The insulin receptor: a new anticancer target for peroxisome proliferator-activated receptor- ...

Endocrine-Related Cancer (2008) 15 325–335

The insulin receptor: a new anticancertarget for peroxisome proliferator-activatedreceptor-g (PPARg) and thiazolidinedione-PPARg agonists

V Costa, D Foti, F Paonessa, E Chiefari, L Palaia, G Brunetti1,E Gulletta, A Fusco2 and A Brunetti

Dipartimento di Medicina Sperimentale e Clinica ‘G. Salvatore’, Universita di Catanzaro ‘Magna Græcia’, 88100 Catanzaro, Italy1Dipartimento di Scienze Biomolecolari e Biotecnologie, Universita di Milano, 20133 Milano, Italy2Dipartimento di Biologia e Patologia Cellulare e Molecolare c/o Istituto di Endocrinologia ed Oncologia Sperimentale del Consiglio

Nazionale delle Ricerche, Universita di Napoli Federico II, e NOGEC-CEINGE, Biotecnologie Avanzate, 80131 Napoli, Italy

(Correspondence should be addressed to A Brunetti; Email: [email protected])

V Costa and D Foti contributed equally to this work

Abstract

The peroxisome proliferator-activated receptor-g (PPARg) is a member of the nuclear hormonereceptor superfamily. Ligand activation of PPARg is associated with differentiation andinhibition of proliferation in the normal and malignant cells. Herein, we studied the effects ofPPARg and the PPARg agonists thiazolidinediones (TZDs) on the insulin receptor (IR), a cellmembrane tyrosine kinase receptor protein, whose role is of paramount importance inmediating the metabolic and growth-promoting effects of the peptide hormone insulin.Overexpression of the PPARg1 in human hepatocellular (HepG2) cells was associated withdecreased IR gene transcription and protein expression levels, and these reductions were moreevident in the presence of TZDs. Since no PPARg response elements were identified on the IRpromoter, we postulated that PPARg adversely affects the IR gene transcription by perturbingthe assembly and stability of the transcriptionally active multiprotein-DNA complex identifiedpreviously, which includes the high-mobility group A1 protein, the ubiquitously expressedtranscription factor (Sp1), the CAAT enhancer-binding protein (C/EBPb), and, in some celllines, the developmentally regulated activator protein-2 (AP-2) transcription factor. Usingglutathione S-transferase pull-down assays combined with electrophoretic mobility shift assayand chromatin immunoprecipitation, we demonstrated that by interacting with Sp1, C/EBPb,and AP-2, PPARg can prevent Sp1/AP-2 protein–protein association and inhibit binding ofSp1 and C/EBPb to DNA, thus reducing IR gene transcription. Our results demonstrate that IRis a new target gene of PPARg, and support a potential use of TZDs as anti-proliferativeagents in selected neoplastic tissues overexpressing IRs.


Introduction

The peroxisome proliferator-activated receptor-g(PPARg) is a member of a subfamily of nuclear

receptors involved in the control of several aspects of

lipid metabolism, including fatty acid transport, uptake

by the cells, intracellular binding and activation, as


1351–0088/08/015–325 q 2008 Society for Endocrinology Printed in Great

well as catabolism and storage (Desvergne & Wahli

1999, Kliewer et al. 2001, Berger & Moller 2002). It is

highly expressed in adipose cells and its induction

precedes the activation of most adipose-specific genes

(Rosen et al. 1999, Keller et al. 1993a,b). Although

PPARg is mainly expressed in the adipose tissue,

its expression also occurs in many normal cells

Britain

DOI: 10.1677/ERC-07-0226

Online version via http://www.endocrinology-journals.org

http://dx.doi.org/doi:10.1677/ERC-07-0226

V Costa, D Foti et al.: Effects of PPARg and TZD-PPARg on IR expression

(Vidal-Puig et al. 1997), and in several cancer cell lines

and tissues, including breast, prostate, colon, lung, and

B-lineage cells (Padilla et al. 2000, Allred & Kilgore

2005, Nunez et al. 2005). Besides a fundamental role in

adipogenesis, PPARg plays a critical role in placental,

cardiac, and embryonic development (Barak et al.

1999, Maloney & Rees 2005) and influences the

production of cytokines, growth factor release, and cell

cycle progression leading to differentiation-inducing,

anti-inflammatory, and anti-proliferative effects

(Kersten et al. 2000, Tan et al. 2005, Chou et al.

2007). The PPARg generally exerts its biological

function as heterodimer with the retinoid X receptor,

which binds to a specific cis-acting sequence on DNA,

a peroxisome proliferator response element, initiating

gene transcription (Desvergne & Wahli 1999, Tan

et al. 2005). The PPARg activators identified so far

include some inflammatory activators, like a prosta-

glandin J2 metabolite (15-deoxy-D12,14-PGJ2), and

oxidized LDL particles (9-and 13-HODE; Desvergne

& Wahli 1999, Kersten et al. 2000), and the synthetic

thiazolidinedione (TZD) compounds (Lehmann et al.

1995). The TZDs are a new class of insulin-sensitizing

drugs widely used in the treatment of human type 2

diabetes mellitus, including rosiglitazone, pioglita-

zone, and ciglitazone (Lehmann et al. 1995, Saltiel &

Olefsky 1996). However, despite the positive role of

TZDs in the control of glucose homeostasis, increasing

evidences support a potential use of these drugs as anti-

proliferative agents.

The peptide hormone insulin is a major regulator of

glucose homeostasis and cell growth. The first step in

insulin action is the binding of the hormone to the

insulin receptor (IR), a phylogenetically ancient

receptor protein embedded in the plasma membrane

of insulin target cells. The IR belongs to the tyrosine

kinase growth factors receptor superfamily and

consists of two identical extracellular a-subunits that

bind insulin, and two transmembrane b-subunits withintrinsic tyrosine kinase activity (Ullrich et al. 1985,

Goldfine 1987, White & Kahn 1994). When insulin

binds to the IR, the receptor is first activated by

tyrosine autophosphorylation and then the IR tyrosine

kinase phosphorylates various effector molecules, like

the insulin receptor substrate-1 (IRS-1), leading to

hormone action (Ullrich et al. 1985, Goldfine 1987,

White & Kahn 1994). In target cells, the IR has been

shown to be under the regulation of hormones,

metabolites, and differentiation (Brunetti et al. 1989,

Mamula et al. 1990). To understand the molecular

mechanisms controlling IR gene regulation, many

groups including our own group have previously

identified and characterized the 5 0-flanking region of

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the IR gene (Araki et al. 1987, Seino et al. 1989,

Brunetti et al. 1993). Later, we have provided evidence

that transcriptional activation of the human IR

promoter requires the assembly of a transcriptionally

active multiprotein-DNA complex which includes, in

addition to the architectural factor high-mobility group

A1 (HMGA1), the transcription factors Sp1 and CAAT

enhancer-binding protein (C/EBPb). Functional integ-rity of this nucleoprotein complex is required for full

transactivation of the IR gene by Sp1 and C/EBPb in

cells readily expressing IRs (Brunetti et al. 2001, Foti

et al. 2003, 2005). Recently, we published data

showing that the nuclear transcription factor activator

protein-2 (AP-2), by interacting with Sp1 and

HMGA1, may stabilize this multiprotein complex at

the level of the IR promoter, leading to IR over-

expression in breast cancer cells (Paonessa et al. 2006).

In this paper, we demonstrate for the first time that

PPARg and its ligands inhibit IR gene transcription.

We show that PPARg physically interacts with Sp1,

AP-2, and C/EBPb in vitro, and this protein–protein

interaction significantly reduces the transactivation

of the IR gene by Sp1, C/EBPb, and AP-2 in vivo, in

the absence of a consensus sequence for PPARg on the

IR promoter.

Materials and methods

Cells and protein extracts

HepG2 human hepatoma cells, 3T3-L1 mouse fibro-

blasts, andMCF-7 human breast cancer cells (American

Type Culture Collection, Manassas, VA, USA) were

maintained in DMEM (GIBCO-BRL) supplemented

with 10% fetal bovine serum. The 3T3-L1 fibroblasts

were differentiated into 3T3-L1 adipocytes as described

elsewhere (Foti et al. 2003). Nuclear and cytoplasmic

extracts were prepared from cultured cells as previously

described (Brunetti et al. 2001). For each nuclear

extract, an equal number of nuclei were homogenized,

and the final protein concentration in the extracts

was determined by the modified Bradford method

(Bio-Rad Laboratories).

RT-PCR, immunoprecipitation, and western

blot analysis

Total cellular RNA was extracted from cells using

the RNAqueous-4PCR kit and subjected to DNase

treatment (Ambion, Austin, TX, USA). The cDNA was

synthesized from total RNA with the RETROscript

first strand synthesis kit (Ambion) and used for

PCR amplification. The PCR products were electro-

phoretically resolved on 2% agarose gel, visualized by

www.endocrinology-journals.org


ethidium bromide staining, and quantified by densito-

metry. The values obtained were then normalized to

those of the tubulin gene. Western blot analyses of IR,

PPARg, HMGA1, Sp1, C/EBPb, and AP-2 were done

on total cellular lysates or nuclear extracts as

previously described (Brunetti et al. 2001, Foti et al.

2003, 2005). For immunoprecipitation studies with IR

antibody, aliquotes of HepG2 cytoplasmic extracts

were incubated for 12 h with rotation at 4 8C with 10 mlIR antibody-coupled protein A beads. Covalent

coupling of antibody was performed as previously

described (Paonessa et al. 2006). Beads were recovered

by gentle centrifugation and washed thrice with 500 mlNETN wash buffer (0.1% NP40, 150 mM NaCl, 1 mM

EDTA, 50 mM Tris–HCl (pH 8.0)) for 5 min. Protein

was removed from the beads by boiling in sample

buffer for 5 min and analyzed by SDS-PAGE and

immunoblotting. Antibodies used for these studies

were as follows: anti-HMGA1 (Brunetti et al. 2001),

anti-PPARg1 (H-100), anti-Sp1 (PEP 2), anti-C/EBPb(C-19), anti-AP-2a (H-79), and anti-IRb (C-19; Santa

Cruz Biotechnology, Santa Cruz, CA, USA).

GST pull-down assay

The 35S-labeled HA-tagged HMGA1, Sp1, C/EBPb,and AP-2 (Foti et al. 2003, Paonessa et al. 2006) were

synthesized in vitro using the TNT-T7 quick-coupled

transcription/translation system (Promega). The gluta-

thione S-transferase (GST) fusion protein expression

vectors pGEX4T-3-human PPARg2 (kindly provided

by H Nishizawa and T Funahashi, University of Osaka;

Nishizawa et al. 2002) and the pGEX-2TK control

vector (Amersham Pharmacia Biotech) were trans-

formed into the BL21(D3) strain of Escherichia coli

(Stratagene, La Jolla, CA, USA), expanded in suspen-

sion culture, and induced for 2 h with 0.5 mM

isopropyl-D-thiogalactopyranoside (Sigma). Bacteria

were pelleted, sonicated in ice-cold PBS lysis buffer

containing 1% NP-40, 10% glycerol, 1 mM dithio-

threitol (DTT), 1 mM phenylmethylsulphonyl fluoride

(PMSF), 10 mg pepstatinA, leupeptin, and aprotinin/ml,

and 0.4 mg lysozyme/ml and centrifuged. The resultant

supernatant was then added to 300 ml glutathione-

agarose beads, mixed on a rotating wheel at 4 8C for 2 h

and centrifuged. The bound GST-fused proteins in the

pellet were washed five times with lysis buffer and

resuspended in 300 ml binding buffer (50 mM NaCl,

20 mM Tris–HCl (pH 8.0), 0.05% NP-40, 0.25% BSA,

1 mM PMSF, 1 mM DTT). The bound protein was

quantitated with the Coomassie protein assay reagent

(Pierce Co., Rockford, IL, USA), and 0.5 mg of each

GST-protein bound to glutathione-agarose beads was


incubated with 7 ml in vitro-translated 35S-labeled

protein in 150 ml binding buffer at 4 8C for 2 h.

Reactions were terminated by centrifugation, the

precipitate was washed three times with protein-

binding buffer and subjected to a 10% PAGE

(SDS-10% PAGE), and proteins were visualized by

autoradiography.

Plasmids, transfections, and electrophoretic

mobility shift assay (EMSA)

Recombinant chloramphenicol acetyltransferase

(CAT) plasmids (pIR-CAT, pCAT-C2 and pCAT-E3)

containing different fragments of the human IR gene

promoter have been described previously (Brunetti

et al. 1993, Foti et al. 2003, Paonessa et al. 2006). The

human PPARg1 expression vector was obtained using

standard techniques after PCR amplification and

cloning of PPARg full-length cDNA into the

expression vector pcDNA3. The pC-hu/AP-2aexpression vector was kindly provided by K Fujimori

(Osaka Bioscience Institute). The IR-CAT reporter

plasmids, together with effector vectors for PPARgand/or AP-2, in the absence or presence of the PPARgagonist rosiglitazone (a kind gift of Glaxo Smith Kline)

or ciglitazone (Vinci-Biochem Alexis, Vinci, Italy)

were transiently transfected into cultured cells by the

calcium phosphate precipitation method, and CAT

activity was assayed 48 h later, as previously described

(Brunetti et al. 1993). The pSV-b-galactosidase controlvector served as an internal control of transfection

efficiency, together with measurements of protein

expression levels (Brunetti et al. 2001). For EMSA,

the C2 sequence (300 bp) of the human IR gene

promoter was generated by PCR amplification, using

the recombinant plasmid pCAT-C2 (Brunetti et al.

1993). The 32P-labeled C2 was used in gel shift assays

as previously described (Brunetti et al. 2001).

Chromatin immunoprecipitation (ChIP)

ChIP assay was done as described previously

(Foti et al. 2005), using HepG2 cells transfected with

the AP-2a expression vector and untransfected MCF-7

cells. Formaldehyde-fixed DNA–protein complex was

immunoprecipitated with anti-HMGA1, anti-Sp1, anti-

C/EBPb, or anti-AP-2a antibody. Primers for the IR

sequence (Paonessa et al. 2006) were used for PCR

amplification of immunoprecipitated DNA (30 cycles)

using PCR ready-to-go beads (Amersham Pharmacia

Biotech). The PCR products were electrophoretically

resolved on 1.5% agarose gel and visualized by

ethidium bromide staining.

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Statistical analysis

All experiments were performed at least three times.

The mean and S.E.M. were calculated for each set of

results and the significance of the difference between

values was assessed by the Student’s t-test. For all

analyses, P!0.05 was considered significant.

Results

PPARg and TZDs reduce IR protein and mRNA

expression in HepG2 cells

To see whether PPARg had any effect on IR

expression, we first measured the IR protein content

in HepG2 cells, before and after forced expression of

exogenous PPARg1. The HepG2 cells were consideredideally suited for studying the effects of PPARg on IR

expression since IRs are relatively abundant in this cell

line, while endogenous PPARg is barely detectable.

Using immunoprecipitation and western blot analyses,

we showed that IR protein content was considerably

reduced in cells overexpressing PPARg (w50% less

than control cells), and this reduction was more

pronounced in PPARg1-overexpressing cells exposed

to 10K5 M TZD (rosiglitazone; Fig. 1). It is notable

that rosiglitazone alone also reduced IR protein

content, and it is likely that this occurred, at least

in part, by stimulating endogenous PPARg (Tontonoz

et al. 1994). As measured by RT-PCR, the abundance

of IR mRNA transcripts in HepG2 cells closely

Figure 1 Inhibition of IR protein and mRNA levels by PPARgand TZDs. (A) Total cellular extracts from untransfected(control) or HepG2 cells transfected with PPARg expressionvector, in the absence or presence of TZD (rosiglitazone,10K7 M), were resolved by SDS-PAGE, and the IR proteincontent was measured by immunoprecipitation and the westernblot (IP/WB) with an anti-IRb antibody. Tubulin was measuredas an internal control. (B) In parallel experiments, IR and tubulinmRNA levels were quantified by RT-PCR.

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correlated with IR protein content in every condition

analyzed, indicating that changes at the protein level

reflected differences in IR mRNA (Fig. 1).

PPARg and TZDs reduce IR gene transcription

in cultured cells

In order to reveal whether PPARg and its agonists

inhibited the transcriptional activation of IR gene, we

performed reporter gene analysis using the pIR-CAT

recombinant vector that contains the entire 50-flanking

region of the human IR gene (Foti et al. 2003). Forced

expression of PPARg1 in HepG2 cells transfected with

pIR-CAT determined a 40–50% reduction in CAT

activity that was similar to the reduction observed in

cells exposed to rosiglitazone alone, and resulted more

marked in PPARg1-overexpressing cells simultaneously

treated with TZD rosiglitazone (Fig. 2A). The possibility

that PPARg could theoretically decrease IR gene

transcription by reducing the intracellular expression of

HMGA1, Sp1 or C/EBPb was excluded by western blot

analysis of nuclear extracts showing that no changeswere

observed in the endogenous levels of these proteins under

these experimental conditions (Fig. 2A). The hypothesis

that PPARg and its agonists could negatively interfere

with the crosstalk between these various transcription

factors in the context of the IR gene was substantiated

further by transiently cotransfecting the AP-2-null cell

lineHepG2with thepIR-CATpromoter, togetherwith an

expression vector encoding the wild-type AP-2a. Asshown in Fig. 2A, overexpressionofAP-2 inHepG2 cells

efficiently induced IR gene transcription. However, this

effect was totally abolished by PPARg and TZDs that

reverted gene transcription to levels similar to those

observed in the absence of AP-2 (Fig. 2A). These

findings were confirmed in PPARg overexpressing

MCF-7 cells, a cell line naturally expressing only

detectable levels of PPARg (Tontonoz et al. 1994), and

3T3-L1 adipocytes producing relatively high amounts of

endogenous PPARg (Mueller et al. 1998). As shown in

Fig. 2B and C, exposure of both cell lines to either

rosiglitazone and/or ciglitazone reduced IR gene

transcription in a dose-dependent manner.

We have previously shown that transcriptional

activation of the human IR gene by HMGA1, Sp1,

and C/EBPb requires the assembly and cooperation

among these various nuclear factors at the levels of two

AT-rich sequences of the IR gene promoter, C2 and E3

(K674 to K874 and K1662 to K1818 bp upstream

of the IR ATG codon respectively), which have a

significant ability to drive transcription when intro-

duced into mammalian cells (Brunetti et al. 1993,

2001, Foti et al. 2003). As demonstrated before, the


Figure 2 Functional significance of PPARg and TZDs for IR gene transcription. (A) HepG2, (B) MCF-7, and (C) 3T3-L1 cells weretransfected with a CAT reporter plasmid containing the IR promoter element (pCAT-IR, 2 mg), in the absence or presence of PPARg(2 mg) and/or AP-2 (1 mg) expression vector, with or without treatment with rosiglitazone or ciglitazone at the indicated molarconcentrations. For each cell line, data represent the meansGS.E.M. for three separate experiments; values are expressed relative tothe CAT activity obtained in transfections with the pCAT-IR alone that is assigned an arbitrary value of 1. *P!0.05 versus cellstransfected with the pCAT-IR alone; #P!0.01 versus cells transfected with the pCAT-IR plus AP-2 effector vector. In someexperiments (D), HepG2 cells were transfected as above, using the CAT reporter vector containing the proximal C2 (pCAT-C2) ordistal E3 (pCAT-E3) element of the IR gene promoter. *P!0.05 versus cells transfected with the pCAT-C2 alone. White bars, mock(no DNA); black bars, pCAT-basic (vector without an insert). Shown are representative western blots of PPARg, Sp1, C/EBPb, andHMGA1 from nuclear extracts of transfected HepG2 cells.


lack of any of these factors, or a scarcity of one or

more, may severely perturb IR gene transcription in

cells and tissues readily expressing IR (Brunetti et al.

2001, Foti et al. 2003, 2005). When the recombinant

plasmid pCAT-C2 was transfected into HepG2 cells

(and other cell lines), PPARg, and TZDs, either alone

or in combination, inhibited pCAT-C2 activity to the

same extent than they did in HepG2 cells transfected

with the full-length pCAT-IR promoter (Fig. 2D),

indicating that interference of PPARg and agonists

with IR gene-transcription machinery occurs at the

level of the proximal promoter region, C2. This

conclusion was supported by the observation that in

experiments using the pCAT-E3 reporter vector,

containing the more distant E3 sequence of the IR


gene, no effects on CAT activity were observed in cells

exposed to PPARg and/or TZDs (Fig. 2D).

PPARg physically interacts with Sp1, C/EBPb,

and AP-2, in vitro, in the absence of DNA

In our studies, no peroxisome proliferator response

elements (PPRE) have been identified on the promoter

region of the IR gene. A similar observation has been

previously provided by us for the transcription factor

AP-2, for which DNA-binding activity was undetectable

with the IR gene promoter, and transactivation of the IR

gene by AP-2 occurred indirectly through physical and

functional cooperation with HMGA1 and Sp1 (Paonessa

et al. 2006). Therefore, in an attempt to identify the

biochemical mechanisms underlying the negative effects

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of PPARg on IR gene transcription, we performed

experiments designed to investigate whether PPARgcould affect this promoter function by physically

interacting with HMGA1, Sp1, C/EBPb or AP-2, thus

preventing the interaction between these factors in the

preinitiation complex at the IR promoter. To this

end, we performed GST pull-down assays, in which in

vitro-translated 35S-labeled proteins were analyzed for

their ability to be specifically retained by a GST-PPARgaffinity resin. As shown in Fig. 3, under our experimental

conditions, no interaction was observed between35S-labeled HA-tagged HMGA1 and GST-PPARg,excluding a physical contact between these two proteins

in vitro. Conversely, the 35S-labeled Sp1was retained by

GST-PPARg but not GST alone, indicating that Sp1

physically interacts with PPARg. Similarly, the35S-labeled AP-2 and to a lesser extent labeled C/EBPbwere also specifically bound by GST-PPARg, but not byGST alone (Fig. 3), indicating that PPARg directly

interacts with these two proteins as well. A bona

fide interaction between these factors and GST-PPARgwas proved by the use of high concentrations of

ethidium bromide (not shown), which has been shown

to disrupt DNA-dependent protein–protein contact (Foti

et al. 2003).

Binding of Sp1, AP-2, and C/EBPb to the IR

promoter is reduced by PPARg

Physical interaction between PPARg, Sp1, AP-2, andC/EBPb has been characterized further in vitro by

EMSA, using the previously described radiolabeled

fragment C2 of the human IR promoter gene as probe

(Brunetti et al. 1993).As shown in Fig. 4, binding of Sp1

and HMGA1 was easily detected in nuclear extracts

Figure 3 Physical association between PPARg and IR-DNAbinding proteins. SDS-PAGE of 35S-labeled HA-taggedHMGA1, Sp1, AP-2, and C/EBPb bound to GST-PPARg resin.In the first lane of each panel, 5% of the labeled protein wasadded directly onto the gel without binding to and elution fromGST protein resin (input). Specifically bound proteins werevisualized by autoradiography.

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from untreated HepG2 cells, in which only trace

amounts of PPARg are normally expressed. Binding

of Sp1 was reduced in nuclear extracts from HepG2

cells overexpressing PPARg1, and was further

decreased after treatment of these cells with TZD

rosiglitazone. Under these conditions, no changes were

observed with HMGA1-DNA binding. When EMSA

was performed by nuclear extracts from HepG2 cells

overexpressing AP-2, a co-complex containing Sp1,

HMGA1, andAP-2was also detected, in addition to Sp1

and HMGA1 complexes (Fig. 4). The reduction in Sp1-

DNA binding, in cells overexpressing PPARg1,paralleled the reduction in the co-complex-DNA

binding formation and these interactions were further

reduced by the addition of rosiglitazone. Similar results

were obtained in the MCF-7 cells transfected with

PPARg expression vector, either in the absence or in the

presence of TZD. Like Sp1 and AP-2, binding of

endogenous C/EBPb to DNA was reduced in nuclear

extracts from cells overexpressing PPARg1 and was

further decreased by treatment of cells with rosiglita-

zone (Fig. 4). The presence of HMGA1 in the more

rapidly migrating complex was verified by its super-

shifting in the presence of the anti-HMGA1 antibody,

whereas the presence of Sp1, AP-2, and C/EBPb in

the slowly migrating complexes was verified by their

supershifting in the presence of Sp1-, AP-2-, and

C/EBPb-specific antibodies respectively. Since no

effect on the electrophoretic migration of DNA–protein

complexes was observed in the presence of control-

unrelated antibodies (data not shown), the reduction of

protein–DNA binding in supershift assays suggests that

these factors are simultaneously binding the same

labeled probe. The above findings were substantiated

in vivo by ChIP analyses. As shown in Fig. 5, binding of

Sp1, AP-2, and C/EBPb to the C2 promoter region of

Figure 4 Nuclear protein–DNA interactions within the C2 regionof the IR promoter. EMSAs of radiolabeled fragment C2 (0.2 ng)with 0.5 mg nuclear protein from (A) wild-type HepG2 cells or(B) AP-2-overexpressing HepG2 cells, and (C) wild-type MCF-7cells, in the presence of 0.2 mg poly (dI-dC) as the competitorDNA. In supershift assays, nuclear protein was preincubatedwith 1 mg polyclonal antibody (Ab) to HMGA1, Sp1, AP-2, orC/EBPb before addition of the probe (none: probe alone).


Figure 5 ChIP assay. ChIP of the IR promoter gene in AP-2a-producing HepG2 cells and MCF-7 cells both induced tooverexpress PPARg, either in the absence or in the presence ofTZD (rosiglitazone). ChIP was done using antibodies (Ab)against either HMGA1, Sp1, AP-2, or C/EBPb.


the IR gene was considerably attenuated in the HepG2

and MCF-7 cells overexpressing PPARg, either in the

absence or presence of the TZD rosiglitazone. Thus,

these data suggest that overexpression and/or

activation of PPARg adversely affects IR gene

transcription in the absence of PPRE on the IR gene

promoter. We propose that, by causing a displacement

of Sp1,AP-2, andC/EBPb, PPARgmay play significant

molecular roles in the transcriptional activities of these

factors in the context of the IR gene, both in physiology

and pathology (Fig. 6).

Discussion

The TZDs are insulin-sensitizing drugs that improve

insulin sensitivity in insulin-resistant states such as type

2 diabetes mellitus and obesity. These drugs are

high-affinity ligands for the nuclear receptor PPARg,which regulate transcription of target genes involved in

the homeostasis of nutrients. In addition to the effects on

lipid and glucose metabolism, many evidences have

shown that PPARg and its ligands play an important

role in modulating many processes, including cell

proliferation and differentiation, as well as inflam-

mation, angiogenesis, and immune system function.

The IR is critical in the insulin-mediated effects on

cell metabolism and cell growth. Various studies have

shown that IRs are increased in most human breast

cancers, and both ligand-dependent malignant


transformation and increased cell growth occur in

cultured breast cells overexpressing the IR (Osborne

et al. 1978, Milazzo et al. 1992, Paonessa et al. 2006).

Overexpression of functional IRs has also been

involved in thyroid carcinogenesis (Farid et al.

1994). The IR can exert its oncogenic potential in

malignant cells via abnormal stimulation of multiple

cellular signaling cascades, enhancing growth factor-

dependent proliferation and/or by directly affecting

cell metabolism. Both breast and thyroid neoplastic

cells express PPARg, and PPARg agonists have been

shown to inhibit proliferation in these and other cell

systems (Tontonoz et al. 1994, Martelli et al. 2002,

Grommes et al. 2004). In this light, we undertook to

investigate whether IR expression could be affected by

PPARg. Our study shows that IR gene transcription

and receptor protein content were reduced in cells with

forced PPARg1 overexpression, or TZD-induced

PPARg activation. Although these results apparently

run contrary to what might have been predicted based

on the known insulin-sensitizing effects of TZD,

seeming to exclude the possibility that TZDs may act

as insulin sensitizers through the IR, they are

compatible with the pleiotropic effects of PPARg. Inthis regard, the IR may be considered a new target gene

that accounts for the anti-mitogenic response to

PPARg and its agonists, and this is the first description

of a tyrosine kinase receptor involved in PPARg-induced anti-proliferative mechanisms. To unravel the

molecular basis underlying the decrease in IR gene

expression produced by PPARg, we performed

protein–protein and DNA–protein interaction studies

together with ChIP analysis, combined with transient

transcription assays in the living cells expressing

variable amounts of the IRs. Since no PPRE have

been detected within the IR gene promoter and no

reduction in the expression of HMGA1, Sp1, or

C/EBPb was observed in cells after PPARg or TZD

stimulation, we propose that PPARg acts as a negative

regulator of IR transcription by adversely affecting

binding of Sp1, C/EBPb, or AP-2 to the IR gene. In

other PPARg-responsive genes that carry a PPRE

consensus in their sequence, a functional cooperation

between Sp1 and PPARg has been described (Krey

et al. 1995). On the other hand, functional cooperation

between C/EBPb (and other members of the C/EBP

family of proteins) and PPARg has been reported in

the nutrient signaling system during fetal development

(Maloney & Rees 2005), regulation of vascular

inflammation (Takata et al. 2002), and inhibition of

adhesive interaction between multiple myeloma

and bone marrow stromal cells (Wang et al. 2007).

Herein, we show that PPARg physically interacts with

331

Figure 6 Proposed model for the action of PPARg in the context of the IR gene promoter (C2 region). (A and B) By binding tosequence-specific sites located in the C2 regulatory region of the IR gene, HMGA1, Sp1, and C/EBPb exert a positive control overthe rates of transcription. The PPARg can displace Sp1 and C/EBPb from their binding sites, decreasing the rate of transcription.(C and D) AP-2 overexpression accounts for increased IR expression in both breast cancer cell lines and breast cancer tissues, inwhich HMGA1 acts by facilitating and stabilizing interactions between AP-2 and Sp1 in the preinitiation complex at the IR promoter(Paonessa et al. 2006). By binding to AP-2 and Sp1, PPARg may attenuate the stimulatory role of AP-2 in IR gene expression inbreast carcinogenesis.


Sp1, C/EBPb, and AP-2 reducing the IR gene

transcription in the absence of PPARg DNA-binding

sites on the IR gene. We suggest that, in the absence of

PPRE in the context of the IR promoter, this nuclear

receptor may produce its adverse effects on IR gene

transcription by interacting physically with these

factors, thus reducing their availability to the basic

transcription machinery of the IR gene. With a similar

mechanism involving Sp1/PPARg protein interaction,

PPARg has been shown to exert an anti-proliferative

role by suppressing transcription of the thromboxane

receptor, the cyclin-dependent kinase inhibitor p21,

and the fibronectin genes (Sugawara et al. 2002, Hong

et al. 2004, Han et al. 2005). The molecular

mechanism herein described is therefore in agreement

with the increasing repertoire of ‘non-canonical’

PPARg target genes that now encompasses non-

PPREs containing genes (Tan et al. 2005).

Many potential target genes of PPARg have been

already reported, including bcl-2, b-catenin, and the

PTEN tumor suppressor gene (Elstner et al. 1998, Patel

et al. 2001, Mulholland et al. 2005). However, how

PPARg and its agonists may induce their anti-

proliferative effects is not fully understood yet.

Recently, non-genomic crosstalks between PPARgand cytoplasmic proteins, like extracellular signal-

regulated kinase (ERK) 1/2 and MAPK kinases, have

been reported in cancer cells and functional importance

has been given to the subcellular localization of PPARg

332

(Burgermeister & Seger 2007, Papageorgiou et al.

2007). However, the last three decades of medical

research examining the molecular pathogenesis of

cancers have provided compelling evidence for the

universal disruption of the cell cycle in human tumors,

and recent studies have demonstrated a critical interface

between hormonal signaling and the cell cycle

(Hilakivi-Clarke et al. 2004). In this context, mitogens

like insulin, via the IR, may promote the progression

through the G1 phase by inducing competence of the

cyclin D1/cyclin-dependant kinase 4 (CDK4) complex.

It has been previously demonstrated that the PPARgagonists inhibit cyclin D1 (Wang et al. 2001). Our

findings support the conclusion that PPARg and TZDs

may interfere with the hormonal control of the cell

cycle, at least in part, through the inhibition of the IR.

Over the last decade, PPARg has emerged as an

important drug target in type 2 diabetes mellitus

(Savkur & Miller 2006), and TZDs are widely used for

treatment of diabetic patients. However, conflicting

results on the pro-carcinogenic and anti-tumorigenic

effects of TZDs in humans with diabetes can be found

in the literature. For instance, whereas a population-

based report showed that TZDs were associated with

reduced risk of lung cancer in patients with diabetes

(Govindarajan et al. 2007), a possible association

between cancer and the use of TZD has been reported

later in type 2 diabetic patients (Ramos-Nino et al.

2007). Taken together, our results consistently support



the conclusion that IR gene may be considered a new

anticancer target for PPARg, providing further

evidence for the use of TZDs as anti-proliferative

agents in selected tumors overexpressing the IR.

Acknowledgements

The authors would like to thank K S Chang, K

Fujimori, T Funahashi, H Nishizawa and M A Lazar

for providing reagents. They would also like to thank

A Malta, G Ceravolo, T Rossano and G Grandinetti

for secretarial help. Grant support: Ministero dell’Uni-

versita e della Ricerca, Italy protocol 2004062059-002

and Telethon, Italy grant GGP04245 (A Brunetti).

The authors declare that there is no conflict of

interest that would prejudice the impartiality of this

scientific work.

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