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ORIGINAL ARTICLE

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The Small Heat-Shock Protein Hsp27 Undergoes ERK-Dependent Phosphorylation and Redistribution to the Cytoskeleton in Response to Dual Leucine Zipper-Bearing Kinase Expression Hubert Robitaille1,2, Carolyne Simard-Bisson1,2, Danielle Larouche1,2, Robert M. Tanguay3, Richard Blouin4 and Lucie Germain1,2 Hsp27, a small heat-shock protein, has important roles in many cellular processes, including cytoskeleton dynamics, cell differentiation, and apoptosis. Its expression in normal epidermis correlates with differentiation; however, little is known about the regulatory mechanisms involved. In this study, we report that Hsp27 undergoes upregulation, phosphorylation, and redistribution to the cytoskeleton during the late phase of epidermal keratinocyte differentiation. Our results also show that the expression of the dual leucine zipper-bearing kinase (DLK), an upstream activator of the MAP kinase pathways, is sufficient by itself to induce Hsp27 phosphorylation, cell periphery localization, and redistribution to the insoluble protein fraction (cytoskeleton) in poorly differentiated keratinocytes. This redistribution correlates with the insolubilization of cornified envelope-associated proteins such as involucrin. Interestingly, the effects of DLK on Hsp27 were blocked by PD98059, a selective inhibitor of the extracellular signal-regulated protein kinase (ERK) pathway. Moreover, downregulation of Hsp27 by small interfering RNA in epithelial cells expressing DLK was accompanied by attenuated expression of involucrin in the cytoskeleton. Thus, these observations suggest that the DLK–ERK signaling pathway may act as a regulator of the interaction that occurs between Hsp27 and the cytoskeleton during the formation of the cornified cell envelope, a process conferring to the skin its crucial barrier function. Journal of Investigative Dermatology (2010) 130, 74–85; doi:10.1038/jid.2009.185; published online 13 August 2009

INTRODUCTION The epidermis is a self-renewing stratified epithelium populated by keratinocytes that undergo a complex program of terminal differentiation (Fuchs, 1990a). This process 1

Laboratoire de Recherche des Grands Bruˆle´s/LOEX, Centre de recherche (FRSQ) du CHA de Que´bec, Que´bec, Que´bec, Canada; 2De´partement de Chirurgie, Universite´ Laval, Que´bec, Que´bec, Canada; 3Laboratoire de Ge´ne´tique Cellulaire et De´veloppementale, Faculte´ de Me´decine, Universite´ Laval, Que´bec, Que´bec, Canada and 4De´partement de Biologie, Faculte´ des Sciences, Universite´ de Sherbrooke, Sherbrooke, Que´bec, Canada Correspondence: Dr Lucie Germain, Laboratoire de Recherche des Grands Bruˆle´s/LOEX, Hoˆpital du Saint-Sacrement du CHA, 1050 Chemin Sainte-Foy, Que´bec, Que´bec, Canada G1S 4L8. E-mail: [email protected] Abbreviations: AdDLK, recombinant adenovirus expressing the green fluorescent protein together with a T7 epitope-tagged form of wild-type DLK; AdGFP, recombinant adenovirus expressing the green fluorescent protein; DLK, dual leucine zipper-bearing kinase; ERK, extracellular signal-regulated protein kinase; Hsp, heat-shock protein; JNK, c-jun N-terminal protein kinase; MAPK, mitogen-activated protein kinase; MAPKK, mitogen-activated protein kinase kinase; MK2/3, mitogen-activated protein kinase-activated kinases 2 and 3; MOI, multiplicity of infection; NHK, normal human keratinocyte; siRNA, small interfering RNA; TG1, transglutaminase 1 Received 4 February 2009; revised 8 April 2009; accepted 12 May 2009; published online 13 August 2009

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culminates in the formation of the stratum corneum, which constitutes a barrier protecting the body from penetration by environmental toxins and loss of vital fluids. In the granular layer, keratinocytes undergo irreversible modifications leading to the flattened, enucleated dead cornified cells (corneocytes) that ensure the skin barrier function (Watt, 1989; Fuchs, 1990b; Nemes and Steinert, 1999; Candi et al., 2005). Several molecular events associated with this process remain to be characterized. Heat-shock protein (Hsp) 27 (also known as Hsp25 in the mouse), a member of the small-Hsp family, is highly expressed in many cell types, at different stages of differentiation/development (Ciocca et al., 1993; Parcellier et al., 2003). In addition to its role in protein folding, Hsp27 intervenes in the modulation of differentiation as well as in apoptosis (Beere, 2001; Parcellier et al., 2003). Furthermore, it has been identified as a potent regulator of the cytoskeleton as a result of its ability to inhibit actin polymerization (Lavoie et al., 1993). This property is dependent on the phosphorylation state of Hsp27 and its structural organization (Lavoie et al., 1993; Guay et al., 1997). Hsp27 can be reversibly phosphorylated on three serine residues in humans (two & 2010 The Society for Investigative Dermatology

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for Hsp25 in mice) by the mitogen-activated protein kinase-activated kinases 2 and 3 (MK2/3), which are themselves activated by phosphorylation through either the p38 or the extracellular signal-regulated protein kinase (ERK) signaling pathway (Gandour-Edwards et al., 1994; Guay et al., 1997; Jantschitsch et al., 1998; Geum et al., 2002; Morrow and Tanguay, 2003; Duverger et al., 2004; O’Shaughnessy et al., 2007). Upstream signals, such as differentiating agents and mitogens, that activate MK2/3 also have the ability to induce Hsp27 phosphorylation. In general, it is believed that MK2/3 are the main mediators of Hsp27 phosphorylation, but other kinases such as PKC delta can also phosphorylate Hsp27 (Kato et al., 1998; Maizels et al., 1998). In both human and mouse epidermis, Hsp27 is highly expressed in keratinocytes of the granular layer (GandourEdwards et al., 1994), and its phosphorylation in the mouse keratinocyte cell line PAM 212 is modulated on calciuminduced differentiation (Duverger et al., 2004). Moreover, Hsp27 is phosphorylated on serine 82 by a signaling mechanism involving Akt and p38 kinases (Konishi et al., 1997; Rane et al., 2003; O’Shaughnessy et al., 2007). Interactions between small Hsps and intermediate filaments or other elements of the cytoskeleton occur in many cell types and several pathologies (Kato et al., 1992; Lowe et al., 1992; Vicart et al., 1998; Wisniewski and Goldman, 1998; Perng et al., 1999). Hsp27 colocalizes with keratin filaments and other proteins of the cornified envelope in differentiated keratinocytes as revealed by immunogold labeling (Jonak et al., 2002; O’Shaughnessy et al., 2007). Furthermore, in differentiated PAM212 keratinocytes, Hsp25 colocalizes with aggregated keratin 5, suggesting a role in the disorganization of the keratin 5–keratin 14 network occurring during their differentiation (Duverger et al., 2004). The dual leucine zipper-bearing kinase (DLK) is a mitogen-activated protein kinase kinase kinase expressed in a variety of tissues (Nadeau et al., 1997). In skin, its expression correlates with differentiation (Nadeau et al., 1997; Germain et al., 2000). Recent studies from our laboratory have shown that DLK has an active role in the induction of terminal differentiation of human keratinocytes (Germain et al., 2000; Robitaille et al., 2005). DLK preferentially activates the c-Jun N-terminal kinases (JNKs), a subgroup of MAPKs involved in the regulation of cellular processes such as proliferation, differentiation, and apoptosis (Xu et al., 2001; Eckert et al., 2002; Nishina et al., 2004). In the epidermis, JNK activation induces the differentiation marker cystatin A. In vivo, JNK activation occurs in the granular layer, the last transcriptionally active cell layer of the epidermis (Takahashi et al., 2001, 2002). On the other hand, the ERK1/2 MAPK pathway acts as a positive regulator of proliferation/survival in many cell lines and keratinocytes. For instance, the ERK1/2 pathway is strongly activated by mitogens and integrins, whereas it decreases the expression of cystatin A (Takahashi et al., 2001). Although ERK1/2 is involved in keratinocyte proliferation, it is also activated during differentiation (Schmidt et al., 2000). In the present study, we investigated the dynamics of Hsp27 in human keratinocyte differentiation and whether

DLK, as a result of its ability to induce cornification, may modulate Hsp27 regulation. Our results show that Hsp27 undergoes phosphorylation and redistribution to the cytoskeletal protein fraction in the highly differentiated granular keratinocytes of native and tissue-engineered skin. Moreover, we report that expression of DLK alone in poorly differentiated normal human keratinocytes (NHKs) promotes the phosphorylation, cytoskeletal association, and redistribution of Hsp27 from the cytoplasm to the cell periphery in an ERKdependent manner. These results provide strong evidence that phosphorylation of Hsp27 through the DLK–ERK signaling pathway may promote its interaction with the cytoskeleton and other components of the cornified cell envelope. RESULTS Hsp27 dynamics in normal human keratinocyte differentiation

Immunofluorescence analysis, using a specific antibody, has revealed the localization of Hsp27 in normal human skin. Hsp27 was expressed in the first layers of the stratum spinosum and its expression increased in the granular layer (Figure 1a and b). Using an in vitro model in which cultured NHKs were induced to differentiate by prolonging culture, we observed an apparent increase in Hsp27 expression at day 3 to day 6 following confluence compared with the expression level in poorly differentiated subconfluent NHKs (Figure 1d and e), but the difference was not significant. The presence of a lower band (±22 kDa) was observed only after confluence and could be due to the recognition of another protein by the antibody (such as Hsp22, which is homologous to Hsp27) or a posttranslational modification of Hsp27. Other Hsps, such as Hsp70 and Hsp60, were found to be regulated differently during keratinocyte differentiation (Laplante et al., 1998; Ghoreishi et al., 2000). Hsp60 and Hsp70 expression levels were not significantly changed under the same conditions (Figure 1d and e). We used an in vitro model of cultured NHKs induced to cornify by the elevation of intracellular calcium levels with the A23187 calcium carrier, known to induce keratinocyte terminal differentiation (Takahashi et al., 2000), to further analyze the regulation of Hsp27 during the late phase of differentiation. Hsp27, Hsp60, and Hsp70 were not significantly increased in total protein extract after the addition of A23187 to the culture medium. The absence of an increase in the Hsps in total protein extracts indicates that this is not a stress response. In contrast, the addition of A23187 resulted in the appearance of a proportion of Hsp27 in the Tritoninsoluble (cytoskeletal) fraction (Figure 1f). The absence of caspase 3 in this fraction indicated that it is not contaminated with cytosol (Figure 1f). Involucrin was also strongly increased in the cytoskeletal fraction (Figure 1f). The increase in involucrin and Hsp27 in the cytoskeletal fraction indicates that this is a differentiation-associated response. Hsp27 is phosphorylated in the late phase of epidermal differentiation

To determine the correlation between epidermal differentiation and phosphorylation of Hsp27 in vivo, we used a www.jidonline.org

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Figure 1. Dynamics of Hsp27 in keratinocyte differentiation. Immunofluorescence analysis of total Hsp27 (red staining) and the phosphorylated (phospho-serine 82) form of Hsp27 (green staining) in normal human skin (a and b). (b) An enlargement of the boxed area in (a). Negative control is provided in the right panel c. Hoechst dye was applied to visualize nuclei. Bars ¼ 50 mm. (d) Western blot analysis of Hsp60, Hsp70, Hsp27, and actin expression in total protein extracts of subconfluent (85% confluence) and postconfluent (3 and 6 days after confluence) normal human keratinocytes (NHKs). (e) Densitometric analysis of the data corresponding to panel d and two additional independent experiments. The data are expressed as relative intensity values normalized to b-actin intensity and are expressed graphically for 3 and 6 days after confluence relative to the level measured at 85% confluence that is considered to be 1. (f) Western blot analysis of involucrin, Hsp70, and Hsp27, in total and insoluble (cytoskeletal) protein extracts of NHKs treated with the calcium carrier A23187 or with DMSO. Western blot analysis of cytosolic protein caspase-3 in total and insoluble (cytoskeletal) protein extracts indicating that the cytoskeletal fraction is not contaminated with cytosolic proteins. Immunodetection of actin and Ponceau red staining of the protein transferred to the nitrocellulose membrane were used as loading controls. Representative results of two distinct experiments. Densitometric analysis of Hsp27 and Hsp70 corresponding to total protein extracts above. The number of western blots analyzed is indicated (n). Data represent the mean±SD. *Pp0.05 was considered statistically significant. Statistical analysis did not reveal significant difference in total protein extract in (e) and (f).

specific mAb directed against the phosphorylated form (on serine 82) of the human Hsp27. As shown in Figure 1a, the labeling of the phosphorylated form of Hsp27 occurs only in the most differentiated living keratinocytes in the granular layer, that is consistent with the previous report on phosphorylation of serine 86 in mouse keratinocytes (O’Shaughnessy et al., 2007). Moreover, only a subset of keratinocytes expressing Hsp27 is positive for the phosphorylated Hsp27 labeling (Figure 1a and b). 76

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DLK expression and Hsp27 dynamics in tissue-engineered skin

We took advantage of an in vitro model of human tissue–engineered skin that undergoes complete cornification to further analyze the pattern of DLK expression and Hsp27 dynamics. First, immunolocalization of DLK and filaggrin, a marker of terminal differentiation expressed in the granular layer of the epidermis (Dale et al., 1985), was performed. DLK and filaggrin were not expressed at day 5 of maturation (Figure 2a), a time at which the most differentiated layers

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Hsp 27 AE3 Cytoskeletal fraction Hsp 27 PhosphoHsp27 Actin Total extract Figure 2. Dual leucine zipper-bearing kinase (DLK) expression and Hsp27 distribution in human reconstructed skin. (a) Immunofluorescence labeling of DLK and filaggrin in human tissue-engineered skin cultured for 0, 5, 7, and 14 days at the air–liquid interface. Nuclei were stained with Hoechst dye. Phase contrast microscopy is also provided. (b) Masson’s trichrome staining of human tissue–engineered skin cultured for 0, 5, 7, and 14 days at the air–liquid interface. Bars ¼ 50 mm. (c) Western blot analysis of Hsp27 and phospho-Hsp27 in total protein extracts as well as Hsp27 in insoluble (cytoskeletal) protein extracts of epidermis isolated from human tissue-engineered skin cultured for 0, 5, 7, and 14 days at the air–liquid interface. Actin and pan-keratin AE3 antibodies were used as loading controls for total and insoluble extracts, respectively. Results are representative of three distinct experiments.

have not yet appeared (Figure 2b). These proteins were induced after day 7 of culture at the air–liquid interface, when the stratum granulosum appeared, and their expression was extended to the entire granular layer at day 14 (Figure 2a), a time at which a thick cornified layer was present (Figure 2b). To assess Hsp27 expression during epidermal differentiation, total and cytoskeletal protein extracts were prepared from tissue-engineered skin at different times of culture at the air–liquid interface (0, 5, 7, and 14 days) and processed for immunoblotting. As shown in Figure 2c, Hsp27 was detected from day 5 and increased gradually with keratinocyte differentiation in total protein extracts. In contrast, the

phosphorylated forms of Hsp27 were detected only at day 14. At the same time, Hsp27 appeared in the cytoskeletal fraction of epidermal proteins (Figure 2c) but caspase 3 was absent (data not shown). These results suggest that phosphorylation of Hsp27 and its presence in the cytoskeletal fraction occurring during the late phase of keratinocyte differentiation are closely linked. DLK expression in NHKs induces the redistribution of Hsp27 to the cytoskeletal protein fraction

Previous studies have reported that DLK acts as a positive regulator of keratinocyte terminal differentiation (Germain www.jidonline.org

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et al., 2000; Robitaille et al., 2005). Thus, to examine the influence of DLK on Hsp27 dynamics during this process, NHKs were infected with adenoviruses expressing either green fluorescent protein (GFP) alone (AdGFP) or GFP and DLK (AdDLK). As shown in Figure 3a, DLK expression did not interfere with the amount of Hsp27 in total protein extracts. In contrast, a strong increase of Hsp27 was noted in the cytoskeletal protein fraction (insoluble protein extracts) of NHKs infected at a multiplicity of infection (MOI) of 50 with AdDLK (Figure 3b). On the other hand, Hsp27 was barely detectable in the cytoskeletal protein fraction of noninfected and GFP-expressing NHKs (Figure 3b). The purity of the cytoskeletal fraction was monitored by immunoblotting this protein extract with antibodies against keratins (Pan keratin AE3); actin; a cytosolic protein, caspase 3; a membrane protein, Na þ /K þ ATPase a-1; and a nuclear protein, histone H1 (Figure 3c). Furthermore, Ponceau Red staining is provided as a loading control and shows the keratin cluster in the cytoskeletal fraction (Figure 3c). As expected, keratins were very abundant in the cytoskeletal fraction and actin was found only at a very low level. Caspase 3, Na þ /K þ ATPase, and histone H1 were absent from the cytoskeletal fraction but were found in the total protein extract, as well as keratins and actin, indicating that the cytoskeletal fraction was very enriched in keratins but not contaminated with cytosolic, membrane, and nuclear proteins. These results show that DLK expression is sufficient to induce the redistribution of Hsp27 from the soluble to the insoluble cytoskeletal fraction in the late phase of keratinocyte differentiation. To evaluate the link between DLK, Hsp27, and epidermal cell differentiation, we downregulated Hsp27 in HaCaT keratinocytes with Hsp27-specific small interfering RNA (siRNA). HaCaT keratinocytes were transfected with siRNA more efficiently than NHKs, in which the low rate of siRNA transfection (±10% efficiency, data not shown) was not sufficient to perform conclusive biochemical experiments. Twenty-four hours after the siRNA transfection, HaCaT cells were infected with AdDLK or AdGFP. Control (untreated) and Lipofectamine-only-treated HaCaT cells were also infected with AdDLK or AdGFP. Forty-eight hours later, the levels of Hsp27, DLK, and involucrin were determined by western blotting of total and cytoskeletal extracts. In AdDLKand Hsp27 siRNA-transfected cells, Hsp27 expression was downregulated by 91% in the cytoskeletal fraction compared

with control siRNA (scrambled siRNA) (Figure 3d right panel). In these cells double-transfected with AdDLK and Hsp27siRNA, the amount of involucrin in the cytoskeletal fraction was decreased by 38% compared with cells transfected with AdDLK and scrambled siRNA (Figure 3d right panel). Therefore, the reduced expression of involucrin when Hsp27 was downregulated in HaCaT cells overexpressing DLK indicates a direct link between Hsp27 and DLK in the regulation of involucrin expression or stability in the differentiation process of keratinocytes. Regulation of Hsp27 redistribution by DLK is independent of transglutaminase activity

DLK expression in NHKs induces transglutaminase (TG) activity (Robitaille et al., 2005). TGs, especially TG1, are responsible for the insolubilization of most cornified envelope precursors occurring during the late phase of keratinocyte differentiation (Candi et al., 2005; Eckert et al., 2005). To determine the role of TG activity in the insolubilization of Hsp27, as suggested by its presence in the cytoskeletal fraction, NHKs expressing either GFP alone or GFP and DLK were incubated with the TG inhibitor putrescine before the analysis of cytoskeletal extracts by western blot with the antiHsp27 antibody. As expected, cells expressing GFP (control) had no Hsp27 associated with the cytoskeletal fraction before or after putrescine treatment (Figure 4a). Band quantification confirmed that inhibition of the TG activity reduced by about 50% the transfer of involucrin to the cytoskeletal fraction in the DLK-expressing cells. In contrast, the amount of Hsp27 in the cytoskeletal fraction in DLK-expressing cells was not reduced by the inhibition of TG activity. In DLK-expressing NHKs, however, a substantial amount of Hsp27 was present in the cytoskeletal protein extract and putrescine had no effect on its relocalization to this fraction. Caspase 3 was absent from this cytoskeletal fraction but was found in the total protein fraction, indicating that the cytoskeletal fraction was not contaminated with cytosolic proteins. As expected, involucrin, which is among the first cornified envelope precursors to be cross-linked during keratinocyte differentiation, was detected in the cytoskeletal fraction after DLK expression in NHKs, and its insolubilization was partially prevented by the inhibition of TG activity (Figure 4a). Under the same conditions, involucrin and Hsp27 levels remained unaffected in total protein extracts (data not shown).

Figure 3. Insolubilization of Hsp27 by dual leucine zipper-bearing kinase (DLK) expression in normal human and HaCaT keratinocytes. Western blot analysis of Hsp27 in total (40 mg) (a) and cytoskeletal (5 mg) (b) protein extracts prepared from mock-, AdGFP- (MOI of 10 and 50), or AdDLK- (MOI of 10 and 50) infected normal human keratinocytes (NHKs). Actin and keratin 14 were used as loading controls for total and cytoskeletal fractions, respectively. (c) Evaluation of the cytoskeletal fraction purity. Western blot analysis of the membrane-associated Na þ /K þ ATPase, the cytosolic protein caspase 3, the nuclear protein histone H1, actin, and keratins (pan-keratin AE3) in total and insoluble (cytoskeletal) protein extracts (30 and 15 mg) of NHKs. *Equal exposures were taken for total extract and cytoskeletal fraction except for AE3 where longer exposure time was necessary to visualize AE3 keratin protein bands in the total extract (overnight) compared to cytoskeletal fraction (2 seconds). Immunodetection of actin and Ponceau red staining of the protein transferred to the nitrocellulose membrane (right panel) were used as loading controls. Representative results of three distinct experiments. (d) Silencing of Hsp27 by RNA interference. HaCaT cells were transfected with siRNA specific for Hsp27 or scramble siRNA using Lipofectamine. When indicated, untreated control (c), or siRNA-transfected HaCaT cells were subsequently infected with AdDLK or AdGFP. Total (15 mg, left panel) and insoluble (5 mg, right panel) proteins were prepared 72 hours after siRNA transfection and analyzed by western blotting to monitor involucrin, DLK, and Hsp27 expression. Actin expression (left panel) or Ponceau red staining of the protein transferred to the nitrocellulose membrane (right panel) was provided as loading controls. Representative results of four distinct experiments.

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AE3 Cytoskeletal fraction Figure 4. Redistribution of Hsp27 to the cytoskeletal fraction in DLK expressing cells is not dependent on transglutaminase activity. (a) Western blot analysis of Hsp27 and involucrin in insoluble protein extracts isolated from normal human keratinocytes (NHKs) expressing green fluorescent protein (GFP) or DLK in the presence or absence of putrescine to inhibit the transglutaminase activity. Pan-keratin AE3 antibody was used as loading control. Graphics are the quantification of band intensity for involucrin and Hsp27 on the AE3 loading control representing the mean±SD. *Pp0.05 was considered statistically significant. (b) Western blot analysis of Hsp27 in insoluble (cytoskeletal) protein extracts isolated from NHKs expressing either GFP, a kinase inactive form of DLK (AdK185R), or wild-type DLK. Pan keratin AE3 antibody was also used as a loading control. Results are representative of three distinct experiments.

Furthermore, the expression of a kinase-inactive form of DLK (DLK[K185R]) did not lead to the accumulation of Hsp27 in cytoskeletal protein extracts, indicating that the kinase activity of DLK was necessary to induce Hsp27 relocalization and insolubilization in NHKs (Figure 4b). These results suggest that DLK-induced redistribution of Hsp27 depends on DLK activity and is not dependent on TG activity.

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Figure 5. Dual leucine zipper-bearing kinase (DLK) expression induces ERK and JNK activation in normal human keratinocytes. Western blot analysis of DLK, phosphorylated, and total forms of ERK, JNK, and p38 and actin in total protein extracts prepared from mock-, AdGFP-, or AdDLK-infected (MOI of 50) normal human keratinocytes. Actin is provided as a loading control. Representative results of at least three independent experiments.

DLK expression in NHKs induces ERK and JNK activation

DLK induces Hsp27 phosphorylation and redistribution by an ERK-dependent mechanism in NHKs

DLK is an upstream activator of MAPK pathways in many cell types (Xu et al., 2001; Robitaille et al., 2005). To determine which of the MAPK pathways are activated by the expression of DLK in NHKs, we performed western blot analysis with antibodies raised against the phosphorylated forms of ERK, JNK, and p38 after infection of the cells with control GFP and DLK adenoviral vectors. DLK expression (MOI of 50) induced phosphorylation of ERK and JNK MAPKs but not p38 (Figure 5). DLK-mediated activation of the JNK pathway was greater than the activation of the ERK pathway; this may be due to the basal activation of ERK in the noninfected cells or the greater affinity of DLK for JNK than for ERK. Thus, ERK and JNK are potential pathways by which DLK regulates Hsp27 redistribution in NHKs undergoing terminal differentiation.

To better target which signaling pathway was involved in Hsp27 phosphorylation and redistribution, specific inhibitors of the ERK and JNK pathways—PD98059 and SP600125, respectively—were added 6 hours after infection of NHKs with GFP (control) or DLK. Forty-eight hours after infection, the phosphorylated forms of ERK, JNK, and Hsp27 were analyzed by western blotting on total protein extracts (Figure 6a). In addition, western blot analysis of Hsp27 was also performed on cytoskeletal protein extracts from NHKs cultured in the same conditions (Figure 6b). Inhibition of the ERK pathway with the MEK1 inhibitor PD98059 resulted in a decrease of ERK phosphorylation in DLK-expressing NHKs and a reduction of phosphorylated Hsp27 in total protein extract (Figure 6a). Moreover, Hsp27 in the cytoskeletal extract was also reduced (Figure 6b). In contrast, inhibition of

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JNK activation with SP600125 led to an increased phosphorylation of ERK and Hsp27 in DLK-expressing NHKs. It also increased the amount of Hsp27 in the cytoskeletal fraction (Figure 6b), probably as a result of ERK activation. These results showed that DLK-induced Hsp27 phosphorylation and redistribution in the cytoskeletal pool of proteins were dependent on the ERK pathway and also suggested that the JNK pathway counteracts Hsp27 phosphorylation by reducing ERK activity. DLK expression induces cell periphery localization of Hsp27 in NHKs by an ERK-dependent pathway

To evaluate whether DLK expression could influence the subcellular localization of Hsp27 in NHKs, confocal microscopy analysis of Hsp27 and GFP double labeling was performed (Figure 7) on NHKs infected with adenoviruses expressing GFP only (Figure 7a and b) or GFP and DLK (Figure 7c–h). In control GFP-expressing NHKs, Hsp27 was localized to the entire cytoplasm of the cells (Figure 7b). In contrast, a cell periphery-associated Hsp27 localization was detected in DLK-expressing NHKs (Figure 7d). Inhibition of the ERK pathway with PD98059 blocked this translocation to the cell periphery and Hsp27 remained in the cytoplasm (Figure 7f). Inhibition of the JNK pathway with SP600125 did not inhibit the DLK-mediated cell periphery translocation of Hsp27 in NHKs (Figure 7h). Thus, these results showed that DLK promoted Hsp27 translocation from the cytosol to the cell periphery by an ERK-dependent mechanism in NHKs.

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Figure 7. DLK expression induces Hsp27 relocalization to the cell periphery in an ERK dependent manner. Confocal analysis of a GFP (a, c, e, and g) or Hsp27 (b, d, f, and h) immunofluorescence labeling in green fluorescent protein- (a and b) and DLK- (c–h) expressing (MOI of 50) normal human keratinocytes incubated in the presence or absence of 5 mM PD98059 (e and f) or 40 mM SP600125 (g and h) to inhibit the ERK and c-jun N-terminal protein kinase pathways, respectively. Representative results of at least three independent experiments. Bar ¼ 50 mm.

DISCUSSION The terminal differentiation of epidermal cells is a complex, multistep program culminating in the formation of the cornified layer. Because this process ultimately leads to cell death, it is expected that cornification must be tightly regulated in vivo, and that its regulators should be expressed mainly in the most differentiated layers of the epidermis. Although our understanding of the mechanisms underlying cornification is still limited, the available data suggest a role for the different MAPK signaling cascades in regulating keratinocyte proliferation, survival, differentiation, and death (Eckert et al., 2002). In keratinocytes undergoing differentiation, the expression of Hsp27, a stress-inducible protein, is constitutively induced. To our knowledge, it has been previously unreported that the ERK signaling pathway, as a result of its activation by the mitogen-activated protein kinase kinase kinase DLK, may modulate Hsp27 phosphorylation in human skin keratinocytes, resulting in its redistribution to the cytoskeletal protein fraction and the cell periphery during www.jidonline.org

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terminal differentiation. These results are consistent with previous studies suggesting an active role for Hsp27 in keratin filament and cornified envelope assembly during keratinocyte terminal differentiation (Jonak et al., 2002; Duverger et al., 2004). Furthermore, the relevance of Hsp27 in keratinocyte terminal differentiation is also supported by the demonstration that Hsp27 inhibition by RNA interference in rat keratinocytes results in a cornification defect (O’Shaughnessy et al., 2007). Changes of Hsp27 expression level, phosphorylation state, and subcellular localization occur during epidermal differentiation

We showed that Hsp27 is expressed during human epidermal differentiation and that its expression level increases during the late phase of this differentiation program. Furthermore, using an in vitro model of human tissue-engineered skin, we showed that Hsp27 is phosphorylated in the very late phase of epidermal differentiation. Our results obtained with human keratinocytes are consistent with the findings of Duverger et al. (2004), who reported Hsp25 phosphorylation in the mouse keratinocyte cell line PAM212 induced to differentiate by increasing the extracellular calcium level (Duverger et al., 2004), and those of O’Shaughnessy et al. (2007) showing an AKT-dependent phosphorylation of Hsp27 in murine keratinocyte differentiation (O’Shaughnessy et al., 2007). Given that calcium is an important regulator of epidermal differentiation, Hsp27 phosphorylation could be an important event during the keratinocyte terminal differentiation process in vivo. We also found that Hsp27 was present in the pool of cytoskeletal proteins in the late phase of epidermal differentiation when cornification occurs in a tissue-engineered skin model and in NHKs treated with the calcium carrier A23187. A23187 induces cornification of NHKs in vitro as shown by the presence of involucrin in the cytoskeletal protein fraction. Moreover, A23187 is not a source of cell stress—Hsp70 and Hsp60 are not induced in treated cells— thus, this phenomenon is related mainly to keratinocyte differentiation. These findings suggest that Hsp27 interacts with insoluble proteins in the upper suprabasal layers of the epidermis. Accordingly, the murine analog Hsp25 interacts with filaggrin (O’Shaughnessy et al., 2007). Among these insoluble proteins, keratins are the most abundant. Indeed, keratins occupy the major part of the epithelial cell cytoskeleton and are found in a very insoluble state when packed in their filamentous form. Moreover, keratin filaments are covalently cross-linked to the cornified envelope during the keratinocyte differentiation process. Induction of Hsp27 redistribution to the cytoskeletal pool of proteins by DLK

Insolubilization of proteins is an important phenomenon in epidermal differentiation. The functional barrier of the epidermis, the cornified layer, is established after the TGdependent covalent cross-linking of several proteins and lipids resulting in the formation of the cornified envelope (Dale, 1985; Nemes and Steinert, 1999; Candi et al., 2005; Eckert et al., 2005). In a previous study, we reported that the expression of DLK alone in human keratinocytes is sufficient 82

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to induce TG activation and cornification (Robitaille et al., 2005). Here we showed, using putrescine as a competitor substrate, that redistribution of Hsp27 to the cytoskeletal pool of protein in DLK-expressing keratinocytes is not TG-dependent. The major portion of insoluble proteins in keratinocytes is occupied by keratins that resist the sustained proteasemediated degradation occurring during the formation of the cornified layer. An interaction of Hsp27 with keratins during keratinocyte differentiation is possible given that similar interactions between intermediate filaments and small Hsps have previously been reported in astrocytoma disease as well as in healthy conditions (Wisniewski and Goldman, 1998; Perng et al., 1999). The cornified cell envelope of the differentiated keratinocytes is also very insoluble. In cornifying keratinocytes, cornified envelope precursors are directed toward the cell periphery and covalently bound by a TG1dependent mechanism. Thus, the cornified envelope precursors are other potential targets for Hsp27 during keratinocyte terminal differentiation. Results shown in Figure 7 indicate that DLK expression led to a redistribution of Hsp27 from the cytosol to the cell periphery, suggesting an interaction between Hsp27 and the cornified envelope components. This result is supported by previous findings from electron microscopy immunogold labeling showing that Hsp27 colocalizes with keratins and cornified envelope precursors in the granular layer of epidermis (Jonak et al., 2002; O’Shaughnessy et al., 2007). Interestingly, murine Hsp27 was also shown to interact with filaggrin in its phosphorylated state (O’Shaughnessy et al., 2007). These results combined with ours strongly suggest that the chaperone activity of Hsp27 in epidermal differentiation is linked to its phosphorylation state. Phosphorylation and insolubilization of Hsp27 by an ERK-dependent pathway

Here we report that overexpression of DLK in human keratinocytes induces Hsp27 phosphorylation and relocalization by an ERK-dependent pathway. Several stimuli are known to induce Hsp27 phosphorylation in different cell lines. Furthermore, several studies have reported that the p38–MK2/3 pathway is a major inducer of Hsp27 phosphorylation. For instance, desmosome signaling induces Hsp27 phosphorylation by a p38–MK2 pathway (Berkowitz et al., 2005). Phosphorylation of the three serine residues in human Hsp27 is mediated by MK2/3 and PKC (Stokoe et al., 1992; McLaughlin et al., 1996; Maizels et al., 1998). Interestingly, another member of the small-Hsp family, aB-crystallin, is phosphorylated on serine 45 by ERK1/2 (Ito et al., 1997; Kato et al., 1998). In keratinocytes, few studies have reported activation of the ERK1/2 pathway during differentiation. The ERK pathway is well known for its promitotic properties as its activation correlates with the induction of several proliferation-associated proteins. On the other hand, it was also shown that ERK is transiently activated when keratinocytes are induced to differentiate by the increasing extracellular calcium level (Schmidt et al., 2000). It is now also documented that not only is the ERK pathway involved in

H Robitaille et al. Hsp27 in Keratinocyte Differentiation

proliferation but it can also contribute to the differentiation process (Schmidt et al., 2000; Ebisuya et al., 2005; Yoon and Seger, 2006). JNK is activated in the granular layer of the human epidermis (Takahashi et al., 2001) and is thought to be actively involved in keratinocyte differentiation. Here we show that DLK activates both ERK and JNK in human keratinocytes and that the inhibition of the JNK pathway with the specific inhibitor SP600125 leads to an increased ERK activation, suggesting a negative feedback of JNK on ERK activation. Such negative feedback between MAPK pathways has already been described in other systems (Chen et al., 2000; Shen et al., 2003). As discussed above, the expression of DLK in NHKs leads to a relocalization of Hsp27 to the cell periphery. Our results also show that Hsp27 redistribution is dependent on the activation of the ERK pathway. Taken together, our results shed light on the molecular events occurring during the late phase of the keratinocyte differentiation program. These results suggest that the insolubilization, phosphorylation, and redistribution of Hsp27 are closely linked and also dependent on the ERK pathway, which is activated in response to DLK expression. MATERIALS AND METHODS Cell culture Normal human keratinocytes were isolated from newborn foreskin as described (Germain et al., 2000). NHKs were cultured on irradiated 3T3 fibroblasts in DME-Ham’s F12 medium (Invitrogen, Ontario, Canada) containing 5% FetalCloneII serum (Hyclone, Logan, UT), insulin (5 mg/ml), hydrocortisone (0.4 mg/ml) (Calbiochem, LaJolla, CA), 10#10 M cholera toxin, epidermal growth factor (10 ng/ml) (Austral Biologicals, San Ramon, CA), and antibiotics (penicillin G (100 IU/ml) and gentamicin (25 mg/ml)). HaCaT cells (Boukamp et al., 1988) were cultured in DME containing 10% fetal calf serum (Hyclone) and antibiotics. In some experiments, 5 mM PD98059 (Calbiochem) and 40 mM SP600125 (Calbiochem) were added to the culture medium 6 hours after the adenoviral infection for a 42 hour incubation, to inhibit ERK and JNK, respectively. For induction of differentiation with the A23187 calcium carrier, DMSO-diluted calcimycin (A23187 (10 mg/ml)) (Calbiochem) was added to the culture medium of 85% confluent NHKs. An equal volume of DMSO was added in controls.

Immunofluorescence staining Acetone-fixed frozen sections (5 mm thick) of normal human skin and tissue-engineered skin, or formaldehyde (3.7%)–methanol (100%)fixed cells, were analyzed by immunofluorescence staining as described elsewhere (Germain et al., 2000). A rabbit antiserum directed against the N-terminal portion of recombinant mouse DLK (Germain et al., 2000), phosphorylated Hsp27 (S82) (Cell Signaling Technology, Beverly, MA) and Hsp27, 1/200 (Tanguay et al., 1993), and mouse mAbs raised against filaggrin (BTI, Stoughton, MA) and Hsp27 (Stressgen, San Diego, CA) were used as primary antibodies, and FITC- or tetramethylrhodamine-conjugated goat anti-mouse and anti-rabbit IgG were used as secondary antibodies (Chemicon, Temecula, CA). For the double immunofluorescence staining of Hsp27 and phospho-Hsp27, deparaffinized formol-fixed sections treated with 0.01 mM citrate (10 minutes, 90 1C), anti-rabbit biotinylated antibody (Jackson IRL, West Grove, PA), and Alexa-488-

conjugated-streptavidin (Molecular Probes, West Baltimore Pike, PA) were used. Negative controls consisted of replacing mAbs with isotypic immunoglobulins, or polyclonal antibodies with albumin (Sigma). Nuclei were labeled with Hoechst dye (Sigma, St Louis, MO).

Production of in vitro tissue-engineered skin Tissue-engineered skin specimens were produced as described (Michel et al., 1999). Briefly, human fibroblasts were cultured for 35 days in 25 cm2 culture flasks (BD Biosciences, Mississauga, Ontario, Canada) with fibroblast medium supplemented with 50 mg/ ml of ascorbate (Sigma). The resulting sheets (cells–extracellular matrix) were superimposed, and NHKs were seeded on top to form tissue-engineered skin by culturing 7 days in submerged conditions, followed by 21 days at the air–liquid interface. For immunoblot experiments, the epidermis was separated after overnight (4 1C) thermolysin incubation.

Adenoviruses and infection of NHKs Recombinant adenoviruses expressing the control GFP (AdGFP), alone or together with the T7 epitope-tagged wild-type (AdDLK) or the kinase-defective (AdDLK[K185R]) DLK, were used (Robitaille et al., 2004). Subconfluent cultures of NHKs or HaCaT cells cultured on plastic substrate were infected with one or two recombinant adenoviruses at an MOI of 10 (for Figure 3 only) or 50 in DMEM supplemented with 2% (v/v) serum, 0.8 mM L-arginine, antibiotics. After 1 hour, the medium was replaced with DMEM containing 5% serum for 48 hours.

Silencing of Hsp27 by RNA interference SiRNA duplex oligonucleotides were synthesized by Thermo Scientific Dharmacon Products (Lafayette, CO). For Hsp27, the strands were sense, 50 -UGAGACUGCCGCCAAGUAAUU-30 , and antisense, 50 -UUACUUGGCGGCAGUCUCAUU-30 , and sequences for the scramble siRNA were sense, 50 -UAGCGACUAAACACAUC AAUU-30 , and antisense, 50 -UUGAUGUGUUUAGUCGCUAUU-30 . SiRNA transfection was performed as described (Ouellet et al. 2006). Briefly, HaCaT cells were cultured in six-well plates. Serum was removed from the culture medium just before transfection. HaCaT cells were transfected with Hsp27 siRNA (60 pmol/well) or scramble siRNA (60 pmol/well) using Lipofectamine (Invitrogen). After 1 day, serum-free medium was replaced with standard culture medium or the cells were subsequently infected with adenoviruses as described above. Total or cytoskeletal proteins were extracted 3 days after the siRNA transfection.

Inhibition of TG activity Two hours after adenoviral infection, monolayer-cultured NHKs were incubated for 1 day with 20 mM putrescine (Sigma). The insoluble fraction of keratinocyte proteins was extracted 2 days later (see below).

Preparation of cell lysates and immunoblotting After cells lysis (60 minutes, 4 1C, in Triton buffer (50 mM Tris-HCl, pH 7.4, 1% Triton X-100, 150 mM NaCl, 5 mM EDTA, 0.2 mM sodium orthovanadate, 0.2 mM sodium fluoride, 1 mM phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, and 1 mg/ml aprotinin)), total extracts were sonicated. To extract the cytoskeletal fraction (insoluble pool of protein), cultured cells or tissue-engineered epidermis was rinsed www.jidonline.org

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with phosphate-buffered saline and incubated for 15 minutes in Triton buffer followed by 90 minutes in the same buffer containing 1.5 M KCl. After centrifugation, the pellet containing the cytoskeletal fraction was solubilized in Tris-EDTA buffer (Tris-HCL 10 mM, EDTA 1 mM) containing 1% SDS. Protein concentration was quantified using the MicroBCA protein assay kit (Pierce, Rockford, IL). For immunoblotting, equal amounts of proteins were fractionated by SDS–PAGE and transferred onto nitrocellulose membranes (Amersham Pharmacia Biotech, Baie d’Urfe´, Que´bec, Canada) using a transfer apparatus (Bio-Rad, Mississauga, Ontario, Canada). The membranes were blocked in 20 mM Tris, pH 7.5, 150 mM NaCl, 0.1% Tween-20 containing 5% dry milk before addition of the primary antibody (1 hour at room temperature or overnight at 4 1C). The primary antibodies were mouse monoclonal anti-phospho-JNK, anti-phospho-p38, anti-phospho-p44/42 ERK (Cell Signaling Technology), anti-Hsp27 (Stressgen), anti-involucrin (Sigma), anti-keratin 10 (MONOSAN, Uden, The Netherlands), anti-Na þ /K þ ATPase a-1 (clone C464.6, Upstate, Temecula, CA), anti-caspase 3 (Clone 3CSP03, Chemicon), anti-AE3 pan-keratin (ICN, Auroa, OH), antiactin (Cedarlane, Burlington, Ontario, Canada), rabbit polyclonal anti-JNK total, anti-p38 total, anti-p44/42 ERK total, anti-phosphoHsp27 (Cell Signaling), anti-Hsp60 and anti-Hsp70 (Tanguay et al., 1993), and anti-keratin 14. The secondary antibodies were antimouse HRP-conjugated (Sigma) and anti-rabbit HRP-conjugated (Chemicon). Chemiluminescence of immunoreactive bands was detected with the ECL Plus western blotting kit (Amersham Pharmacia Biotech). Band quantification of triplicate experiments was performed using ImageJ software (Bethesda, MD). Statistical analysis was performed using Student’s t-test. Po0.05 was considered statistically significant. CONFLICT OF INTEREST The authors state no conflict of interest.

ACKNOWLEDGMENTS We thank Sylvain Gue´rin and Jacques Landry for their help with the siRNA experiment, as well as Jacques Huot for providing HaCaT cells and Ve´ronique Moulin for having given us materials. We also also thank Ve´ronique Couture, Marie-France Champigny, Claudia Fuge`re, Israel Martel, Francis Bisson, Herman Lambert, Yan Nadeau, and Claudie Paquet for technical assistance. This study was supported by grants from the Canadian Institutes of Health Research (CIHR) to LG and RMT and from the Natural Science and Engineering Research Council of Canada to RB. LG holds the Canadian Research Chair in Stem Cells and Tissue Engineering from CIHR, and HR and CS-B are recipients of a studentship from the Fonds de la Recherche en Sante´ du Que´bec.

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