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Research Article

RIP2: A novel player in the regulation of keratinocyteproliferation and cutaneous wound repair?

Stephanie Adams1,2, Ralitsa S. Valchanova2, Barbara Munz⁎

Charité-University Medicine Berlin, Institute of Physiology, Arnimallee 22, D-14195 Berlin, Germany

A R T I C L E I N F O R M A T I O N

⁎ Corresponding author. Fax: +49 30 8445 163E-mail address: barbara.munz@charite.de (B

1 Present Address: Experimental and ClinicalLindenberger Weg 80, D-13125 Berlin, Germany2 Both authors contributed equally to this wo

0014-4827/$ – see front matter © 2009 Elseviedoi:10.1016/j.yexcr.2009.12.001

A B S T R A C T

Article Chronology:

Received 15 May 2009Revised version received1 December 2009Accepted 1 December 2009Available online 16 December 2009

We could recently demonstrate an important role of receptor interacting protein 4 (RIP4) in theregulation of keratinocyte differentiation. Now, we analyzed a potential role of the RIP4 homologRIP2 in keratinocytes. Specifically, we demonstrate here that rip2 expression is induced by scratch-wounding and after the induction of differentiation in these cells. Furthermore, serum growthfactors and cytokines can induce rip2, with TNF-α-dependent induction being dependent on p38MAPK. In addition, we demonstrate that scratch-induced upregulation of rip2 expression iscompletely blocked by the steroid dexamethasone. Since we also show that RIP2 is an importantplayer in the regulation of keratinocyte proliferation, these data suggest that inhibition of rip2upregulation after wounding might contribute to the reduced and delayed wound re-epithelialization phenotype seen in glucocorticoid-treated patients.

© 2009 Elsevier Inc. All rights reserved.

Keywords:

RIP2 (receptor interacting protein 2)Keratinocyte proliferationCutaneous wound repair

Introduction

The receptor interacting proteins (RIPs) are mediators of signaltransduction pathways initiated by receptors of the TNFR (tumornecrosis factor receptor) type. After receptor activation, theybecome activated by the receptor's cytoplasmic domain and thustransduce the signal into the cell, which eventually leads to theactivation of transcription factors, such as nuclear factor kappa B(NF-κB), the induction of apoptotic processes, or activation of themitogen activated protein kinase (MAPK) or the protein kinase C(PKC) signal transduction cascades (for review, see [20]).

To date, five RIP proteins (RIP1-5) are known. RIP1-4 share ahighly homologous aminoterminal serine–threonine kinase do-main, their carboxyterminal domains, however, differ: WhereasRIP1 harbours a death domain (DD), RIP2 is characterized by acarboxyterminal CARD (caspase activation and recruitment do-

4.. Munz).Research Center, Charité.

rk.

r Inc. All rights reserved.

main), both of which are involved in the induction of apoptoticprocesses. By contrast, the RIP3 carboxyterminal domain does notdisplay any significant homology to known proteins. RIP4, on theother hand, is characterized by a carboxyterminal ankyrin repeatdomain, a motif which is found in many proteins of variousfunctional subclasses. RIP5 is unique in a way, since its serine-threonine kinase domain is not located at the amino-, but at thecarboxyterminus (for review, see [20]).

All rip genes are expressed in a broad variety of tissues andorgans, their individual temporal and spatial expression patterns,however, are unique (for review, see [20]).

We could recently demonstrate that RIP2 is an importantmodulator of myoblast proliferation and differentiation. Specifi-cally, we could show that downregulation of rip2 expression isabsolutely necessary for myogenic differentiation to proceed.Remarkably, differentiation is abolished by overexpression of the

- University Medicine Berlin and MDC Berlin, Muscle Research Unit,

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rip2 gene alone and the maintenance of high levels of RIP2 proteinafter the induction of differentiation is associated with absent orincomplete differentiation, such as in rhabdomyosarcoma tumorcells [13,22]. These data suggest that RIP2 is an importantcheckpoint in the regulation of proliferation and differentiationin these cells. Thus, we hypothesized that it might also exert thisfunction in other cell types.

Since we could recently demonstrate an important role of theRIP2 homolog RIP4 in epidermal re-epithelialization, specifically inkeratinocyte differentiation [1,29], we aimed at analyzing apotential role of RIP2 in this process.

Cutaneous wound repair is a complex and highly organizedprocess: after skin injury, keratinocytes at the wound edge areinduced to proliferate, migrate into the lesion, and eventuallydifferentiate to form a new epidermis. The whole process is tightlycontrolled by a complex interplay of various factors, such asgrowth factors, cytokines, or proteins of the extracellular matrix,which activate a broad variety of signal transduction pathways (forreview, see [19,28]).

Here, we present evidence that RIP2 is a modulator ofkeratinocyte proliferation and might be an important player inwound re-epithelialization, which might have interesting thera-peutic implications.

Materials and methods

Tissue culture

HumanHaCaT keratinocytes [3]were cultured inDulbecco'smodifiedEagle's medium containing 10% fetal bovine serum supplementedwith penicillin/streptomycin (100 U/ml each). Prior to treatmentwith the respective factors, cells were serum-starved for 16 h.

Primary human keratinocytes (foreskin, Gibco) were cultured inspecial low-Calcium medium (complete defined K-SFM, Gibco)supplemented with penicillin/streptomycin (20 U/ml each). Cellswere passaged before reaching 70% confluency; experiments wereperformed with passages 3 and 4. All tissue culture dishes were pre-coated with human collagen IV (1 μg/cm2, Sigma) according to theinstructions of the manufacturer. Differentiation was induced byaddingCaCl2 (Roth) at a final concentrationof 1.2mMto thedescribedmedium. Prior to induction studies, cells were transferred into non-supplemented defined K-SFM containing penicillin/streptomycin.

For gene expression studies, cells were treated with thefollowing agents: The p38 MAPK inhibitors SB202190 andSB203580, and the negative control SB202474 (10 μM, Calbio-chem), dexamethasone (10−6 M, Sigma), fetal bovine serum (10%,Biochrom), EGF (20 ng/ml, BD Biosciences), KGF (10 ng/ml,Roche), TNF-α (200 U/ml, Sigma), and PMA (100 nM, Sigma).

Scratch-wounding

Confluent keratinocyte monolayers were serum-starved for 16 h.Subsequently, theywere scratchedwith a sterile disposable scalpelto yield wounds with a standardized pattern.

To examine re-epithelialization of wounded keratinocytes invitro, cells were first rinsed with PBS and then fixed with a pre-chilled mix of Methanol and Acetone for 10minutes (1:1,−20 °C).After air-drying, samples were stored at 4 °C and analyzed using aZeiss Axioscope microscope.

BrdU labeling

Cells were seeded in chamber slides, pulse-labeled with bromo-deoxyuridine (BrdU) for 20 min, and stained with the 5′-Bromo-2′-Deoxy Labeling and Detection Kit II (Roche) according to theinstructions of the manufacturer. For nuclear staining, cells weretreated with 4′-6-Diamidino-2-phenylindole (DAPI).

siRNA treatment

For the transfection of HaCaT cells, siRNA specific for the humanrip2 gene was purchased from Ambion. Cells were transfectedaccording to the manufacturer's instructions.

RNA isolation

Total cellular RNAwas extracted with the RNeasy RNA isolation kit(Qiagen) according to the instructions of the manufacturer.

Northern blot analysis

One to eight micrograms of total RNA isolated as described abovewas separated on a 1% agarose gel containing formaldehyde (2%).Subsequently, RNA was transferred to a nylon membrane. Filterswere hybridized overnight with antisense riboprobes, which hadbeen labeled with the digoxigenin RNA labeling kit (Roche). Afterwashing, blots were incubated with an alkaline phosphatase-coupled antidigoxigenin antibody (Roche), washed, and devel-oped with CDP-Star (Roche) as a chemiluminescent substrate foralkaline phosphatase. Signals were detected via exposure to X-rayfilms (ranging from 2 s to 5 min).

Preparation of protein lysates and Western blot analysis

Cultured cells were lysed in lysis buffer (1% Triton X-100, 20 mMTris–HCl pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA pH 8.0).Ten micrograms of total protein were loaded on a sodium dodecylsulfate-polyacrylamide gel and transferred to nitrocellulosemembranes. Membranes were pre-blocked in 3% powdered milkin Tris-buffered saline containing 0.5% Tween 20 (TBS-T) for30 min; incubated with a dilution of the first antibody in blockingsolution for at least 1 h, washed three times with TBS-T; incubatedfor 30 min with a 1:5000 dilution of the second antibody, aperoxidase-conjugated monoclonal antibody (Amersham) inblocking solution; washed three times with TBS-T; and developedwith the ECL Western blot detection system (Amersham).

Results

Rip2 is induced by scratch-wounding and after the inductionof differentiation in keratinocytes

To study rip2 expression after injury, cultured HaCaT keratinocyteswere scratch-wounded and analyzed viaNorthern andWesternBlot.As shown in Fig. 1A, panel (1), rip2 gene expression was induced inthese cells both at the transcriptional and at the translational level,withmRNA levels peaking around 5 h after injury and protein levelspeaking around 24 h. Induction occurred both in the presence and inthe absence of serum (Fig. 1A, panel (2)), and the kinetics of

Fig. 1 – Rip2 is induced after scratch-wounding and during keratinocyte differentiation. (A) HaCaT cells ((1), (2)), and primarykeratinocytes (NHEK, (3)) were scratch-wounded in the presence or in the absence of serum as indicated. At the indicated timepoints, total cellular RNA was extracted from the cells and analyzed for rip2 expression via Northern blot. The results of twoindependent experiments were densitometrically quantificated, normalized to the 28S rRNA, and are displayed schematically.(B) To induce differentiation, HaCaT cells were grown to 100% confluency. At this point, serum was withdrawn from the cells andthey were grown for a further 4 days under these conditions. Total RNA and total cellular protein were extracted at the indicatedtime points and rip2 expression was analyzed via Northern blot (top left panel), and Western blot (left bottom panel). Detection ofthe 28S rRNA band or a Ponceau S stain of the Western blot were used as controls for equal loading, respectively. In addition,the results of two independent Northern blot experiments were densitometrically quantificated, normalized to the 28S rRNA,and are displayed schematically (top right panel). The bottom right panel shows expression of the differentiation markersinvolucrin and TGI in the cells, demonstrating progression of the differentiation process.

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induction was similar. To assess the physiological significance of ourresults, we also scratch-wounded human primary keratinocytes(NHEK). As shown in Fig. 1A, panel (3), similarly as in the HaCaTcells, rip2 expression was induced after scratch-wounding. Thekinetics of induction, however, was different, with upregulation ofrip2 expression persisting for a longer time period in the primarycells. These data demonstrate that scratch-wounding leads to aprominent induction of rip2 gene expression, suggesting that RIP2might be a modulator of the healing process.

Since re-epithelialization of keratinocyte monolayers involvescell proliferation, migration, and differentiation, we first studiedthe question whether rip2 is differentially expressed in keratino-cyte differentiation. Similar to primary keratinocytes, differentia-tion of HaCaT cells can be induced by a switch of low to highcalcium levels in culture media [3], however, this cell type alsoexpresses specific differentiation-associated genes upon reachingconfluency alone [26]. Thus, cultured HaCaT cells were grown toconfluency. Subsequently, serum was withdrawn and cells wereculturedunder these conditions for another 4 days. As shown in Fig.1B, both at the RNA and at the protein level, we could observe a

Fig. 2 – Rip2 is induced by serum growth factors and cytokines in keand then stimulated with whole serum, single growth factors or cyRNA was extracted from the cells and analyzed for rip2 expressionwere densitometrically quantificated, normalized to the 28S rRNA,

prominent downregulation of rip2 expression during the earlystages of the differentiation process. However, at least at themRNAlevel, rip2was induced again during later stages, specifically 3 and4 days after the induction of differentiation. A similar induction ofrip2 expression was observed in primary keratinocytes afterinduction of differentiation via high calcium levels (data notshown), indicating physiological relevance of our results. Thesedata suggest that rip2 gene expression ismodulated in keratinocytedifferentiation, suggesting a role of the RIP2 protein in this process.

Serum growth factors and cytokines can induce rip2expression in keratinocytes

To determine the factors that regulate rip2 expression in keratino-cytes, serum-starved HaCaT cells were treated with whole serum,different growth factors and cytokines, as well as with the tumorpromoter PMA. As shown in Fig. 2, whole serum as well as TNF-αepidermal growth factor (EGF), and PMA led to an induction of rip2expression in these cells, the kinetics and the degree of induction,however,were slightly different. Keratinocyte growth factor (KGF), by

ratinocytes. Confluent HaCaT cells were serum-starved for 16 h,tokines, or PMA as depicted. At the indicated time points, totalvia Northern blot. The results of two independent experimentsand are displayed schematically.

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contrast, was only aweak inducer of rip2 gene expression. In addition,several other growth factors and cytokines, which are important incutaneouswound repair, such as hepatocyte growth factor (HGF), hadno effect on rip2 gene expression (data not shown). These datademonstrate that rip2 is induced by a broad variety of growth factorsand cytokines, as well as by PMA, in quiescent keratinocytes.

TNF-α-mediated rip2 induction is dependent on p38 MAPK

To get insight into the signaling pathways that play a role in theregulation of rip2 gene expression, quiescent HaCaT cells wereagain stimulated with TNF-α, EGF, or whole serum, or they werescratch-wounded to induce rip2 expression. Prior to treatment,cells were treated with specific inhibitors of the p38 MAPKpathway. As shown in Fig. 3, specific p38 MAPK inhibitors indeedexerted an inhibitory effect on TNF-α-mediated rip2 induction.Remarkably, however, the effect of the inhibitor SB202190 wasstronger than that of SB203580, suggesting differential effects ofthe two inhibitors on keratinocytes. By contrast, they had no effecton EGF-, serum-, or scratch-induced rip2 induction. These datasuggest that TNF-α-, but not EGF-, serum-, or scratch-induced rip2induction is p38 MAPK-dependent.

Dexamethasone completely abolishes scratch-inducedupregulation of rip2 expression

From the background that RIP2 might be involved in theregulation of wound re-epithelialization, we wondered if condi-tions of reduced and delayed re-epithelialization might be

Fig. 3 – Rip2 induction by TNF-α and serum is p38MAPK-dependent.then treated with the p38MAPK inhibitors SB202190 or SB203580, owere stimulated with TNF-α, EGF, or whole serum, or they were scextracted from the cells and analyzed for rip2 expression via Northdensitometrically quantificated, normalized to the 28S rRNA, and a

associated with a failure to upregulate rip2 expression afterinjury. To study this question, we chose dexamethasone-treatedHaCaT keratinocytes as a model system. It has been known for along time that topical as well as systemical glucocorticoidtreatment leads to reduced and delayed wound re-epithelializa-tion in human patients, the underlying mechanism, however, isstill not completely understood. As shown in Fig. 4, dexameth-asone treatment indeed completely abolished rip2 induction inresponse to scratch-wounding. These data suggest that thephenotype of reduced and delayed wound re-epithelializationseen in glucocorticoid-treated patients might be associated witha failure to upregulate rip2 expression in response to injury. Asshown in Fig. 4, we have also analyzed expression of anothergene, which is not repressed by glucocorticoids, in dexametha-sone-treated, scratch-wounded HaCaT cells. For this purpose, wechose the ZFP36L1 gene, which encodes a zinc finger proteininvolved in the regulation of cytokine and growth factortranscript stability [4,8,17]. We show that this gene–similarly asthe rip2 gene–is induced by scratchwounding (Hacker, C., Adams,S., and Munz, B., submitted), however–in contrast to the rip2gene–not repressed by parallel treatment of the cells withdexamethasone, indicating that repression by glucocorticoids isnot a general phenomenon affecting all genes that are normallyinduced by scratching. In addition, we show that expression ofthe rip4 gene, which is normally downregulated by scratch-wounding [1], is no longer repressed when cells are treated withglucocorticoids, indicating that different rip genes are regulatedby scratch-wounding and by dexamethasone in a unique andspecific manner (Adams and Munz, Exp. Cell Res., in revision).

Confluent HaCaT cells were serum-starved overnight. Theywerer the negative control SB22474, respectively. Subsequently, cellsratch-wounded as depicted. After 5 h, total cellular RNA wasern blot. The results of two independent experiments werere displayed schematically.

Fig. 4 – Dexamethasone inhibits rip2 induction upon scratch-wounding. Confluent HaCaT cells were scratch-wounded. Prior totreatment, they were stimulated with the glucocorticoid dexamethasone. (1) At the indicated time points, total cellular RNA wasextracted from the cells and analyzed for rip2 expression via Northern blot. Expressions of the rip4 and the ZFP36L1 gene wereanalyzed in the same cells. The 28S rRNA band is shown as a control for equal loading. (2) Histological examination of the cellsrevealed that dexamethasone completely blocked wound closure (right hand side), whereas in untreated control cells, the woundwas almost completely healed after 48 h (left photograph).

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RIP2 is a stimulator of keratinocyte proliferation

Wound re-epithelialization involves keratinocyte proliferation,differentiation, andmigration. Thus, to further determine a potentialrole of RIP2 in this process, we functionally analyzed the effects ofRIP2 depletion in keratinocytes. For this purpose, cultured HaCaTcells were treated with rip2-specific siRNAs. As shown in Fig. 5A,RIP2protein levelswere reducedbyaround90%within24–48haftertreatment, whereas transfection of a control (“scrambled”) siRNAhad no effect. To study the effects of RIP2 depletion on keratinocyteproliferation, siRNA-treated cells were analyzed for expression ofthe proliferationmarker Ki-67 byWestern blot analysis. As shown inFig. 5B, Ki-67 levels were strongly diminished 48 h after siRNAtreatment in comparison with untransfected controls or cells thathad been transfected with the scrambled siRNA. To quantify theeffects of RIP2depletion onkeratinocyteproliferation, siRNA-treatedcells were pulse-labeled with bromodeoxyuridine (BrdU) 24 and48 h after the transfection procedure. As shown in Fig. 5C andconsistent with the Ki-67 Western blot, statistical analysis showedthat RIP2 depletion indeed strongly inhibited keratinocyte prolifer-ation. Since we could recently demonstrate an important role of theRIP2 homolog RIP4 in the regulation of keratinocyte differentiation[1], and since the rip2 gene was differentially during HaCaT celldifferentiation in vitro (our data), we hypothesized that RIP2 mightalso be a crucial player in this process. To test this hypothesis, siRNA-treated cells were analyzed for the expression of a broad variety of“early” and “late” keratinocyte differentiation markers, such as

different keratins, involucrin, or transglutaminase. However, wecould not detect any effect of RIP2 depletion on the expression ofthese markers (Fig. 5D and data not shown). However, we coulddetect a prominent effect of lowRIP2 levels onwoundclosure in vitro(Fig. 5E), which was reminiscent of the inhibitory effect ofdexamethasone (Fig. 4)—and indicates that this effect might atleast in part be mediated by RIP2. However, when compared to thedexamethasone-treated cells, wound healing in the rip2 siRNA-transfected cells was more “irregular,” with regions of almost noreepithelialization after 1–2 days, but also regions which resembledthe scrambled- or the mock transfected cells. Also, in contrast to thedexamethasone-treated cells, the effects were not detectable anymore at later time points. These findings are probably due to the factthat (a) siRNA transfection never reaches all cells, (b) a positiveselection for untransfected cells (due to their higher proliferationrate) is likely, and (c) to the fact that the inhibitory effect of thesiRNA ceaseswith time. Taken together, these data suggest that RIP2is a stimulator of keratinocyte proliferation, however, has no effecton the differentiation behaviour of these cells.

Discussion

Previous results from us and others could demonstrate animportant role of RIP4 in keratinocyte differentiation and incutaneous wound repair [1,15,29]. Here, we analyze the role of theRIP4 homolog RIP2 in keratinocytes.

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Whereas rip4 expression is downregulated after scratch-wounding of keratinocyte monolayers, we demonstrate here thatthe rip2 gene shows an inverse expression kinetics: whereas RIP2

Fig. 5 – Inhibition of rip2 expression inhibits keratinocyte proliferatkeratinocytes were treated with a rip2-specific siRNA, a scrambledtransfection, cells were analyzed for rip2 expression byWestern blotblot is shown top panels). Furthermore, rip2 expression after siRNArRNA band is shown as a control for equal loading (bottom panels).proliferation marker Ki67 was determined in the cells by Westerncontrol for equal loading. (C) Cellular proliferation rate was determthe rip2-specific siRNA or the scrambled control. The total percentBrdU-labeled nuclei vs. the total number of nuclei as determined vkeratin K14, and the differentiation-associated keratin K10was anatransfection via Northern blot analysis. The 28S rRNA band is showkeratinocytes were transfected with a rip2-specific siRNA or a “scrwound closure was analyzed histologically after 45 h and quantita

levels are low in quiescent keratinocytes, they are strongly inducedafter scratching and remain high for several hours thereafter. Thesedata suggest that RIP2 might be important for the healing process,

ion, but has no effect on keratinocyte differentiation. (A) HaCaTcontrol, or left untreated as indicated. 1 day and 2 days after. As a control for equal loading, a Ponceau S staining of the sametreatment was analyzed by Northern blot after 1 day. The 28S

(B) 1 day and 2 days after transfection, the concentration of theblot analysis. A Ponceau S stain of the same blot is shown as ained via BrdU labeling 1 day and 2 days after transfection withage of proliferating cells was calculated as the proportion ofia DAPI staining. (D) Expression of the proliferation-associatedlyzed in siRNA-treated HaCaT cells and in control cells 1 day aftern as a control for equal loading as indicated. (E) HaCaTambled” control. Transfected cells were scratch-wounded andted using the method described in ref. [14].

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either as a regulator of keratinocyte proliferation, migration ordifferentiation.

A role of RIP2 in the regulation of keratinocyte proliferationor differentiation is further supported by the finding that RIP2 levelsare strongly reduced after serumwithdrawal in confluent keratinocytes.

Since we could demonstrate that a broad variety of serumgrowth factors and cytokines canmodulate rip4 gene expression inkeratinocytes, we hypothesized that these factors, which arepresent at high concentrations in wound tissue, might also beregulators of rip2 expression in these cells. However, when wetested a broad variety of different factors, we could identify onlyEGF and the proinflammatory cytokine TNF-α as potent inducersof rip2 expression in these cells. These data suggest that rip2expression is controlled only by very few, quite specific, factors, incontrast to rip4, whose expression is regulated by most of theclassical wound healing growth factors and cytokines.

From this background, we aimed at identifying the pathwayswhich might be responsible for the induction of rip2 expression inkeratinocytes. Primarily, we focused on the p38 MAPK pathwaywhich is an important modulator of the stress-induced cellularresponse in keratinocytes [11]. Surprisingly, we could demonstratethat only the TNF-α-dependent, but not the EGF- or scratch-wounding-induced rip2 upregulation was dependent on p38 MAPK.In addition, it is of note that the inhibitory effect of the p38 MAPKinhibitor SB202190 on rip2 expression was much stronger than thatof SB203580. Most likely, this is due to the fact that in contrast toSB202190, SB203580 is an inhibitor of p38 α and β, but does notinhibit p38 δ [9,16]. In keratinocytes, the genes encoding the p38isoforms α β and δ are expressed [10]. Thus, our data suggest thatparticularly p38 δ might be involved in the induction of rip2expression by TNF-α. Interestingly, a similar picture is seen inkeratinocyte differentiation,which is exclusively dependent onp38 δwith no activation of the other p38 isoforms [12].

In the future, it will be interesting to determine which otherpathways are responsible for the modulation of rip2 expression inepidermal cells.

Based on these results, we speculated that aberrant regulation ofrip2 expression after injury might be associated with conditions ofreduced and delayed re-epithelialization. To test this hypothesis,HaCaT keratinocyteswere treatedwith the steroid dexamethasone.The wound healing phenotype of glucocorticoid-treated patients,specifically the inhibition of re-epithelialization, have been knownfor a long time, the underlying mechanisms, however, are still notcompletely understood. We demonstrate here that in dexameth-asone-treated keratinocytes, rip2 gene expression is not inducedafter scratch wounding. These data suggest that elevated levels ofRIP2might be important for normal re-epithelialization after injury.In the future, it will be interesting to test whether overexpression ofthe rip2 gene in keratinocytes might lead to at least partialcompensation of the re-epithelialization failure after glucocorticoidtreatment, which might have interesting therapeutic implications.

With respect to the function of RIP2 in keratinocytes–specifically after wounding–we hypothesized that the proteinmight be involved either in the regulation of keratinocyteproliferation and/or differentiation. To test this hypothesis,cultured keratinocytes were transfected with rip2-specific siRNAsand the proliferation and differentiation behaviour of the cells wasanalyzed. Most interestingly, we could detect a strong inhibitoryeffect of RIP2 depletion on keratinocyte proliferation. By contrast,keratinocyte differentiation was not affected. These data do not

exclude a role of RIP2 in the regulation of differentiation, theymerely suggest that downregulation is not sufficient to inducedifferentiation. For example, it is possible that the loss of RIP2protein is compensated for by the upregulation of functionallysimilar proteins. Nevertheless, this result is different from the dataobtained for RIP4 with a corresponding approach: RIP4 depletionaffected the expression of specific keratinocyte differentiationmarkers, however, cellular proliferation was not altered [1], whichis again consistent with the in vivo approach of [15], who describedisturbed epidermal differentiation, but normal keratinocyteproliferation, in RIP4-deficient mice. These data suggest that RIP2and RIP4 are not functionally redundant, but have clearly distinctfunctions in keratinocytes and in epidermal repair.

The kinetics of rip2 upregulation very early after skin injury andits induction after scratch-wounding suggest that this proteinmightalso be a regulator of keratinocyte migration. Interestingly, severalreports demonstrate that the growth factor EGF, which can inducerip2 expression in HaCaT cells (our study) also stimulates keratino-cytemigration in vitro and in vivo [2,7,23]. Furthermore, the cytokineTNF-α, which also induces rip2 expression in HaCaT cells (thisstudy), had a positive effect on keratinocytemigration in vitro and invivo in healing wounds [27]. Thus, it is possible that rip2, as a targetgene of these factors, is important for proper keratinocytemigrationin healing wounds, thereby modulating re-epithelialization.

In the future, it will be interesting to study the signaltransduction pathways which are controlled by RIP2 in keratino-cytes and eventually lead to the protein's effects on keratinocyteproliferation and perhaps migration. An interesting candidate isthe NF-κB pathway: previous studies could show that RIP2 is anactivator of NF-κB when overexpressed in vitro (for review, see[20]). Nevertheless, we did not find altered NF-κB activity in rip2siRNA-transfected HaCaT cells (S. Adams and B. Munz, unpub-lished results). However, these studies will have to be repeated inprimary keratinocytes, since immortalized keratinocytes, such asHaCaT cells, are characterized by abnormal NF-κB signaling invarious experimental settings [5,6,18,21,24,25]. Furthermore,since RIP2 is also a Raf1-activated MAPKK (for review, see [20]),it will be interesting to determine whether this pathway also playsa role in keratinocytes. Specifically, we are currently analyzingERK1/2 activity in keratinocytes that had been treated with rip2-specific siRNAs. Remarkably, Fitsialos et al. [14], could alreadydemonstrate an important role of ERK1/2 activation in theregulation of wound closure after in vitro scratching.

Taken together, our data indicate that RIP2 is an importantfactor in the regulation of keratinocyte proliferation and epidermalrepair, which might have interesting therapeutic implications. Inthe future, it will be interesting to determine whether induction ofrip2 gene expression is important for normal healing. Because ofthe very complex immunological phenotype of completely rip2-deficient animals (for review, see [20], the generation of skin-specific rip2 knockouts will be necessary for these studies.

Acknowledgments

We thank Renate Noske-Reimers and Gabi Beyer for excellenttechnical assistance, and Sabine Werner, ETH Zurich, for stimulat-ing discussions, suggestions, and advice. This work was supportedby grants from the Deutsche Forschungsgemeinschaft (Mu 1556 /3-1) and from the Sonnenfeld-Stiftung, Berlin, Germany (to BM).

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R E F E R E N C E S

[1] S. Adams, S. Pankow, S. Werner, B. Munz, Regulation ofNF-kappaB activity and keratinocyte differentiation by the RIP4protein: implications for cutaneous wound repair, J. Invest.Dermatol. 127 (2007) 538–544.

[2] Y. Ando, P.J. Jensen, Epidermal growth factor and insulin-likegrowth factor I enhance keratinocyte migration, J. Invest.Dermatol. 100 (1993) 633–639.

[3] P. Boukamp, R. Petrussevska, D. Breitkreutz, J. Hornung, A.Markham, N.. Fusenig, Normal keratinization in a spontaneouslyimmortalized aneuploid human keratinocyte cell line, J. Cell Biol.106 (1988) 761–771.

[4] E. Carballo, W.S. Lai, P.J.. Blackshear, Evidence that tristetraprolinis a physiological regulator of granulocyte-macrophagecolony-stimulating factor messenger RNA deadenylation andstability, Blood 95 (2000) 1891–1899.

[5] V. Chaturvedi, J.-Z. Qin, M.-F. Denning, D. Choubey, M.O. Diaz, B.J.Nickoloff, Apoptosis in proliferating, senescent, and immortalizedkeratinocytes, J. Biol. Chem. 274 (1999) 23358–23380.

[6] V. Chaturvedi, J.-Z. Qin, M.-F. Denning, D. Choubey, M.O. Diaz, B.J.Nickoloff, Abnormal NF-kB signalling pathway with enhancedsusceptibility to apoptosis in immortalized keratinocytes,J. Dermatol. Sci. 26 (2001) 67–78.

[7] J.D. Chen, J.P. Kim, K. Zhang, Y. Sarret, K.C. Wynn, R.H. Kramer, D.T.Woodley, Epidermal growth factor (EGF) promotes humankeratinocyte locomotion on collagen by increasing the alpha 2integrin subunit, Exp. Cell Res. 209 (1993) 216–223.

[8] D. Ciais, N. Cherradi, S. Bailly, E. Grenier, E. Berra, J. Pouyssegur, J.Lamarre, J.J. Feige, Destabilization of vascular endothelial growthfactor mRNA by the zinc-finger protein TIS11b, Oncogene 23(2004) 8673–8680.

[9] A. Cuenda, P. Cohen, V. Buee Scherrer, M. Goedert, Activation ofstress-activated protein kinase-3 (SAPK3) by cytokines andcellular stresses is mediated via SAPKK3 (MKK6); comparison ofthe specificities of SAPK3 and SAPK2 (RK / p38), EMBO J. 16(1997) 295–305.

[10] S.R. Dashti, T. Efimova, R.L.. Eckert, MEK7-dependent activation ofp38 MAP kinase in keratinocytes, J. Biol. Chem. 276 (2001)8059–8063.

[11] R.L. Eckert, T. Efimova, S. Balasubramanian, J.F. Crish, F. Bone, S.Dashti, p38 Mitogen-activated protein kinases on the bodysurface—a function for p38[delta], J. Invest. Dermatol. 120 (2003)823–828.

[12] T. Efimova, A.M. Broome, R.L. Eckert, A regulatory role for p38delta MAPK in keratinocyte differentiation. Evidence for p38delta–ERK 1/2 complex formation, J. Biol. Chem. 278 (2003)34277–34285.

[13] S. Ehlers, T. Mueck, S. Adams, L. Landuzzi, P.L. Lollini, B. Munz,RIP2 regulates growth and differentiation of normal myoblastsand of rhabdomyosarcoma cells, Eur. J. Cell. Biol. 87 (2008)163–172.

[14] G. Fitsialos, A.A. Chassot, L. Turchi, M.A. Dayem, K. LeBrigand, C.Moreilhon, G. Meneguzzi, R. Buscà, B. Mari, P. Barbry, G. Ponzio,Transcriptional signature of epidermal keratinocytes subjected toin vitro scratch wounding reveals selective roles for ERK1/2, p38,

and phosphatidylinositol 3-kinase signaling pathways, J. Biol.Chem. 282 (2007) 15090–15102.

[15] P. Holland, C. Willis, S. Kanaly, M. Glaccum, A. Warren, K. Charrier,J. Murison, J. Derry, G. Virca, T. Bird, J. Peschon, RIP4 is anankyrin repeat-containing kinase essential for keratinocytedifferentiation, Curr. Biol. 12 (2002) 1424–1428.

[16] S. Kumar, P.C. McDonnell, R.J. Gum, A.T. Hand, J.C. Lee, P.R. Young,Novel homologues of CSBP / p38 MAP kinase: activation,substrate specificity and sensitivity to inhibition by pyridinylimidazoles, Biochem. Biophys. Res. Commun. 235 (1997)533–538.

[17] W.S. Lai, E. Carballo, J.M. Thorn, E.A. Kennington, P.J. Blackshear,Interactions of CCCH zinc finger proteins with mRNA. Binding oftristetraprolin-related zinc finger proteins to Au-rich elementsand destabilization of mRNA, J. Biol. Chem. 275 (2000)17827–17837.

[18] D.A. Lewis, S.F. Hengeltraub, F.C. Gao, M.A. Leivant, D.F. Spandau,Aberrant NF-κB activity in HaCaT cells alters their response toUVB signalling, J. Invest. Dermatol. 126 (2006) 1885–1892.

[19] P. Martin, Wound healing—aiming for perfect skin regeneration,Science 276 (1997) 75–81.

[20] E. Meylan, J. Tschopp, The RIP kinases: crucial integrators ofcellular stress, Trends Biochem. Sci. 30 (2005) 151–159.

[21] C. Muller, M. Bektas, C.C. Geilen, Differential involvement ofceramide in TNFalpha-mediated activation of NF kappa B inprimary human keratinocytes and HaCaT keratinocytes, Cell. Mol.Biol. 49 (2003) 399–407.

[22] B. Munz, E. Hildt, M.L. Springer, H.M. Blau, RIP2, a checkpoint inmyogenic differentiation, Mol. Cell. Biol. 22 (2002) 5879–5886.

[23] B.J. Nickoloff, R.S. Mitra, B.L. Riser, V.M. Dixit, J. Varani, Modulationof keratinocyte motility. Correlation with production ofextracellular matrix molecules in response to growth promotingand antiproliferative factors, Am. J. Pathol. 132 (1988)543–551.

[24] J.-Z. Qin, V. Chaturvedi, M.F. Denning, D. Choubey, M.O. Diaz, B.J.Nickoloff, Role of NF-κB in the apoptotic-resistant phenotype ofkeratinocytes, J. Biol. Chem. 274 (1999) 37957–37964.

[25] Q. Ren, C. Kari, M.R.D. Quadros, R. Burd, P. McCue, A.P. Dicker,Malignant transformation of immortalized HaCaT keratinocytesthrough deregulated nuclear factor kB signalling, Cancer Res. 66(2006) 5209–5215.

[26] C.M. Ryle, D. Breitkreutz, H.J. Stark, I.M. Leigh, P.M. Steinert, D.Roop, N.E. Fusenig, Density-dependent modulation of synthesis ofkeratins 1 and 10 in the human keratinocyte line HACAT and inras-transfected tumorigenic clones, Differentiation 40 (1989)42–54.

[27] K.A. Scott, C.H. Arnott, S.C. Robinson, R.J. Moore, R.G. Thompson,J.F. Marshall, F.R. Balkwill, TNF-alpha regulates epithelialexpression of MMP-9 and integrin alphavbeta6 during tumourpromotion. A role for TNF-alpha in keratinocyte migration,Oncogene 23 (2004) 6954–6966.

[28] S. Werner, R. Grose, Regulation of wound healing by growthfactors and cytokines, Physiol. Rev. 83 (2003) 835–870.

[29] S. Adams, B. Munz, RIP4 is a target of multiple signal transductionpathways in keratinocytes: implications for epidermal differen-tiation and cutaneous wound repair, Exp. Cell Res. 316 (2010)126–137.