HSP25 Is Involved in Two Steps of the Differentiation ofPAM212 Keratinocytes*

Received for publication, September 5, 2003, and in revised form, December 5, 2003Published, JBC Papers in Press, December 8, 2003, DOI 10.1074/jbc.M309906200

Olivier Duverger‡§, Liliana Paslaru¶, and Michel Morange‡�

From the ‡Departement de Biologie, Unite de Genetique Moleculaire, Ecole Normale Superieure, 46 rue d’Ulm,Paris 75230, France, and the ¶University of Medecine and Pharmacy Carol Davila, Postgraduate Department ofBiochemistry, Fundeni Hospital, 258 Soseaua Fundeni Street, Bucharest 2, Romania

HSP25 is a member of the small heat shock proteinfamily. This 25-kDa protein exhibits a highly specificdistribution during mouse embryonic development. Al-though multiple functions have been proposed forHSP25, the role it plays during differentiation is stillunknown. High levels of HSP25 can be detected in em-bryonic and adult skin. During epidermis differentia-tion, the concentration of HSP25 increases with the dis-tance of keratinocytes from the basal layer, in parallelwith the extent of keratinization. We used an ex vivocellular system, PAM212 cells, to analyze quantitativelyand qualitatively the dynamics of HSP25 productionand phosphorylation during the differentiation of kera-tinocytes. Our observations suggest that HSP25 is in-volved in two steps of PAM212 keratinocyte differentia-tion. Shortly after the induction of differentiation, atransient hyperphosphorylation of HSP25 seems to beessential for the expression of differentiation markers.Later, the chaperone-active form of HSP25 is organizedprogressively into characteristic aggregates involved inthe dynamics of keratin filament networks.

HSP25 (called HSP27 in human cells) belongs to the smallheat shock protein (HSP)1 family. All members of this familyare small (20–30 kDa) and contain a highly conserved carbox-yl-terminal region, called the �-crystallin domain (1). HSP25was the first of the 10 mammalian members of this family to bedescribed. Its synthesis is strongly up-regulated in response toheat and chemical and oxidative stresses. Two residues fromHSP25 can be phosphorylated via the p38 MAP kinase path-way: Ser-15 and Ser-86. p38 phosphorylates MAPKAP kinase2/3 which in turn phosphorylates HSP25 (2). �A- and �B-crystallin were initially described as eye lens structural pro-teins. However, �B-crystallin has a function and sequence sim-ilar to those of HSP25. It can be induced by heat and chemicaland oxidative stresses. These proteins are well known chaper-ones (3, 4), although no substrate specificity has been describedso far.

During embryonic development, HSP25 appears to bestrongly produced in specific organs or tissues such as heart,skeletal muscles, smooth muscles, epidermis, eye lens, carti-lage, and bone (5–9). Immunohistochemical analyses have de-tected HSP25 in some areas of the developing central nervoussystem, even though global Western and Northern blottingfailed to detect this protein (10, 11). This specific pattern sug-gests that this small heat shock protein is involved in celldifferentiation, which is further supported by the results ob-tained by a few cellular approaches.

The ex vivo differentiation of HL-60 promyelocytic leukemiccells into macrophages or granulocytes is accompanied by thetransient phosphorylation of HSP27 and by an increase in theproduction of the protein and a decrease in proliferation (12,13). The use of antisense oligonucleotides to reduce the produc-tion of HSP27 in HL-60 cells leads to a less pronounced reduc-tion in cell growth after induction with retinoic acid and alterssome parameters of granulocytic differentiation (14). However,HSP25 phosphorylation is not essential for HL-60 cell differ-entiation (15). During the pluripotent differentiation of embry-onic stem cells, HSP27 is dephosphorylated rapidly after induc-tion, which precedes the transient accumulation andoligomerization of the protein. In these cells, the overexpres-sion of HSP27 reduces the rate of cell proliferation, whereas thedown-regulation of the protein by stable antisense RNA com-promises the differentiation program, causing massive cell apo-ptosis (16). Thus, HSP27 may act as a switch between differ-entiation and apoptosis. The ex vivo differentiation of ratolfactory progenitors is accompanied by a transient increase inHSP27 production. A down-regulation of HSP27 leads to thefailure of differentiation and a massive commitment to celldeath, whereas the overexpression of this protein strongly de-creases the proportion of dying cells. Thus, HSP27 is probablya key element in the control of cell death during neuronaldifferentiation (17). The production of HSP25 and the activa-tion of p38 MAP kinase are essential for the differentiation ofP19 cells into cardiomyocytes. However, the effects of p38 MAPkinase activation seem to be independent of HSP25 accumula-tion (18). The overexpression of HSP25 in C1 cells, which areable to differentiate into chondrocytes, showed that elevatedHSP25 levels decrease the cellular adhesion of C1 cells andinterfere with their differentiation into chondrocytes (19).

In vitro studies and cell system analysis suggested severalroles for HSP25. HSP25 can interfere with actin polymeriza-tion and the stabilization of microfilaments (20–22). HSP25phosphorylation (through the p38 kinase pathway) is necessaryfor the regulation of stress fibers and actin filament dynamics(23–28). It also interacts with intermediate filaments. Moststudies of the interaction between small HSPs and intermedi-ate filaments concerned �B-crystallin in pathological (29–32)or physiological (33) conditions. However, HSP27 is also in-

* This work was supported in part by Grant ARC 5939 from theAssociation pour la Recherche contre le Cancer. The costs of publicationof this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked “advertisement” in ac-cordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§ Supported by a grant from the Association pour la Recherche contrele Cancer.

� To whom correspondence should be addressed. Tel.: 33-1-4432-3946Fax: 33-1-4432-3941; E-mail: morange@wotan.ens.fr.

1 The abbreviations used are: HSP, heat shock protein; BSA, bovineserum albumin; ERK, extracellular signal-regulated kinase; MAP ki-nase, mitogen-activated protein kinase; MAPKAP, MAP kinase-acti-vated protein; MEK, MAP/ERK kinase; MEKK, MEK kinase; MK,mouse keratin; PBS, phosphate-buffered saline.

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 279, No. 11, Issue of March 12, pp. 10252–10260, 2004© 2004 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A.

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volved in such interactions (34, 35). HSP25 can directly inhibitapoptosis (36–38) and interfere with translation (39).

In this study, we concentrated on the role of HSP25 in thedifferentiation of mouse epidermis. In the adult mouse, theepidermis consists of several cell layers. The degree of differ-entiation increases from the basement membrane to the out-side of the body: basal layer, spinous layer, granular layer, andthe stratum corneum. The cells in the basal layer expressmainly mouse keratins 5 (MK5) and 14 (MK14), whereas thosein the suprabasal layers (from the spinous layer) expressmainly keratins 1 (MK1) and 10 (MK10). HSP27 can be de-tected in the human epidermis from week 14 of gestation.HSP27 staining increases with the distance of the keratino-cytes from the basal layer, in parallel with the extent of kera-tinization (40). In normal human skin, HSP27 colocalizes withkeratins and other markers of the cornified layer, suggestingthat it plays a role in the assembly of keratin filaments (41).

We used PAM212 cells as a model for keratinocyte differen-tiation. This malignantly transformed mouse cell line can pro-liferate in a medium with a low calcium concentration, andterminal differentiation of these keratinocytes can be inducedby increasing the calcium concentration of the culture medium(42). The overall pattern of keratin synthesis in these cells issimilar to that in the newborn epidermis, whereas primarycultures of newborn mouse epidermis fail to produce ex vivo thesame keratins (43). Therefore, the PAM212 cell line is anappropriate model for studying the role of HSP25 in the differ-entiation of the epidermis.

We show that HSP25 is involved in two stages of PAM212keratinocyte differentiation. First, a transient accumulation ofthe phosphorylated form of HSP25 seems to be essential for theinduction of differentiation. Second, the nonphosphorylatedchaperone-active form of HSP25 contributes to the reorganiza-tion of the keratin network by driving the disassembly of theMK5/MK14 network.

MATERIALS AND METHODS

Cell Culture and Differentiation—PAM212 cells were cultured intissue culture dishes (Falcon) at 37 °C in 7.5% CO2 water-saturatedatmosphere in calcium-free Dulbecco’s modified Eagle’s medium (In-vitrogen) supplemented with 10% heat-inactivated fetal calf serum, 1mM pyruvate, 2 mM glutamine, 50 �g/ml streptomycin, 50 units/mlpenicillin, and 0.05 mM CaCl2 (low calcium medium). To carry outterminal differentiation, PAM212 cells were grown until confluence andthen incubated in the same medium containing 0.5 mM CaCl2 (highcalcium medium). High calcium medium was replaced every day. Forp38 inhibition assays, 10 �M SB203580 (Calbiochem) was added to theculture medium.

Gel Electrophoresis and Immunoblotting—Protein extracts were pre-pared by washing cells in 4 °C PBS, scraping into Laemmli samplebuffer with 5% �-mercaptoethanol, and then heating for 10 min at 95 °C(44). Protein concentration was determined using the Bio-Rad proteinassay kit, and equal amounts of protein were loaded on an SDS-poly-acrylamide gel (15%). Prestained low range SDS-PAGE standards fromBio-Rad were included in each gel. Proteins were transferred ontoHybond-ECL nitrocellulose membranes (Amersham Biosciences). Im-munoblotting was performed using the ECL kit (Amersham Bio-sciences). The primary antibodies were MK1 (Babco), MK10 (Babco),and HSP25 (Stressgen). The secondary antibody was horseradish per-oxidase-conjugated anti-rabbit IgG (Promega).

Two-dimensional Gel Electrophoresis—Aliquots of protein extracts,containing equivalent amounts of HSP25, saturated with urea, andcompleted with 2% Nonidet P-40, were separated by isoelectric focusingin 4% acrylamide gels containing 8 M urea, 2% Nonidet P-40, 0.02%Ampholine 3–10, and 0.02% Ampholine 4–6, in glass tubes. The uppertank was filled with 20 mM NaOH and the lower tank with 10 mM

phosphoric acid. Samples were subjected to electrophoresis at 800 Vovernight. First dimension gels were then removed from the tubes,soaked in Laemmli buffer, laid on the top of a 15% SDS-PAGE, andsubjected to second dimension electrophoresis in classical conditions forsize separation. Proteins were electrotransferred onto Hybond-ECL

nitrocellulose membranes. HSP25 was detected by immunoblotting asdescribed above.

Immunocytochemistry—Cells were grown and allowed to differenti-ate on gelatin-coated sterile glass coverslips. Cells at each stage werewashed with PBS at 4 °C and fixed in 4% paraformaldehyde for 15 minat room temperature. Coverslips were washed in PBS and stored at 4 °Cin PBS. The cells were subjected to immunocytochemistry in a humidchamber. To permeabilize the cells, coverslips were incubated in 0.1%Triton X-100 in PBS for 5 min. After blocking in 3% BSA in PBS for 1 h,cells were incubated with primary antibodies in 3% BSA and PBS for1 h. The coverslips were washed three times in PBS and once in 3% BSAand PBS (5 min), then incubated with secondary antibodies, Hoechst33342 (Sigma) and fluorescein isothiocyanate-conjugated phalloidinwhen necessary (Sigma) for 30 min in the dark. After four washes inPBS, coverslips were mounted on slides with Mowiol, dried overnight,and examined on a Leica DMRB microscope or a Leica TCS/SP2 confo-cal microscope. The primary antibodies were HSP25 (Stressgen), MK1(Babco), MK5 (gift from Dr. Oulad, IGBMC, Strasbourg, France), andMK10 (DAKO). The secondary antibodies were Cy®3-conjugated goatanti-rabbit IgG (Jackson,) and Alexa Fluor® 488-conjugated goat anti-mouse IgG (Molecular Probes).

Immunohistochemistry—For immunohistochemical analyses em-bryos were fixed in 4% paraformaldehyde in PBS overnight at 4 °C.They were embedded in Paraplast® Plus (Sherwood Medical). 10-�msections cut on a Leica microtome were laid on treated slides (Super-Frost Plus) in water, dried at 37 °C, and stored at 4 °C. Before immu-nohistochemistry, slides were cleared of Paraplast in two Histo-Clear®(National Diagnostics) baths of 15 min, then gradually rehydratedthrough an ethanol series and soaked in PBS for 5 min. Immunohisto-chemistry was performed in a humid chamber. Free aldehydes weresaturated with 50 mM NH4Cl in PBS for 30 min. Sections were sub-merged for 30 min in a blocking solution (3% BSA in PBS), containing0.5% Triton X-100 for permeabilization. Slides were washed three timesfor 10 min in 3% BSA, 0.1% Triton X-100 in PBS (dilution buffer), thenincubated with primary antibodies in dilution buffer for 1 h. Afterwashing three times in dilution buffer, slides were incubated withsecondary antibodies in dilution buffer for 30 min. Sections werewashed three times in PBS and mounted in Mowiol. Slides were exam-ined on a Leica TCS/SP2 confocal microscope. Each picture was theresult of an average performed from a series of 12 scans separated by0.2 �m. The same antibodies were used as for immunocytochemistry.

Electron Microscopy—PAM212 cells grown on Petri dishes were fixedwith 2.5% glutaraldehyde and 1% tannic acid in 0.1 M cacodylate bufferfor 1 h at pH 7.4, washed in the same buffer, postfixed in 1% OsO4 for45 min, and dehydrated with increasing concentrations of ethanol in-cluding a 30-min treatment in 0.4% uranyl acetate in 70% ethanol.Then, a monolayer of EPONTM was deposited on the cells and incubatedfor 1 h at 37 °C and then at 60 °C for 2 days. Thin strip of cellsembedded in EPON were cut and embedded in cross-section. Thinsections were cut and stained with uranyl acetate and lead citrate andthen examined in a Philips Tecnai 12 electron microscope.

RESULTS

The Morphology of PAM212 Cells Changes during TerminalDifferentiation—PAM212 keratinocytes were cultured in lowcalcium medium until confluence. On day 0, terminal differen-tiation was induced by increasing the concentration of calciumin the medium. The morphology of the cells was followed bydirect observation in the culture dish. Half-confluent cells,observed 2 days before induction (day �2), were stretched andoften exhibited cytoplasmic extensions (Fig. 1A, a). At day 0,PAM212 cells were apposed, but spaces were visible betweenthe cells (Fig. 1A, b). On day 1, the cells were already organizedinto two layers, but spaces could still be seen between cells (Fig.1A, c). From day 2 onward, the cells in the upper layer formeda flat surface of tightly sealed cells, making it difficult tolocalize cell boundaries. Only nuclei were clearly visible atthese stages (Fig. 1A, d and e). The stratification of PAM212cells could easily be visualized after staining with Hoechst.Indeed, at day 0, nuclei were well separated from each other,showing that there is only one layer of cells at this stage (Fig.1B, a). On day 1, the nuclei density had significantly increased,and some nuclei overlapped each other, demonstrating thatcells had begun to form a second layer (Fig. 1B, b). The density

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of the nuclei continued to increase with time such that threeand sometimes four nuclei overlapped, suggesting the presenceof at least three cell layers (Fig. 1B, c). Electron microscopyanalysis of transverse cell sections on day 4 was consistent withthese observations. On day 4, three or four cell layers werevisible. The nuclear region appeared to be rather thick, but thecells exhibit very thin cytoplasmic extensions that make themcreep between neighboring cells (Fig. 1C).

The Production of HSP25 Increases during the Differentia-tion of PAM212 Cells—We used Western blotting to analyze theexpression of specific differentiation markers to confirm thefinal differentiation of the keratinocytes (Fig. 2). MK1 andMK10 are the two main keratins produced by the suprabasallayers of the epidermis in vivo. In differentiating PAM212 cells,MK1 could be detected as early as day 1 and then increased2-fold during the following steps. However, MK10 could not bedetected before day 2 and increased until day 4.

The level of HSP25 changed considerably during the differ-entiation process (Fig. 2). Western blot analysis detected a lowlevel of HSP25 in proliferating cells cultured in a low calciummedium (days �2 and 0). During differentiation in high cal-cium medium, the level of HSP25 increased (�6-fold) until day3. The amount of HSP25 decreased slightly at day 4.

These results confirm that PAM212 cells are a good model forepidermal differentiation and that this system is appropriatefor studying the role of HSP25 in the differentiation ofkeratinocytes.

HSP25 Aggregates Progressively during the Differentiation ofthe Keratinocytes—We used immunocytochemistry to analyzethe distribution of HSP25 in PAM212 cells before and duringterminal differentiation. Actin filaments were stained in thesame experiments to (i) distinguish all the cell layers regard-less of whether they contain HSP25; (ii) follow changes in cellmorphology; and (iii) look for possible interactions betweenHSP25 and actin filaments.

We found that HSP25 aggregates progressively during thedifferentiation of PAM212 cells (Fig. 3). At days �2 and 0,before induction, the protein was distributed homogeneously inthe cytoplasm (Fig. 3, a and d), and the cells (one layer) dis-

played a typical actin network (Fig. 3, b and e). The patterns ofHSP25 and actin did not overlap at these stages (Fig. 3, c andf). From day 1 onward, the cells became stratified, and those inthe bottom layer exhibited a bundle of stretched actin filamentscrossing the cell in a preferential direction (Fig. 3t). HSP25 wasno longer expressed in these cells (Fig. 3s). At day 1, thedistribution of actin filaments was very characteristic in thecells in the newly formed upper layer. Indeed, actin formedgranular structures, concentrated mainly at the cytoplasm pe-riphery (Fig. 3, h and h�). In these cells, HSP25 had alreadystarted to form thin, elongated, tortuous aggregates. Thesestructures were located mainly at the cell periphery andaround the nuclei (Fig. 3, g and g�). The HSP25 and actinsignals seemed to overlap partially at the plasma membrane(Fig. 3, i and i�). At day 2, the actin filaments in upper layercells were mainly flattened along the cortical cytoplasm, thusrevealing the borders between the cells, and granular struc-tures were still found throughout the cytoplasm (Fig. 3k).These cells were polygonal and tightly adhered to each other,giving the whole surface a paved appearance. This pattern ofactin filaments did not change during the following steps ofdifferentiation (Fig. 3, n, q, and q�). At day 2, the aggregatedHSP25 began to concentrate in some parts of the cell, but otherparts tended to be free of HSP25 (Fig. 3j). Interestingly, highamounts of HSP25 were detected around the nuclei of thesecells. At day 3, HSP25 formed large (ø 1–2 �m), very compactaggregates distributed in the whole cytoplasm and in the nuclei(Fig. 3m). At day 4, the aggregates were the same shape, but ina large proportion of the cells they were located mainly at theperiphery, just below the cytoplasmic membrane, and in thenucleus (Fig. 3, p and p�). Interestingly, these aggregates werering-shaped. Indeed, staining revealed spherical structures inperipheral regions. These structures sometimes formed dou-blets (Fig. 3r�) but often formed single rings (see Fig. 4, p–r).This characteristic pattern suggests that HSP25 is located atthe periphery of areas that do not contain HSP25 and that mayor may not contain other elements. These aggregates and actindid not overlap in any part of the cell (Fig. 3, l, o, r, and r�). Atday 4, HSP25 was again distributed diffusely in the cytoplasmof some cells (see Fig. 4p).

Thus, HSP25 forms progressively large aggregates that tendto concentrate at the edges of the cells and in the nuclei. Thishighly dynamic pattern, plus the increase in the amount ofprotein, strongly suggested that HSP25 plays a specific role inthe differentiation of PAM212 cells.

HSP25 Is Involved in the Dynamics of Cytokeratin Filamentsduring PAM212 Cell Differentiation—During the terminal dif-ferentiation of keratinocyte, the keratin MK5 and MK14 net-work, visible in basal cells in vivo, is replaced by a keratin MK1and MK10 network, visible in suprabasal cells in vivo. We havealready confirmed that MK1 and MK10 are actually inducedduring the differentiation of PAM212 cells. Given that HSP25interacts with intermediate filaments in other systems, weanalyzed whether HSP25 is involved in this switch. For this,

FIG. 1. Morphological differentiation of PAM212 cells. A, phase-contrast observations of PAM212 cells at day �2 (a), day 0 (b), day 1 (c),day 2 (d), and day 3 (e). B, PAM212 nuclei stained with Hoechst at day0 (a), day 1 (b), and day 4 (c). C, transversal section of PAM212 cellslayers embedded in EPON resin analyzed by transmission electronmicroscopy. Bo, bottom side of the cells stack; Up, upper side of the cellsstack.

FIG. 2. Western blot analysis of differentiation marker andHSP25. At each stage of differentiation (days �2, 0, 1, 2, 3, and 4),proteins were extracted from PAM212 cells by treatment with Laemmlibuffer. Equal amounts of proteins were used to analyze the levels ofMK1, MK10, and HSP25 throughout differentiation.

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we compared the pattern of HSP25 with that of each of thesecytokeratins.

We compared the pattern of HSP25 with that of MK5, whichis expressed in PAM212 cells before induction. At days �2 and0, MK5 formed a typical network of cytokeratin filamentsthroughout the cytoplasm (Fig. 4, b and e). As described above,HSP25 was present in the whole cytoplasm at these stages(Fig. 4, a and c). The patterns of HSP25 and MK5 did not seemto overlap at these stages (Fig. 4, c and f). From day 1 onward,the cells in the bottom layer no longer contained either HSP25or MK5. The distribution of MK5 and HSP25 overlapped in theupper layer cells from this stage, both forming similar aggre-gates (Fig. 4, g–r). The aggregates of HSP25 and MK5 are bothring-shaped (singlets or doublets) (Fig. 4, p–r). Immunocyto-chemical analysis showed that MK14 exhibits the same aggre-gation pattern as MK5 and thus HSP25 (data not shown).Thus, HSP25 interacts with the main keratins present in thebasal layer in vivo. HSP25 seems to be involved in the reorga-nization of the keratin network during the differentiation ofPAM212 cells.

MK1 and MK10 were not expressed in PAM212 cells beforeinduction but appeared during differentiation. For technicalreasons, we did not directly compare MK1 and HSP25 patterns(both antibodies raised in rabbit). Instead, we compared theexpression patterns of MK1 and MK5, the pattern of which issimilar to that of HSP25 from day 1 onward. At day 1, MK1 wasexpressed in cells expressing MK5 that had started to aggre-gate (Fig. 5A, a–c). At day 2, more cells expressed MK1, andthese cells no longer expressed MK5. The cells in the upperlayers were very flat and intertwined, which sometimes madeit difficult to distinguish the borders between the cells. Exam-

ination of a series of sections (averaged) from the upper layershowed that the cells expressing MK5 were distinct from thoseexpressing MK1 (Fig. 5A, d–f). The same was observed at day 4,when a large proportion of the cells expressed MK1 and werestill closely intertwined with cells expressing MK5, aggregatedor not (Fig. 5A, g–i). We also compared the expression patternsof MK1 and MK10. Basically, cells expressing MK10 also ex-pressed MK1, and some cells expressed MK1 without express-ing MK10 (Fig. 5B). These observations are consistent with thekinetics of differentiation markers expression revealed byWestern blot analysis (Fig. 2).

Thus, MK1 is initially induced in cells containing HSP25that has started to aggregate, but at subsequent stages cellsexpressing MK1 no longer contain HSP25.

Interactions between HSP25 and MK5 Are Likely to Occur inDeveloping Epidermis, at the Beginning of Stratification—Theinteraction described above between HSP25 and MK5 wasquite puzzling. Indeed, these two proteins are not produced inthe same cells in adult epidermis; MK5 is specific for the basallayer, whereas HSP25 is expressed mainly in the suprabasallayers. We wondered whether these two proteins could be de-tected in the same cells during epidermis development. There-fore, we analyzed by immunohistochemistry the expressionpatterns of HSP25 and MK5 in mouse epidermis (dorsal side ofthe embryo), before and after the beginning of stratification.We observed that HSP25 and MK5 are both present in themonolayer constituting the epidermis at E12.5 (Fig. 6, a–c). AtE15.5, when the epidermis is already stratified and keratin-ized, HSP25 was present in the suprabasal layers known toexpress MK1 and MK10, whereas the basal layer expressingMK5 did not contain HSP25 (Fig. 6, d–f).

FIG. 3. Patterns of HSP25 and actinfilaments during differentiation ofPAM212 cells. PAM212 cells grown onglass coverslips were fixed with 4%paraformaldehyde on day �2 (a–c), day 0(d–f), day 1 (g–i), day 2 (j–l), day 3 (m–o),and day 4 (p–r). HSP25 was detected byimmunocytochemistry (Cy3, red; a, d, g, j,m, and p), and actin microfilaments werestained with fluorescein isothiocyanate-conjugated phalloidin (green; b, e, h, k, n,and q). c, f, i, l, o, and r, overlay of thesignals. For days 1 and 4, areas delimitedby white squares were magnified threetimes (g�–i� and p�–r�, respectively). Allobservations were carried out on a confo-cal microscope (single scans). At days �2and 0 (a–f), only a single cell layer existed.From day 1 to day 4 (g–r), several celllayers existed, and we focused on the up-per layers. Cells in the bottom layer werealso observed at day 1 for HSP25 (s) andactin (t). The white arrow indicates a nu-cleus containing HSP25. The white circledelimits a nucleus at day 4.

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Thus, interactions between HSP25 and MK5, such as thoseobserved in PAM212 cells, are likely to occur between the timewhen epidermis forms a monolayer and the time when it isstratified, during embryonic development.

HSP25 Is Transiently Phosphorylated a Few Hours afterInduction, and the Inhibition of p38 MAP Kinase PreventsDifferentiation—We wondered whether the increase in theHSP25 level during the differentiation of PAM212 cells and itshighly dynamic pattern were accompanied by a change in thephosphorylation state of the protein.

The phosphorylation of HSP25 is known to be dependent onthe p38 MAP kinase pathway. p38 phosphorylates MAPKAPkinase 2/3, which subsequently phosphorylates HSP25. p38

can be specifically inhibited by the pyridyl imidazoleSB203580. We tested the effects of this inhibitor on the differ-entiation of PAM212 cells (Fig. 7A). SB203580 was added to theculture medium on day �2, 0, 2, 4 or 6. In each case, cells werecultured until day 8, and proteins were extracted every otherday (day �2, 0, 2, 4, 6, and 8). The addition of SB203580 at day�2 or 0 prevented the terminal differentiation of PAM212 cells,as revealed by the absence of MK10. When added at day 2, 4, or6, SB203580 did not prevent the expression of MK10, suggest-ing that PAM212 cells differentiate properly in these condi-tions. Given that MK1 is expressed before MK10 in normalconditions, we analyzed the expression of MK1 in cells treatedwith SB203580 from day �2. The expression of MK1 inSB203580-treated cells was strongly delayed and very slightcompared with that of MK1 in nontreated cells analyzed in thesame conditions (Fig. 7B).

These results suggest that p38 is activated early in thedifferentiation process and that this activation is essential forthe expression of MK1 and MK10. Given that MK1 was de-tected on day 1 (see Fig. 2), p38 must be involved before thisstage. Thus, we studied the kinetics of the expression of thismarker. MK1 was expressed faintly 10 h after induction andstrongly from 16 h (Fig. 7C).

Given that the expression of MK1 and MK10 is dependent onthe activation of p38 a few hours after induction, we followedthe dynamics of HSP25 phosphorylation during the hours afterinduction. HSP25 was partially phosphorylated (one phospho-rylated isoform) before induction (0 h). Between 4 and 6 h afterinduction, two phosphorylated isoforms of HSP25 appeared,the proportion of which increased dramatically between 6 and12 h after induction (Fig. 7D). At day 1, the two phosphorylatedisoforms were still detected, although in a lower proportion,whereas later, the phosphorylated isoforms could no longer bedetected in these conditions (data not shown).

Thus, p38 is essential for the differentiation of PAM212 cells,and HSP25 is transiently hyperphosphorylated a few hoursafter induction, before accumulating in its nonphosphorylatedform.

Specific Inhibition of p38 MAP Kinase Alters the AggregationPattern of HSP25—We used immunocytochemistry to analyzePAM212 cells treated with SB203580 from day �2 to seewhether HSP25 aggregates when p38 is inhibited. At day 4, inthe presence of SB203580, PAM212 cells were stratified, andthe morphology of the actin network was similar to that ob-served in normal conditions. Indeed, the cells in the bottomlayer exhibited typical transversal fibers (Fig. 8b), and, in thecells in the upper layers, actin was distributed below theplasma membrane and in granular structures within the cyto-plasm (Fig. 8, e and e�). However, the pattern of HSP25 wasstrongly altered by the inhibition of p38. Indeed, contrary towhat was observed in normal conditions at day 4, the cells inthe bottom layer still expressed HSP25 (Fig. 8a), and those inthe upper layers did not exhibit the typical aggregates de-scribed above (Fig. 8d). The pattern of HSP25 looked ratherlike that observed on day 1 (small granular aggregates), withthe exception of around the cell borders (Fig. 8d�; see Fig. 3, iand i�). The patterns of HSP25 and actin did not overlap (Fig.8, c, f, and f�). In the same conditions, the distribution of MK5was the same as that of HSP25 (Fig. 8, g–l). As expected fromthe Western blot analysis (see Fig. 7B), MK1 was expressed ina few isolated cells in the upper layers, less numerous thanthose observed at day 1 in normal conditions (Fig. 8, m and m�).Thus, the inhibition of p38 MAP kinase not only impairs dif-ferentiation, but also alters the aggregation pattern of HSP25.

FIG. 4. Patterns of HSP25 and MK5 during the differentiationof PAM212 cells. PAM212 cells were treated as in Fig. 3 at day �2(a–c), day 0 (d–f), day 1 (g–i), day 2 (j–l), day 3 (m–o), and day 4 (p–r).HSP25 (Cy3, red; a, d, g, j, m, and p) and MK5 (Alexa Fluor 488, green;b, e, h, k, n, and q) were detected by immunocytochemistry. c, f, i, l, o,and r, overlay of the signals. All observations were carried out on aconfocal microscope (single scans). At days �2 and 0 (a–f), only a singlecell layer existed. From day 1 to day 4 (g–r), several layers existed, andwe focused on the upper layers. The insets in p, q, and r show anenlargement of an aggregate (bar, 2 �m).

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DISCUSSION

HSP25 and the Dynamics of Cytokeratin Filaments—Weshowed that aggregates containing HSP25, MK5, and MK14form progressively during the differentiation of PAM212 cells.These observations clearly demonstrate that HSP25 interactsspecifically with the two keratins constituting the keratin net-work present in these cells before induction.

Several studies have already reported interactions betweensmall HSPs and intermediate filaments. The first evidence ofsuch interactions came from the study of Alexander’s diseaseinvolving the formation of characteristic cellular inclusions(Rosenthal fibers) containing glial fibrillary acidic protein as-sociated with �B-crystallin (29) plus HSP27 (34). Other dis-eases were subsequently shown to be characterized by theaggregation of �B-crystallin with intermediate filaments (30–32). Independently of disease conditions, a study performed oneye lens extracts demonstrated that �A-crystallin and �B-crys-tallin interact directly with vimentin and their soluble sub-units (33). The same study showed that �B-crystallin can in-hibit intermediate filaments assembly in vitro. HSP27 waslater shown to interact with glial fibrillary acidic protein and�B-crystallin in astrocytoma cells producing both small HSPs,and to interact with keratin 18 in epithelial cells that do not

contain �B-crystallin (35). According to these observations, thepattern of the small HSPs perfectly overlaps the intermediatefilament network. Even though several models have been pro-posed, the physiological significance of this interaction has notbeen elucidated. The discovery that a point mutation in �B-crystallin causes desmin filament aggregation in certain my-opathies (45) provided evidence of a physiological role for smallHSPs with intermediate filaments. We did not find any corre-lation between the pattern of HSP25 and the intact keratinnetwork before induction of PAM212 cells. The interaction wedescribe here involves keratins 5 and 14, dissociated from theirinitially intact network and involved in the formation of char-acteristic aggregates. These aggregates are totally differentfrom those involving small HSPs and intermediate filaments indisease contexts. This is the first time that such structurescontaining HSP25 and intermediate filaments have been de-scribed. A previous study found an interaction between keratin18 and HSP27 in epithelial cells (35), but these cells exhibiteda stable and well established keratin network. The type ofinteraction we describe here may be typical of the differentia-tion of stratified epithelia, involving a switch from a keratinnetwork to a new one.

Our observations suggest that HSP25 drives the disassemblyof the keratin network through the sequestration of keratin 5and 14 subunits. Moreover, we noticed that MK1 is expressedinitially in cells positive for HSP25/MK5/MK14 aggregates be-fore being limited to another kind of cell that does not containthese aggregates. Thus, it is possible that the keratin networkof undifferentiated cells (MK5/MK14) has to be disassembledbefore the keratin network of differentiated cells can be built.The link between the aggregation of HSP25/MK5/MK14 andthe expression of MK1 and MK10 is strengthened by the factthat SB203580 both inhibits differentiation and preventsaggregation. However, it is not clear whether the beginningof the aggregation process is a consequence of differentiationinduction or whether it drives differentiation. Interestingly,in PAM212 cells containing MK5/MK14 starting to aggregateand newly synthesized MK1, HSP25 did not interact withMK1, which is an intermediate filament of the same type asMK5 (type I). However, in epithelial cells normally express-ing keratins in interaction with HSP27, HSP27 interactswith glial fibrillary acidic protein (type III intermediate fil-ament), the expression of which was triggered by transfection(35). This observation further confirms the high specificity ofthe interaction among HSP27, MK5, and MK14 in thissystem.

FIG. 5. Patterns of MK5 and MK1during the differentiation of PAM212cells. PAM212 cells were treated as inFig. 3. A, at day 1 (a–c), day 2 (d–f), andday 4 (g–i), MK5 (Alexa Fluor 488, green;a, d, and g) and MK1 (Cy3, red; b, e, andh) were detected by immunocytochemis-try. c, f, and i, overlay of the signals. Allobservations were carried out on a confo-cal microscope, and the results are themean of a series of 12 images separatedby 0.2 �m. B, at day 4, MK1 (Cy3, red; a)and MK10 (Alexa Fluor 488, green; b)were detected by immunocytochemistry.c, overlay of the signals. The observationswere carried out on a confocal microscope(single scan).

FIG. 6. Patterns of HSP25 and MK5 in developing mouse epi-dermis, at E12.5 and E15.5. Mouse embryos were fixed with 4%paraformaldehyde at E12.5 (a–c) and E15.5 (d–f). HSP25 (Cy3, red; aand d) and MK5 (Alexa Fluor 488, green; b and e) were detected byimmunohistochemistry on paraffin sections. c and f, overlay of thesignals. Observations were carried out on a confocal microscope, andthe results are the average of a series of 12 scans separated by 0.2 �m.White arrowheads indicate the basal lamina.

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MK5 and MK14 Are Physiological Substrates of HSP25Chaperone Activity—According to the dynamics of HSP25 phos-phorylation throughout differentiation, the aggregation proc-ess involves the nonphosphorylated form of HSP25, whichshould form large oligomers known to be associated with thechaperone activity of HSP25 (46). A recent study showed thatthe level of HSP27 is increased during the keratinocyte differ-entiation of human Hacat cells and that this HSP27 accumu-lates in the form of large oligomers (47). However, the authorsconsidered the accumulation of the chaperone-active form ofHSP27 to be a marker of endogenous stress and claimed that itwas not to be related to a particular differentiation process.

Our results clearly show that during PAM212 cells differenti-ation HSP25 interacts with proteins that are specifically re-lated to the differentiation process. The chaperone-active formof HSP25 interacts with denatured proteins. Thus, the disrup-tion of the MK5/MK14 network may involve a partial unfoldingof keratin subunits that are sequestered by HSP25 to preventundesirable interactions and aggregation. MK5 and MK14 arethe first physiological substrates of the HSP25 chaperone com-plex described so far. This is the first report showing thatHSP25 chaperone activity is involved in a specific differentia-tion process, with specific substrates.

However, these considerations do not explain the fate of the

FIG. 7. Differentiation and phosphorylation. A, effects of the specific inhibition of p38 MAP kinase by SB203580 on the expression of MK10.SB203580 was added to the culture medium at different stages (days �2, 0, 2, 4, and 6), and cells were treated as in Fig. 2 for Western blot analysisof HSP25 and MK10 expression. B, Western blot analysis of MK1 expression during the differentiation of control and SB203580-treated (from day�2) PAM212 cells. C, kinetics of MK1 expression during the 24 h after induction. D, phosphorylation of HSP25 analyzed by two-dimensional gelelectrophoresis 0, 2, 4, 6, and 12 h after induction. IEF, isoelectric focusing; MW, molecular weight.

FIG. 8. Effects of p38 MAP kinaseinhibition on the patterns of HSP25,actin, MK5, and MK1 at day 4.PAM212 cells incubated with SB203580from day �2 were treated as in Fig. 3 andanalyzed at day 4. The bottom cell layer(a–c and g–i) and the upper layers (d–fand j–l) were analyzed by confocal micros-copy. HSP25 (Cy3, red; a and d) and actinmicrofilaments (fluorescein isothiocya-nate-conjugated phalloidin, green; b ande) were detected. c and f, overlay of thesignals. For the upper layer, areas delim-ited with white are magnified three times(d�–f �). HSP25 (Cy3, red; g and j) andMK5 (Alexa Fluor 488, green; h and k)were detected. i and l, overlay of the sig-nals. MK1 (Cy3, red) was detected (m),and the area delimited with a whitesquare was magnified three times (m�).

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aggregates. We thought that the formation of aggregates mightbe a first step before degradation by the proteasome, but thishypothesis is not in agreement with the oriented motility of thestructures. Moreover, the finding of cells exhibiting a typicalMK5/MK14 network among the cells in which these keratinshad aggregated suggests that sequestered keratins can still beused to build an intact network. This hypothesis is in agree-ment with the demonstration that the substrates interactingwith the chaperone-active form of HSP25 are not irreversiblysequestered but can readopt their native state (48). The natureof the ring-shaped structures and the significance of their lo-calization remain to be elucidated.

HSP25 Phosphorylation, p38 Activation, and Terminal Dif-ferentiation of PAM212 Cells—HSP25 is transiently hyper-phosphorylated a few hours after induction of PAM212 cellterminal differentiation. Indeed, the phosphorylation of HSP25increased dramatically between 6 and 12 h after induction. Thetwo phosphorylated isoforms were still detected 24 h afterinduction, but subsequently they were much rarer than thenonphosphorylated isoform.

The phosphorylation of HSP25 is dependent on the p38 MAPkinase pathway (2). The role of MAP kinases in keratinocytedifferentiation has already been addressed in other systems,generating different conclusions. For example, human foreskinterminal differentiation induced with 12-O-tetradecanoylphor-bol-13-acetate depends on a MAP kinase pathway includingprotein kinase C, Ras, MEKK1, MEK3, p38 and activator pro-tein 1 (49). In another system, the induction of terminal differ-entiation after an increase in calcium concentration was shownto be independent of protein kinase C and Ras and to involvethe activation of Raf and MEK, the transient activation of ERK,and an increase in p21 associated with cell cycle arrest. How-ever, in this system, ERK was shown not to be sufficient aloneto induce differentiation, suggesting that other pathways act inconcert with the Raf/MEK/ERK pathway (50). Here, we showthat p38 MAP kinase activity is required early in the differen-tiation of PAM212 cells.

These data show that HSP25 is transiently hyperphospho-rylated a few hours after induction and that the activation ofp38 MAP kinase is essential for terminal differentiation. Thus,the phosphorylation of HSP25 may be involved in the differen-tiation process. Of course, other targets such as transcriptionfactor (51, 52) or other protein kinases (53–55) may also beaffected by the inhibition of p38. Nevertheless, the fact that thephosphorylation of HSP25 (6–12 h) is followed rapidly by theinduction of MK1 (10–16 h) strengthens the hypothesis thatHSP25 phosphorylation is involved in this induction process,probably in concert with other pathways. HSP25 phosphoryla-tion may modulate actin polymerization (22, 56) in a way thatcould affect differentiation. This hypothesis is likely to be truebecause the regulation of actin dynamics is essential for epi-thelial cell-cell adhesion (57). Moreover, at day 1, HSP25seemed to colocalize partially with actin at the plasma mem-brane. A more detailed analysis of the dynamics of HSP25localization during the early steps of the differentiation mayprovide a clearer idea of what happens during these steps.

PAM212 Cells and Epidermis Differentiation—The expres-sion patterns of the differentiation markers that we analyzedfurther characterize PAM212 cells as a model for epidermisdifferentiation.

During terminal differentiation, PAM212 cells express MK1before MK10, which is surprising as the cytokeratin network ofdifferentiated cells consists of a MK1/MK10 heteropolymer.Moreover, all cytokeratin filaments are made of both a type Icytokeratin and a type II cytokeratin. Thus, the filaments con-taining MK1 at day 1 must contain another type II cytokeratin.

Given that PAM212 cells stratify, one could expect a parallelbetween this pattern of stratification and the organization ofthe cell layers in the epidermis: lower cells constituting thebasal layer and upper cells the suprabasal layers. In fact, eventhough PAM212 cells express the MK5/MK14 network beforeinduction, the bottom cells no longer expressed either MK5/MK14 or MK1/MK10 on day 1, suggesting that they do notcorrespond to either basal or suprabasal layers, respectively.These cells may serve as a matrix for the upper keratinocytesinvolved in the differentiation process, separating them fromthe surface of the culture dish.

What particularly puzzled us was the lack of correlationbetween the distribution of HSP25 in the different layers of theskin and the distribution of HSP25 in undifferentiated anddifferentiated PAM212 cells. On the one hand, in vivo, basalcells mainly expressing MK5/MK14 keratins do not containHSP25, whereas suprabasal cells expressing mainly MK1/MK10 keratins contain high levels of HSP25. On the otherhand, we showed that HSP25 is present in PAM212 cells con-taining MK5/MK14 and closely interacts with these keratinsand that it is present in differentiated cells (MK1/MK10) onlywhen they emerge. Thus, it is difficult to consider that theobservations made in PAM212 cells correspond to a physiolog-ical phenomenon taking place in skin at a time when it isalready composed of several layers. The analysis of the expres-sion patterns of HSP25 and MK5 in developing mouse epider-mis shows that the two proteins are present in the same cells inthe monolayered epidermis, before stratification. Thus, the in-teraction between HSP25 and MK5 observed in PAM212 cellsis likely to correspond to a phenomenon happening at thebeginning of epidermis stratification and not at a time whenthe epidermis already consists of several cell layers.

These observations suggest that what we observed withPAM212 cells is not really a model of what happens in newbornor adult epidermis, which is involved in a continuous renewalprocess. Instead, this model reflects what happens at the be-ginning of stratification during embryonic development. Theseresults also show that HSP25 has distinct roles in the supra-basal layers of the epidermis (from E15.5, in newborn and adultmice), where it acts with other partners.

HSP25 and Cellular Differentiation—Studies on HL-60 pro-myelocytic leukemic cell differentiation have shown that cellproliferation decreases as HSP27 levels increase (12, 13) andthat HSP27 is essential for the correct differentiation of HL-60cells (14). Studies on the pluripotent differentiation of embry-onic stem cells (16) and on rat olfactory neurons differentiation(17) have suggested that HSP27 acts as a switch betweendifferentiation and apoptosis. Other studies have demon-strated the importance of HSP25 in cell differentiation, withoutelucidating the function of the protein in the processes ana-lyzed. Thus, apart from possibly being involved in the balancebetween cell proliferation, apoptosis, and differentiation, thespecific role of HSP25 in the organs where the protein is pro-duced in vivo remains unknown.

Here we show that HSP25 chaperone complexes drive thedisassembly of the MK5/MK14 network during PAM212 kera-tinocytes differentiation. This is obviously specific to epithelialcell differentiation. Thus, we believe that HSP25 has differentfunctions in cell differentiation according to the system stud-ied. This protein probably interacts with different specific part-ners in each tissue producing HSP25 during embryo develop-ment. Moreover, in vivo observations clearly suggest thatHSP25 is involved in the differentiation of a subset of tissues,but not in all differentiation processes. It is therefore difficultto draw general conclusions about the role of HSP25 in celldifferentiation.

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Acknowledgments—We are grateful to Dr. Y. Barrandon (Universityof Munster, Germany) for advice on the choice of the system, Dr. S.Yuspa (NCI, National Institutes of Health, Bethesda, MD) for thePAM212 cells, Dr. M. Oulad (Institut de Genetique et de BiologieMoleculaire et Cellulaire, Strasbourg, France) for providing the mono-clonal antibody against mouse keratin 5, Dr. S. Lepanse (InstitutJacques Monod, Paris, France) for electron microscopy analysis, Dr. J.P. Laporte for help in image processing, and S. Uzan, who did a prac-tical training in the laboratory.

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Olivier Duverger, Liliana Paslaru and Michel MorangeHSP25 Is Involved in Two Steps of the Differentiation of PAM212 Keratinocytes

doi: 10.1074/jbc.M309906200 originally published online December 8, 20032004, 279:10252-10260.J. Biol. Chem. 

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