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1 ORIGINAL ARTICLE See related ommentary on pg 1 The Small Heat-Shok Protein Undergoes ERK-Dependent Phosphorylation and Redistribution to the Cytoskeleton in Response to Dual Leuine Zipper-Bearing Kinase Expression Hubert Robitaille 1,2, Carolyne Simard-Bisson 1,2, Danielle Larouhe 1,2, Robert M. Tanguay, Rihard Blouin 4 and Luie Germain 1,2, a small heat-shok protein, has important roles in many ellular proesses, inluding ytoskeleton dynamis, ell differentiation, and apoptosis. Its expression in normal epidermis orrelates with differentiation; however, little is known about the regulatory mehanisms involved. In this study, we report that undergoes upregulation, phosphorylation, and redistribution to the ytoskeleton during the late phase of epidermal keratinoyte differentiation. Our results also show that the expression of the dual leuine zipper-bearing kinase (DLK), an upstream ativator of the MAP kinase pathways, is suffiient by itself to indue phosphorylation, ell periphery loalization, and redistribution to the insoluble protein fration (ytoskeleton) in poorly differentiated keratinoytes. This redistribution orrelates with the insolubilization of ornified envelope-assoiated proteins suh as involurin. Interestingly, the effets of DLK on were bloked by PD9859, a seletive inhibitor of the extraellular signal-regulated protein kinase (ERK) pathway. Moreover, downregulation of by small interfering RNA in epithelial ells expressing DLK was aompanied by attenuated expression of involurin in the ytoskeleton. Thus, these observations suggest that the DLK ERK signaling pathway may at as a regulator of the interation that ours between and the ytoskeleton during the formation of the ornified ell envelope, a proess onferring to the skin its ruial barrier funtion. Journal of Investigative Dermatology (21) 1, 74 85; doi:1.18/jid ; published online 1 August 29 INTRODUCTION The epidermis is a self-renewing stratified epithelium populated by keratinoytes that undergo a omplex program of terminal differentiation (Fuhs, 199a). This proess 1 Laboratoire de Reherhe des Grands Brûlés/LOEX, Centre de reherhe (FRSQ) du CHA de Québe, Québe, Québe, Canada; 2 Département de Chirurgie, Université Laval, Québe, Québe, Canada; Laboratoire de Génétique Cellulaire et Développementale, Faulté de Médeine, Université Laval, Québe, Québe, Canada and 4 Département de Biologie, Faulté des Sienes, Université de Sherbrooke, Sherbrooke, Québe, Canada Correspondene: Dr Luie Germain, Laboratoire de Reherhe des Grands Brûlés/LOEX, Hôpital du Saint-Sarement du CHA, 15 Chemin Sainte-Foy, Québe, Québe, Canada G1S 4L8. luie.germain@hg.ulaval.a Abbreviations:, reombinant adenovirus expressing the green fluoresent protein together with a T7 epitope-tagged form of wild-type DLK;, reombinant adenovirus expressing the green fluoresent protein; DLK, dual leuine zipper-bearing kinase; ERK, extraellular signal-regulated protein kinase; Hsp, heat-shok protein; JNK, -jun N-terminal protein kinase; MAPK, mitogen-ativated protein kinase; MAPKK, mitogen-ativated protein kinase kinase; MK2/, mitogen-ativated protein kinase-ativated kinases 2 and ; MOI, multipliity of infetion; NHK, normal human keratinoyte; sirna, small interfering RNA; TG1, transglutaminase 1 Reeived 4 February 29; revised 8 April 29; aepted 12 May 29; published online 1 August 29 ulminates in the formation of the stratum orneum, whih onstitutes a barrier proteting the body from penetration by environmental toxins and loss of vital fluids. In the granular layer, keratinoytes undergo irreversible modifiations leading to the flattened, enuleated dead ornified ells (orneoytes) that ensure the skin barrier funtion (Watt, 1989; Fuhs, 199b; Nemes and Steinert, 1999; Candi et al., 25). Several moleular events assoiated with this proess remain to be haraterized. Heat-shok protein (Hsp) 27 (also known as Hsp25 in the mouse), a member of the small-hsp family, is highly expressed in many ell types, at different stages of differentiation/development (Cioa et al., 199; Parellier et al., 2). In addition to its role in protein folding, intervenes in the modulation of differentiation as well as in apoptosis (Beere, 21; Parellier et al., 2). Furthermore, it has been identified as a potent regulator of the ytoskeleton as a result of its ability to inhibit atin polymerization (Lavoie et al., 199). This property is dependent on the phosphorylation state of and its strutural organization (Lavoie et al., 199; Guay et al., 1997). an be reversibly phosphorylated on three serine residues in humans (two 74 Journal of Investigative Dermatology (21), Volume 1 & 21 The Soiety for Investigative Dermatology

2 in Keratinoyte Differentiation for Hsp25 in mie) by the mitogen-ativated protein kinase-ativated kinases 2 and (MK2/), whih are themselves ativated by phosphorylation through either the p8 or the extraellular signal-regulated protein kinase (ERK) signaling pathway (Gandour-Edwards et al., 1994; Guay et al., 1997; Jantshitsh et al., 1998; Geum et al., 22; Morrow and Tanguay, 2; Duverger et al., 24; O Shaughnessy et al., 27). Upstream signals, suh as differentiating agents and mitogens, that ativate MK2/ also have the ability to indue phosphorylation. In general, it is believed that MK2/ are the main mediators of phosphorylation, but other kinases suh as PKC delta an also phosphorylate (Kato et al., 1998; Maizels et al., 1998). In both human and mouse epidermis, is highly expressed in keratinoytes of the granular layer (Gandour- Edwards et al., 1994), and its phosphorylation in the mouse keratinoyte ell line PAM 212 is modulated on aliumindued differentiation (Duverger et al., 24). Moreover, is phosphorylated on serine 82 by a signaling mehanism involving Akt and p8 kinases (Konishi et al., 1997; Rane et al., 2; O Shaughnessy et al., 27). Interations between small Hsps and intermediate filaments or other elements of the ytoskeleton our in many ell types and several pathologies (Kato et al., 1992; Lowe et al., 1992; Viart et al., 1998; Wisniewski and Goldman, 1998; Perng et al., 1999). oloalizes with keratin filaments and other proteins of the ornified envelope in differentiated keratinoytes as revealed by immunogold labeling (Jonak et al., 22; O Shaughnessy et al., 27). Furthermore, in differentiated PAM212 keratinoytes, Hsp25 oloalizes with aggregated keratin 5, suggesting a role in the disorganization of the keratin 5 keratin 14 network ourring during their differentiation (Duverger et al., 24). The dual leuine zipper-bearing kinase (DLK) is a mitogen-ativated protein kinase kinase kinase expressed in a variety of tissues (Nadeau et al., 1997). In skin, its expression orrelates with differentiation (Nadeau et al., 1997; Germain et al., 2). Reent studies from our laboratory have shown that DLK has an ative role in the indution of terminal differentiation of human keratinoytes (Germain et al., 2; Robitaille et al., 25). DLK preferentially ativates the -Jun N-terminal kinases (JNKs), a subgroup of MAPKs involved in the regulation of ellular proesses suh as proliferation, differentiation, and apoptosis (Xu et al., 21; Ekert et al., 22; Nishina et al., 24). In the epidermis, JNK ativation indues the differentiation marker ystatin A. In vivo, JNK ativation ours in the granular layer, the last transriptionally ative ell layer of the epidermis (Takahashi et al., 21, 22). On the other hand, the ERK1/2 MAPK pathway ats as a positive regulator of proliferation/survival in many ell lines and keratinoytes. For instane, the ERK1/2 pathway is strongly ativated by mitogens and integrins, whereas it dereases the expression of ystatin A (Takahashi et al., 21). Although ERK1/2 is involved in keratinoyte proliferation, it is also ativated during differentiation (Shmidt et al., 2). In the present study, we investigated the dynamis of in human keratinoyte differentiation and whether DLK, as a result of its ability to indue ornifiation, may modulate regulation. Our results show that undergoes phosphorylation and redistribution to the ytoskeletal protein fration in the highly differentiated granular keratinoytes of native and tissue-engineered skin. Moreover, we report that expression of DLK alone in poorly differentiated normal human keratinoytes (NHKs) promotes the phosphorylation, ytoskeletal assoiation, and redistribution of from the ytoplasm to the ell periphery in an ERKdependent manner. These results provide strong evidene that phosphorylation of through the DLK ERK signaling pathway may promote its interation with the ytoskeleton and other omponents of the ornified ell envelope. RESULTS dynamis in normal human keratinoyte differentiation Immunofluoresene analysis, using a speifi antibody, has revealed the loalization of in normal human skin. was expressed in the first layers of the stratum spinosum and its expression inreased in the granular layer (Figure 1a and b). Using an in vitro model in whih ultured NHKs were indued to differentiate by prolonging ulture, we observed an apparent inrease in expression at day to day 6 following onfluene ompared with the expression level in poorly differentiated subonfluent NHKs (Figure 1d and e), but the differene was not signifiant. The presene of a lower band (±22 kda) was observed only after onfluene and ould be due to the reognition of another protein by the antibody (suh as Hsp22, whih is homologous to ) or a posttranslational modifiation of. Other Hsps, suh as Hsp7 and Hsp6, were found to be regulated differently during keratinoyte differentiation (Laplante et al., 1998; Ghoreishi et al., 2). Hsp6 and Hsp7 expression levels were not signifiantly hanged under the same onditions (Figure 1d and e). We used an in vitro model of ultured NHKs indued to ornify by the elevation of intraellular alium levels with the A2187 alium arrier, known to indue keratinoyte terminal differentiation (Takahashi et al., 2), to further analyze the regulation of during the late phase of differentiation., Hsp6, and Hsp7 were not signifiantly inreased in total protein extrat after the addition of A2187 to the ulture medium. The absene of an inrease in the Hsps in total protein extrats indiates that this is not a stress response. In ontrast, the addition of A2187 resulted in the appearane of a proportion of in the Tritoninsoluble (ytoskeletal) fration (Figure 1f). The absene of aspase in this fration indiated that it is not ontaminated with ytosol (Figure 1f). Involurin was also strongly inreased in the ytoskeletal fration (Figure 1f). The inrease in involurin and in the ytoskeletal fration indiates that this is a differentiation-assoiated response. is phosphorylated in the late phase of epidermal differentiation To determine the orrelation between epidermal differentiation and phosphorylation of in vivo, we used a 75

3 H Robitaille et al. Hsp7 A2187 Cytoskeletal fration DMSO A2187 DMSO Control Total extrat Control 6 Days postonfluene Days postonfluene 85% Confluene in Keratinoyte Differentiation Involurin Hsp7 Hsp6 Caspase % C n= 2 n= n=2 87 SO n = n= n= n = 1 on flu en e D o ay nf s lu po en s t6 D e o ay nf s lu po en s e t- n= n= n = n= n = n = 85 Hsp7 M 2 4 Hsp7 Hsp6 A2 Poneau s red stain D 4 Figure 1. Dynamis of in keratinoyte differentiation. Immunofluoresene analysis of total (red staining) and the phosphorylated (phospho-serine 82) form of (green staining) in normal human skin (a and b). (b) An enlargement of the boxed area in (a). Negative ontrol is provided in the right panel. Hoehst dye was applied to visualize nulei. Bars ¼ 5 mm. (d) Western blot analysis of Hsp6, Hsp7,, and atin expression in total protein extrats of subonfluent (85% onfluene) and postonfluent ( and 6 days after onfluene) normal human keratinoytes (NHKs). (e) Densitometri analysis of the data orresponding to panel d and two additional independent experiments. The data are expressed as relative intensity values normalized to b-atin intensity and are expressed graphially for and 6 days after onfluene relative to the level measured at 85% onfluene that is onsidered to be 1. (f) Western blot analysis of involurin, Hsp7, and, in total and insoluble (ytoskeletal) protein extrats of NHKs treated with the alium arrier A2187 or with DMSO. Western blot analysis of ytosoli protein aspase- in total and insoluble (ytoskeletal) protein extrats indiating that the ytoskeletal fration is not ontaminated with ytosoli proteins. Immunodetetion of atin and Poneau red staining of the protein transferred to the nitroellulose membrane were used as loading ontrols. Representative results of two distint experiments. Densitometri analysis of and Hsp7 orresponding to total protein extrats above. The number of western blots analyzed is indiated (n). Data represent the mean±sd. *Pp.5 was onsidered statistially signifiant. Statistial analysis did not reveal signifiant differene in total protein extrat in (e) and (f). speifi mab direted against the phosphorylated form (on serine 82) of the human. As shown in Figure 1a, the labeling of the phosphorylated form of ours only in the most differentiated living keratinoytes in the granular layer, that is onsistent with the previous report on phosphorylation of serine 86 in mouse keratinoytes (O Shaughnessy et al., 27). Moreover, only a subset of keratinoytes expressing is positive for the phosphorylated labeling (Figure 1a and b). 76 Journal of Investigative Dermatology (21), Volume 1 DLK expression and dynamis in tissue-engineered skin We took advantage of an in vitro model of human tissue engineered skin that undergoes omplete ornifiation to further analyze the pattern of DLK expression and dynamis. First, immunoloalization 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 whih the most differentiated layers

4 H Robitaille et al. in Keratinoyte Differentiation DLK Filaggrin Hoehst Phase ontrast Day Day Day 5 Day 5 Day 7 Day 7 Day 14 Day Air liquid interfae (days) Hsp 27 AE Cytoskeletal fration Hsp 27 Phospho Total extrat Figure 2. Dual leuine zipper-bearing kinase (DLK) expression and distribution in human reonstruted skin. (a) Immunofluoresene labeling of DLK and filaggrin in human tissue-engineered skin ultured for, 5, 7, and 14 days at the air liquid interfae. Nulei were stained with Hoehst dye. Phase ontrast mirosopy is also provided. (b) Masson s trihrome staining of human tissue engineered skin ultured for, 5, 7, and 14 days at the air liquid interfae. Bars ¼ 5 mm. () Western blot analysis of and phospho- in total protein extrats as well as in insoluble (ytoskeletal) protein extrats of epidermis isolated from human tissue-engineered skin ultured for, 5, 7, and 14 days at the air liquid interfae. and pan-keratin AE antibodies were used as loading ontrols for total and insoluble extrats, respetively. Results are representative of three distint experiments. have not yet appeared (Figure 2b). These proteins were indued after day 7 of ulture at the air liquid interfae, when the stratum granulosum appeared, and their expression was extended to the entire granular layer at day 14 (Figure 2a), a time at whih a thik ornified layer was present (Figure 2b). To assess expression during epidermal differentiation, total and ytoskeletal protein extrats were prepared from tissue-engineered skin at different times of ulture at the air liquid interfae (, 5, 7, and 14 days) and proessed for immunoblotting. As shown in Figure 2, was deteted from day 5 and inreased gradually with keratinoyte differentiation in total protein extrats. In ontrast, the phosphorylated forms of were deteted only at day 14. At the same time, appeared in the ytoskeletal fration of epidermal proteins (Figure 2) but aspase was absent (data not shown). These results suggest that phosphorylation of and its presene in the ytoskeletal fration ourring during the late phase of keratinoyte differentiation are losely linked. DLK expression in NHKs indues the redistribution of to the ytoskeletal protein fration Previous studies have reported that DLK ats as a positive regulator of keratinoyte terminal differentiation (Germain 77

5 in Keratinoyte Differentiation et al., 2; Robitaille et al., 25). Thus, to examine the influene of DLK on dynamis during this proess, NHKs were infeted with adenoviruses expressing either green fluoresent protein (GFP) alone () or GFP and DLK (). As shown in Figure a, DLK expression did not interfere with the amount of in total protein extrats. In ontrast, a strong inrease of was noted in the ytoskeletal protein fration (insoluble protein extrats) of NHKs infeted at a multipliity of infetion (MOI) of 5 with (Figure b). On the other hand, was barely detetable in the ytoskeletal protein fration of noninfeted and GFP-expressing NHKs (Figure b). The purity of the ytoskeletal fration was monitored by immunoblotting this protein extrat with antibodies against keratins (Pan keratin AE); atin; a ytosoli protein, aspase ; a membrane protein, Na þ /K þ ATPase a-1; and a nulear protein, histone H1 (Figure ). Furthermore, Poneau Red staining is provided as a loading ontrol and shows the keratin luster in the ytoskeletal fration (Figure ). As expeted, keratins were very abundant in the ytoskeletal fration and atin was found only at a very low level. Caspase, Na þ /K þ ATPase, and histone H1 were absent from the ytoskeletal fration but were found in the total protein extrat, as well as keratins and atin, indiating that the ytoskeletal fration was very enrihed in keratins but not ontaminated with ytosoli, membrane, and nulear proteins. These results show that DLK expression is suffiient to indue the redistribution of from the soluble to the insoluble ytoskeletal fration in the late phase of keratinoyte differentiation. To evaluate the link between DLK,, and epidermal ell differentiation, we downregulated in HaCaT keratinoytes with -speifi small interfering RNA (sirna). HaCaT keratinoytes were transfeted with sirna more effiiently than NHKs, in whih the low rate of sirna transfetion (±1% effiieny, data not shown) was not suffiient to perform onlusive biohemial experiments. Twenty-four hours after the sirna transfetion, HaCaT ells were infeted with or. Control (untreated) and Lipofetamine-only-treated HaCaT ells were also infeted with or. Forty-eight hours later, the levels of, DLK, and involurin were determined by western blotting of total and ytoskeletal extrats. In and sirna-transfeted ells, expression was downregulated by 91% in the ytoskeletal fration ompared with ontrol sirna (srambled sirna) (Figure d right panel). In these ells double-transfeted with and - sirna, the amount of involurin in the ytoskeletal fration was dereased by 8% ompared with ells transfeted with and srambled sirna (Figure d right panel). Therefore, the redued expression of involurin when was downregulated in HaCaT ells overexpressing DLK indiates a diret link between and DLK in the regulation of involurin expression or stability in the differentiation proess of keratinoytes. Regulation of redistribution by DLK is independent of transglutaminase ativity DLK expression in NHKs indues transglutaminase (TG) ativity (Robitaille et al., 25). TGs, espeially TG1, are responsible for the insolubilization of most ornified envelope preursors ourring during the late phase of keratinoyte differentiation (Candi et al., 25; Ekert et al., 25). To determine the role of TG ativity in the insolubilization of, as suggested by its presene in the ytoskeletal fration, NHKs expressing either GFP alone or GFP and DLK were inubated with the TG inhibitor putresine before the analysis of ytoskeletal extrats by western blot with the anti- antibody. As expeted, ells expressing GFP (ontrol) had no assoiated with the ytoskeletal fration before or after putresine treatment (Figure 4a). Band quantifiation onfirmed that inhibition of the TG ativity redued by about 5% the transfer of involurin to the ytoskeletal fration in the DLK-expressing ells. In ontrast, the amount of in the ytoskeletal fration in DLK-expressing ells was not redued by the inhibition of TG ativity. In DLK-expressing NHKs, however, a substantial amount of was present in the ytoskeletal protein extrat and putresine had no effet on its reloalization to this fration. Caspase was absent from this ytoskeletal fration but was found in the total protein fration, indiating that the ytoskeletal fration was not ontaminated with ytosoli proteins. As expeted, involurin, whih is among the first ornified envelope preursors to be ross-linked during keratinoyte differentiation, was deteted in the ytoskeletal fration after DLK expression in NHKs, and its insolubilization was partially prevented by the inhibition of TG ativity (Figure 4a). Under the same onditions, involurin and levels remained unaffeted in total protein extrats (data not shown). Figure. Insolubilization of by dual leuine zipper-bearing kinase (DLK) expression in normal human and HaCaT keratinoytes. Western blot analysis of in total (4 mg) (a) and ytoskeletal (5 mg) (b) protein extrats prepared from mok-, - (MOI of 1 and 5), or - (MOI of 1 and 5) infeted normal human keratinoytes (NHKs). and keratin 14 were used as loading ontrols for total and ytoskeletal frations, respetively. () Evaluation of the ytoskeletal fration purity. Western blot analysis of the membrane-assoiated Na þ /K þ ATPase, the ytosoli protein aspase, the nulear protein histone H1, atin, and keratins (pan-keratin AE) in total and insoluble (ytoskeletal) protein extrats ( and 15 mg) of NHKs. *Equal exposures were taken for total extrat and ytoskeletal fration exept for AE where longer exposure time was neessary to visualize AE keratin protein bands in the total extrat (overnight) ompared to ytoskeletal fration (2 seonds). Immunodetetion of atin and Poneau red staining of the protein transferred to the nitroellulose membrane (right panel) were used as loading ontrols. Representative results of three distint experiments. (d) Silening of by RNA interferene. HaCaT ells were transfeted with sirna speifi for or sramble sirna using Lipofetamine. When indiated, untreated ontrol (), or sirna-transfeted HaCaT ells were subsequently infeted with or. Total (15 mg, left panel) and insoluble (5 mg, right panel) proteins were prepared 72 hours after sirna transfetion and analyzed by western blotting to monitor involurin, DLK, and expression. expression (left panel) or Poneau red staining of the protein transferred to the nitroellulose membrane (right panel) was provided as loading ontrols. Representative results of four distint experiments. 78 Journal of Investigative Dermatology (21), Volume 1

6 in Keratinoyte Differentiation Total extrat Cytoskeletal fration Western blot Poneau s red stain Cytoskeletal Total extrat Cytoskeletal fration Total extrat fration Total extrat Cytoskeletal fration sirna sramble sirna sirna sramble sirna sirna sramble sirna sirna sramble sirna sirna sramble sirna sirna sramble sirna Non-infeted MOI of 1 MOI of 5 MOI of 1 MOI of 5 Non-infeted MOI of 1 MOI of 5 MOI of 1 MOI of 5 Keratin 14 Na + /K + ATPase Caspase Histone H AE * 4 26 Involurin DLK Poneau s red stain

7 in Keratinoyte Differentiation Putresine + + Involurin Ad[K185R] Non-infeted MOI of 5 MOI of * * AE DLK Cytoskeletal fration Phospho-ERK * Total ERK AE Cytoskeletal fration Phospho-JNK Figure 4. Redistribution of to the ytoskeletal fration in DLK expressing ells is not dependent on transglutaminase ativity. (a) Western blot analysis of and involurin in insoluble protein extrats isolated from normal human keratinoytes (NHKs) expressing green fluoresent protein (GFP) or DLK in the presene or absene of putresine to inhibit the transglutaminase ativity. Pan-keratin AE antibody was used as loading ontrol. Graphis are the quantifiation of band intensity for involurin and on the AE loading ontrol representing the mean±sd. *Pp.5 was onsidered statistially signifiant. (b) Western blot analysis of in insoluble (ytoskeletal) protein extrats isolated from NHKs expressing either GFP, a kinase inative form of DLK (AdK185R), or wild-type DLK. Pan keratin AE antibody was also used as a loading ontrol. Results are representative of three distint experiments. Total JNK Phospho-p8 Total p8 Furthermore, the expression of a kinase-inative form of DLK (DLK[K185R]) did not lead to the aumulation of in ytoskeletal protein extrats, indiating that the kinase ativity of DLK was neessary to indue reloalization and insolubilization in NHKs (Figure 4b). These results suggest that DLK-indued redistribution of depends on DLK ativity and is not dependent on TG ativity. DLK expression in NHKs indues ERK and JNK ativation DLK is an upstream ativator of MAPK pathways in many ell types (Xu et al., 21; Robitaille et al., 25). To determine whih of the MAPK pathways are ativated by the expression of DLK in NHKs, we performed western blot analysis with antibodies raised against the phosphorylated forms of ERK, JNK, and p8 after infetion of the ells with ontrol GFP and DLK adenoviral vetors. DLK expression (MOI of 5) indued phosphorylation of ERK and JNK MAPKs but not p8 (Figure 5). DLK-mediated ativation of the JNK pathway was greater than the ativation of the ERK pathway; this may be due to the basal ativation of ERK in the noninfeted ells or the greater affinity of DLK for JNK than for ERK. Thus, ERK and JNK are potential pathways by whih DLK regulates redistribution in NHKs undergoing terminal differentiation. Figure 5. Dual leuine zipper-bearing kinase (DLK) expression indues ERK and JNK ativation in normal human keratinoytes. Western blot analysis of DLK, phosphorylated, and total forms of ERK, JNK, and p8 and atin in total protein extrats prepared from mok-, -, or -infeted (MOI of 5) normal human keratinoytes. is provided as a loading ontrol. Representative results of at least three independent experiments. DLK indues phosphorylation and redistribution by an ERK-dependent mehanism in NHKs To better target whih signaling pathway was involved in phosphorylation and redistribution, speifi inhibitors of the ERK and JNK pathways PD9859 and SP6125, respetively were added 6 hours after infetion of NHKs with GFP (ontrol) or DLK. Forty-eight hours after infetion, the phosphorylated forms of ERK, JNK, and were analyzed by western blotting on total protein extrats (Figure 6a). In addition, western blot analysis of was also performed on ytoskeletal protein extrats from NHKs ultured in the same onditions (Figure 6b). Inhibition of the ERK pathway with the MEK1 inhibitor PD9859 resulted in a derease of ERK phosphorylation in DLK-expressing NHKs and a redution of phosphorylated in total protein extrat (Figure 6a). Moreover, in the ytoskeletal extrat was also redued (Figure 6b). In ontrast, inhibition of 8 Journal of Investigative Dermatology (21), Volume 1

8 H Robitaille et al. in Keratinoyte Differentiation GFP SP µm + Phospho-ERK Total ERK 2 1 Phospho- Phospho- fold indution Total JNK ytoskeletal fration fold indution Phospho-JNK Total extrats AE Cytoskeletal fration * * ontrol 2 1 b Hsp 27 PD9859 SP * PD9859 SP * * 1 PD9859 SP6125 PD µm Phospho-ERK fold indution a Figure 6. DLK expression promotes ERK dependent insolubilization and phosphorylation of. Western blot analysis of phosphorylated and total forms of ERK, -jun N-terminal protein kinase (JNK), and in total protein extrats (a) and insoluble protein extrats (b) isolated from and -infeted (MOI of 5) normal human keratinoytes ultured in the presene or absene of the ERK pathway inhibitor PD9859 (5 mm) and the JNK pathway inhibitor SP6125 (4 mm). () Quantifiation of immunoblotting for phospho-erk, phospho-, and in the ytoskeletal fration, representing the mean±sd of the relative band intensity on the loading ontrol of three independent experiments. *Pp.5 was onsidered statistially signifiant. JNK ativation with SP6125 led to an inreased phosphorylation of ERK and in DLK-expressing NHKs. It also inreased the amount of in the ytoskeletal fration (Figure 6b), probably as a result of ERK ativation. These results showed that DLK-indued phosphorylation and redistribution in the ytoskeletal pool of proteins were dependent on the ERK pathway and also suggested that the JNK pathway ounterats phosphorylation by reduing ERK ativity. DLK expression indues ell periphery loalization of in NHKs by an ERK-dependent pathway To evaluate whether DLK expression ould influene the subellular loalization of in NHKs, onfoal mirosopy analysis of and GFP double labeling was performed (Figure 7) on NHKs infeted with adenoviruses expressing GFP only (Figure 7a and b) or GFP and DLK (Figure 7 h). In ontrol GFP-expressing NHKs, was loalized to the entire ytoplasm of the ells (Figure 7b). In ontrast, a ell periphery-assoiated loalization was deteted in DLK-expressing NHKs (Figure 7d). Inhibition of the ERK pathway with PD9859 bloked this transloation to the ell periphery and remained in the ytoplasm (Figure 7f). Inhibition of the JNK pathway with SP6125 did not inhibit the DLK-mediated ell periphery transloation of in NHKs (Figure 7h). Thus, these results showed that DLK promoted transloation from the ytosol to the ell periphery by an ERK-dependent mehanism in NHKs. PD9859 SP6125 Figure 7. DLK expression indues reloalization to the ell periphery in an ERK dependent manner. Confoal analysis of a GFP (a,, e, and g) or (b, d, f, and h) immunofluoresene labeling in green fluoresent protein- (a and b) and DLK- ( h) expressing (MOI of 5) normal human keratinoytes inubated in the presene or absene of 5 mm PD9859 (e and f) or 4 mm SP6125 (g and h) to inhibit the ERK and -jun N-terminal protein kinase pathways, respetively. Representative results of at least three independent experiments. Bar ¼ 5 mm. DISCUSSION The terminal differentiation of epidermal ells is a omplex, multistep program ulminating in the formation of the ornified layer. Beause this proess ultimately leads to ell death, it is expeted that ornifiation 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 mehanisms underlying ornifiation is still limited, the available data suggest a role for the different MAPK signaling asades in regulating keratinoyte proliferation, survival, differentiation, and death (Ekert et al., 22). In keratinoytes undergoing differentiation, the expression of, a stress-induible protein, is onstitutively indued. To our knowledge, it has been previously unreported that the ERK signaling pathway, as a result of its ativation by the mitogen-ativated protein kinase kinase kinase DLK, may modulate phosphorylation in human skin keratinoytes, resulting in its redistribution to the ytoskeletal protein fration and the ell periphery during 81

9 in Keratinoyte Differentiation terminal differentiation. These results are onsistent with previous studies suggesting an ative role for in keratin filament and ornified envelope assembly during keratinoyte terminal differentiation (Jonak et al., 22; Duverger et al., 24). Furthermore, the relevane of in keratinoyte terminal differentiation is also supported by the demonstration that inhibition by RNA interferene in rat keratinoytes results in a ornifiation defet (O Shaughnessy et al., 27). Changes of expression level, phosphorylation state, and subellular loalization our during epidermal differentiation We showed that is expressed during human epidermal differentiation and that its expression level inreases during the late phase of this differentiation program. Furthermore, using an in vitro model of human tissue-engineered skin, we showed that is phosphorylated in the very late phase of epidermal differentiation. Our results obtained with human keratinoytes are onsistent with the findings of Duverger et al. (24), who reported Hsp25 phosphorylation in the mouse keratinoyte ell line PAM212 indued to differentiate by inreasing the extraellular alium level (Duverger et al., 24), and those of O Shaughnessy et al. (27) showing an AKT-dependent phosphorylation of in murine keratinoyte differentiation (O Shaughnessy et al., 27). Given that alium is an important regulator of epidermal differentiation, phosphorylation ould be an important event during the keratinoyte terminal differentiation proess in vivo. We also found that was present in the pool of ytoskeletal proteins in the late phase of epidermal differentiation when ornifiation ours in a tissue-engineered skin model and in NHKs treated with the alium arrier A2187. A2187 indues ornifiation of NHKs in vitro as shown by the presene of involurin in the ytoskeletal protein fration. Moreover, A2187 is not a soure of ell stress Hsp7 and Hsp6 are not indued in treated ells thus, this phenomenon is related mainly to keratinoyte differentiation. These findings suggest that interats with insoluble proteins in the upper suprabasal layers of the epidermis. Aordingly, the murine analog Hsp25 interats with filaggrin (O Shaughnessy et al., 27). Among these insoluble proteins, keratins are the most abundant. Indeed, keratins oupy the major part of the epithelial ell ytoskeleton and are found in a very insoluble state when paked in their filamentous form. Moreover, keratin filaments are ovalently ross-linked to the ornified envelope during the keratinoyte differentiation proess. Indution of redistribution to the ytoskeletal pool of proteins by DLK Insolubilization of proteins is an important phenomenon in epidermal differentiation. The funtional barrier of the epidermis, the ornified layer, is established after the TGdependent ovalent ross-linking of several proteins and lipids resulting in the formation of the ornified envelope (Dale, 1985; Nemes and Steinert, 1999; Candi et al., 25; Ekert et al., 25). In a previous study, we reported that the expression of DLK alone in human keratinoytes is suffiient to indue TG ativation and ornifiation (Robitaille et al., 25). Here we showed, using putresine as a ompetitor substrate, that redistribution of to the ytoskeletal pool of protein in DLK-expressing keratinoytes is not TG-dependent. The major portion of insoluble proteins in keratinoytes is oupied by keratins that resist the sustained proteasemediated degradation ourring during the formation of the ornified layer. An interation of with keratins during keratinoyte differentiation is possible given that similar interations between intermediate filaments and small Hsps have previously been reported in astroytoma disease as well as in healthy onditions (Wisniewski and Goldman, 1998; Perng et al., 1999). The ornified ell envelope of the differentiated keratinoytes is also very insoluble. In ornifying keratinoytes, ornified envelope preursors are direted toward the ell periphery and ovalently bound by a TG1- dependent mehanism. Thus, the ornified envelope preursors are other potential targets for during keratinoyte terminal differentiation. Results shown in Figure 7 indiate that DLK expression led to a redistribution of from the ytosol to the ell periphery, suggesting an interation between and the ornified envelope omponents. This result is supported by previous findings from eletron mirosopy immunogold labeling showing that oloalizes with keratins and ornified envelope preursors in the granular layer of epidermis (Jonak et al., 22; O Shaughnessy et al., 27). Interestingly, murine was also shown to interat with filaggrin in its phosphorylated state (O Shaughnessy et al., 27). These results ombined with ours strongly suggest that the haperone ativity of in epidermal differentiation is linked to its phosphorylation state. Phosphorylation and insolubilization of by an ERK-dependent pathway Here we report that overexpression of DLK in human keratinoytes indues phosphorylation and reloalization by an ERK-dependent pathway. Several stimuli are known to indue phosphorylation in different ell lines. Furthermore, several studies have reported that the p8 MK2/ pathway is a major induer of phosphorylation. For instane, desmosome signaling indues phosphorylation by a p8 MK2 pathway (Berkowitz et al., 25). Phosphorylation of the three serine residues in human is mediated by MK2/ and PKC (Stokoe et al., 1992; MLaughlin et al., 1996; Maizels et al., 1998). Interestingly, another member of the small-hsp family, ab-rystallin, is phosphorylated on serine 45 by ERK1/2 (Ito et al., 1997; Kato et al., 1998). In keratinoytes, few studies have reported ativation of the ERK1/2 pathway during differentiation. The ERK pathway is well known for its promitoti properties as its ativation orrelates with the indution of several proliferation-assoiated proteins. On the other hand, it was also shown that ERK is transiently ativated when keratinoytes are indued to differentiate by the inreasing extraellular alium level (Shmidt et al., 2). It is now also doumented that not only is the ERK pathway involved in 82 Journal of Investigative Dermatology (21), Volume 1

10 in Keratinoyte Differentiation proliferation but it an also ontribute to the differentiation proess (Shmidt et al., 2; Ebisuya et al., 25; Yoon and Seger, 26). JNK is ativated in the granular layer of the human epidermis (Takahashi et al., 21) and is thought to be atively involved in keratinoyte differentiation. Here we show that DLK ativates both ERK and JNK in human keratinoytes and that the inhibition of the JNK pathway with the speifi inhibitor SP6125 leads to an inreased ERK ativation, suggesting a negative feedbak of JNK on ERK ativation. Suh negative feedbak between MAPK pathways has already been desribed in other systems (Chen et al., 2; Shen et al., 2). As disussed above, the expression of DLK in NHKs leads to a reloalization of to the ell periphery. Our results also show that redistribution is dependent on the ativation of the ERK pathway. Taken together, our results shed light on the moleular events ourring during the late phase of the keratinoyte differentiation program. These results suggest that the insolubilization, phosphorylation, and redistribution of are losely linked and also dependent on the ERK pathway, whih is ativated in response to DLK expression. MATERIALS AND METHODS Cell ulture Normal human keratinoytes were isolated from newborn foreskin as desribed (Germain et al., 2). NHKs were ultured on irradiated T fibroblasts in DME-Ham s F12 medium (Invitrogen, Ontario, Canada) ontaining 5% FetalCloneII serum (Hylone, Logan, UT), insulin (5 mg/ml), hydroortisone (.4 mg/ml) (Calbiohem, LaJolla, CA), 1 1 M holera toxin, epidermal growth fator (1 ng/ml) (Austral Biologials, San Ramon, CA), and antibiotis (peniillin G (1 IU/ml) and gentamiin (25 mg/ml)). HaCaT ells (Boukamp et al., 1988) were ultured in DME ontaining 1% fetal alf serum (Hylone) and antibiotis. In some experiments, 5 mm PD9859 (Calbiohem) and 4 mm SP6125 (Calbiohem) were added to the ulture medium 6 hours after the adenoviral infetion for a 42 hour inubation, to inhibit ERK and JNK, respetively. For indution of differentiation with the A2187 alium arrier, DMSO-diluted alimyin (A2187 (1 mg/ml)) (Calbiohem) was added to the ulture medium of 85% onfluent NHKs. An equal volume of DMSO was added in ontrols. Immunofluoresene staining Aetone-fixed frozen setions (5 mm thik) of normal human skin and tissue-engineered skin, or formaldehyde (.7%) methanol (1%)- fixed ells, were analyzed by immunofluoresene staining as desribed elsewhere (Germain et al., 2). A rabbit antiserum direted against the N-terminal portion of reombinant mouse DLK (Germain et al., 2), phosphorylated (S82) (Cell Signaling Tehnology, Beverly, MA) and, 1/2 (Tanguay et al., 199), and mouse mabs raised against filaggrin (BTI, Stoughton, MA) and (Stressgen, San Diego, CA) were used as primary antibodies, and FITC- or tetramethylrhodamine-onjugated goat anti-mouse and anti-rabbit IgG were used as seondary antibodies (Chemion, Temeula, CA). For the double immunofluoresene staining of and phospho-, deparaffinized formol-fixed setions treated with.1 mm itrate (1 minutes, 9 1C), anti-rabbit biotinylated antibody (Jakson IRL, West Grove, PA), and Alexa-488- onjugated-streptavidin (Moleular Probes, West Baltimore Pike, PA) were used. Negative ontrols onsisted of replaing mabs with isotypi immunoglobulins, or polylonal antibodies with albumin (Sigma). Nulei were labeled with Hoehst dye (Sigma, St Louis, MO). Prodution of in vitro tissue-engineered skin Tissue-engineered skin speimens were produed as desribed (Mihel et al., 1999). Briefly, human fibroblasts were ultured for 5 days in 25 m 2 ulture flasks (BD Biosienes, Mississauga, Ontario, Canada) with fibroblast medium supplemented with 5 mg/ ml of asorbate (Sigma). The resulting sheets (ells extraellular matrix) were superimposed, and NHKs were seeded on top to form tissue-engineered skin by ulturing 7 days in submerged onditions, followed by 21 days at the air liquid interfae. For immunoblot experiments, the epidermis was separated after overnight (4 1C) thermolysin inubation. Adenoviruses and infetion of NHKs Reombinant adenoviruses expressing the ontrol GFP (), alone or together with the T7 epitope-tagged wild-type () or the kinase-defetive ([K185R]) DLK, were used (Robitaille et al., 24). Subonfluent ultures of NHKs or HaCaT ells ultured on plasti substrate were infeted with one or two reombinant adenoviruses at an MOI of 1 (for Figure only) or 5 in DMEM supplemented with 2% (v/v) serum,.8 mm L-arginine, antibiotis. After 1 hour, the medium was replaed with DMEM ontaining 5% serum for 48 hours. Silening of by RNA interferene SiRNA duplex oligonuleotides were synthesized by Thermo Sientifi Dharmaon Produts (Lafayette, CO). For, the strands were sense, 5 -UGAGACUGCCGCCAAGUAAUU-, and antisense, 5 -UUACUUGGCGGCAGUCUCAUU-, and sequenes for the sramble sirna were sense, 5 -UAGCGACUAAACACAUC AAUU-, and antisense, 5 -UUGAUGUGUUUAGUCGCUAUU-. SiRNA transfetion was performed as desribed (Ouellet et al. 26). Briefly, HaCaT ells were ultured in six-well plates. Serum was removed from the ulture medium just before transfetion. HaCaT ells were transfeted with sirna (6 pmol/well) or sramble sirna (6 pmol/well) using Lipofetamine (Invitrogen). After 1 day, serum-free medium was replaed with standard ulture medium or the ells were subsequently infeted with adenoviruses as desribed above. Total or ytoskeletal proteins were extrated days after the sirna transfetion. Inhibition of TG ativity Two hours after adenoviral infetion, monolayer-ultured NHKs were inubated for 1 day with 2 mm putresine (Sigma). The insoluble fration of keratinoyte proteins was extrated 2 days later (see below). Preparation of ell lysates and immunoblotting After ells lysis (6 minutes, 4 1C, in Triton buffer (5 mm Tris-HCl, ph 7.4, 1% Triton X-1, 15 mm NaCl, 5 mm EDTA,.2 mm sodium orthovanadate,.2 mm sodium fluoride, 1 mm phenylmethylsulfonyl fluoride, 1 mg/ml leupeptin, and 1 mg/ml aprotinin)), total extrats were soniated. To extrat the ytoskeletal fration (insoluble pool of protein), ultured ells or tissue-engineered epidermis was rinsed 8

11 in Keratinoyte Differentiation with phosphate-buffered saline and inubated for 15 minutes in Triton buffer followed by 9 minutes in the same buffer ontaining 1.5 M KCl. After entrifugation, the pellet ontaining the ytoskeletal fration was solubilized in Tris-EDTA buffer (Tris-HCL 1 mm, EDTA 1mM) ontaining 1% SDS. Protein onentration was quantified using the MiroBCA protein assay kit (Piere, Rokford, IL). For immunoblotting, equal amounts of proteins were frationated by SDS PAGE and transferred onto nitroellulose membranes (Amersham Pharmaia Bioteh, Baie d Urfé, Québe, Canada) using a transfer apparatus (Bio-Rad, Mississauga, Ontario, Canada). The membranes were bloked in 2 mm Tris, ph 7.5, 15 mm NaCl,.1% Tween-2 ontaining 5% dry milk before addition of the primary antibody (1 hour at room temperature or overnight at 4 1C). The primary antibodies were mouse monolonal anti-phospho-jnk, anti-phospho-p8, anti-phospho-p44/42 ERK (Cell Signaling Tehnology), anti- (Stressgen), anti-involurin (Sigma), anti-keratin 1 (MONOSAN, Uden, The Netherlands), anti-na þ /K þ ATPase a-1 (lone C464.6, Upstate, Temeula, CA), anti-aspase (Clone CSP, Chemion), anti-ae pan-keratin (ICN, Auroa, OH), antiatin (Cedarlane, Burlington, Ontario, Canada), rabbit polylonal anti-jnk total, anti-p8 total, anti-p44/42 ERK total, anti-phospho- (Cell Signaling), anti-hsp6 and anti-hsp7 (Tanguay et al., 199), and anti-keratin 14. The seondary antibodies were antimouse HRP-onjugated (Sigma) and anti-rabbit HRP-onjugated (Chemion). Chemiluminesene of immunoreative bands was deteted with the ECL Plus western blotting kit (Amersham Pharmaia Bioteh). Band quantifiation of tripliate experiments was performed using ImageJ software (Bethesda, MD). Statistial analysis was performed using Student s t-test. Po.5 was onsidered statistially signifiant. CONFLICT OF INTEREST The authors state no onflit of interest. ACKNOWLEDGMENTS We thank Sylvain Guérin and Jaques Landry for their help with the sirna experiment, as well as Jaques Huot for providing HaCaT ells and Véronique Moulin for having given us materials. We also also thank Véronique Couture, Marie-Frane Champigny, Claudia Fugère, Israel Martel, Franis Bisson, Herman Lambert, Yan Nadeau, and Claudie Paquet for tehnial assistane. This study was supported by grants from the Canadian Institutes of Health Researh (CIHR) to LG and RMT and from the Natural Siene and Engineering Researh Counil of Canada to RB. LG holds the Canadian Researh Chair in Stem Cells and Tissue Engineering from CIHR, and HR and CS-B are reipients of a studentship from the Fonds de la Reherhe en Santé du Québe. REFERENCES Beere HM (21) Stressed to death: regulation of apoptoti signaling pathways by the heat shok proteins. Si STKE 21:RE1 Berkowitz P, Hu P, Liu Z, Diaz LA, Enghild JJ, Chua MP et al. 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