Peroxisome proliferator-activated receptor gamma induces apoptosis and inhibits autophagy of human monocyte-derived macrophages via induction of cathepsin L: potential role in atherosclerosis.
ABSTRACT Macrophages play a pivotal role in the pathophysiology of atherosclerosis. These cells express cathepsin L (CatL), a cysteine protease that has been implicated in atherogenesis and the associated arterial remodeling. In addition, macrophages highly express peroxisome proliferator-activated receptor (PPAR) γ, a transcription factor that regulates numerous genes important for lipid and lipoprotein metabolism, for glucose homeostasis, and inflammation. Hence, PPARγ might affect macrophage function in the context of chronic inflammation such as atherogenesis. In the present study, we examined the effect of PPARγ activation on the expression of CatL in human monocyte-derived macrophages (HMDM). Activation of PPARγ by the specific agonist GW929 concentration-dependently increased the levels of CatL mRNA and protein in HMDM. By promoter analysis, we identified a functional PPAR response element-like sequence that positively regulates CatL expression. In addition, we found that PPARγ-induced CatL promotes the degradation of Bcl2 without affecting Bax protein levels. Consistently, degradation of Bcl2 could be prevented by a specific CatL inhibitor, confirming the causative role of CatL. PPARγ-induced CatL was found to decrease autophagy through reduction of beclin 1 and LC3 protein levels. The reduction of these proteins involved in autophagic cell death was antagonized either by the CatL inhibitor or by CatL knockdown. In conclusion, our data show that PPARγ can specifically induce CatL, a proatherogenic protease, in HMDM. In turn, CatL inhibits autophagy and induces apoptosis. Thus, the proatherogenic effect of CatL could be neutralized by apoptosis, a beneficial phenomenon, at least in the early stages of atherosclerosis.
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ABSTRACT: The formation of an atherosclerotic lesion is mediated by lipid-laden macrophages (foam cells), which also establish chronic inflammation associated with lesion progression. The peroxisome proliferator-activated receptor (PPAR) gamma promotes lipid uptake and efflux in these atherogenic cells. In contrast, we found that the closely related receptor PPARdelta controls the inflammatory status of the macrophage. Deletion of PPARdelta from foam cells increased the availability of inflammatory suppressors, which in turn reduced atherosclerotic lesion area by more than 50%. We propose an unconventional ligand-dependent transcriptional pathway in which PPARdelta controls an inflammatory switch through its association and disassociation with transcriptional repressors. PPARdelta and its ligands may thus serve as therapeutic targets to attenuate inflammation and slow the progression of atherosclerosis.Science 11/2003; 302(5644):453-7. · 31.20 Impact Factor
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ABSTRACT: An early event in acute and chronic inflammation and associated diseases such as atherosclerosis and rheumatoid arthritis is the induced expression of specific adhesion molecules on the surface of endothelial cells (ECs), which subsequently bind leukocytes. Peroxisome proliferator-activated receptors (PPARs), members of the nuclear receptor superfamily of transcription factors, are activated by fatty acid metabolites, peroxisome proliferators, and thiazolidinediones and are now recognized as important mediators in the inflammatory response. Whether PPAR activators influence the inflammatory responses of ECs is unknown. We show that the PPAR activators 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), Wyeth 14643, ciglitazone, and troglitazone, but not BRL 49653, partially inhibit the induced expression of vascular cell adhesion molecule-1 (VCAM-1), as measured by ELISA, and monocyte binding to human aortic endothelial cells (HAECs) activated by phorbol 12-myristate 13-acetate (PMA) or lipopolysaccharide. The "natural" PPAR activator 15d-PGJ(2) had the greatest potency and was the only tested molecule capable of partially inhibiting the induced expression of E-selectin and neutrophil-like HL60 cell binding to PMA-activated HAECs. Intracellular adhesion molecule-1 induction by PMA was unaffected by any of the molecules tested. Both PPAR-alpha and PPAR-gamma mRNAs were detected in HAECs by using reverse transcription-polymerase chain reaction and a ribonuclease protection assay; however, we have yet to determine which, if any, of the PPARs are mediating this process. These results suggest that certain PPAR activators may help limit chronic inflammation mediated by VCAM-1 and monocytes without affecting acute inflammation mediated by E-selectin and neutrophil binding.Arteriosclerosis Thrombosis and Vascular Biology 10/1999; 19(9):2094-104. · 6.34 Impact Factor
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ABSTRACT: Atherosclerotic coronary heart disease is a common complication of the insulin resistance syndrome that can occur with or without diabetes mellitus. Thiazolidinediones (TZDs), which are insulin-sensitizing antidiabetic agents, can modulate the development of atherosclerosis not only by changing the systemic metabolic conditions associated with insulin resistance but also by exerting direct effects on vascular wall cells that express peroxisome proliferator-activated receptor-gamma (PPAR-gamma), a nuclear receptor for TZDs. Here we show that troglitazone, a TZD, significantly inhibited fatty streak lesion formation in apolipoprotein E-knockout mice fed a high-fat diet (en face aortic surface lesion areas were 6.9+/-2.5% vs 12.7+/-4.7%, P<0.05; cross-sectional lesion areas were 191 974+/-102 911 micrometer(2) vs 351 738+/-175 597 micrometer(2), P<0.05; n=10). Troglitazone attenuated hyperinsulinemic hyperglycemia and increased high density lipoprotein cholesterol levels. In the aorta, troglitazone markedly increased the mRNA levels of CD36, a scavenger receptor for oxidized low density lipoprotein, presumably by upregulating its expression, at least in part, in the macrophage foam cells. These results indicate that troglitazone potently inhibits fatty streak lesion formation by modulating both metabolic extracellular environments and arterial wall cell functions.Arteriosclerosis Thrombosis and Vascular Biology 04/2001; 21(3):372-7. · 6.34 Impact Factor
Peroxisome Proliferator-activated Receptor ? Induces
Dler Faieeq Darweesh Mahmood‡1, Imene Jguirim-Souissi‡1, El-Hadri Khadija‡, Nicolas Blondeau§, Vimala Diderot‡,
Souliman Amrani¶, Mohamed-Naceur Slimane?, Tatiana Syrovets**, Thomas Simmet**, and Mustapha Rouis‡2
Fromthe‡Unite ´ deRecherche,Vieillissement,StressetInflammation,Universite ´ PierreetMarieCurie,75252Paris,Cedex5,France,
the§InstitutdePharmacologieMole ´culaireetCellulaire,UMR6097,CNRS/Universite ´ deNiceSophiaAntipolis,06560Valbonne,
France,the¶LaboratoiredeBiochimie,Faculte ´ desSciencesdeOujda,60000Oujda,Morocco,the?LaboratoiredeBiochimie,
Faculte ´ deMe ´decinedeMonastir,5000Monastir,Tunisia,andthe**InstituteofPharmacologyofNaturalProductsandClinical
Pharmacology,UlmUniversity,D-89081 Ulm, Germany
erosclerosis. These cells express cathepsin L (CatL), a cysteine
ciated arterial remodeling. In addition, macrophages highly
express peroxisome proliferator-activated receptor (PPAR) ?, a
transcription factor that regulates numerous genes important
for lipid and lipoprotein metabolism, for glucose homeostasis,
and inflammation. Hence, PPAR? might affect macrophage
genesis. In the present study, we examined the effect of PPAR?
activation on the expression of CatL in human monocyte-de-
rived macrophages (HMDM). Activation of PPAR? by the spe-
cific agonist GW929 concentration-dependently increased the
levels of CatL mRNA and protein in HMDM. By promoter anal-
ysis, we identified a functional PPAR response element-like
sequence that positively regulates CatL expression. In addition,
we found that PPAR?-induced CatL promotes the degradation
of Bcl2 without affecting Bax protein levels. Consistently, deg-
found to decrease autophagy through reduction of beclin 1 and
LC3 protein levels. The reduction of these proteins involved in
itor or by CatL knockdown. In conclusion, our data show that
in HMDM. In turn, CatL inhibits autophagy and induces apo-
ized by apoptosis, a beneficial phenomenon, at least in the early
stages of atherosclerosis.
involves interaction of a variety of cells within the vessel wall,
including smooth muscle cells, endothelial cells, and mono-
genesis, mainly through accumulation of oxidatively modified
LDL and production of a variety of inflammatory mediators,
cytokines, and extracellular matrix-degrading enzymes (1).
Rupture of atherosclerotic plaques is the most common event
triggering formation of occlusive thrombi in arteries and sub-
sequent acute ischemic events, such as acute coronary syn-
dromes and stroke (2).
Several cysteine proteases have been implicated in athero-
genesis and the associated arterial remodeling. Cysteine cathe-
psins are members of the papain family of proteases that
degrade elastin and collagen (3). Among them, cathepsin L
(CatL)3is one of the most potent collagenases and elastases
expression is enhanced in human coronary lesions, but also in
human carotid lesions and abdominal aortic aneurysms (8).
CatK, CatS, and CatF are also expressed in human atheroscle-
rotic lesions (9, 10), and CatB, CatD, and CatL were found in
macrophage-derived foam cells in lipid-rich plaque areas (11).
that deficiency of CatS and CatL in LDL receptor knock-out
mice reduced atherosclerotic lesions (6, 12). Of note, unstable
plaque regions contain increased levels of active legumain, a
cysteine protease that promotes intracellular processing of
CatL to its mature 25-kDa form (13). The role of cathepsins in
apoptosis is now widely recognized in a variety of cell types
including macrophages (14, 15).
Peroxisome proliferator-activated receptor (PPAR) ? is a
transcription factor that regulates a large number of genes
important for lipid and lipoprotein metabolism, for glucose
* This work was supported by Fondation Coeur et Arte `res, Fondation de
France,andtheComite ´ MixtedeCoope ´rationUniversitaireexchangepro-
gram between Tunisia and France.
1Both authors contributed equally to this work.
2To whom correspondence should be addressed: UR-04 Vieillissement,
StressetInflammation,Universite ´ PierreetMarieCurie-Paris6,Ba ˆt.A.5e `me
e ´tage/Case courrier 256. 7, Quai St-Bernard, 75252 Paris Cedex 5, France.
3The abbreviations used are: Cat, cathepsin; PPAR, peroxisome proliferator-
activated receptor; HMDM, human monocyte-derived macrophage(s);
ODN, phosphorothioate-modified oligonucleotides; PTEN, Phosphatase
tor; Trx, thioredoxin.
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 286, NO. 33, pp. 28858–28866, August 19, 2011
© 2011 by The American Society for Biochemistry and Molecular Biology, Inc.Printed in the U.S.A.
at CNRS, on February 6, 2013
the retinoid X receptor (RXR) and their subsequent binding to
PPAR response elements (PPRE) in the promoter regions of
target genes (17). PPAR? can also repress gene expression
either in a DNA binding-independent manner by interfering
with other signaling pathways or in a DNA binding-dependent
way through the recruitment of corepressors (18). Similar to
rotic lesions and macrophage-derived foam cells (19–21),
where it may affect macrophage function and consequently
of atherosclerosis in apolipoprotein E-deficient (apoE?/?) (22)
and in LDL receptor-deficient (LDLR?/?) mice (23, 24).
In addition, mice transplanted with bone morrow from
PPAR??/?chimera mice exhibited a significantly increased
atherosclerosis (25, 26) suggesting an antiatherogenic role of
PPAR? in macrophages.
PPAR? could inhibit atherosclerosis either by direct inhibi-
receptor ? (28), further, by negatively interfering with AP-1,
necrosis factor-?, IL-1, and IL-6 secretion (30). In addition,
inhibits CC chemokine receptor 2 (CCR2) and suppresses
MCP-1-mediated chemotaxis (31) Atheroprotective effects of
induce reduction of endothelial adhesion molecules by reduc-
tion of monocyte/macrophage recruitment in a mouse model
of atherosclerosis (24, 32, 33). Moreover, it has been reported
apoptosis of nonactivated or activated human macrophages
(34). Concomitantly, it was observed that PPAR? inhibits the
transcriptional activity of the p65/RelA subunit of NF?B, sug-
gesting that PPAR activators induce macrophage apoptosis by
negatively interfering with anti-apoptotic NF?B signaling (34).
Up-regulation of phosphatase and tensin homolog by PPAR?
may be another mechanism by which PPAR? could induce
macrophage apoptosis (35). Only recently, we reported a new
mechanistic explanation for the PPAR?-mediated induction of
macrophage apoptosis. We demonstrated that PPAR? activa-
tion stimulates apoptosis in human macrophages by altering
the cellular redox balance via regulation of thioredoxin-1
(Trx-1) and its endogenous inhibitor, TXNIP (the thioredoxin
ulated protein-1) (36).
In the present study, we examined the effect of PPAR? acti-
vation on the expression of CatL in human monocyte-derived
macrophages. Because several studies indicated that PPAR?
activation exerts antiatherogenic effects, we hypothesized
that PPAR? activation might inhibit the expression of CatL
in human monocyte-derived macrophages. Surprisingly,
we found that PPAR? concentration-dependently increases
Isolation and Culture of Human Monocytes—Mononuclear
cells were isolated by Ficoll gradient centrifugation from the
buffy coats of healthy normolipidemic donors. Cells were
cultured in RPMI 1640 supplemented with gentamycin (40
?g/ml), glutamine (0.05%) (Sigma), 10% pooled human serum
(Promocell, Heidelberg, Germany) at a density of 6 ? 106cells/
well in 60-mm well Primaria-plastic culture dishes (Polylabo,
Strasbourg, France). Differentiation of monocytes into human
monocyte-derived macrophages (HMDM) was allowed to
occur spontaneously by adhesion of cells to the culture dishes
and by continued maturation for the subsequent 8–12 days.
RNA Extraction and Analysis—Total cellular RNA was
extracted using TRIzol (Invitrogen). For quantitative PCR,
reverse transcribed CatL mRNA was quantified by real time
PCR on a MX4000 apparatus (Stratagene, La Jolla, CA), using
the forward primer (5?-GCATAATCCATTAGGCCACC-
ATT-3?) and the reverse primer (5?-CAGATCTGTGGATTG-
GAGAGA-3?). PCR amplification was performed in a volume
of 25 ?l containing 100 nM of each paired primer, 4 mM MgCl2,
and the Brilliant quantitative PCR core reagent kit mix as rec-
at 95, 55, and 72 °C. Levels of CatL were normalized to the
internal control, 36B4 mRNA, using the primer set (forward:
5?-CATGCTCAACATCTCCCCCTTCTCC-3? and reverse:
Western Blotting Analyses—HMDM cells were treated with
PPAR?, ?, or ?/? agonists or with CatL inhibitor (Merck, ref.
of protease inhibitors (Sigma). Protein concentrations were
measured by Peterson’s method with BSA as standard. The
samples were denatured with SDS loading buffer and sub-
jected to SDS-PAGE (Invitrogen SARL, Cergy-Pontoise,
France). The samples were transferred to nitrocellulose
membranes blocked with nonfat dried milk. The blots were
incubated with monoclonal anti-human CatL antibody (Sig-
ma; 1:500), rabbit anti-human beclin 1 antibody (Cell Signal-
ing; 1:1000), rabbit anti-human LC3 antibody (Cell Signal-
ing; 1:1000), rabbit anti-human Bax antibody (Cell Signaling;
1:1000), or rabbit anti-human Bcl2 antibody (Cell Signaling;
1:1000); after washing, the blots were incubated with perox-
idase-conjugated goat anti-mouse IgG (Bio-Rad) (1:500 dilu-
tion). Human ?-actin (Santa Cruz; 1:1000) served as loading
kit (Amersham Biosciences).
Immunocytochemistry—HMDM cells were cultured in
chamber slides (LabTec). After treatment, HMDM were fixed
with 4% paraformaldehyde/PBS, permeabilized in 0.3% Tween
20, and blocked with 5% goat serum/PBS at room temperature.
HMDM were then incubated with the following rabbit poly-
clonal antibodies from Cell Signaling: anti-beclin-1 (1:400),
antibodies (1:1000; Molecular Probes, Leiden, Netherlands) in
5% normal goat serum. Hoechst 33342 stain was used to label
To ensure comparable immunostaining, HMDM cultures
were processed together under the same conditions. Confocal
microscopy was performed with a laser scanning confocal
at CNRS, on February 6, 2013
tive. Quantification was done on pictures acquired with the
same settings (such as the laser power, scanning speed, and
photomultiplier gain). The fluorescence intensity was digitally
measured for each cell of five randomly selected fields from
each culture condition (Image J; National Institutes of Health).
Quantification was carried out by investigators blinded to the
Plasmids and Transient Transfection Assays—The human
CatL promoter constructs were generated by PCR using Pfu
polymerase (Stratagene) with human genomic DNA as a tem-
plate (GenBank AF163338.1). A SmaI site-linked reverse
5?-primer (5?-CGCACCCcgGGATGCCGCTC-3?) was used
with NheI site-linked forward 5?-primer (5?-ACCAAAAAT-
gCtAGcACTAAGGAATAG-3?) to amplify the ?370/?36
fragment, with NheI site-linked forward 5?-primer (5?-ATAA-
TCCATAGgCTAGcGAGAG-3?) to amplify the ?783/?36
fragment or with KpnI site-linked forward 5?-primer (5?-TTC-
TAAAGGTACcTAATGCTAC-3?) to amplify the ?1250/?36
fragment. Inserted nucleotides used to create restriction sites
erless firefly luciferase reporter vector pGL3-Basic (Promega,
Mannheim, Germany) in the correct orientation. One firefly
CatL promoter (?1250/?36 bp) with mutated PPRE-like (5?-
GCAGGCCAGcCcCCTCCCTCC-3?) was generated using the
QuikChange site-directed mutagenesis kit (Stratagene). Point
mutations were introduced in the human CatLPPRE-like using
Pfu DNA polymerase, double-stranded plasmid DNA, and
ified by automated sequencing (Applied Biosystems Inc.,
Courtaboeuf, France). Plasmid DNA was prepared using the
Qiagen Maxi Prep kit (Qiagen).
THP-1 cells, grown in 6-well culture dishes in RPMI 1640
transfected with these luciferase reporter plasmids or with
empty vector, using jetPEI-Man transfection reagent (Qbio-
cytometry with a GFP expression control plasmid, was ?30%.
?-Galactosidase expression vector (100 ng of a pCH110-?-Gal;
GE Healthcare) was cotransfected as control for transfection
efficiency. 20 h post-transfection, the medium was changed
(RPMI 1640 with 1% Nutridoma HU) and supplemented with
or without PPAR? agonist. After 36 h, the cells were washed
with PBS, lysed in 100 ?l of passive lysis buffer (Promega), and
were normalized to ?-galactosidase and presented as fold
ing promoterless control vector (basic pGL3).
Knockdown of CatL and CatB—For in vitro knockdown of
CatL and CatB, phosphorothioate-modified oligonucleotides
(ODN) (ThermoHybaid, Ulm, Germany) were used. The ODN
sequences complementary to corresponding mRNA sequences
devoid of secondary structures, i.e. the loops, were selected
using available algorithms (38). The antisense ODN for CatL
corresponded to the nucleotides 544–565 of human CatL
mRNA (NM_001912.4) 5?-AATACAGGGAAGGGAAACAC-
AG6–3?, and for human CatB, they corresponded to nucleo-
tides 1087–1105 (NM_001908.3) 5?-TTCTTTAAAATACTC-
base pairs in a scrambled order. The sequences were analyzed
for a lack of secondary structure and oligonucleotide pairing.
According to blast search, the selected sequences did not show
any similarity to coding mRNA. Macrophages were treated for
72 h every 24 h with 5 ?M of the ODN in RPMI 1640 supple-
mented with 10% FCS (39). The cells were kept in the presence
or absence of the PPAR? agonist for 24 h, lysed, and analyzed
for protein expression by Western immunoblotting using anti-
bodies against CatL, CatB, beclin 1, LC3, or ?-actin.
Electrophoretic Mobility Shift Assays—Human PPAR?,
PPAR?, PPAR?/?, and RXR? were synthesized in vitro using
the TNT quick coupled transcription/translation system (Pro-
stranded oligonucleotides containing the consensus PPRE
sequence were used as control PPRE (40). The putative CatL
PPRE-like (5?-GCAGCCAGTCTCCTCCCTCC-3?) and its
mutated form (5?-GCAGGCCAGCCCCTCCCTCC-3?) were
end-labeled with [?-32P]ATP with T4-polynucleotide kinase.
Either protein (2.5 ?l) was incubated for 15 min at room tem-
perature in a total volume of 20 ?l with 2.5 ?g of poly(dI-dC)
and 1 ?g of herring sperm DNA in binding buffer before the
radiolabeled probe was added. Binding reactions were incu-
bated for a further 15 min and then resolved by 4% nondena-
turing PAGE. For competition experiments, a 50-fold excess
of unlabeled oligonucleotides over the labeled probe were
included in the binding reaction.
ChIP Assays—Experiments were performed with a ChIP
min at 37 °C. Subsequent procedures were performed on ice in
the presence of protease inhibitors. Cells cross-linked by form-
aldehyde treatment were harvested, washed, and lysed. Chro-
matin was sonicated with five 10-s pulses at 30% amplitude
(sonifier; Branson Ultrasonic Corp). After centrifugation, the
supernatant was diluted 10-fold with ChIP dilution. Diluted
protein A-agarose. One-tenth of the diluted extract was kept
for direct PCR (Input). The remaining extracts were incubated
for 16 h at 4 °C in the presence of 1 ?g of specific PPAR? anti-
body (Santa Cruz Biotechnology) per ml, followed by 1 h of
incubation with salmon sperm DNA protein A-agarose beads.
Following extensive washing, bound DNA fragments were
eluted with elution buffer (1% SDS, 0.1 M NaHCO3). DNA was
recovered in elution buffer containing 200 mM NaCl and then
incubated in the presence of proteinase K (20 ?g/ml) for 1 h at
and chloroform isoamyl alcohol and ethanol-precipitated
before being subjected to PCR using specific oligonucleotide
primers that amplify a fragment containing the human CatL
PPRE-like site (forward, 5?-ACGCCCGCGCTCTCTGGAG-
3?; and reverse, 5?-TTTAGAAGAATCTGGTAAG-3?).
Statistical Analysis—Quantitative results are expressed as
the means ? S.D. The results were analyzed by multi-group
comparison Mann-Whitney U and Newman-Keul’s tests. The
differences were considered statistically significant at p ? 0.05.
at CNRS, on February 6, 2013
PPAR? Induces Expression of Human CatL in HMDM—To
determine whether CatL is regulated by PPAR?, PPAR?, or
PPAR?/?, we treated HMDM with 0.06, 0.6, and 6 ?M of each
agonist. Of the tested agonists, only the selective PPAR? ago-
nist GW929 significantly increased CatL mRNA expression
(100 ? 10% at 0.06 ?M; 170 ? 50%, p ? 0.05 at 0.6 ?M; and
320 ? 49%, p ? 0.01 at 6 ?M versus control cells (100%)) (Fig.
1A). In addition, we investigated the effects of the PPAR? ago-
and aP2 and correlated their induction to that of CatL. Indeed,
we observed induction of both genes with the agonist GW929,
and the expression was aP2 ? CD36 ? CatL (aP2: 550 ? 28%,
225%, p ? 0.001 at 6 ?M versus control; and CD36: 200 ? 100%
0.05 at 6 ?M versus control) (Fig. 1B). In contrast, the PPAR?
agonist Wy14643 did not induce mRNA. However, similar to
GW929, another PPAR? agonist, BRL49653, induced a signifi-
cant increase in mRNA expression of CatL, aP2, and CD36
(data not shown).
FIGURE 1. PPAR? activation induces CatL, aP2, and CD36 mRNA expres-
sion. HMDM cells were treated with either vehicle (control), GW647, GW929,
or GW516 for 24 h. A, effects of PPAR activation on CatL mRNA and 36B4
mRNA. Compounds were used at 0.06, 0.6, and 6 ?M, and their effects
are the means ? S.D. of four separate experiments; each was performed in
FIGURE 2. PPAR? activation induces protein expression of CatL. HMDM
cells were treated either with vehicle (control), GW647, GW929, or GW516
(each at 0.06, 0.6, and 6 ?M) for 24 h. CatL protein levels were evaluated by
three independent Western immunoblots. The results are the means ? S.D.;
0.05; **, p ? 0.01; NS, not significant, compared with control.
FIGURE 3. PPAR? activation elicits CatL promoter activity. HMDM were
cotransfected with one of three constructs of the CatL promoter
(pGL3?1250/?36, pGL3?783/?36, pGL3?370/?36, or PPRE-like mutated
RXR? expression vectors and pCH110, a ?-galactosidase vector for 20 h. The
cells were then incubated in the presence or absence of GW929 (0.6 ?M) for
36 h. The results are the means ? S.D. of three separate experiments, each
AUGUST19,2011•VOLUME286•NUMBER33 JOURNALOFBIOLOGICALCHEMISTRY 28861
at CNRS, on February 6, 2013
To determine whether the GW929-induced changes in
mRNA expressions would also occur at the protein level,
we performed immunoblot experiments. These experiments
revealed a significant increase in CatL expression (180 ? 68%,
specific PPAR? GW647 or the PPAR ?/? agonist GW516 did
study on the effects of the PPAR? agonist GW929 on CatL
Effects of PPAR? Activation on the CatL Promoter—Because
expression of CatL was increased upon treatment of HMDM
with the specific PPAR? agonist (Figs. 1 and 2), we determined
whether this effect was mediated by the promoter region prox-
imal upstream of the CatL gene. First, HMDM cells were tran-
siently transfected with a luciferase reporter vector driven by
?783/?36 and ?370/?36). Cotransfection with PPAR? and
RXR? significantly stimulated the ?783/?36 CatL promoter
tion of the CatL gene by PPAR? indicated the presence of a
functional PPRE in the CatL promoter located between ?783
and ?370. A detailed computer analysis was performed in the
presence of a putative PPRE-like motif, located between ?652
and ?662, with 91% homology with the PPRE consensus
defined for PPARs (40). To prove that this putative PPRE-like
sequence could be involved in the transcriptional activation of
CatL by PPAR?, we next analyzed the activity of a mutated
the mutant CatL promoter did not respond to GW929 (Fig. 3).
CatL Promoter Contains a PPRE-like Motif That Confers
Responsiveness to PPAR?—To demonstrate that the putative
heterodimer, we performed an electrophoretic mobility shift
assay. First, we verified that the in vitro translated RXR? and
tion/translation system bind the consensus PPRE by het-
FIGURE 4. PPAR? and RXR? bind as heterodimer to a PPRE-like site located in the 5?-flanking region of the CatL gene. A, gel retardation assays with in
vitro translated hPPAR? and hRXR? were performed with end-labeled oligonucleotides representing the consensus DR1 PPRE, the human putative CatL
PPRE-like, and a mutated version of the human putative PPRE-like. The consensus DR1 PPRE was used as unlabeled competitor. Supershift analysis was
positive control, input chromatin of the cell lysate was also amplified.
at CNRS, on February 6, 2013
erodimerization. Second, in agreement with our previous data
(36), incubation of the labeled PPRE-like sequence from the
CatL promoter with in vitro translated PPAR? and RXR?
complex was not observed with the mutated PPRE-like oligo-
nucleotide and disappeared when it was incubated with a
50-molar excess of unlabeled CatL-PPRE oligonucleotide as a
specific competitor (Fig. 4A). Moreover, incubation of labeled
PPRE-like oligonucleotide from CatL promoter with in vitro
PPAR? resulted in a supershifted band (Fig. 4A). Finally, the
result of the ChIP experiment showed an increased fixation of
the complex on the PPRE-like site upon activation of PPAR?
with GW929 (Fig. 4B). Taken together, these results indicate
that the PPAR? agonist GW929 induced CatL expression in
HMDM, at least in part, through binding of PPAR? to the
PPRE-like motif located between ?783 and ?1250 of the
human CatL promoter.
CatL Increases Apoptosis in HMDM—Next we analyzed the
effects of PPAR? activation on the induction of autophagy and
apoptosis in HMDM. By quantitative immunocytochemistry,
tion of autophagosomes during autophagy (42). At the same
time, treatment with GW929 concentration-dependently
sis in HMDM (Fig. 5).
were determined using Western immunoblots. The results
showed no significant changes in the protein levels of Bax after
normalization with ?-actin (Fig. 6). In contrast, a concentra-
a consequence, the Bax/Bcl-2 ratio was decreased (76 ? 13%,
p ? 0.05 and 51 ? 12%, p ? 0.05 by 0.6 and 6 nM of the CatL
consistent with a decreased susceptibility to apoptosis, indicat-
ing that CatL is involved in the PPAR?-mediated induction of
The cells were then washed, fixed, permeabilized, and blocked. HMDM were then incubated overnight with anti-cleaved caspase-3 (Asp175) antibody,
followed by anti-IgG Alexa 488-coupled antibodies. Hoechst 33342 staining was used to label HMDM nuclei. Confocal microscopy was performed with a
p ? 0.01; ***, p ? 0.001, compared with control cells.
FIGURE 6. CatL inhibitor reduces the Bax/Bcl2 ratio in PPAR?-activated
macrophages. HMDM were treated with PPAR? agonist GW929 (0.6 ?M) for
24 h, followed by treatment with CatL inhibitor at 0.6 or 6 nM for additional
Bax, Bcl2, and ?-actin protein levels were analyzed, and the results are pre-
sented as the means ? S.D. of four independent Western immunoblots. One
representative Western blot for each protein was inserted. *, p ? 0.05 com-
pared with control.
at CNRS, on February 6, 2013
Cathepsin L Decreases Autophagy in HMDM—Because
bilization, selective induction of macrophage death is increas-
ingly gaining attention in cardiovascular medicine because it
could stabilize vulnerable, rupture-prone lesions (43). Com-
pared with apoptosis or necrosis, autophagy might be a favor-
able type of cell death leading to elimination of macrophages
in atherosclerotic plaques, because autophagic macrophages
literally digest themselves to death (43). As a consequence of
autophagy, the cytoplasmic content progressively decreases so
postautophagic necrosis are minimal. Therefore, we examined
whether autophagy is involved in mechanisms of cell death
induced by PPAR? treatment. To do so, we examined the pro-
tein levels of beclin 1 (Bcl2-interacting protein) and microtu-
bule-associated protein 1 LC3 (light chain 3), which had previ-
ously been shown to promote autophagy (42). Our results
indicate that PPAR? decreases the protein expression of beclin
1 (87 ? 13% at 0.06 ?M; 49 ? 21% at 0.6 ?M, p ? 0.05) (Figs. 7A
and 8). In addition, in the presence of the CatL inhibitor, we
any changes in the mRNA levels of beclin 1 following either
Similar experiments were conducted to evaluate the level of
LC3 protein. Fig. 9A shows a concentration-dependent inhibi-
tory effect of PPAR? on LC3 expression (48 ? 15%, p ? 0.01
and 39 ? 25% p ? 0.01 by 0.06 and 0.6 ?M GW929, respec-
tion-dependently prevented the degradation of LC3 (162 ?
14%, p ? 0.05 and 220 ? 25%, p ? 0.01 by 0.6 and 6 nM of the
CatL inhibitor, respectively) (Fig. 9B). Finally, knockdown of
CatL in PPAR? agonist-treated HMDM (Fig. 10A) protects
beclin 1 and LC3 from degradation (Fig. 10B). In contrast,
expression of both proteins (Fig. 10B), confirming the specific
effect of CatL on beclin 1 and LC3 degradation.
Our study shows that PPAR? activation increases the levels
of CatL mRNA and protein in HMDM. Using promoter analy-
sis, we identified a functional PPRE-like sequence in the CatL
promoter region that confers increased CatL regulation by
PPAR?. In addition, we show that PPAR?-induced CatL
decreases the levels of Bcl2 and increases those of BAX, indi-
cating involvement of CatL in the PPAR?-mediated induction
of macrophage apoptosis. We also found that PPAR?-induced
CatL decreases autophagy through the reduction of beclin 1
and LC3. The reduction of proteins involved in autophagic cell
FIGURE 7. PPAR?-induced decrease in beclin 1 levels is reversed by CatL
inhibitor. A, HMDM were treated with the PPAR? agonist GW929 (0.06 and
0.6 ?M) for 24 h. The cells were washed and lysed, and the protein levels of
beclin 1 and ?-actin (for normalization) were analyzed by Western immuno-
representative Western blot for each protein was inserted. *, p ? 0.05 com-
pared with control. B, HMDM were treated with the PPAR? agonist GW929
(0.6 ?M) for 24 h. The cells were incubated for an additional 24 h in the pres-
lysed, and beclin 1 and ?-actin (for normalization) protein levels were ana-
lyzed. The results are presented as the means ? S.D. of four independent
inserted. *, p ? 0.05 compared with control.
FIGURE 8. PPAR? activation reduces beclin 1 expression in HMDM.
HMDM, cultured in chamber slides for 7 days, were rinsed and treated with
the PPAR? agonist GW929 for 24 h. HMDM were incubated overnight with
anti-beclin-1 antibody followed by anti-IgG Alexa 488-coupled antibodies.
Hoechst 33342 staining was used to label HMDM nuclei, and all of the cham-
ber slides were rinsed and mounted with Fluoprep mounting medium. Con-
Plan Apo 63?/1.4 NA oil immersion objective. Quantification was done on
condition and presented as the means ? S.D. ***, p ? 0.001 compared with
at CNRS, on February 6, 2013
death was antagonized by a CatL inhibitor following CatL
knockdown in PPAR? agonist-treated HMDM. To the best of
our knowledge, this is the first study showing that PPAR?
of CatL in human macrophages. Previous studies have shown
that PPAR? can induce apoptosis through other pathways.
Thus, Trx-1, a 12-kDa protein, which plays a major role in the
regulation of the cellular redox balance, can interact with
ASK-1 and inhibits apoptosis (44). In addition, we have previ-
ously shown that the PPAR? agonist GW929 concentration-
dependently increased HMDM expression of VDUP-1, a spe-
cific endogenous inhibitor for Trx-1, suggesting that PPAR?
activation stimulates apoptosis in human macrophages by
(36). Trx-1 may also antagonize apoptosis by binding to PTEN
and the subsequent inhibition of its phosphatase activity (45).
The reduced expression/activity of Trx-1 in cells treated with
PPAR? agonists would increase PTEN activity and contribute
to decreased cell survival by inhibition of Akt signaling. Fur-
thermore, it has been demonstrated that activation of PPAR?
by its selective ligand rosiglitazone directly up-regulates PTEN
expression in several cell types including macrophages and
reduces the rate of macrophage proliferation (35). Taken
ways, such as direct up-regulation of PTEN gene expression,
through reduction of Trx-1 activity or, as we have demon-
strated in the present study, by increasing the CatL expression,
or by a combination of these mechanisms.
It is important to note that death of lesional macrophages by
very different in early versus late atherosclerotic lesions (46).
One explanation based on in vivo investigations is that phago-
cytic clearance of apoptotic macrophages in early atheroscle-
rotic lesions is highly efficient (47–50), whereas phagocytic
clearance in advanced lesions is less effective because of the
high number of apoptotic macrophages in advanced athero-
sclerotic lesions (47, 51–53).
Recently, Liu et al. (54) reported that macrophage apoptosis
suppresses the development of atherosclerosis in LDLR?/?
mice. On this background, our results would imply that the
PPAR?-mediated increase in CatL expression in macrophages
may represent a new approach for the inhibition of atheroscle-
than to reduce the progression of atherosclerosis (6, 12).
atherogenic and anti-atherogenic effects. In general, the bene-
ficial effects of PPAR? activation are considered to be greater
than its atherogenic effects (55). We therefore hypothesized
act atherogenesis. Autophagy is a catabolic pathway for bulk
turnover of long-lived proteins and organelles via lysosomal
degradation. A growing body of evidence suggests that
autophagy is playing a role in many diseases by promoting or
preventing their progression (56). In atherosclerotic plaques,
basal autophagy is a survival mechanism safeguarding plaque
cells against oxidative injury, metabolic stress, and inflamma-
tion, by removing harmful oxidatively modified proteins and
damaged components. Hence, autophagy is anti-apoptotic
and contributes to cellular recovery. Basal autophagy can be
intensified by appropriate drugs. Hence, pharmacological
approaches have been developed to stabilize rupture-prone
death, without affecting the plaque stabilizing smooth muscle
cells (for review see Ref. 56).
FIGURE 9. PPAR? activation induces a CatL-dependent decrease of
(0.06 and 0.6 ?M) for 24 h. The cells were analyzed for LC3 and ?-actin (for
normalization) expression using Western immunoblotting. The results are
presented as the means ? S.D. of four independent Western blots. One rep-
with control. B, HMDM were treated with the PPAR? agonist GW929 (0.6 ?M)
?-actin (for normalization) protein levels were evaluated. The results are the
FIGURE 10. In vitro knockdown of CatL reverses the PPAR?-mediated
decrease of autophagy markers. A, down-regulation of CatL by phospho-
thioate ODN. HMDM were treated with 5 ?M ODN complementary to the
corresponding mRNA sequence devoid of secondary structures for 72 h.
blot is shown. The control ODN contained the same set of the bases in a
scrambled order. B, HDMD were treated for 72 h with 5 ?M of either ODN
24 h. Expression of CatL, beclin 1, LC3, or ?-actin was analyzed by Western
immunoblotting. Representative blots are shown.
at CNRS, on February 6, 2013
Because macrophages play a central role in atherosclerotic
plaque destabilization, selective induction of macrophage
death aiming at stabilization of vulnerable, rupture-prone
proteases, the deposition of necrotic debris, and the activation
of inflammatory responses would be minimal.
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