critical roles of ppar in keratinocyte response to...

Critical roles of PPAR�/� in keratinocyteresponse to inflammationNguan Soon Tan,1 Liliane Michalik,1 Noa Noy,1,3 Rubina Yasmin,1 Corinne Pacot,1,4

Manuel Heim,2 Beat Flühmann,2 Beatrice Desvergne,1 and Walter Wahli1,5

1Institut de Biologie Animale, Université de Lausanne, CH-1015 Lausanne, Switzerland; 2F. Hoffmann-La Roche Ltd,Vitamins and Fine Chemicals Division, Human Nutrition and Health, 4070 Basel, Switzerland

The immediate response to skin injury is the release of inflammatory signals. It is shown here, by use ofcultures of primary keratinocytes from wild-type and PPAR�/�−/− mice, that such signals including TNF-� andIFN-�, induce keratinocyte differentiation. This cytokine-dependent cell differentiation pathway requiresup-regulation of the PPAR�/� gene via the stress-associated kinase cascade, which targets an AP-1 site in thePPAR�/� promoter. In addition, the pro-inflammatory cytokines also initiate the production of endogenousPPAR�/� ligands, which are essential for PPAR�/� activation and action. Activated PPAR�/� regulates theexpression of genes associated with apoptosis resulting in an increased resistance of cultured keratinocytes tocell death. This effect is also observed in vivo during wound healing after an injury, as shown in dorsal skin ofPPAR�/�+/+ and PPAR�/�+/− mice.

[Key Words: PPARs; inflammation; apoptosis; differentiation; keratinocytes]

Received May 7, 2001; revised version accepted October 25, 2001.

Peroxisome proliferator-activated receptors (PPARs),which control many cellular and metabolic processes,are members of the superfamily of ligand-inducible tran-scription factors known as nuclear receptors. Three iso-types called PPAR� (NR1C1), PPAR�/� (NR1C2), andPPAR� (NR1C3) (Nuclear Receptors NomenclatureCommittee 1999) have been identified in vertebrates.They display differential tissue distribution, suggestingthat each of them fulfills specific functions. PPAR� andPPAR� play important roles in lipid homeostasis and ininflammation (Desvergne and Wahli 1999). In contrast,the exact functions of PPAR� remain an enigma. Al-though fatty acids can bind and activate PPAR�, studieson this isotype have so far been impeded by the lack ofinformation about the nature of its physiological ligandsand by its remarkably broad tissue distribution. Recentlyhowever, PPAR� was implicated in reverse cholesteroltransport (Oliver et al. 2001), in oligodendrocyte matu-ration and in membrane sheet formation (Saluja et al.2001).

In addition, PPARs may play an important role in skindevelopment, as PPAR� ligands can accelerate fetal ratepidermal development (Hanley et al. 1998). In epider-mis, the three PPAR isotypes are expressed during devel-

opment, but their levels decrease to become undetect-able in the interfollicular keratinocytes 5–9 d after birth(Michalik et al. 2001). However, the expression of bothPPAR� and PPAR� is reactivated upon proliferativestimuli, such as treatment with the phorbol ester TPA orhair plucking and at the wound edges after skin injury.Although PPAR�−/− and PPAR�+/− mice are not affectedby apparent skin defects, they display an increased hy-perplasic response to TPA treatment (Peters et al. 2000;Michalik et al. 2001). More interestingly, wound closureis delayed in PPAR�+/− mice as compared with wild-typeanimals (Michalik et al. 2001). Taken together, these ob-servations suggest that PPAR� may play an importantrole in skin, particularly in stress situations.

The epidermis in which keratinocyte is the predomi-nant cell type is characterized by a lifelong polarizedpattern of epithelial growth and cell differentiation inassociation with normal skin renewal (Spellberg 2000).In pathological conditions, for example, psoriasis, or af-ter epidermal injury, keratinocytes produce and respondto growth factors and cytokines, they become migratory,and synthesize components of the basement membrane.These activated wound keratinocytes express a specificset of keratins that are distinct from those present innormal epidermis. The expression of keratin 1 (K1) andkeratin 10 (K10), normally produced in the suprabasaland differentiating keratinocytes, is suppressed and re-placed by the expression of keratin 6 (K6) and keratin 16(K16) (Mansbridge and Knapp 1987). These responses areorchestrated by a number of signaling pathways, includ-ing the stress-associated kinase pathway (SAPK/JNK),

Present addresses: 3Division of Nutritional Sciences, Cornell University,Ithaca, NY 14853, USA; 4Laboratoire de Biologie Comparée, UMR-CNRS6023, Université Blaise Pascal, 63117 Aubière Cedex, France.5Corresponding author.E-MAIL [email protected]; FAX 41-21-692-4115.Article and publication are at http://www.genesdev.org/cgi/doi/10.1101/gad.207501.

GENES & DEVELOPMENT 15:3263–3277 © 2001 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/01 $5.00; www.genesdev.org 3263

Cold Spring Harbor Laboratory Press on August 19, 2020 - Published by genesdev.cshlp.orgDownloaded from

http://genesdev.cshlp.org/

http://www.cshlpress.com

which function both in sequential and parallel fashions(Dotto 1999).

Interestingly, whereas keratinocyte differentiation oc-curs during both normal epidermis renewal and woundrepair, PPAR� up-regulation is observed only in the lat-ter. Hence, this study was undertaken to unveil the rolesof PPAR� during wound healing. The results obtainedindicate that the up-regulation of the PPAR� gene isclosely associated with necrosis. Moreover, pro-inflam-matory cytokines, for example, TNF-�, can both increasePPAR� expression via the stress kinases signaling path-way and trigger the production of ligands for this receptor.Consistent with an important role of PPAR� in mediatinginflammation-induced keratinocyte differentiation, kera-tinocytes derived from PPAR�−/− mice are both severelydelayed in inflammatory cytokine-stimulated differentia-tion and more sensitive to TNF-�-induced apoptosis.

Results

Reactivation of PPAR� is associated with cell deathby necrosis

The initial approach to this study was to identify thecascade of events leading to the injury-dependent activa-tion of the PPAR� gene, which is normally silent inadult interfollicular keratinocytes (Michalik et al. 2001).Because the response to tissue injury can be mimicked inmixed leukocyte reactions (MLR), we used this assay toidentify potential signals that trigger PPAR� expression(Gallucci et al. 1999). In MLR, immature bone-marrow-derived dendritic cells (DCs) are activated by exposure tonecrotic cells. The activated DCs are, in turn, potentstimulators of T-cell proliferation (Banchereau andSteinman 1998). The factors secreted by these DCs/T-cells are representative of the signals produced after in-jury and comprise pro-inflammatory cytokines. In thisstudy, necrosis was achieved by freeze thawing primaryfibroblasts or by mincing skin. As a control, MLR wasalso performed with DCs exposed to UVB-induced apop-totic fibroblasts. Conditioned medium (CM) collectedfrom the activated DCs/T-cell cocultures was used toculture mouse primary keratinocytes. Its effects on theirdifferentiation and PPAR� expression were analyzed.

Keratinocytes cultured in control medium — not ex-posed to DCs/T-cells — did not differentiate over theexperimental time period (data not shown), whereas ke-ratinocytes cultured in apoptosis-derived CM started toexpress keratinocyte differentiation markers, such as in-volucrin (INV), transglutaminase type I (Tgase I), andkeratin 10 (K10) (Fig. 1A, first panel). There was nochange in the expression of PPAR� and PPAR�. In par-allel cultures, primary keratinocytes isolated fromPPAR�−/− mice and exposed to apoptosis-derived CMdisplayed a differentiation pattern similar to that of wild-type cells, showing that apoptosis-induced differentiationis a PPAR�-independent process (Fig. 1A, second panel).

In contrast, exposure of keratinocytes to necrosis-de-rived CM resulted in a rapid and intense up-regulation ofPPAR� (Fig. 1A, third panel). This was accompanied by

accelerated keratinocyte differentiation and cell cycle ar-rest, as indicated by both an early onset of INV and TgaseI and a decrease in cyclin A (Cyc A) expression. Theexpression of the wound-repair-associated keratins, K6and K17, was dramatically increased, whereas the weakexpression of K10 was suppressed (Fig. 1A, third panel).Strikingly, the differentiation pattern induced by necro-sis-derived CM was dramatically delayed in PPAR�−/−

keratinocytes (Fig. 1A, fourth panel), as revealed by theexpression of the differentiation markers. In these cells,the transient and weak PPAR� up-regulation seen inwild-type keratinocyte was unaltered. Together, theseobservations clearly show that PPAR� is essential formediating necrosis-associated signals that stimulate ke-ratinocyte differentiation.

Both in vivo and in the MLR model, necrosis triggersan inflammatory response mediated by cytokines (Mess-mer and Pfeilschifter 2000). Hence, we tested whetherpro-inflammatory cytokines can induce PPAR� expres-sion and keratinocyte differentiation similarly to the ne-crosis-derived CM. We first assessed the effect of TNF-�,which is considered as the main coordinator of the in-flammation response. TNF-� alone (5 ng/ml) inducedPPAR�, INV, Tgase I, and K6 expression, and inhibitedCyc A expression (Fig. 1B, first panel). In contrast,PPAR�−/− keratinocytes exposed to TNF-� exhibited aweak and delayed expression of the differentiation mark-ers accompanied by a gradual decrease in Cyc A level(Fig. 1B, second panel). This delayed response by thesemutant cells is unlikely to be due to defective TNF-�signaling, because PPAR�−/− cells, like their wild-typecounterparts, expressed high levels of the TNF-� recep-tor (p55TNF-R1) (data not shown).

To assess the contribution of TNF-� signaling in thenecrosis-induced differentiation, we precleared the ne-crosis-derived CM of TNF-� by incubation with excessanti-TNF-� antibodies prior to addition to the keratino-cytes. This pretreatment significantly reduced the po-tency of the CM to induce PPAR�, INV, Tgase I, and K6expression. Thus, TNF-� is a trigger of PPAR� expres-sion and a major player in inflammatory-induced kera-tinocyte differentiation (Fig. 1A, fifth panel). However,the anti-TNF-� antibody in the CM did not totally abro-gate the differentiation process, nor did it alter the ex-pression of K17 (Fig. 1A, fifth panel), indicating that ad-ditional factors in necrosis-derived CM participate toPPAR�-induced keratinocyte differentiation. Anotherimportant inflammatory cytokine, IFN-�, also produceda similar differentiation profile as necrosis-derived CMor TNF-�. Interestingly, IFN-� strongly induced K17 butnot K6 expression (Fig. 1B, third and fourth panels). Fi-nally, we also investigated the effect of the phorbol esterTPA, known to induce inflammation in skin (Puigneroand Queralt 1997). TPA treatment resulted in a ninefoldincrease in the expression of PPAR�, associated with in-creased K6, but not K17 expression (Fig. 1B, fifth andsixth panels). Both IFN-� and TPA-induced differentia-tion patterns are PPAR� dependent, as seen from theresults of the parallel experiments performed withPPAR�−/− keratinocytes.

Tan et al.

3264 GENES & DEVELOPMENT




Figu

re1.

Ext

ern

alsi

gnal

sst

imu

late

PP

AR

�ex

pres

sion

and

cell

diff

eren

tiat

ion

inpr

imar

ym

ouse

ker

atin

ocyt

ecu

ltu

res.

(A)A

popt

osis

-der

ived

CM

and

nec

rosi

s-de

rive

dC

Min

duce

ker

atin

ocyt

edi

ffer

enti

atio

n.P

rim

ary

ker

atin

ocyt

esfr

omw

ild-

type

(fir

st,t

hir

d,an

dfi

fth

pan

els)

orfr

omP

PA

R�

−/−

mic

e(s

econ

dan

dfo

urt

hpa

nel

s)w

ere

expo

sed

atti

me

0(u

sual

lyaf

ter

two

orth

ree

pass

ages

)to

con

diti

oned

med

ium

(CM

)fro

mm

ixed

leu

koc

yte

reac

tion

s(M

LR

),pr

epar

edei

ther

wit

hap

opto

tic

fibr

obla

sts

(fir

stan

dse

con

dpa

nel

s)or

wit

hn

ecro

tic

cell

s(t

hir

d,fo

urt

h,

and

fift

hpa

nel

s).

Ker

atin

ocyt

esw

ere

lyse

daf

ter

diff

eren

tti

mes

ofex

posu

reto

CM

asin

dica

ted,

and

the

expr

essi

onof

PP

AR

�w

asev

alu

ated

byR

PA

.T

he

expr

essi

onof

invo

lucr

in(I

NV

),tr

ansg

luta

min

ase

I(T

gase

I),w

asu

sed

asm

ark

ers

ofk

erat

inoc

yte

diff

eren

tiat

ion

.Th

eex

pres

sion

ofk

erat

in(K

)K6,

K10

,an

dK

17re

flec

tsdi

ffer

ent

path

way

sof

ker

atin

ocyt

edi

ffer

enti

atio

n.

Cyc

lin

A(C

ycA

)ex

pres

sion

indi

cate

dth

est

atu

sof

the

cell

wit

hre

spec

tto

cell

cycl

e.In

the

fift

hpa

nel

,th

en

ecro

sis-

deri

ved

CM

was

prec

lear

edof

TN

F-�

.(B

)Th

em

ajor

pro-

infl

amm

ator

ym

edia

tors

TN

F-�

,IFN

-�,a

nd

TP

Au

p-re

gula

teP

PA

R�

expr

essi

on.P

rim

ary

ker

atin

ocyt

esfr

omw

ild-

type

(fir

st,t

hir

d,an

dfi

fth

pan

els)

orfr

omP

PA

R�

−/−

mic

e(s

econ

d,fo

urt

h,

and

sixt

hpa

nel

s)w

ere

cult

ure

din

KSF

Man

dex

pose

dat

tim

e0

toT

NF-

�5

ng/

ml

(fir

stan

dse

con

dpa

nel

s)IF

N-�

5n

g/m

l(t

hir

dan

dfo

urt

hpa

nel

s).o

rT

PA

20n

g/m

l(fi

fth

and

sixt

hpa

nel

s).I

nal

lth

ree

trea

tmen

ts,t

he

expr

essi

onof

alld

iffe

ren

tiat

ion

mar

ker

sis

stro

ngl

yre

duce

dan

dde

laye

din

PP

AR

�−/−

cell

s.

Control of PPAR �/� action by inflammatory cytokines

GENES & DEVELOPMENT 3265




These observations show that necrosis-derived differ-entiation signals, such as produced after injury, lead toelevated PPAR� expression. The peak of PPAR� expres-sion typically coincides with the switch to cell cyclearrest and the onset of differentiation. Western blotanalyses, performed as reported by Michalik et al. (2001),verified that corresponding changes occurred in PPAR�protein levels (data not shown). Thus, pro-inflammatorycytokines that are produced in response to necrosis act asprimary triggers of elevated PPAR� expression. Upon ex-posure to necrosis-derived CM or pro-inflammatory cy-tokines, PPAR�−/− keratinocytes displayed dramaticallyimpaired differentiation, showing that PPAR� plays acritical role in enabling keratinocytes to differentiate inresponse to inflammation.

Reactivation of PPAR� in keratinocytes is mediatedby an AP-1 site in the PPAR� promoter.

Treatment of cultured primary keratinocytes withTNF-� resulted in an expression profile of PPAR�, whichwas closest to that observed after injury in an in vivomodel (Michalik et al. 2001). Therefore, the molecularevents by which TNF-� regulates the promoter of thePPAR� gene were studied next. The mouse PPAR� pro-moter was isolated (accession no. AF187850) and a 1.9-kb fragment containing the transcription initiation sitewas subcloned into a CAT reporter gene. The effectof TNF-� on the activity of a series of truncations of this1.9-kb PPAR� promoter region were analyzed by useof transfection into primary keratinocytes. The effectsof ceramide (Cer), a downstream effector of TNF-�(Hannun and Luberto 2000), and TPA were also stud-ied. The addition of each of the three agents increasedthe promoter activity of pPPAR�(−1880), as well as thatof the shorter promoter constructs pPPAR�(−864),pPPAR�(−587), and pPPAR�(−445), by two to threefold(Fig. 2A). In contrast, pPPAR�(−277), pPPAR�(−223), andpPPAR�(−11) were not responsive to TNF-�, TPA, orCer, suggesting that the response elements are locatedbetween −445 and −277.

This proximal part of the PPAR� promoter containssequences related to AP-1, Ets, and C/EBP transcriptionfactor binding sites (Fig. 2A), as identified by using Mat-Inspector (Wingender et al. 2000). Mutations were intro-duced in each of these sites and their relative importancewas assessed via transactivation studies (Fig. 2B).Whereas mutation of the Ets or C/EBP sites did not affectregulation by the three agents, mutation of the AP-1 site,located at −414, completely abolished responsiveness toall three inducers. Hence, the signaling pathways thatare activated by TPA, TNF-�, or Cer terminate at theAP-1(−414) site in the PPAR� promoter.

TNF-�-induced expression of PPAR� is mediated bythe factor-associated-with-neutral-sphingomyelinaseand the stress-associated kinase cascade

Next, experiments were designed to identify the indi-vidual steps relaying the signal initiated from TNF-� to

the AP-1(−414) site in the PPAR� promoter. TNF�-in-duced trimerization of its receptor (p55TNF-R1) leads tothe stimulation of several signaling cascades. One ofthese pathways involves the production of bioactive Cer,which, in turn, acts as second messengers to initiatedownstream effects (Kronke 1999). Cer is of particularinterest in this study as it is a component of the epider-mal stratum corneum. Adam-Klages et al. (1996) re-ported that FAN (factor associated to neutral sphingo-myelinase) couples the p55TNF-R1 to neutral sphingo-myelinase (N-Smase), which is the major enzymeresponsible for Cer generation via sphingomyelin hydro-lysis (Fig 3A). Thus, the involvement of FAN in trans-ducing the effect of TNF-� to the PPAR� promoter wasexplored. Keratinocytes were transfected with wild-type(wt) or a dominant-negative (dn) FAN and treated withTNF-�. Overexpression of wtFAN potentiated the abilityof TNF-� to induce PPAR� expression (Fig. 3B, secondpanel) and triggered an early onset of keratinocyte differ-entiation as indicated by the induction of INV expres-sion. In contrast, overexpression of dnFAN abolished theeffect of TNF-� on PPAR� expression and resulted indelayed cell differentiation. The repressive effect of dn-FAN could be rescued by addition of exogenous Cer (Fig.3B, third and fourth panels), in accordance with the roleof Cer as a downstream effector of FAN. Treatment ofuntransfected keratinocytes with Cer alone resulted inan elevated PPAR� expression and an early expression ofINV.

Transmission of the TNF-� signal downstream of Cerinvolves the mitogen-activated protein (MAP) kinasepathways. These pathways lead to the phosphorylationof transcription factors, such as those belonging to theAP-1 family, and consequently to a modulation of geneexpression (Kyriakis and Avruch 1996). TNF-� and Cerare known to activate MEKK1, which is a key player ofthe MAPK cascade. MEKK1, in turn, can phosphorylateseveral downstream kinases including SEK1 (Davis 2000;Fig. 3A). To evaluate the role of these kinases in regulat-ing PPAR� expression, the pPPAR�(−445) construct,which harbors the AP-1 site that is responsive to TNF-�,was cotransfected with expression vectors encoding ei-ther constitutively active (ca) or dominant-negativeforms of several kinases. Alternatively, specific knownkinase inhibitors were used. Transfection withdnMEKK1 abolished TNF�-, TPA-, and Cer-dependentpromoter activity (Fig. 3C). In contrast, caMEKK1 in-creased the basal activity by two to threefold and a fur-ther twofold upon addition of the cytokines. The cyto-kine-mediated induction of the PPAR� promoter is alsosuppressed by dnSEK1.

Downstream of SEK1, both JNK/SAPK and p38 havebeen shown to modulate AP-1 expression and activity(Davis 2000). The dnJNK/SAPK completely abolishedthe responsiveness of the PPAR� promoter to the threeinducers. Interestingly, SB20358, a specific inhibitor ofp38, only partially attenuated the effect of TPA stimu-lation and had no effect on stimulation by TNF-� or Cer(Fig. 3C). Finally, additional experiments ruled out thepossible involvement of S6K, PKA, and MEK1 signaling

Tan et al.





Figure 2. Identification of the region in the PPAR� promoter that mediates the TNF-�, TPA, and ceramide responses. (A) PPAR�

promoter 5�-deletion mutants. Plasmids are named according to the length of the promoter region they contain upstream from thetranscription start site. Keratinocytes in culture were cotransfected with a CAT reporter driven by the various PPAR� promoterconstructs and pCMV-�-galactosidase as a control of transfection efficiency. Keratinocyte cultures were treated for 24 h with theinducers indicated. Results were normalized to �-galactosidase activity and are presented as relative CAT activity compared with thatof the promoterless vector pBLCAT alone. (B) TNF�-, TPA-, and Cer-signaling target an AP-1(−414) of the PPAR� promoter. Thehatched bar depicts site-directed mutations of the transcription factor binding sites. The mutations introduced into the site areindicated as underlined nucleotides above the respective hatched bar. The numbers associated with the sites indicate their positionrelative to the transcription start site. Data are means of six independent experiments. Vehicles for TPA, TNF-�, and Cer were ethanol,PBS, and DMSO, respectively.






in regulating PPAR� expression (Fig. 3C). Hence, activa-tion of the PPAR� promoter by TNF-�, TPA, and Cer ismediated through MEKK1, SEK1, and SAPK/JNK (Fig.3C).

Taken together, these observations show that TNF-�,TPA, and Cer utilize overlapping signal transductioncascades that converge to JNK/SAPK and AP-1. Inciden-tally, the cascades that are activated by the three agentsare stress-associated pathways and are invoked in re-sponse to inflammation (Kyriakis and Avruch 1996).

Hence, these data attest to an important role for PPAR�in the stress- and injury-associated responses of keratino-cytes.

Expression and activation of PPAR� leadto keratinocyte differentiation

The results presented so far show that expression ofPPAR�, induced by inflammatory cytokines, correlateswith keratinocyte cell cycle arrest and differentiation.

Figure 3. Signaling cascades that mediate the effects of TNF-�, TPA, and Cer on the PPAR� gene in keratinocytes. (A) (Overallscheme) The solid arrows represent the major TNF�-activated, FAN-mediated, pathway regulating PPAR� expression. The cascadeinvoked by TPA is depicted by the dashed arrows. (B) TNF-� induces PPAR� gene expression through FAN and Cer. Keratinocytes werecotransfected with expression vectors for wild-type FAN (wtFAN, second panel) or a dominant-negative form of FAN (dnFAN, thirdand fourth panels) and pCMV-�-galactosidase as internal control. Transfected keratinocytes were treated with TNF-� and RPA wereperformed at the indicated times. (fourth panel) Suppression of PPAR� expression by dnFAN was rescued by addition of exogenousceramide at 12 h post-transfection. (first panel) Control experiment with TNF-� alone; (fifth panel) effect of ceramide in absence ofTNF-� treatment. (C) Regulation of PPAR� via the stress-associated kinase pathway. Keratinocytes were transfected with a CATreporter driven by the pPPAR�(−445) promoter region together with expression vectors encoding dominant negative (dn) or constitu-tively active (ca) kinases. The empty vector pCDNA 3.1 was used as a control (vector). Transfected keratinocytes were treated withan inducer (TNF-�, ceramide, or TPA) and CAT activity was measured 24 h post-treatment. SB203580 (10 µM) is a specific inhibitorof p38 and was added 1 h prior to treatment with the inducers. Values are means of six independent experiments.

Tan et al.





Furthermore, experiments with PPAR�−/− keratinocyteshave shown that PPAR� is required for these processesto take place (Fig. 1). Therefore, we hypothesized thatPPAR� overexpression would be sufficient to induce ke-ratinocyte differentiation in our primary cell culture as-say. To test this possibility, primary keratinocytes over-expressing PPAR� were treated or not with the syntheticPPAR� ligand L165041 (LD). A high level of PPAR�alone was not sufficient to trigger keratinocyte differen-tiation, whereas in conjunction with a 6-h LD treatment,these cells expressed the differentiation marker, INV(Fig. 4A, first and second panels). Repeated LD exposureof the PPAR� overexpressing cells led both to cell cyclearrest and to keratinocyte differentiation (Fig. 4A, thirdpanel). However, the same treatment applied to untrans-fected keratinocytes resulted only in a mild expression ofINV (Fig. 4A, fourth panel). Thus, the induction of kera-tinocyte differentiation requires both an elevated expres-sion of PPAR� and the presence of a PPAR� ligand thatactivates the receptor.

TNF-�, TPA, and ceramide can generate endogenousligands of PPAR�

Together, our results suggest that signaling by inflam-matory molecules result not only in stimulation of thePPAR� gene but also leads to the production of an en-dogenous ligand. To test this hypothesis, an expressionvector encoding a fusion protein comprising the GAL4–DNA-binding domain (DBD) and the PPAR� ligand-binding domain (LBD) was constructed. This fusion pro-tein allows for monitoring the activation of the PPAR�LBD independently of the expression of the endogenousPPAR� gene. Keratinocytes were cotransfected with thisfusion construct and a CAT reporter gene controlled byGAL4 response elements. The responsiveness of this sys-tem was validated with the synthetic PPAR� ligandL165041, which activated the fusion protein in a dose-dependent manner (Fig. 4B). Treatment of the transfectedcells with either IFN-�, TNF-�, TPA, or Cer that wereshown above to up-regulate PPAR� gene expression, alsoresulted in a dose-dependent increase in the transcrip-tional activity of the fusion protein. Interestingly, boththe bioactive lipid Cer and arachidonic acid (AA) led to adramatic increase in CAT activity (Fig. 4B). In contrast,TGF-� had no effect on CAT activity.

To verify that the activation of the PPAR� LBD wasnot due to its phosphorylation but to the production of aligand, we tested the ability of an organic extract from cy-tokine-activated keratinocytes to transactivate PPAR�.This experiment was performed in keratinocytes trans-fected either with the GAL4 DBD/PPAR� LBD fusion pro-tein and its corresponding reporter CAT gene (Fig 4C, left),or with wild-type PPAR� and a PPRE-containing reporterluciferase gene (Fig 4C, right). In both systems, a dose-de-pendent increase in reporter activity was observed, show-ing the presence of a ligand in the organic extracts. Thus, inaddition to elevating PPAR� gene expression, inflamma-tory signals also trigger the production of endogenous li-gand(s) necessary for PPAR� activation.

PPAR� regulates genes associated with apoptosis

We have established that inflammation stimulatesPPAR� expression and produces ligand(s) that activatethe receptor, which together induce keratinocyte differ-entiation. Interestingly, amidst the TNF-�-induced kera-tinocyte differentiation experiments, we observed thatPPAR�−/− keratinocytes, upon a prolonged exposure toTNF-�, died at a higher rate than wild-type cells. Thisdifferential response to this cytokine, which is an in-ducer of apoptosis (Natoli et al. 1998), led us to testwhether PPAR� controls genes involved in apoptosis.The expression of such genes was analyzed by RPA inPPAR�−/− versus wild-type keratinocytes. Expression ofthe proapoptotic genes CIDE-A and CIDE-B (cell death-inducing DFF45-like effector A and B), involved innuclear condensation and DNA fragmentation (Inoharaet al. 1998), was 1.8–2.5-fold higher in the PPAR�−/− cells(Fig. 5). In contrast, the expression of the anti-apoptoticICAD-S and ICAD-L (inhibitor of caspase-activated de-oxyribonuclease short and long forms), which are highlyhomologous to the human DFF45, were reduced in thePPAR�−/− keratinocytes (Fig. 5). This is consistent withthe known role of DFF45 (DNA fragmentation factor 45)in the inhibition of the DNase/apoptotic activity of CI-DEs (Inohara et al. 1998). Thus, PPAR� appears to up-regulate antiapoptotic and down-regulate proapoptoticgenes in keratinocytes. Whether this regulation is director indirect, that is, does not or does require de novo pro-tein synthesis, was analyzed using keratinocytes treatedfor 12 and 24 h with the PPAR� ligand LD, in the pres-ence or absence of cycloheximide (CH), a protein synthe-sis inhibitor. Consistent with the results presentedabove, all the genes tested responded to the PPAR� ago-nist in the absence of the protein synthesis inhibitor (Fig.5). However, in the presence of CH, the down-regulationof CIDE-A and CIDE-B was lost, whereas the up-regula-tion of ICAD-S and ICAD-L was maintained. This resultindicates that de novo protein synthesis is required forPPAR�-dependent down-regulation of CIDE-A andCIDE-B, whereas up-regulation of ICAD-S and ICAD-L isa direct effect of PPAR�. A further analysis of the mo-lecular mechanisms by which PPAR� modulates apop-tosis is underway. In addition to the genes involved inapoptosis, the expression of �-actin in the PPAR�−/− cellswas ∼2.3-fold higher than in wild-type cells, a fact worthmentioning because of the frequent use of �-actinmRNA as a internal control in expression studies. Fur-thermore, this result is consistent with our previous ob-servation that PPAR�−/− keratinocytes exhibited alteredcell morphology (Michalik et al. 2001).

Elevated PPAR� confers increased resistanceto TNF-�-induced apoptosis

The above results implicate a role of PPAR� in the re-pression and stimulation of pro-apoptotic and anti-apop-totic genes, respectively. Regardless of the origin of theapoptotic stimuli, the commitment to apoptosis occursthrough the activation of caspases and the fragmentation






Figure 4. PPAR� accelerates keratinocyte differentiation. (A) Overexpression and activation of PPAR� stimulates keratinocytedifferentiation. Keratinocytes were transfected with a wtPPAR� expression vector and lysed for RPA at various time points post-transfection, as indicated. Overexpression of PPAR� alone is not sufficient to trigger keratinocyte differentiation (first panel), whichrequires a 6-h exposure to a PPAR�-specific ligand L165041 (LD) (second panel). Better results were obtained with a repeated exposureto LD [6 h, followed by an additional 18 h with fresh medium containing the second dose (third panel)]. This latter dose regime wasalso able to slightly induce the expression of differentiation markers in absence of ectopical expression of PPAR� (fourth panel). (B)Activation of the PPAR� LBD by cytokines. Keratinocytes were cotransfected with an expression vector for a GAL4 DBD–PPAR� LBD,the Gal4 responsive reporter vector pG5CAT (Clontech), and the pCMV-�-galactosidase vector. CAT and �-galactosidase activitieswere measured after 24-h exposure to two different concentrations (open and solid bars) of PPAR� selective ligand L165041 (LD; 1, 5µM), IFN-� (10, 25 ng/ml), TNF-� (10, 25 ng/ml), TGF-� (10, 25 ng/ml), TPA (10, 20 ng/ml), Ceramide (Cer; 10, 20 µM), or arachidonicacid (AA; 10, 20 µM). Exposure to IFN-�, TNF-�, TPA, and Cer resulted in a dose-dependent increase of the CAT reporter activity. (C)Total lipid extract of cytokine-activated keratinocytes activates PPAR�. Total lipid extract were prepared from keratinocytes un-treated (control), or exposed to vehicle or to various pro-inflammatory mediators, as indicated. (Left) Transfections were performed asin A. Cells were then exposed to 1 or 2 µL of a total lipid extract for 24 h and CAT activity was measured. (Right) Keratinocytes weretransfected with the wtPPAR� expression vector, pGL-3xPPRE-tk-luc reporter gene, and pCMV-�-galactosidase. Normalized reporteractivity is shown as fold increase as compared with control.

Tan et al.





of nuclear DNA. In accordance with this process, TNF-�receptors can trigger apoptosis via activation of caspase8, followed by DNA fragmentation. This prompted usto measure this enzymatic activity and to monitorDNA fragmentation in wild-type and PPAR�−/− kera-tinocytes.

Primary keratinocytes displayed minimal caspase 8 ac-tivity (Fig. 6A). Upon treatment with TNF-�, the caspaseactivity increased by 12- and 14-fold after 6 and 24 h oftreatment, respectively. Interestingly, keratinocytes ex-pressing PPAR� ectopically showed a reduced TNF�-in-duced caspase activity (Fig. 6A). Incubation of these cellswith a PPAR� selective ligand LD further reduced thecaspase activity by 2.5-fold, when compared with un-transfected wild-type keratinocytes. The importance ofPPAR� in controlling caspase activity levels was furtherexamined in PPAR�+/− and PPAR�−/− keratinocytes. Inunchallenged keratinocytes, only PPAR�−/− cells dis-played higher basal levels of caspase 8 activity. However,both mutant keratinocytes exhibited significantly en-hanced TNF�-induced caspase activity when comparedwith wild-type cells (Fig. 6A). Finally, the reduced re-sponse to TNF-� seen in wild-type cells could be attrib-uted to PPAR� expression, as the response of PPAR�−/−

keratinocytes to TNF-� could be normalized by transfec-tion of a PPAR�-expression vector.

The fragmentation of genomic DNA is also a hallmarkof apoptosis (Duke 1983) and, hence, was also assessed inPPAR�−/− and PPAR�+/+ keratinocytes upon TNF�-in-duced apoptosis. As expected from the caspase 8 activitymeasurements presented above, PPAR�−/− keratino-cytes showed higher levels of TNF-�-induced DNA frag-mentation than wild-type cells over the time coursemonitored (Fig. 6B).

The observed higher caspase 8 activity, higher DNAfragmentation rate, and resulting reduced resistance toapoptosis of PPAR�−/− cells, provide evidence for an im-portant role of PPAR� in the control of keratinocytedeath during inflammation.

Ablation of a PPAR� allele in vivo results in increasedproliferation and apoptosis in mouse epidermis

The relevance of the antiapoptotic role of PPAR�, ob-served in primary keratinocyte culture, was also exam-ined in vivo during wound repair. We used a model ofepidermal repair in PPAR� mutant mice (Michalik et al.2001). Epidermal repair after a skin wound includes ke-ratinocyte proliferation, migration, and differentiation.For wound closure and efficient healing, the balance be-tween proliferation and cell death has to be tightly con-trolled. Recently, we have shown that PPAR� expressionis up-regulated in the keratinocytes at the wound edges,and that the deletion of one PPAR� allele (PPAR�+/− ani-mals) resulted in delayed wound healing (Michalik et al.2001).

Apoptosis and proliferation were assessed in thismodel by use of TUNEL and immunofluorescent label-ing of the Ki67 nuclear proliferation marker, respec-tively, on dorsal mouse skin sections from days 4 and 7after an injury. Figure 7A and Table 1 summarize theobservations made at the epidermal wound edges (seeFig. 7B) of PPAR�+/− and wild-type mice. Quantificationof the Ki67-positive cells revealed a twofold increase inthe number of proliferating keratinocytes in thePPAR�+/− mice at 4 and 7 d post injury. This observationshows that in a physiological situation such as woundhealing, PPAR� is involved in the control of epithelialcell proliferation. Furthermore, it is in agreement withresults obtained previously by us and others revealinghigher keratinocyte proliferation rates in the epidermisof mutant mice after TPA treatment (Peters at al. 2000;Michalik et al. 2001). Even more importantly and con-sistent with the result obtained here with primary kera-tinocytes culture, the number of TUNEL-positive kera-tinocytes in the same region was 10-fold higher in thePPAR� mutant epidermis at day 7 post injury (Fig. 7;Table 1). These data show that in vivo as well as in vitro,PPAR� has an anti-apoptotic role. In the context of skinwound healing, these findings show that PPAR� is nec-

Figure 5. PPAR� target genes are associated withapoptosis. RPA was performed using total RNA(2.5 µg) isolated from keratinocytes derived fromPPAR�+/+ and PPAR�−/− mice (left); or from wild-type keratinocytes not treated (−) or treated for 12and 24 h with 1µM of the PPAR� specific ligandL165041 (LD; middle); and with 5 or 10 µg/ml ofcycloheximide (CH; right). Two representativesamples are shown per condition. Numbers beloweach band represent the fold differences in mRNAexpression levels with respect to the wild-type oruntreated keratinocytes.






essary for the control of the keratinocyte life span, and,thus, for a well-tuned balance between cell death andproliferation.

Discussion

The results presented herein show that, in keratinocytes,PPAR� is an important transcription factor relaying in-flammatory signals at the cell surface into specific geneexpression patterns, which define appropriate cellular re-sponses in sudden stress situations. Firstly, signals such

as TNF-� or IFN-� activate the stress-associated signal-ing pathway leading to the stimulation of the PPAR�gene via an AP-1 site in its promoter. Secondly, theytrigger the production of PPAR� ligands. Thirdly, theresulting increase in PPAR� transcriptional activitystrongly accelerates the differentiation of keratinocytesand increases their resistance to apoptotic signals. Fi-nally, increased proliferation and death of keratinocytesat the edges of epidermal wounds in PPAR�+/− mice arelikely to participate in the healing delay observed previ-ously in these animals.

Figure 6. Inflammation-induced PPAR� expression protects keratinocytes from apoptosis. (A) Elevated PPAR� expression inhibitsTNF�-induced apoptosis. Keratinocytes prepared from PPAR�+/+,PPAR�+/−, and PPAR�−/− mice were either transfected with expres-sion vectors harboring the wtPPAR� and/or cultured in the absence or presence of TNF-� and L165041 (LD, 5 µM) as indicated. Thelevel of TNF�-induced apoptosis was monitored by caspase 8 activity. Keratinocytes expressing wtPPAR� were more resistant toapoptosis signals, whereas PPAR�+/− cells showed an increase susceptibility. PPAR�−/− keratinocytes exhibited higher basal caspase 8activity and were more sensitive to TNF�-induced apoptosis, but could be rescued by transfection with the vector expressing wtP-PAR�. Values are means of three independent experiments. (B) PPAR�−/− keratinocytes are more susceptible to TNF�-inducedapoptosis. Culture of primary keratinocytes from wild-type and from PPAR�−/− mice were exposed to TNF-�. Apoptosis was measuredby radioactive DNA fragmentation assay over indicated periods of time. Values are means of three independent experiments.

Tan et al.





Figure 7. PPAR� controls the balance between apoptosis and proliferation of keratinocytes in vivo. (A) Increased proliferation andelevated apoptosis in the PPAR�+/− mice. The Ki67 and TUNEL-positive keratinocytes were revealed by fluorescent labeling on skinsections obtained at days 4 and 7 after a dorsal skin injury. The figure shows representative fields of the labelings obtained at the woundedges (region c/c’ of the epithelium, according to B). PPAR�+/+ (a–e) and PPAR�+/− (f–j) mice skin sections after hematoxylin/eosinstaining (HE) (a,f), Ki67 immunostaining (b,g), TUNEL (d,i) and DAPI staining (c,h,e,j). Magnification bar, 50 µm. (B) Regions of a skinwound at days 4 and 7 after a full-thickness biopsy. (C) Clot; (WB) wound bed. The positions of the 10 microscope fields in whichapoptosis and cell proliferation were quantified is indicated as regions a–e and a’ –e’. The quantification (summarized in Table 1) is amean of the number of proliferative or apoptotic positive cells counted in regions e to e’ of the epithelium. A shows pictures taken inregion c and c’, as representative fields.






PPAR� promotes terminal differentiationof keratinocytes

As mentioned above, IFN-� and TNF-�-induced activa-tion of PPAR� accelerates keratinocyte differentiation inaddition to the antiapoptotic effect of the receptor. Theinflammatory cytokines trigger both elevated expressionand ligand-induced activation of PPAR�, which is re-quired to achieve their effects. We were able to recapitu-late the cytokine-dependent differentiation simply byboth high-ectopic expression of PPAR� in undifferenti-ated keratinocytes and its activation by a specific syn-thetic ligand. With respect to keratinocyte differentia-tion, it is unclear as yet why Peters et al. (2000) did notobserve any difference in INV expression upon TPAtreatment between PPAR�+/+ and PPAR�−/− mice. Pos-sible explanations are that their expression data werenormalized to the �-actin expression level that, as shownhere, is increased in PPAR�−/− cells. An increase in INVexpression, if paralleled by an elevation of �-actin syn-thesis would obviously have remained undetected usingthis normalization procedure. Alternatively, the pleio-tropic effects of TPA treatment might activate bothPPAR�-dependent and independent pathways of kera-tinocyte differentiation (Dotto 1999). Finally, topical ap-plication of TPA on skin may also have effects on thedermal fibroblasts that might modulate the keratinocyteresponse (Szabowski et al. 2000).

Whether the PPAR�-dependent cell differentiationnoted here is keratinocyte specific or if activation ofPPAR� in other tissues can accelerate cell differentiationas well, remains uncertain. In support of the latter, ec-topic expression of PPAR� in NIH-3T3 fibroblasts wasreported to promote early adipocyte differentiation thatwas dependent on the addition of PPAR�-selective li-gands (Amri et al. 1995; Hansen et al. 2000). Also,PPAR� agonists were shown to stimulate oligodendro-cyte differentiation and regulate the size of membranesheets (Saluja et al. 2001).

PPAR� expression is regulated by pro-inflammatorycytokines

The epidermis is the first line of defense of the organismagainst aggressions from the environment, and thusmust often respond to various types of injuries. As such,

epidermal injury is also typically associated with inflam-mation, particularly during wound repair, which in-volves modulation of cell growth and differentiation ofkeratinocytes. Although PPAR� is undetectable by insitu hybridization in adult mouse interfollicular epider-mis, its reactivation after a topical application of TPA,during wound repair and the healing delay observed inPPAR�+/− mice (Michalik et al. 2001), suggested a linkbetween this receptor and keratinocyte biology duringinflammation. The findings reported herein reveal thatpro-inflammatory cytokines, such as TNF-� and IFN-�produced during skin injury, can increase expression ofPPAR� and produce its ligands. The inflammatory re-sponse is one of the major differences between normalskin renewal and wound repair (Savill and Fadok 2000).The response of keratinocytes to inflammatory signalswas tested by use of necrotic cells in the MLR assay, andalso by treating them with the noncytokine inflamma-tory agent, TPA. The stimulation of the PPAR� gene bythe inflammatory molecules, as studied herein usingTNF-�, comprises the recruitment of FAN by p55TNF-R1, which, via activation of N-Smase, induces the pro-duction of ceramide. In turn, ceramide activates a stress-associated kinase cascade terminating at an AP-1 site inthe PPAR� promoter. It is worth noting that FAN-defi-cient mice, which show an impaired N-Smase activa-tion, have a delayed cutaneous barrier homeostasis andrepair phenotype (Kreder et al. 1999). Hence, PPAR� up-regulation and activation is clearly a stress-associatedevent, such as it occurs after injury.

Inflammation-induced PPAR� expressionand increased resistance to apoptosis

Certainly, the up-regulation of PPAR� in any biologicalsystem can lead to several outcomes. Here, these effectswill be considered in the context of keratinocytes ex-posed to TNF-�. This cytokine induces oligomerizationof the receptor p55TNF-R1, which leads to the formationof a multimolecular signal-transducing complex, fromwhich several pathways originate rapidly. Paradoxically,activation of p55TNF-R1 initiates two different types ofsignals, the first leading to cell death, and the secondprotects the cell from it. p55TNF-R1 interacts withTRADD, which recruits the downstream transducersFADD and TRAF2. FADD interacts with apoptotic pro-teases triggering cell death (Natoli et al. 1998). In con-trast, TRAF2 can activate the transcription factorsNF�B, c-Jun, ATF, TCF/Elk, or SAPK/JNK, which arenecessary, but not sufficient to confer protection againstTNF�-induced apoptosis (Natoli et al. 1998). This pointsto a critical role for additional p55TNF-R1 complex com-ponents that are involved in cytoprotection againstTNF�-induced apoptosis. We showed herein that suchan event is the stimulation of PPAR� expression and itsligand-dependent activation, which confer increased re-sistance to TNF�-induced apoptosis, as measured bycaspase activity and DNA fragmentation. PPAR� up-regulates antiapoptotic and down-regulates proapoptoticgenes, in response to inflammatory signals in keratino-

Table 1. Number of Ki67 or TUNEL positive cellsquantified based on respective fluorescent labelings

Day 4 Day 7

Ki67 TUNEL Ki67 TUNEL

PPAR�+/+ 12.3 ± 1.28 9.0 ± 2.0 5.1 ± 1.4 1.3 ± 0.8PPAR�+/− 24.1 ± 3.13** 7.5 ± 1.2 10.3 ± 1.7* 15.1 ± 2.3*

Values represent the mean cell number counted from 10 stan-dardized microscope fields per section (Fig. 7B, regions a to e anda� to e�), performed on three different animals for each genotype± SEM. Asterisks indicate that the difference is statistically sig-nificant (*P < 0.05; **P < 0.01).

Tan et al.





cytes. Thus, up-regulation of PPAR� by the FAN-medi-ated pathway, which is independent of the TRAF2-asso-ciated signaling (Adam-Klages et al. 1996), is an addi-tional way of directing TNF-� signaling away fromapoptosis and toward cell survival and differentiation.This novel pathway should contribute to avoid excessivecell death during inflammation (see below). Further sup-port to this anti-apoptotic role of PPAR� comes from theobservation that increased PPAR� levels in colorectalcarcinoma confers resistance to NSAID-induced apopto-sis (He et al. 1999).

Roles of elevated PPAR� expression duringwound repair

Wound repair requires the integration of interdependentprocesses that involve, among others, soluble mediators,inflammatory cytokines produced by a variety of celltypes, and extracellular matrix components. It comprisesthree successive major phases, that is, inflammation, re-epithelization, and tissue remodeling (Martin 1997).During the initial phase of inflammation, keratinocytesare exposed to a battery of pro-inflammatory cytokinesand bioactive lipids. PPAR� expression is activated inthe very early phase of the injury and remains high untilwound closure, whereas PPAR� expression is reactivatedonly transiently during the inflammation phase (Micha-lik et al. 2001). In agreement with the in vivo study, wealso observed a transient increase in PPAR� expressionin cultured primary keratinocytes upon exposure to in-flammatory signals. This transient PPAR� expression,together with the fact that inflammatory eicosanoids, forexample, LTB4 and 8S-HETE are PPAR� ligands(Devchand et al. 1996), and an alteration in the recruit-ment of inflammatory cells to the wound bed inPPAR�−/− mice (Michalik et al. 2001), reinforces the hy-pothesis that PPAR� participates in the control of theinflammatory response. It is worth noting, with respectto the present work on the role of PPAR�, that the ex-pression of PPAR� in response to inflammatory signalsis very similar in wild-type and PPAR�−/− keratinocytes,indicating that PPAR� does not compensate for the lackof PPAR�. It also indicates that PPAR� expression is notcontrolled by PPAR�.

The injury-triggered release of pro-inflammatory cyto-kines serves as an instant danger signal. Paradoxically,whereas these factors are important for hemostasis, re-cruitment of macrophages and removal of infectiousagents, they are also apoptotic signals (Hannun andLuberto 2000). A role of PPAR� expression, induced bycytokines and bioactive lipids, would be to confer resis-tance against apoptotic signals as discussed above,thereby providing a critical window for the action ofother factors, such as KGF, which modulate keratino-cytes behavior (Werner 1998). A deficiency in PPAR�during this phase would result in an increased apoptosisof keratinocytes, hence, reducing the number of prolif-erating and migrating cells that are vital for wound clo-sure. The results obtained in this study at wound edgesof PPAR�+/− mice are in agreement with this hypothesis.

Although we observed higher keratinocyte proliferationrates at the wound edges in mutant mice, the number ofapoptotic cells was increased dramatically. Also consis-tent with data herein, Michalik et al. (2001) reported thatthe most significant differences in the rate of wound clo-sure between wild-type and PPAR�+/− mice are observedduring the first week of wound repair.

As wound repair enters into the re-epithelizationphase, migrating keratinocytes have a major importantrole in rapid wound closure. The signals for the contin-ued PPAR� activation at that stage might be related toT-cell activation that occurs at that time. The acceler-ated keratinocyte differentiation sustained by elevatedand prolonged activation of PPAR� as seen in keratino-cyte cultures, is likely to be important during the re-epithelialization phase. Furthermore, the role of PPAR�might then also be related to a different, specific spatio-temporal role. For example, we have reported thatPPAR�+/− keratinocytes in culture are defective in sub-strate adhesion (Michalik et al. 2001). At present, it isunclear which are the PPAR� target genes that contrib-ute to this phenotype.

In conclusion, PPAR� expression and activation mightparticipate in the sequential regulatory events takingplace during wound healing or inflammatory challenges.As deviation from this pattern may result in skin disor-ders, for example, psoriasis (Nickoloff et al. 2000), theexploration of the role of PPAR� in skin disorders mightopen important therapeutic perspectives and lead to thediscovery of additional, so far unknown functions of thisPPAR isotype.

Materials and methods

Reagents

All cytokines were purchased from PeproTech, Inc. N-acetyl-D-erythro-sphingosine (ceramide) and SB203580 were from Cal-Biochem. Anti-mouse-TNF-� antibodies were from R & D Sys-tems. Keratinocyte serum-free culture medium (KSFM), Is-cove’s Modified Eagles Medium (IMDM), and Dispase wereobtained from GIBCO-BRL. Fetal calf serum (FCS, <10 EU/mL)was from Sigma. Sterile nylon wool for enrichment of T-cellswas from Robbins Scientifics. The in situ cell death detectionkit was from Roche.

Cell cultures

Dendritic cells (DCs) were cultured as described by Gallucci etal. (1999). Splenic T lymphocytes were isolated as described byPeterson et al. (2000). Mouse keratinocytes were obtained from3-day-old pups from PPAR�+/+, PPAR�+/−, and PPAR�−/− (Mi-chalik et al. 2001) and isolated from epidermis as reported byHager et al. (1999) with some modifications; epidermis wasseparated from the dermis following overnight incubation at4°C in 2.5 U/mL of Dispase. Epidermis was placed in a 50-mLcentrifuge tube with 10 mL of KSFM, and the tube was given 50firm shakes. Keratinocytes were resuspended in KSFM contain-ing 0.05 mM Ca2+ and 0.1 ng/ml epidermal growth factor, andseeded at 5 × 104 cells/cm2. Keratinocytes were used after twoto three passages. Fibroblasts were similarly isolated from thedermal pieces. Fibroblasts were plated in IMDM containing 5%FCS and were used after three to five passages.






Mixed leukocyte reaction

Induction of apoptosis in fibroblasts was performed by UV irra-diation as reported (Fadok et al. 1998). Apoptotic efficiency wasmonitored by caspase 8 activity. Necrosis was achieved by re-peated freezing at −80°C and thawing at 37°C, or by mincingnewborn mouse skin (1g/mL in PBS). Large cellular debris andany remaining viable cells were removed by centrifugation at2000g. Necrosis was assessed by trypan blue uptake.

MLR were carried out as reported (Gallucci et al. 1999).Briefly, DCs were exposed for 24 h to supernatant of necroticcells or medium from apoptotic cells in culture, after which themedium was discarded and the activated DCs were coculturedwith T-cells for 48 h. Conditioned medium obtained from thisMLR (CM) was clarified of cells by centrifugation and desaltedvia ultrafiltration with 3000 MW cutoff YM membrane (Am-bion), prior to addition to keratinocytes. Ca2+ concentration ofultrafiltered medium was below 0.02 mM.

For TNF-� neutralization experiment, necrosis-derived CMwas preincubated at 37°C for 1 h with 20 µg/mL of anti-TNF-�antibody. After treatment, the CM was added onto the keratino-cytes. The 50% neutralization dose of anti-TNF-� was 0.6µg/mL in the presence of 0.25 ng/mL of TNF-�.

RNase protection assay

The cDNA corresponding to the mouse PPAR� and PPAR� A/Bdomain, as well as gene-specific probes corresponding to Cyc A,INV, TgaseI, K6, K10, K17, and L27 were subcloned intopGEM3Zf(+) (Promega). Gene-specific antisense riboprobeswere synthesized by in vitro transcription with either T7 or Sp6RNA polymerase (Ambion). For all riboprobes, except L27, aratio of 1:1 of �32P-UTP to cold UTP was used, whereas a ratio1:20 was used for L27 probe.

RPA was carried out as described by the manufacturer (Am-bion) with the following modifications: 1–2 × 106 keratinocyteswere lysed in 200 µL of Lysis/Denaturation buffer and clarifiedby centrifugation through a Qiashredder (QIAGEN). A total of45 µL of lysate was incubated with 1 ng of gene-specific ribo-probes (1 × 109 cpm/µg) and 10 ng of L27 probe (1 × 107 cpm/µg).RPA products were resolved in a 6% electrolyte-gradient dena-turing polyacrylamide gel. Gels were dried and exposed to X-rayfilm or to Phosphor screen.

Transfection of primary keratinocytes

Keratinocytes were trypsinized and replated into 12-well cul-ture plates, and incubated for 24 h to reach 40%–50% conflu-ence. Cells were transfected using Superfect (QIAGEN). At theindicated time, cells were rinsed with phosphate buffer andlysed in Reporter Lysis buffer (Promega) for �-galactosidase andCAT assay or in Lysis/Denaturation Solution for RPA. Activityor mRNA of cotransfected pCMV-�-galactosidase reporter wasused to monitor transfection efficiency. The various MKK7 andSEK1 expression vectors were from E. Nishida (Kyoto Univer-sity, Japan) and the various MEKK1 and SAPK/JNK expressionconstructs were from R.J. Davis (University of MassachusettsMedical School, Worcester) and D.J. Templeton (University ofVirginia Medical School, Charlottesville).

The proximal 1.9-kb mouse PPAR� promoter region was sub-cloned upstream of the CAT reporter gene in pBLCAT. The 5�

deletion promoter mutants were generated using available re-striction enzyme sites. Site-directed mutagenesis of transcrip-tion factor binding sites was achieved using the QuikChangeSite-Directed mutagenesis kit (Stratagene).

The LBD of PPAR�, corresponding to amino acids 136–440,was PCR amplified, and subcloned into EcoRI/HindIII sites ofpMGAL4 (Clontech). The PPRE-TK-Luc reporter was from R.

Evans (Salk Institute for Biological Sciences, La Jolla, CA). Lip-ids of cytokine-induced keratinocytes were extracted as re-ported (Buck et al. 1991). Briefly, 5 × 106 keratinocytes weretreated with indicated agents for 12 h. The organic extract wasvacuum dried and the pellet dissolved in 10 µL of ethanol.

Apoptosis assays

TNF-�-induced apoptosis was carried out as reported (Reinartzet al. 1996). The apoptotic keratinocytes were measured usingApoAlert Caspase 8 Colorimetric assays (Clontech). Apoptosiswas also monitored using radioactive DNA fragmentation assay(Duke 1983). Briefly, the cells, at a density of 2 × 104 cells/cm2,were prelabeled via incubation with 5 µCi/mL of [3H]thymidinefor 24 h. TNF�-induced apoptosis were performed on these la-beled cells. As background control, labeled cells were unchal-lenged. At indicated time points, the cells were lysed in 150 µLof Cell Lysis buffer (Boehringer Mannheim), and subsequently,100 µL of supernatant were counted.

In vivo proliferation and apoptosis assays

The number of proliferative and apoptotic keratinocytes in thePPAR�+/+ and PPAR�+/− mice epidermis was analyzed on frozensections of skin wounds, 4 and 7 d after a dorsal full-thicknesswound. The detection of the Ki67 proliferation marker was per-formed as described (Michalik et al. 2001). The apoptotic kera-tinocytes were detected using the TUNEL assay according tomanufacturer’s protocol (Roche). Briefly, 7-µm cryosections ofdorsal wounds were fixed (4% formaldehyde/PBS, 20 min atroom temperature), treated with Proteinase K (20 mg/ml, 10min at room temperature), and the fragmented DNA was la-beled by use of fluorescent nucleotides. The slides were subse-quently counterstained (DAPI), washed, and mounted beforemicroscopic observation.

Acknowledgments

We thank E. Nishida, R.J. Davis, D.J. Templeton, and R. Evansfor the gift of plasmids; J. Tschopp for critical reading of themanuscript; and N. Deriaz, T. Favez, and M. Narce for theirassistance. This work was supported by grants from the SNSF,the Etat de Vaud, and the HFSP to B.D. and W.W. and by theNovartis Foundation to N.N.

The publication costs of this article were defrayed in part bypayment of page charges. This article must therefore be herebymarked “advertisement” in accordance with 18 USC section1734 solely to indicate this fact.

References

Adam-Klages, S., Adam, D., Wiegmann, K., Struve, S., Kolanus,W., Schneider-Mergener, J., and Kronke, M. 1996. FAN, anovel WD-repeat protein, couples the p55 TNF-receptor toneutral sphingomyelinase. Cell 86: 937–947.

Amri, E.Z., Bonino, F., Ailhaud, G., Abumrad, N.A., andGrimaldi, P.A. 1995. Cloning of a protein that mediates tran-scriptional effects of fatty acids in preadipocytes. Homologyto peroxisome proliferator-activated receptors. J. Biol.Chem. 270: 2367–2371.

Banchereau, J. and Steinman, R.M. 1998. Dendritic cells and thecontrol of immunity. Nature 392: 245–252.

Buck, J., Myc, A., Garbe, A., and Cathomas, G. 1991. Differ-ences in the action and metabolism between retinol and ret-inoic acid in B lymphocytes. J. Cell Biol. 115: 851–859.

Davis, R.J. 2000. Signal transduction by the JNK group of MAP

Tan et al.





kinases. Cell 103: 239–252.Devchand, P.R., Keller, H., Peters, J.M., Vazquez, M., Gonzalez,

F.J., and Wahli, W. 1996. The PPARalpha-leukotriene B4pathway to inflammation control. Nature 384: 39–43.

Desvergne, B. and Wahli, W. 1999. Peroxisome proliferator-ac-tivated receptors: nuclear control of metabolism. Endocr.Rev. 20: 649–688.

Dotto, G.P. 1999. Signal transduction pathways controlling theswitch between keratinocyte growth and differentiation.Crit. Rev. Oral Biol. Med. 10: 442–457.

Duke, R.C. 1983. Endogenous endonuclease-induced DNA frag-mentation: An early event in cell-mediated cytolysis. Proc.Natl. Acad. Sci. 80: 6361–6365.

Fadok, V.A., Bratton, D.L., Konowal, A., Freed, P.W., Westcott,J.Y., and Henson, P.M. 1998. Macrophages that have in-gested apoptotic cells in vitro inhibit pro-inflammatory cy-tokine production through autocrine/paracrine mechanismsinvolving TGF-beta, PGE2, and PAF. J. Clin. Invest.101: 890–898.

Gallucci, S., Lolkema, M., and Matzinger, P. 1999. Natural ad-juvants: Endogenous activators of dendritic cells. Nat. Med.5: 1249–1255.

Hager, B., Bickenbach, J.R., and Fleckman, P. 1999. Long-termculture of murine epidermal keratinocytes. J. Invest. Derma-tol. 112: 971–976.

Hanley, K., Jiang, Y., He, S.S., Friedman, M., Elias, P.M., Bikle,D.D., Williams, M.L., and Feingold, K.R. 1998. Keratinocytedifferentiation is stimulated by activators of the nuclear hor-mone receptor PPARalpha. J. Invest. Dermatol. 110: 368–375.

Hannun, Y.A. and Luberto, C. 2000. Ceramide in the eukaryoticstress response. Trends Cell Biol. 10: 73–80.

Hansen, J.B., Zhang, H., Rasmussen, T.H., Petersen, R.K.,Flindt, E.N., and Kristiansen, K. 2000. PPARdelta-mediatedregulation of preadipocyte proliferation and gene expressionis dependent on cAMP signaling. J. Biol. Chem. 276: 3175–3182.

He, T.C., Chan, T.A., Vogelstein, B., and Kinzler, K.W. 1999.PPARdelta is an APC-regulated target of nonsteroidal anti-inflammatory drugs. Cell 99: 335–345.

Inohara, N., Koseki, T., Chen, S., Wu, X., and Nunez, G. 1998.CIDE, a novel family of cell death activators with homologyto the 45kDa subunit of the DNA fragmentation factor.EMBO J. 17: 2526–2533.

Kreder, D., Krut, O., Adam-Klages, S., Wiegmann, K., Scherer,G., Plitz, T., Jensen, J.M., Proksch, E., Steinmann, J., Pfeffer,K., et al. 1999. Impaired neutral sphingomyelinase activa-tion and cutaneous barrier repair in FAN-deficient mice.EMBO J. 18: 2472–2479.

Kronke, M. 1999. Involvement of sphingomyelinases in TNFsignaling pathways. Chem. Phys. Lipids 102: 157–166.

Kyriakis, J.M. and Avruch, J. 1996. Protein kinase cascades ac-tivated by stress and inflammatory cytokines. BioEssays18: 567–577.

Mansbridge, J.N. and Knapp, A.M. 1987. Changes in keratino-cyte maturation during wound healing. J. Invest. Dermatol.89: 253–263.

Martin, P. 1997. Wound healing–aiming for perfect skin regen-eration. Science 276: 75–81.

Messmer, U.K. and Pfeilschifter, J. 2000. New insights into themechanism for clearance of apoptotic cells. BioEssays22: 878–881.

Michalik, L., Desvergne, B., Tan, N.S., Basu-Modak, S., Escher,P., Rieusset, J., Peters, J.M., Kaya, G., Gonzalez, F.J., Zakany,J., et al. 2001. Impaired skin wound healing in PPAR� andPPAR� mutant mice. J. Cell. Biol. 154: 799–814.

Natoli, G., Costanzo, A., Guido, F., Moretti, F., and Levrero, M.1998. Apoptotic, non-apoptotic, and anti-apoptotic path-ways of tumor necrosis factor signalling. Biochem. Pharma-col. 56: 915–920.

Nickoloff, B.J., Schroder, J.M., von den Driesch, P., Raych-audhuri, S.P., Farber, E.M., Boehncke, W.H., Morhenn, V.B.,Rosenberg, E.W., Schon, M.P., and Holick, M.F. 2000. Is pso-riasis a T-cell disease? Exp. Dermatol. 9: 359–375.

Nuclear Receptors Nomenclature Committee. 1999. A unifiednomenclature system for the nuclear receptor superfamily.Cell 97: 161–163.

Oliver, Jr., W.R., Shenk, J.L., Snaith, M.R., Russell, C.S., Plun-ket, K.D., Bodkin, N.L., Lewis, M.C., Winegar, D.A., Sznaid-man, M.L., Lambert, M.H., et al. 2001. A selective per-oxisome proliferator-activated receptor delta agonist pro-motes reverse cholesterol transport. Proc. Natl. Acad. Sci.98: 5306–5311.

Peters, J.M., Lee, S.S., Li, W., Ward, J.M., Gavrilova, O., Everett,C., Reitman, M.L., Hudson, L.D., and Gonzalez, F.J. 2000.Growth, adipose, brain, and skin alterations resultingfrom targeted disruption of the mouse peroxisome prolifera-tor-activated receptor beta(delta). Mol. Cell. Biol. 20: 5119–5128.

Peterson, K.E., Iwashiro, M., Hasenkrug, K.J., and Chesebro, B.2000. Major histocompatibility complex class I gene con-trols the generation of gamma interferon-producing CD4(+)and CD8(+) T cells important for recovery from friend retro-virus-induced leukemia. J. Virol. 74: 5363–5367.

Puignero, V. and Queralt, J. 1997. Effect of topically appliedcyclosporin A on arachidonic acid (AA)- and tetradec-anoylphorbol acetate (TPA)-induced dermal inflammation inmouse ear. Inflammation 21: 357–369.

Reinartz, J., Bechtel, M.J., and Kramer, M.D. 1996. Tumor ne-crosis factor-alpha-induced apoptosis in a human keratino-cyte cell line (HaCaT) is counteracted by transforminggrowth factor-alpha. Exp. Cell Res. 228: 334–340.

Saluja, I., Granneman, J.G., and Skoff, R.P. 2001. PPARdeltaagonists stimulate oligodendrocyte differentiation in tissueculture. Glia 33: 191–204.

Savill, J. and Fadok, V. 2000. Corpse clearance defines the mean-ing of cell death. Nature 407: 784–788.

Spellberg, B. 2000. The cutaneous citadel: A holistic view ofskin and immunity. Life Sci. 67: 477–502.

Szabowski, W., Mass-Szabowsk, N., Andrecht, S., Kolbus, A.,Schorpp-Kistner, M., Fusenig, N.E., and Angel, P. 2000. c-Junand JunB antagonistically control cytokine-regulated mesen-chymal-epidermal interaction in skin. Cell 103: 745–755.

Werner, S. 1998. Keratinocyte growth factor: A unique player inepithelial repair processes. Cytokine Growth Factor Rev.9: 153–165.

Wingender, E., Chen, X., Hehl, R., Karas, H., Liebich, I., Matys,V., Meinhardt, T., Prüß, M., Reuter, I., and Schacherer, F.2000. TRANSFAC: An integrated system for gene expressionregulation. Nucleic Acids Res. 28: 316–319.






10.1101/gad.207501Access the most recent version at doi: 15:2001, Genes Dev.

Nguan Soon Tan, Liliane Michalik, Noa Noy, et al.

in keratinocyte response to inflammationδ/βCritical roles of PPAR

References

http://genesdev.cshlp.org/content/15/24/3263.full.html#ref-list-1

This article cites 38 articles, 11 of which can be accessed free at:

License

ServiceEmail Alerting

click here.right corner of the article or

Receive free email alerts when new articles cite this article - sign up in the box at the top

Cold Spring Harbor Laboratory Press


http://genesdev.cshlp.org/lookup/doi/10.1101/gad.207501

http://genesdev.cshlp.org/content/15/24/3263.full.html#ref-list-1

http://genesdev.cshlp.org/cgi/alerts/ctalert?alertType=citedby&addAlert=cited_by&saveAlert=no&cited_by_criteria_resid=protocols;10.1101/gad.207501&return_type=article&return_url=http://genesdev.cshlp.org/content/10.1101/gad.207501.full.pdf



critical roles of ppar in keratinocyte response to...

Documents