rip4 is a target of multiple signal transduction pathways in keratinocytes: implications for...

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Research Article

RIP4 is a target of multiple signal transduction pathways inkeratinocytes: Implications for epidermal differentiation andcutaneous wound repair

Stephanie Adams1, 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: [email protected] (B

1 Present address: Experimental and ClinicalLindenberger Weg 80, D-13125 Berlin, Germany

0014-4827/$ – see front matter © 2009 Elseviedoi:10.1016/j.yexcr.2009.10.006

A B S T R A C T

Article Chronology:

Received 17 June 2009Revised version received

2 October 2009Accepted 2 October 2009Available online 8 October 2009

Receptor interacting protein 4 (RIP4) is an important regulator of epidermal morphogenesisduring embryonic development. We could previously show that expression of the rip4 gene isstrongly downregulated in cutaneous wound repair, which might be initiated by a broad variety of

growth factors and cytokines. Here, we demonstrate that in keratinocytes, rip4 expression iscontrolled by a multitude of different signal transduction pathways, such as the p38 mitogen-activated protein kinase (MAPK) and the nuclear factor kappa B (NF-κB) cascade, in a unique andspecific manner. Furthermore, we show that the steroid dexamethasone abolishes thephysiological rip4 downregulation after injury and might thus contribute to the phenotype ofreduced and delayed wound reepithelialization seen in glucocorticoid-treated patients. As awhole, our data indicate that rip4 expression is regulated in a complex manner, which might havetherapeutic implications

© 2009 Elsevier Inc. All rights reserved.

Keywords:

Receptor interacting protein 4 (RIP4)KeratinocytesCutaneous wound repair

Introduction

The receptor interacting proteins (RIPs) were initially identifiedas adapter proteins of receptors of the tumor necrosis factor-1(TNFR-1) type. Currently, five RIP proteins have been identified.Whereas they all share a homologous N-terminal serine-threoninekinase domain, their C-terminal domains, however, differ and arecharacteristic for each individual RIP protein: Specifically, RIP1harbours a death domain (DD) and RIP2 a caspase activation andrecruitment domain (CARD), which are both known to be involvedin the modulation of apoptotic processes. By contrast, the C-terminal domain of RIP3 shows no obvious homology to otherproteins. RIP4 is characterized by a carboxyterminal ankyrin repeatdomain. RIP5 is unique in a way, since this recently identifiedfamily member also harbours an ankyrin repeat domain, which is,

4.. Munz).Research Center, Charité,.

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however, localized at the aminoterminus, whereas the kinasedomain shows C-terminal localization. All RIP proteins have beenimplicated in the activation of the transcription factor nuclearfactor kappa B (NF-κB) as well as the induction of apoptoticprocesses (for review, see [1]).

In addition, RIP proteins are regulators of a broad variety ofother signal transduction cascades, such as the mitogen-activatedprotein kinase (MAPK) pathway [2–4]. Hence, they modulate cellproliferation and differentiation in a broad variety of cell types andtissues [1]. Specifically, RIP1 could be identified as an essentialintegrator in the anti-apoptotic signal transduction pathwayinitiated by TNFR activation [5]. By contrast, RIP2 appears to bean important modulator of the innate and adaptive immuneresponse [6,7], and furthermore plays a fundamental role inmyogenic differentiation [8,9]. Whereas the precise function of

University Medicine Berlin and MDC Berlin, Muscle Research Unit,

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RIP3 still remains unclear, RIP4 is a crucial factor in embryonicdevelopment of the epidermis, which [10] could demonstrateusing rip4-deficient mice. By contrast, with respect to RIP5, nofunctional data are available to date [11].

Cutaneous wound repair requires a tight regulation ofkeratinocyte proliferation, migration and differentiation. This isperformed by a complex system of growth factors and cytokines,which bind to various types of specific receptors and thus initiate acomplex network of characteristic signal transduction pathways[12,13]. Since RIP proteins are important modulators of specificsignal transduction pathways, we had previously analyzed thepattern of rip gene expression in cutaneous wound healing. Inthese studies, we demonstrated an important participation of RIP4in the regeneration process of wounded skin as well as inregulation of keratinocyte differentiation. In particular, we couldshow that interference of rip4-RNA results in a disorderedexpression of genes which are associated with keratinocytedifferentiation [14].

To get further insight into the role of RIP4 in keratinocytes, wenow analyzed the temporal and spatial expression pattern of therip4 gene in differentiating keratinocytes in vivo and in vitro. Inaddition, since our previous studies had shown that rip4 geneexpression is regulated by a broad variety of growth factors andcytokines in keratinocytes, we also studied the question whichsignal transduction pathways might be major players in thiscomplex network. As a whole, we present in vivo and in vitro datawhich suggest that RIP4 might play a role in keratinocytedifferentiation. Furthermore, we demonstrate that rip4 geneexpression is regulated by a finely tuned network of signaltransduction pathways in these cells. Finally, we provide evidencethat aberrant regulation of rip4 gene expression might beassociated with an abnormal pattern of epidermal reepithelializa-tion after injury.

Results

Expression of the rip4 gene is associated with keratinocytedifferentiation

Previously, we reported a strong expression of the rip4 gene inintact murine back skin [14]. However, the exact localization of theRIP4 protein within the epidermis was not known yet. Sincemurine back skin is very thin and displays no obvious stratifica-tion, we used tail skin, which more closely resembles the multi-layered human epidermis, as a model system. As shown in Fig. 1A,using a RIP4-specific antibody, we could detect a strong signal bothin the basal and suprabasal layers of the epidermis. In addition, theRIP4-specific signal was detected mostly in the cytoplasmiccompartment of keratinocytes in all epidermal layers, suggestingpredominant or exclusive cytoplasmic localization of the respec-tive protein (Fig. 1A). As a whole, these data suggest that RIP4might play an important role in the regulation of keratinocyteproliferation and/or differentiation, however, further functionalstudies have to be carried out to prove this.

From this background, we aimed at getting further insight intothe pattern of rip4 gene expression during keratinocyte differen-tiation. As a model system, we used human primary keratinocytes(NHEK), which can be induced to differentiate in vitro by highcalcium levels. As shown in Fig. 1B, we found that this treatment

gradually repressed rip4 expression, suggesting that this gene isindeed downregulated during epidermal differentiation.

Rip4 gene expression is modulated by the p38-MAPKpathway in keratinocytes

We have previously demonstrated regulation of rip4 geneexpression by growth factors and cytokines in keratinocytes.Specifically, rip4 expression was high in quiescent cells and low inthe presence of serum or single growth factors and cytokines [14].Now, we aimed at analyzing the underlying signal transductionmechanisms. In particular, we studied two different pathwayswhich play a central role in keratinocytes, for their potential tomodulate rip4 gene expression: On the one hand, we tested theprotein kinase C (PKC)-signaling pathwaywhich plays a key role inkeratinocyte differentiation [15], on the other hand, we focused onthe p38-MAPK pathway which is an important modulator of thestress-induced cellular response in these cells [16].

For this purpose we used the p38 inhibitor SB202190 (and thestructurally similar, but inactive control substance SB202474), andthe PKC inhibitor GF 109203X (and the structurally similar, butinactive control substance RO-31-6045).

First, using these inhibitors, we studied participation of bothpathways in the induction of rip4 gene expression, which isobserved after serum withdrawal [14]. As shown in Fig. 2A, wecould demonstrate a prominent effect of p38MAPK in this process.By contrast, PKC does not appear to be involved (Fig. 2A).

Previously, we could also demonstrate extensive regulation ofrip4 gene expression in keratinocytes in response to scratch-wounding. Specifically, rip4 expression is strongly downregulatedafter wounding of these cells [14]. Thus, we next investigated apossible participation of the PKC- and the p38-MAPK signalingpathways in this process. As shown in Fig. 2B, in quiescent aswell asin serum-stimulated cells, the p38-specific inhibitor SB 202190 hada moderate effect on rip4 expression after injury. However, thiseffect was confined to the early stages (up to 2 h) after woundingand was no longer detectable at later stages (data not shown).

To further define the role of p38-MAPK in the regulation of rip4gene expression,we focused on the effects of this kinase on rip4 generepression induced by growth factors and cytokines. We hadpreviously shown that a broad variety of these factors can repressrip4 gene expression in keratinocytes [14]. As shown in Fig. 2C, wecould demonstrate an important role of the p38-MAPK pathway onserum-induced as well as on tumor necrosis factor-alpha (TNF-α)-induced rip4 gene repression. By contrast, p38-MAPK inhibitors hadno effect on epidermal growth factor (EGF)- and transforminggrowth factor beta (TGF-β)-mediated repression of rip4 geneexpression (Fig. 2C). Furthermore, we could not demonstrate a roleof the PKC-signal transductionpathway in the cytokine- andgrowth-factor induced regulation of rip4 gene expression (Fig. 2D). Bycontrast, phorbol 12-myristate 13-acetate (PMA)-induced repres-sion of the rip4 genewasmediated via PKC-signaling, demonstratingspecificity and activity of the respective inhibitor. However, p38-MAPK was dispensable for gene regulation here (Fig. 2E).

Repression of rip4 gene expression can be abolished byCurcumin in a dose-dependent manner

Since the ubiquitous transcription factor NF-κB is a target ofmultiple signal transduction pathways, we next analyzed the

Fig. 1 – Rip4 gene expression in differentiating keratinocytes in vivo and in vitro. (A) Localization of RIP4 in murine tail and ear skinwas determined by immunofluorescence analysis using a specific antibody raised against the C-terminal domain of themurine RIP4protein. Strong cytoplasmic RIP4 staining was detected throughout all epidermal layers. (B) Expression of the rip4 gene in humanprimary keratinocytes in vitro was studied by Northern blot analysis. Cells were cultured until reaching 90% of confluency, then,differentiation was induced by application of 1.2mMCaCl2. After isolation of total RNA at defined time points, equal amounts (2 μg)were analyzed for the expression of the rip4 gene. Staining of the 28S rRNA was used as a loading control in all Northern blots asindicated and used for normalization of signal intensity when quantifying individual bands of two independent experiments(bottom panel).

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role of this factor in the regulation of rip4 gene expression.Inhibition of NF-κB activity was obtained using the inhibitorCurcumin, a pharmacologically active extract of curcuma longa

Fig. 2 – Participation of the p38 MAPK and the PKC pathway in thep38-MAPK- or the PKC signalling pathway in the regulation of rip4202190 is a specific inhibitor for p38 MAPK, SB 202474 is a non-funRO-31-6045 is a non-functional control. DMSO is a solvent control.HaCaT-cells (60%) were transferred into serum-freemedium. After 2pathways on expression of the rip4 gene. To analyze repression of thwith selected growth factors and cytokines (C, D). Participation of trip4 gene (B) as well as after serum- and TNF-α- induction (C I, II),(D I–V). By contrast, PMA-induced repression of the rip4 gene wasfor this effect (E). Gene expression was determined via Northern blblots, Staining of the 28S rRNA was used as a loading control as indquantifying individual bands of two independent experiments (dis

[17]. As shown in Fig. 3A, using this inhibitor, we coulddemonstrate a dose-dependent inhibition of NF-κB activity inHaCaT-cells. Specifically, concentrations above 25 μM Curcumin

regulation of rip4 gene expression. Participation of either thegene expression was analyzed using specific inhibitors. SBctional control. GF 109203X is a specific PKC inhibitor,(A) To induce expression of the rip4 gene, subconfluent4 h, total RNAwas obtained and analyzed for the effects of bothe rip4 gene, confluent HaCaT-cells were scratched (B) or treatedhe p38 MAPKwas observed in scratch-induced repression of thewhereas the PKC-pathway was dispensable for these effectsmediated by PKC isoforms, whereas p38 MAPK was dispensableot analysis using a rip4-specific antisense probe. In all Northernicated and used for normalization of signal intensity whenplayed as bar graphs).


exerted potent inhibitory effects on TNF-α-induced NF-κBactivation. However, cytotoxic effects were observed alreadyat a concentration range as low as 70 μM (data not shown).With respect to the impact of NF-κB activity on the repressionof rip4 gene expression normally seen after TNF-α-treatment, adistinct correlation between NF-κB activity and rip4 gene

expression could be determined (Fig. 3B). Whereas very lowconcentrations of Curcumin interestingly even enhanced theTNF-α-induced decrease of rip4 gene expression, repressionwas completely abrogated by 25–50 μM Curcumin. Further-more, as shown in Fig. 3C, H2O2-induced repression of rip4gene expression was also abolished after Curcumin treatment.

Fig. 2 (continued).


These data suggest involvement of NF-κB in the regulation ofrip4 gene expression.

Dexamethasone attenuates scratch-induced repression ofrip4 gene expression as well as reepithelialization ofwounded epidermis in vitro

Previously, we could demonstrate that in intact keratinocytemonolayers, the glucocorticoid dexamethasone enhances rip4gene expression. Furthermore, we could show that scratch-wounding represses rip4 expression in these cells [14]. Thus,since the adverse clinical effects of high endogenous cortisol levelsas well as of the exogenous application of glucocorticoids onwound healing are well-known but still not completely under-stood, we analyzed the effects of dexamethasone on rip4expression after wounding. For this purpose, keratinocyte mono-layers were treated with dexamethasone and wounded in parallel.Subsequently, the reepithelialization was monitored histologically

and in parallel, rip4 expression was analyzed via Northern blot. Asshown in Fig. 4A, in contrast to untreated cells, dexamethasone-treated HaCaT-cells were unable to reepithelialize the woundedarea. In addition, we could not demonstrate an activated cellularphenotype in dexamethasone-treated cells, confirming an inter-ference of glucocorticoids with the activation of keratinocytes inresponse to injury [18] (Fig. 4B). In addition, the rip4 down-regulation normally seen after woundingwas completely absent inthese cells (Fig. 4B). These data suggest a potential involvement ofdysregulated rip4 expression in the complex phenotype of delayedreepithelialization as seen in glucocorticoid-treated patients.

Discussion

Even though RIP4 is known to be a crucial modulator of bothembryonic development of the epidermal tissue [10], and theregulation of epidermal differentiation and potentially cutaneous

Fig. 2 (continued).


Fig. 2 (continued).


wound repair in the adult [14], the mechanisms which areinvolved in the regulation of rip4 gene expression in keratinocytesare poorly understood.

To identify rip4-expressing cells, we first determined thespatial pattern of rip4 gene expression within the adultepidermis. High levels of RIP4 were observed throughout allepidermal layers, indicating that RIP4 might play a role in theregulation of keratinocyte proliferation, differentiation, and/orepidermal homeostasis. This is consistent with our previousresults: We could recently demonstrate differential expression ofgenes which are associated with keratinocyte differentiation as aconsequence of rip4-mRNA interference and thus confirm acrucial role of the RIP4-protein in keratinocyte differentiation[14]. When we analyzed rip4 expression during differentiation ofprimary keratinocytes in vitro, we could furthermore demon-strate high expression levels of this gene at early stages of thedifferentiation process, and a gradual downregulation withproceeding differentiation. Taken together, these data suggest arole of the RIP4 protein in keratinocyte differentiation. However,since our in vitro model system of keratinocyte differentiationmechanistically differs from the in vivo situation in many aspects,it will be necessary to analyze rip4 expression in morephysiological systems of keratinocyte differentiation (such asthree-dimensional culture systems) in the future.

In addition, it will be interesting to study rip4 expression inphysiological and pathological situations of “abnormal” keratino-cyte differentiation, specifically a comparison of ortho- andparakeratotic regions of murine tail skin or an analysis of psoriaticskin. This will help to elucidate the exact function of RIP4 inkeratinocyte differentiation in a more refined manner.

Since cultured HaCaT cells in our studies consistently showedthe same pattern of rip4 gene expression as primary keratinocytes,

further analyses were performed using the established non-tumorigenic HaCaT cell line.

Specifically, we analyzed the signal transduction pathways thatcontrol rip4 gene expression in these cells. In particular, westudied two different pathways which play a central role inkeratinocytes: on one hand, we focused on the p38-MAPKpathway which is an important modulator of the stress-inducedcellular response in keratinocytes [16], on the other hand, wetested the PKC-signaling pathway which plays a key role inkeratinocyte differentiation [15].

Since we could previously show that repression of rip4 geneexpression in keratinocytes can be achieved by scratch-woundingas well as by various growth factors and cytokines [14], weparticularly investigated a potential contribution of the p38 andthe PKC pathways in the underlying regulatory mechanisms:

Interestingly, we could indeed detect participation of the p38MAPK pathway in the downregulation of rip4 gene expression (I)by scratch-wounding as well as (II) by serum and (III) TNF-α.Finding (I) is particularly interesting, since it provides mechanisticinsight into the cell-autonomous, scratch-induced regulation ofrip4 gene expression described in our previous paper [14]. In thiscontext, it appears particularly interesting that two recentpublications could demonstrate a role of p38 in the healing ofscratch-induced keratinocyte monolayers [19,20]. In the future, itwill be particularly interesting to study the role of different p38isoforms in scratch-induced regulation of rip4 gene expression.Interestingly, preliminary data from our laboratory suggest thatthe effect of the p38 inhibitor SB202190 on scratch-inducedregulation of rip4 expression is much stronger than that of thestructurally similar compound SB203580, which is also a p38inhibitor. This differential effect of the two inhibitors SB202190and SB203580 is most likely due to the fact that these two

Fig. 3 – The NF-κB inhibitor Curcumin attenuates repression ofthe rip4 gene in a dose-dependent manner. (A) To monitorthe effect of Curcumin on NF-κB activity, cellular IκBαconcentrations were analyzed via Western blot. Incubationwitch an antibody against α-tubulin served as a loadingcontrol. Note the strong effect of 25–50 μM Curcumin on IκBαexpression. (B) To analyze the role of NF-κB in the regulation ofrip4 gene expression, HaCaT-cells were incubated with varyingdoses of Curcumin as indicated. Effects of this treatment onTNF-α-induced repression of rip4 gene expression were studiedvia Northern blotting. (C) The effect of Curcumin treatment onH2O2-induced rip4 gene expression was also determined.


inhibitors exert differential effects on different p38 isoforms[21,22], and that at least in keratinocyte differentiation, p38 δ,which is not inhibited by SB203580, appears particularly impor-tant [23].

With respect to finding III, it seems interesting to mention thatin vivo, initial activation of keratinocytes after wounding ismediated by the proinflammatory cytokines TNF-α and interleu-kin-1 (IL-1) [24,25]. Since (I) both cytokines lead to an activationof the p38-MAPK cascade [26], since (II) the contribution of p38MAPK to the TNF-α-dependent repression of rip4 expression is inagreement with current knowledge on TNFR I signaling, whichinvolves p38 activation [27], and since (III) we could previouslydemonstrate early downregulation of rip4 expression in woundtissue [14], our data are consistent with a role of a TNF-α-induced,

p38-mediated repression of rip4 gene expression at the very earlystages of wound repair.

However, since we could also demonstrate that regulation ofrip4 expression by EGF and TGF-β1–growth factors that are alsopresent at high concentration in a healing wound (for review, see[12])–occurred independently from the p38 MAPK cascade, thereis strong evidence that further signal transduction pathwaysmightcontribute to the regulation of rip4 gene expression in keratino-cytes. Thus, it might be interesting to focus on further MAPKs aswell as on a potential implication of Smad proteins in theregulation of rip4 gene expression.

Surprisingly, however, we could not detect any involvement ofthe PKC pathway in the downregulation of rip4 gene expression ,either by scratch-wounding or by growth factors and cytokines,even though this pathway is known to be a very importantmodulator of keratinocyte differentiation [15]. However, the PKCenzymes represent a complex family of diverse classical, novel,and atypical isoforms (for review, see [28], whereas the inhibitorwe used (GF 109203X) shows high selectivity for PKCα and -β1isoforms. However, in keratinocytes, also other PKC isoforms areimportant in regulating proliferation, differentiation, and apopto-sis [15,29]. This point is particularly important, since RIP4 itselfmight again be involved in the regulation of PKC signalling, since itassociates with PKCβ and PKCδ and has thus earlier been describedas DIK (PKCdelta-interacting kinase) or PKK (protein kinase C-associated kinase). [30,31].

Furthermore, we focused on a potential implication of the NF-κB pathway in the regulation of rip4 gene expression.

First, we analyzed the effects of Curcumin, an inhibitor of NF-κBactivity, on TNF-α-induced rip4 gene repression. Interestingly, wecould demonstrate a consistent and dose-dependent effect ofCurcumin on rip4 gene expression, or, in other words, we coulddemonstrate that NF-κB activity was absolutely necessary for TNF-α-induced repression of rip4 gene expression. A similar effect wasseen after treatment with Curcumin and H2O2, which is alsoknown to downregulate rip4 gene expression in keratinocytes (ourdata). However, it is important to note that the anti-inflammatory,anti-tumorigenic and anti-oxidative effects of Curcumin [32,33]are only partially mediated by the inhibition of NF-κB activity, butalso by other pathways, such as the extracellular signal-regulatedkinase (ERK)-, the c-Jun N-terminal kinase (JNK)-MAPK, and thephosphoinositide 3-kinase (PI3K) [34], as well as the activatorprotein 1 (AP-1) pathway [35]. In addition, Curcumin also exertsdirect anti-oxidative effects [32]. Thus, the further downregulationof rip4 expression by low doses of curcumin appears onlysurprising at first glance, particularly from the background thatdifferential (and partially contradicting effects) of curcumin atdifferent dosages, particularly in the ranges of <10 μM, 10–150 μM, and above 150 μM, have been described, specifically inkeratinocytes. In particular, curcumin can exert pro- and anti-oxidative, pro- and antiproliferative, as well as pro- and anti-genotoxic effects on keratinocytes at different dosages, with theunderlying mechanisms and signal transduction pathways beingonly partially understood ([36], and references therein).

Since our previous studies had revealed a very tight regulationof rip4 gene expression upon wounding, we finally analyzed theeffects of dexamethasone–a synthetic glucocorticoid-derivativewhich has inhibitory effects on epidermal reepithelialization–onthe regulation of rip4 gene expression in scratch-woundedkeratinocytes. Interestingly, we could show that the long-term

Fig. 4 – Dexamethasone abolishes repression of rip4 gene expression in response to wounding, accompanied by the absence ofkeratinocyte reepithelialization in vitro. HaCaT-cells were incubated with Dexamethasone (10−5M) for 1 h and subsequentlyscratch-wounded. (A) The reepithelialization processwas examined histologically 2 days after wounding. (B) To examine the impacton rip4 expression, total RNA was isolated at defined time points early after wounding and analyzed by Northern blotting. Stainingof the 28S rRNA band served as a control for equal loading andwas used for normalizationwhen quantifying individual bands of twoindependent experiments (bar graph).


downregulation of rip4 expression normally seen in response towounding was completely abolished by the steroid, indicating arole of elevated RIP4 levels in the delayed reepithelialization seenin glucocorticoid-treated patients, which might have importanttherapeutic implications. Although the negative side effects ofendogenous or exogenous glucocorticoids on cutaneous woundhealing are not completely understood, however, besides the directeffects of activated glucocorticoid-receptors on gene expression,novel findings also point to indirect effects on NF-κB activity [37],which might be mediated by the NF-κB activator RIP4.

Taken together, our data suggest an essential role of RIP4 in theregeneration of wounded epidermis and indicate participation ofthe p38-MAPK in the early repression of rip4 gene expression,whereas long-term repression of this gene seems to be at leastpartially dependent on the activity of the NF-κB pathway. Inaddition, regulation of rip4 expression by glucocorticoids mightmediate at least in part the reepithelialization defects induced bythe latter.

In vivo, a tight balance between keratinocyte proliferation,differentiation, and death is important in both epidermal


homeostasis and in cutaneous wound repair. Our data (thisstudy and [14] as well as the skin phenotype of RIP4-deficientmice [10] point to a role for RIP4 in the regulation ofkeratinocyte differentiation, which argues for a function ofthis protein in both processes–homeostasis and repair–andoffers an explanation for the differential expression of the rip4gene after wounding. Interestingly, a recent publication [38]also shows involvement of the RIP4 protein in the regulation ofthe inflammatory response—specifically, transgenic mice thatoverexpress the rip4 gene in the epidermis are characterized bydramatic, neutrophilic inflammation. Since this effect is inde-pendent of TNFR-1/p55 receptor activation, it seems likely thatit is at least in part mediated by differential expression of therip4 gene after wounding—in other words, decreased levels ofRIP4 after injury might be important in keeping woundinflammation at bay or in downregulating the inflammatoryresponse at later stages of the healing process.

Materials and methods

Preparation of tail skin tissue

Tail skin was isolated from female Balb/c mice (10 to 12 weeksold). Experiments with animals were performed in the labora-tory of Prof. Dr. Sabine Werner, ETH Zurich, Switzerland, withpermission from the local veterinary authorities, Zurich,Switzerland.

Immunofluorescence

For immunofluorescence analysis, complete tail skin was fixed in1% acetic acid/95% ethanol, embedded in paraffin, and cut at 6 μmthickness. For detection of the endogenous RIP4 protein, depar-affinized sections of paraffin-embedded tissues were subjected toan antigen retrieval protocol (incubation in 5 M urea for 30 min at60 °C), incubated in blocking buffer and subsequently in blockingbuffer containing the first antibody overnight at 4 °C (mousemonoclonal anti-RIP4 (Abnova). Bound antibody was detectedwith an Alexa 594-conjugated goat anti mouse antibody (Invitro-gen), and sections were mounted in 10% glycerol after a finalwashing step. They were analyzed using an Axioscope fluores-cence microscope (Zeiss).

Tissue culture

Human HaCaT keratinocytes [39] were cultured in Dulbecco'smodified Eagle's medium containing 10% fetal bovine serumsupplemented with penicillin/streptomycin (100 U/ml each).Prior to treatment with the respective factors, cells were serum-starved for 16 h.

Primary human keratinocytes (foreskin, Gibco) were culturedin special low-calcium medium (complete defined K-SFM, Gibco)supplemented with penicillin/streptomycin (20 U/ml each). Cellswere passaged before reaching 70% confluence; experiments wereperformed with passages 3 and 4. All tissue culture dishes werepre-coatedwith human collagen IV (1 μg/cm2, Sigma) according tothe instructions of the manufacturer. Differentiation was inducedby adding CaCl2 (Roth) at a final concentration of 1.2 mM to thedescribed medium. Prior to induction studies, cells were trans-

ferred into non-supplemented defined K-SFM containing penicil-lin/streptomycin.

For gene expression studies, cells were treated with thefollowing agents: The p38-MAPK inhibitor SB202190 and thenegative control SB202474 (10 μM,Calbiochem), the PKC inhibitorsGF109103X and the negative control Ro-31-6045 (100 nM,Calbiochem), Curcumin (10–70 μM, Calbiochem), dexamethasone(10−6M, Sigma), fetal bovine serum(10%, Biochrom),H2O2 (1mM,Roth), EGF (20 ng/ml, BD Biosciences), TGF-β (1 ng/ml, Boehrin-ger), 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 reepithelialization of wounded keratinocytes invitro, cells were first rinsed with phosphate-buffered saline (PBS)and then fixed with a pre-chilled mix of Methanol and Acetone for10 minutes (1:1,−20 °C). After air-drying, samples were stored at4°C and analyzed using a Zeiss Axioscope microscope.

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 was 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).

Acknowledgments

We thank Renate Noske-Reimers and Gabi Beyer for excellenttechnical assistance, and SabineWerner, ETHZurich, for stimulating


discussions, suggestions, and advice. This work was supported bygrants from the Deutsche Forschungsgemeinschaft (Mu 1556/3-1)and from the Sonnenfeld-Stiftung, Germany (to BM).

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rip4 is a target of multiple signal transduction pathways in keratinocytes: implications for...

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