathway. They have shown that OPC proliferationand lineage progression were inhibited by glutamate recep-tor-mediated Kþ channels block (Gallo et al., 1996). Theyfurther showed that the intracellular pathway, downstreamof this Kþ channels block, involved the accumulation ofCdkI p27/Kip1 and p21/Cip1, and G1 cell cycle arrestin OPCs (Ghiani et al., 1999a,b). The pH-sensitivity of



various Kþ channels is well documented, particularly forthe Kv1 (Padanilam et al., 2002) and the Kir4.1 subunits(Xu et al., 2000; Pessia et al., 2001) that are both crucialfor oligodendrocyte development (Neusch et al., 2001;Chittajallu et al., 2002). It is thus conceivable that definedchanges in intracellular pH could also affect cell cycle pro-gression and OPC differentiation through the modulationof some of these potassium channels, as shown for themodulation of astrocyte proliferation (Pappas et al., 1994).Further investigations will aim to clarify these pHi-drivenmechanisms in oligodendrocytes.

Given the key role played by MAP kinases in regu-lating the cell cycle, we analyzed the activation profile ofJNK and p38 (data not shown) at the various pHi andcould not detect any pH-dependent activation of theseMAP kinases. However, we cannot rule out a role forthese two subtypes of MAP kinases or any other signallingcascade at any time in the process of oligodendrocyte dif-ferentiation independently of the intracellular pH. Thisaspect is important to fully understand mechanisms in-volved in OPC differentiation but the primary aim of ourstudy was to define the intracellular events responsible forstimulation of OPC differentiation induced by pHi increaseobserved at the onset of differentiation.

Demyelinating lesions of the CNS in diseases such asmultiple sclerosis often fail to repair. This failure of remye-lination seems due mainly to the failure of OPCs, still pres-ent in the adult brain, to differentiate with high myelo-genic potency (Gensert and Goldman, 1997; Wolswijk,2002). Therapeutic strategies designed to stimulate remye-lination should take into account the fine pHi dependenceof OPC differentiation described in the present study.

ACKNOWLEDGMENTS

We wish to thank Dr. J. Caboche for providingP-ERK and P-JNK antibodies and Dr. J.M. Schwab forcritically reading the manuscript.

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