# The Role of PPAR Ligands in Controlling Growth-Related  Gene Expression and their Interaction with  Lipoperoxidation Products

Authors: Giuseppina Barrera; Cristina Toaldo; Stefania Pizzimenti; Angelo Cerbone; Piergiorgio Pettazzoni; Mario Umberto Dianzani; Carlo Ferretti
Journal: PPAR Research
 (2008)

---

## Abstract

Peroxisome proliferators-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor superfamily. The three PPAR isoforms (α, γ and β/δ) have been found to play a pleiotropic role in cell fat metabolism. Furthermore, in recent years, evidence has been found regarding the antiproliferative, proapoptotic, and differentiation-promoting activities displayed by PPAR ligands, particularly by PPARγ ligands. PPAR ligands affect the expression of different growth-related genes through both PPAR-dependent and PPAR-independent mechanisms. Moreover, an interaction between PPAR ligands and other molecules which strengthen the effects of PPAR ligands has been described. Here we review the action of PPAR on the control of gene expression with particular regard to the effect of PPAR ligands on the expression of genes involved in the regulation of cell-cycle, differentiation, and apoptosis. Moreover, the interaction between PPAR ligands and 4-hydroxynonenal (HNE), the major product of the lipid peroxidation, has been reviewed.


---

## Body

## 1. The Role of PPAR in Controlling Gene Transcription

Peroxisome proliferator-activated receptors (PPARs) are members of the steroid hormone
nuclear receptor superfamily which act by altering the transcription of
PPAR-regulated genes by means of a recognition sequence known as a peroxisome
proliferation responsive element (PPRE) [1].The term peroxisome proliferator-activated receptor is
derived from early observations in rodent livers that certain
industrial compounds could cause an increase in size and number of peroxisomes [2, 3]. Subsequently, these compounds, including fibrates, were found to bind to
certain recently identified nuclear receptors [4]; hence, the term “PPAR” arose.
PPAR agonists are not known to induce peroxisome proliferation in primates or
humans, making the term PPARs archaic as well [5]. At least three subtypes of PPARs have been identified: PPARα, the first isolated  from mice liver in
1990 by Issemann and Green [4] and involved in fatty acid oxidation; PPARγ,
identified by Tontonoz and collaborators as a transcription factor associated
with adipocyte determination and differentiation [6]; and
PPARβ/δ, ubiquitously expressed and involved in basic cellular functions [7, 8]. Like other steroid hormone nuclear receptors, PPARs
contain several modulating domains: a ligand binding domain (LBD) to which the
specific PPAR agonist binds; a transactivating domain (activation function 2,
AF 2), which undergoes conformational changes, in response to ligand binding,
allowing the heterodimerization with RXR and facilitating recruitment of
coactivators and release of corepressor; and finally a DNA-binding domain,
which interacts with PPRE [3, 9–11].PPAR coactivator and corepressor are small accessory molecules that are
critical determinants of the transcriptional complex. These accessory molecules include coactivator
proteins, like PPARγ coactivator-1 (PGC-1); steroid receptor coactivator and CREB (cAMP-response
element binding protein)-binding protein, recruited from the activated PPAR; and corepressor
proteins, like nuclear receptor corepressor (N-CoR) and silencing mediator for retinoid and
thyroid hormone receptors (SMRTs1), released upon PPAR
activation [12, 13]. A multimolecule complex
formed by PPAR, PPAR ligand, RXR, RXR-ligand (purportedly 9-cis-retinoic acid)
and accessory proteins ultimately combine to cause the PPAR response through
the binding with PPRE sequences consisting of a direct repeat of the consensus
half-site motif (AGGNCA) spaced by a single nucleotide [14]
(Figure 1).Figure 1
Mechanism of PPAR action. PPARs in response to ligand binding in the cytosol, dimerize with RXR, recruit
coactivators and release corepressor; in the nucleus a multimolecule complex, formed by PPAR, PPAR ligand, RXR, RXR-ligand,
and accessory proteins bind PPRE DNA sequences in the promoters of target genes.Several genes that are selectively upregulated by a given PPAR isotype have
been identified over the years and a majority of these genes is known to play a
central role in energy metabolism. Moreover, microarray technology and genome
wide identification of PPREs suggest the existence of many other target genes
that were not previously known to be regulated by PPAR. The identified PPRE
putative sequences on target genes for PPARs are listed in Table1.Table 1Genes containing PPRE putative sequencesFunction of geneRef.Lipid metabolismP450 4A6Omega oxidation of fatty acids[15]malic enzyme geneFatty acid
synthesis[16]apoA-I and apoA-II.Components of HDL[17]LPL (lipoprotein lipase)Hydrolysis of triglycerides[18]UCP3 (Uncoupling protein 3)Fatty acid transport and  thermogenesis[19]CEH (Cholesteryl ester hydrolase)Hydrolysis of stored cholesterol esters in
macrophage foam cells and release of free cholesterol for high-density
lipoprotein-mediated efflux[20]Aox/ACO (Acyl-CoA
oxidase)Beta-oxidation in peroxisome[21]HD (enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase)Beta oxidation in perixisome[21]ILK (Integrin-linked kinase)Integrin-mediated signaling[22]HMG-CoA (3-hydroxy-3-methylglutaryl coenzyme
A synthase and reductase)Cholesterol biosynthesis[23]LRP (lipoprotein receptor-related protein)Lipoprotein metabolism, neurological function,
tissue remodelling, protease complex clearance, cell signal transduction[24]CPT1beta (human carnitine palmitoyltransferase 1beta)Fatty acid mitochondrial beta-oxidation[25]FABP (fatty acid binding protein)Lipid transport (solubilization of long-chain
fatty acids)[26]ADRP (Adipose differentiation-related
protein)Maintenance of lipid stores in non-adipocytes[27]FIAF (The fasting-induced adipose factorCirculating lipoprotein lipase inhibitor
secreted from adipose tissue[28]Carbohydrate metabolismbetaGK (beta-cell-specific glucokinase)Glucose-sensing apparatus in pancreatic beta-cells[29]GPDH (Glycerol 3-phosphate dehydrogenase)NAD-dependent enzyme that catalyzes the
oxidation of sn-glycerol 3-phosphate to dihydroxyacetone phosphate. It restores NAD+.[30]UGDH (UDP-glucose dehydrogenase)Biosynthesis of complex carbohydrates and
detoxification of toxic compounds in the liver[31]PDK (Pyruvate dehydrogenase kinase)Modulation of pyruvate dehydrogenase complex
activity[32]SHP (Small heterodimer partner)Bile acid-dependent down regulation of
gluconeogenic gene expression in liver[33]InflammationPrm3 (thromboxane receptor (TP) beta
promoter)Thromboxane receptor (TP) beta transcription[34]IL-1ra
(Interleukin-1 receptor antagonist)IL-1 receptor signaling blockage[35]CD36 (scavenger
receptor)Scavenger receptor[36]sPLA2-IIA (Group IIA secretory phospholipase
A2)Proinflammatory effect[37]AhR (Aromatic hydrocarbon receptor)Proinflammatory effect[38]Growth factors and cell cycle regulatorsSSAT (spermidine/spermine N1-acetyltransferase)Polyamine catabolism[39]GOS2 (GO/G1 switch gene 2)Cell cycle
regulation[40]VEGF (Vascular endothelial growth factor)Vasculogenesis[41]IGFBP-1 (Insulin-like growth factor-binding
protein 1)Binding protein  of insulin-like growth factor (IGF)-I and
IGF-II. Biomarker for metabolic and hyperproliferative diseases[42]Detoxification and redox enzymesCYP1A1 (Cytochrome P450 1A1)Degradation of endobiotics and the
bioactivation of numerous environmental procarcinogens[43]GST (glutathione S-transferase gene)Antioxidant function[44]POX
(Proline oxidase)Redox enzyme[45]VDUP-1 (Vitamin D-upregulated protein-1)Inhibition of thioredoxin-1 which plays a
role in the regulation of cellular redox balance (Cellular redox balance)[46]OthersBCM (Beta-carotene
15,15′-monooxygenase)Vitamin A biosynthesis[47]I-BABP (Ileal bile acid-binding protein)Enterohepatic circulation of bile acids[48]PCLN-1 (paracellin-1)Tight-junction protein, exclusively, in the
kidney[49]BACE1(Beta-site amyloid precursor protein
cleaving enzyme)Central
causal role in Alzheimer's disease[50]nephrin promoterNephrin synthesis[51]CIDEA (Cell death-inducing DNA fragmentation
factor alpha-like effector A)Proapoptotic protein[52]TFF2 (Trefoil factor family 2)Defense and repair of gastric mucosa[53]Recent evidence indicates that the PPAR response can result both in gene activation
and repression. As far as it regards gene repression, PPARγ was shown to be unable to bind to
DNA while it is associated with the corepressor complex. In contrast to PPARγ, the
interaction between NCoR/SMRT and PPARβ/δ does not impair its DNA binding
[54, 55]. PPARγ, after ligand binding, dissociates from the corepressor, and binds to
DNA via PPREs. The liberated corepressor protein SMRT interacts with the signal
transducer and activator of transcription-3 (STAT3), which inhibits
STAT-dependent transactivation [56]. Recent data suggest
that PPARγ-mediated transrepression may involve stabilization of corepressor
recruitment after posttranslational PPAR modification by sumoylation [57].In macrophages, PPARβ/δ was shown to function as an activator of the
monocyte chemoattractant protein (MCP-1) gene by sequestering a transcriptional repressor, specifically the
transcriptional repressor B-cell lymphoma-6 (BCL-6)
[37, 58]. The ligand-induced activation of PPARβ/δ releases the corepressor BCL-6,
which is thought to inhibit MCP-1 expression.
Hence, PPARβ/δ can function as an intrinsic transcriptional repressor, a mechanism
that is also shared by other nuclear receptors such as the thyroid hormone
receptor (NR1A1, NR1A2), retinoic acid receptor (NR1B1, NR1B2, NR1B3), Rev-Erb
(NR1D1, NR1D2) and COUP-TF (NRT2F3).The best-documented mechanism by which PPARα can transrepress non-PPREs
containing genes is its ability to physically interact with the p65 subunit of
nuclear factor (NF)-κB, which inhibits NF-κB-dependent transactivation [59]. However,
PPARα activators do not inhibit all NF-κB-driven target genes and their effect is promoter context-dependent. Taken together,
data obtained about PPAR transcriptional regulation
demonstrated that PPARs can also modulate the transcriptional activity of
non-PPRE containing genes via transrepression.


## 2. PPAR Ligands

PPAR ligands are a heterogeneous group that includes both endogenous and exogenous ligands  [60]. Activating
ligands for PPARs are semiselective for the subtype and selectivity depends on ligand concentration and
cell type. Endogenous ligands include unsaturated fatty acids that bind
all three PPARs, with PPARα exhibiting the highest activity, while saturated
fatty acids are weak PPAR ligands in general [61]. Biological modifications of
linoleic acid, linolenic acid, eicosapentanoic acid (EPA), and arachidonic acid
originate PPARα activators [62–64]. Moreover, the oxidized form of EPA,
eicosanoids (15-hydroxy-eicosatetranoic acid, HETE and HODEs), and leukotriene
B4 has also been reported to be PPARα activators [62–66].The natural ligands of PPARγ include
several prostanoids such as 15-deoxy-prostaglandin J2 (15d-PG J2) and
15-hydroxy-eicosatetranoic acid (HETE), which are metabolites of arachidonic
acid [67]. 15d-PG J2 (the most widely used natural ligand for PPARγ) is gamma-selective at low concentrations but also activates
alpha at higher levels [68, 69]. Like PPARα, PPARβ/δ is activated by
long chain fatty acids, including several polyunsaturated fatty acids and
eicosanoids [3]. Erucic acid has been reported to be more selective for PPARβ/δ
than other PPAR subtypes [70].Synthetic ligands of PPARs have been
demonstrated to possess pharmacological activity. Triglyceride-lowering/high-density
lipoprotein (HDL)-raising fibrates (gemfibrozil, fenofibrate, clofibrate,
ciprofibrate) are PPARα agonists used clinically to treat dyslipidemia [71, 72].
The insulin-sensitizing thiazolindinedione (TZD) class (troglitazone,
pioglitazone and rosiglitazone) is PPARγ activators that are used to treat diabetes
mellitus [73, 74]. Several nonsteroidal anti-inflammatory drugs (NSAIDs), in
particular indomethacin and ibuprofen, bind to PPARγ and are weak  PPARγ agonists at high micromolar
concentrations [75, 76].The first PPARβ/δ-selective agonists
(L-165041 and GW501516) were shown to augment HDL-C in diabetic mice as well as
in obese rhesus monkeys, in which they decreased elevated levels of
triglycerides and insulin [77, 78].


## 3. The Role of PPAR Ligands in Affecting Cell Proliferation and Differentiation

Although a direct control of PPAR transcription is limited
to a very small number of growth-related genes (see Table1), the ability of
PPAR ligands to inhibit cell growth by inducing cell differentiation or
apoptosis has long been demonstrated in several cell lines. In general, the
PPARα and the PPARγ ligands display an inhibitory effect on cell growth, while
PPARβ/δ have different effects, strictly dependent on the cell type. Indeed, in murine colorectal cells,
the Apc-β-catenin tumour-suppressor pathway was shown to repress PPARβ/δ expression [79]. More recently it was suggested that ligand activation of PPARβ/δ
induces expression of cyclooxygenase-2 (COX2), which could theoretically promote cell growth and inhibit apoptosis through
mechanisms that involve the production of prostaglandins and/or
inflammation-dependent signalling [80]. However, there are several observations
that are inconsistent with the idea that ligands of PPARβ/δ potentiate cell
growth. For example, inhibition of cell growth is observed in a variety of
different cells and cell lines cultured in the presence of highly specific
PPARβ/δ ligands including human colonocytes [81], a human
lung adenocarcinoma cell line [82], mouse lung fibroblasts [83], rat cardiomyocytes [84], a human keratinocyte cell line
[85], normal human keratinocytes [86], and mouse primary keratinocytes [87]. Some evidence about the effects of PPAR ligands on cell
differentiation, cell cycle progression, and apoptosis induction is illustrated
as follows.


### 3.1. Effect of PPAR Ligands in Differentiation Induction

The first demonstration of PPARγ
involvement in adipocyte differentiation was given by Tontonoz et al. (1993) [6]. Subsequently, PPARγ and
PPARα ligands have been demonstrated to induce differentiation alone or
in association with other differentiation inducers. It has been demonstrated
that clofibrate, a PPARα ligand, increases the
differentiation of HL-60 cells induced by retinoic acid and all-trans retinol
[88]. Other PPARα activators, including
putative endogenous ligands such as fatty acids, induce differentiation and
inhibit proliferation in keratinocytes [89]. The PPARα
ligand, ciprofibrate induces differentiation of HL-60 cells and its effect is
potentiated by phorbol 12-myristate 13-acetate (TPA) [90]. Benzafibrate induces
differentiation of HL-60, U937, and K562 cells [91]. PPARγ ligands induce
terminal differentiation of human liposarcoma cells “in vitro” and in patients
suffering from advanced liposarcoma [92], and promote terminal differentiation
of malignant breast epithelial cells “in vitro” [93]. Our research group
demonstrated that both PPARα (clofibrate and ciprofibrate) and PPARγ ligands (troglitazone and 15d-PG J2) inhibit growth of
HL-60 human leukemic cells and induced the onset of monocytic like
differentiation [94]. In another leukemic cell
line, U937 cells, PPARγ ligands inhibited proliferation but did not induce differentiation (except the higher doses of
15d-PG J2 which induced a poor monocytic differentiation) [94] indicating that the
differentiation induction by PPAR ligands is cell-type specific.Several experimental results
indicate that ligand activation of PPARβ/δ induces terminal differentiation of
keratinocytes [86, 87, 95, 96] and it has also been shown that differentiation
of breast and colon cancer cell lines is associated with increased expression
of PPARβ/δ [97].
PPARβ/δ expression also increases following differentiation in
human primary macrophages or in monocyte/macrophage cell lines [98]. In addition, activation of PPARβ/δ using a
selective agonist promotes oligodendrocyte differentiation in a mouse cell
culture [99].


### 3.2. Effect of PPAR Ligands on Cell Cycle Progression

Evidence has been demonstrated that PPAR ligands inhibit cell growth by acting on cell cycle
progression. Fibrates, in a dose dependent-manner, significantly alter the cell
cycle distribution, mainly leading to G0/G1 phase increase and a G2/M phase
reduction in human leukemic cell lines [91]. In HL-60 human leukemic cells, both PPARα and PPARγ ligands increase the
proportion of G0/G1 cells [100]. PPARγ, ectopically expressed
in nonprecursor fibroblastic cell lines, induces the conversion to adipocytes
and induces the expression of p21 and p18, two cyclin/cyclin-dependent kinase (CDK) inhibitors [101].
Troglitazone arrests U937 cells in the G1 phase of the cell cycle [102] and inhibits cyclin D1 expression in MCF7 cells [103]. PPARγ activation induces cell cycle withdrawal of
preadipocytes via suppression of the transcriptional activity of E2F/DP
DNA-binding complex [104]. E2F activity is regulated by the tumour suppressor retinoblastoma protein (pRb) that,
when hypophosphorylated, binds and inactivates the E2F transcription factor
[105]. Interestingly, PPARγ ligands
inhibit pRb phosphorylation in vascular smooth muscle cells [106–108],
increasing the amount of hypophosphorylated pRb able to bind E2F. Others found
that troglitazone inhibits the growth of six of nine pancreatic cancer cell
lines, by inducing G1 phase cell cycle arrest through the up-regulation of  the expression
of p21 [109].Ligand activation of PPARβ/δ with GW0742
prevents cell cycle progression from G1 to S phase and attenuates cell
proliferation in N/TERT-1 keratinocyte cells [110].


### 3.3. Effect of PPAR Ligands on Apoptosis Induction

Inhibition of cell proliferation by PPAR ligands is
also supported by their effect on apoptosis induction. PPARγ ligands seem to be more effective than
PPARα in inducing apoptosis, since its proapototic activity has been
demonstrated in a wide variety of experimental cancer models [111]. PPARγ
ligands have been reported to reduce levels of FLICE-inhibitory protein (FLIP),
and apoptosis-suppressing protein that blocks early events in TRAIL/TNF (Tumor necrosis factor-related apoptosis inducing
ligand/Tumor necrosis factor) family death receptor signalling [112]. 15d-PG
J2 and troglitazone suppress DNA synthesis and induce apoptosis in a dose-dependent
way in HT-29 colon cancer cells, whereas ligands for PPARα and β/δ had no
significant effect [113]. Troglitazone inhibited growth of liver cancer cells
PLC/PRF/5, HepG2 and HuH-7, by inducing apoptosis through caspase-3 activation
[114]. In breast cancer cells, both troglitazone and 15d-PG J2 induce apoptosis
[115, 116]. Kondo et al. have shown that the 15d-PG J2-induced accumulation of
p53 results in the activation of a death-inducing caspase cascade mediated by
Fas and the Fas ligand in neurons [117]. Activation of PPARγ by troglitazone or
15d-PG J2 inhibits cell growth via apoptosis and blocks cell cycle in human
colorectal cancer [118]. However, in some cell models, both PPARα and PPARγ
displayed proapoptotic activity, as it has been demonstrated in the HL-60 cell
line [100] and in the lymphoblastic leukaemia cell line [119]. In keratinocytes
[120], ovarian cancer cells [121] and in human hepatoma cell line SK-HEP-1
[122], PPARα ligands have been reported to induce apoptosis.Colon  cancer cell lines cultured in the presence of the PPARβ/δ ligand GW501516 exhibit inhibited levels of
apoptosis [123, 124]. It has been postulated that apoptosis
is inhibited by PPARβ/δ-dependent downregulation of the tumour
suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN)
and upregulation of the 3-phosphoinositide-dependent kinase-1 (PDK1) and
integrin-linked kinase-1 (ILK1) [22]. The net effect of this
change in activity would have increased phosphorylation of protein kinase B
(Akt) and inhibition of apoptosis; and these changes were shown in cultured
primary keratinocytes [22]. In mouse
keratinocytes, PPARβ/δ inhibits proliferation and promotes
cell survival and migration [96, 125, 126]. In contrast with these data, prostacyclin (PGI2) was shown to
promote apoptosis in a kidney cell line, most probably through PPARβ/δ activation [127].


## 3.1. Effect of PPAR Ligands in Differentiation Induction

The first demonstration of PPARγ
involvement in adipocyte differentiation was given by Tontonoz et al. (1993) [6]. Subsequently, PPARγ and
PPARα ligands have been demonstrated to induce differentiation alone or
in association with other differentiation inducers. It has been demonstrated
that clofibrate, a PPARα ligand, increases the
differentiation of HL-60 cells induced by retinoic acid and all-trans retinol
[88]. Other PPARα activators, including
putative endogenous ligands such as fatty acids, induce differentiation and
inhibit proliferation in keratinocytes [89]. The PPARα
ligand, ciprofibrate induces differentiation of HL-60 cells and its effect is
potentiated by phorbol 12-myristate 13-acetate (TPA) [90]. Benzafibrate induces
differentiation of HL-60, U937, and K562 cells [91]. PPARγ ligands induce
terminal differentiation of human liposarcoma cells “in vitro” and in patients
suffering from advanced liposarcoma [92], and promote terminal differentiation
of malignant breast epithelial cells “in vitro” [93]. Our research group
demonstrated that both PPARα (clofibrate and ciprofibrate) and PPARγ ligands (troglitazone and 15d-PG J2) inhibit growth of
HL-60 human leukemic cells and induced the onset of monocytic like
differentiation [94]. In another leukemic cell
line, U937 cells, PPARγ ligands inhibited proliferation but did not induce differentiation (except the higher doses of
15d-PG J2 which induced a poor monocytic differentiation) [94] indicating that the
differentiation induction by PPAR ligands is cell-type specific.Several experimental results
indicate that ligand activation of PPARβ/δ induces terminal differentiation of
keratinocytes [86, 87, 95, 96] and it has also been shown that differentiation
of breast and colon cancer cell lines is associated with increased expression
of PPARβ/δ [97].
PPARβ/δ expression also increases following differentiation in
human primary macrophages or in monocyte/macrophage cell lines [98]. In addition, activation of PPARβ/δ using a
selective agonist promotes oligodendrocyte differentiation in a mouse cell
culture [99].


## 3.2. Effect of PPAR Ligands on Cell Cycle Progression

Evidence has been demonstrated that PPAR ligands inhibit cell growth by acting on cell cycle
progression. Fibrates, in a dose dependent-manner, significantly alter the cell
cycle distribution, mainly leading to G0/G1 phase increase and a G2/M phase
reduction in human leukemic cell lines [91]. In HL-60 human leukemic cells, both PPARα and PPARγ ligands increase the
proportion of G0/G1 cells [100]. PPARγ, ectopically expressed
in nonprecursor fibroblastic cell lines, induces the conversion to adipocytes
and induces the expression of p21 and p18, two cyclin/cyclin-dependent kinase (CDK) inhibitors [101].
Troglitazone arrests U937 cells in the G1 phase of the cell cycle [102] and inhibits cyclin D1 expression in MCF7 cells [103]. PPARγ activation induces cell cycle withdrawal of
preadipocytes via suppression of the transcriptional activity of E2F/DP
DNA-binding complex [104]. E2F activity is regulated by the tumour suppressor retinoblastoma protein (pRb) that,
when hypophosphorylated, binds and inactivates the E2F transcription factor
[105]. Interestingly, PPARγ ligands
inhibit pRb phosphorylation in vascular smooth muscle cells [106–108],
increasing the amount of hypophosphorylated pRb able to bind E2F. Others found
that troglitazone inhibits the growth of six of nine pancreatic cancer cell
lines, by inducing G1 phase cell cycle arrest through the up-regulation of  the expression
of p21 [109].Ligand activation of PPARβ/δ with GW0742
prevents cell cycle progression from G1 to S phase and attenuates cell
proliferation in N/TERT-1 keratinocyte cells [110].


## 3.3. Effect of PPAR Ligands on Apoptosis Induction

Inhibition of cell proliferation by PPAR ligands is
also supported by their effect on apoptosis induction. PPARγ ligands seem to be more effective than
PPARα in inducing apoptosis, since its proapototic activity has been
demonstrated in a wide variety of experimental cancer models [111]. PPARγ
ligands have been reported to reduce levels of FLICE-inhibitory protein (FLIP),
and apoptosis-suppressing protein that blocks early events in TRAIL/TNF (Tumor necrosis factor-related apoptosis inducing
ligand/Tumor necrosis factor) family death receptor signalling [112]. 15d-PG
J2 and troglitazone suppress DNA synthesis and induce apoptosis in a dose-dependent
way in HT-29 colon cancer cells, whereas ligands for PPARα and β/δ had no
significant effect [113]. Troglitazone inhibited growth of liver cancer cells
PLC/PRF/5, HepG2 and HuH-7, by inducing apoptosis through caspase-3 activation
[114]. In breast cancer cells, both troglitazone and 15d-PG J2 induce apoptosis
[115, 116]. Kondo et al. have shown that the 15d-PG J2-induced accumulation of
p53 results in the activation of a death-inducing caspase cascade mediated by
Fas and the Fas ligand in neurons [117]. Activation of PPARγ by troglitazone or
15d-PG J2 inhibits cell growth via apoptosis and blocks cell cycle in human
colorectal cancer [118]. However, in some cell models, both PPARα and PPARγ
displayed proapoptotic activity, as it has been demonstrated in the HL-60 cell
line [100] and in the lymphoblastic leukaemia cell line [119]. In keratinocytes
[120], ovarian cancer cells [121] and in human hepatoma cell line SK-HEP-1
[122], PPARα ligands have been reported to induce apoptosis.Colon  cancer cell lines cultured in the presence of the PPARβ/δ ligand GW501516 exhibit inhibited levels of
apoptosis [123, 124]. It has been postulated that apoptosis
is inhibited by PPARβ/δ-dependent downregulation of the tumour
suppressor phosphatase and tensin homologue deleted on chromosome ten (PTEN)
and upregulation of the 3-phosphoinositide-dependent kinase-1 (PDK1) and
integrin-linked kinase-1 (ILK1) [22]. The net effect of this
change in activity would have increased phosphorylation of protein kinase B
(Akt) and inhibition of apoptosis; and these changes were shown in cultured
primary keratinocytes [22]. In mouse
keratinocytes, PPARβ/δ inhibits proliferation and promotes
cell survival and migration [96, 125, 126]. In contrast with these data, prostacyclin (PGI2) was shown to
promote apoptosis in a kidney cell line, most probably through PPARβ/δ activation [127].


## 4. The Role of PPAR Ligands in the Control of Growth-Related Gene Expression

The effect of PPAR ligands in the expression of growth regulatory genes has been in part illustrated in the
previous section. Results obtained until now do not allow the identification of
a precise signalling pathway and the PPAR target genes that mediate the antiproliferative effects
remain elusive, as genomic responses to PPARγ activation in cancer
cells are highly complicated [128]. PPARγ ligands seem to be more effective
than PPARα ligands in inhibiting cell growth, thus the majority of data about
the gene expression following treatment with PPAR ligands is obtained in PPARγ ligand-treated
cells. Recently, some evidence has been found for PPARβ/δ and its ligands in regulating gene
expression. However, the number of growth-regulatory genes, affected by
specific PPARβ/δ ligands, is limited and comprises growth-inducing genes such as COX2
[80], and Akt, via transcriptional upregulation of integrin linked kinase (ILK)
and 3-phosphoinositide-dependent kinase-1 (PDK1) [22] and the decrease in the
level of ERK phosphorylation [110].Reported causal mechanisms for PPARγ growth inhibitory effects include
attenuated expression of protein phosphatase 2A and subsequent
inhibition of E2F/DP DNA binding [129], the inhibition of cyclins D1 and E,
inflammatory cytokines and transcription factors expression [130] and increased
expression of an array of gene products linked to growth regulation
and cell maturation [128]. Moreover, our and other research groups have
demonstrated that the reduction of cell growth by PPAR ligands is accompanied
by the downregulation of  the c-myc gene in myeloid leukaemia cells [131] and in colon cancer cells [132, 133]. In the HL-60 cell line, both PPARα (ciprofibrate and
clofibrate) and PPARγ (troglitazone and 15d-PG J2) ligands inhibit c-myb and
cyclin D2 expressions [100]. In prostate cancer cells PPARγ
ligands omega-6 fatty acids and ciglitazone down-regulated
(1-2-fold) beta-catenin and c-myc expression and the selective PPARγ antagonist
GW9662 abolished the effect of those ligands, demonstrating a PPAR-dependent
mechanism. 15-d PG J2 inhibits N-myc expression in neuroblastoma cells [134] while it does not
decrease c-myc expression in vascular smooth muscle cells [135].The major part of the genes of which expression is modulated by PPARγ ligands does not
contain PPRE putative sequences in their promoter regions. Besides
downregulation of c-myc, c-myb, and cyclin D2 genes, previously reported, an array ofnon-PPARγ
targets has been implicated in the antitumor activities of
troglitazone and/or ciglitazone in several cell systems. These targets include intracellular Ca2+ stores
[136], phosphorylating
activation of extracellular signal-regulated kinases [137, 138],
c-JunN-terminal protein kinase, and p38  [139], upregulationof early
growth response-1 [140], the CDK inhibitors p27 [141] and p21 [142],
the tumor suppressor protein p53 and the p53-responsive stress
protein Gadd45 [135], and altered expression of B-cell
leukemia/lymphoma 2 (Bcl-2)family members [139]. However, some of
these targets appear to be cell-type specific due to differences insignalling pathways regulating cell growth and
survival in differentcell systems.Recent findings demonstrate that part of the above mentioned
growth-regulatory genes are affected by PPAR ligands, mostly by PPARγ through a
PPAR-independent mechanism. The most important evidence of PPAR-independent
effects displayed by PPAR ligands is illustrated in Table 2.Table 2
PPAR-independent effects on tumor-related genes.
PPARs ligandPPAR-independent effectExperimental strategiesRef.PPARγ ligandsTroglitazone  in LNCaP prostate cancer cellsAndrogen receptor (AR) suppression by facilitating the
ubiquitin-dependent proteasomal degradation of the transcriptional factor
Sp-1STG28, a PPARγ-inactive analogue of troglitazone.[143]Troglitazone in
miceRapidly AMP-activated protein kinase (AMPK)
activation  through a yet undefined
PPAR-γ-independent mechanism, leading to
the suppression of insulin-like growth factor-I tumor-promoting activity
(IGF-1)Expression of a dominant-negative AMPK[144]Troglitazone and ciglitazone in MCF-7 breast
cancerRepression of cyclin D1 expression, though a
post transcriptional mechanism, via proteasome-facilitated proteolysisProteasome inhibitors[145]Ciglitazone  in HT1080
human fibrosarcomaIncrease of
MMP-2 expression  through ROS
production and ERK activationPPARγ antagonist GW9662[146]Troglitazone and 15-deoxy-prostaglandin J2
(15dPGJ2) in prostate and bladder cancer cellsTroglitazone
induces G0/G1 growth arrest and PGJ2 induces apoptosisPPARγ antagonist GW9662[147]Troglitazone in B cell acute lymphoblastic
leukemia  cell linesApoptosis and cell growth inhibition
associated with G1 cell cycle arrestPPAR antagonists[148]Thiazolidinediones (TZD) in human breast
cancerInhibition of Cyclin D3 expression by
decreasing cyclin mRNA levels and by inducing its proteasomal degradationA dominant negative mutant of PPARγ[149]Troglitazone in mouse skin keratinocytesInhibition of cyclin D1 expressionPPARγ antagonist  GW9662 and
dominant Dominant negative PPARγ.[150]Thiazolidinediones (TGZ) in human colon
cancer cells HTC-116Egr-1 promoter activity increaseDifferent PPARγ ligands[140]15-deoxy-prostaglandin
J2 (15dPGJ2) in colon carcinoma cellsCOX2 and VEGF
inhibition via  AP-1 activity
repressionDominant negative form of PPARγ and a PPARγ antagonist[151]15-deoxy-prostaglandin J2 (15dPGJ2) induces
apoptosis in human B lymphocytesApoptosis through the induction of ROS and
depletion of glutathioneDominant negative form of PPARγ and a PPARγ antagonist[152]15-deoxy-prostaglandin J2 (15dPGJ2) in Jurkat
human leukemic cells and PC3 human prostate cancer cellsApoptosis
by increasing the mRNA stability of death Receptor 5 (DR5), a specific
receptor for tumor-necrosis factor-related apoptosis-inducing ligand (TRAIL)PPARγ antagonist  GW9662[153]PPARα ligandsDEHP in miceInduction of hepatic tumorigenesisWild-type and PPARα-null mice in comparison[154]WY14,643 in activated splenocytes isolated from C57BL/6 miceApoptosisWild-type and PPARα-null mice in comparison[155]PPARβ/δ ligandsGW0742 in PPARβ-null mouse modelInduction of keratinocyte terminal differentiation and inhibition of
keratinocyte proliferationPPARδ-null mice[86]


## 5. The Products of Lipid Peroxidation in the Control of Growth-Related Gene Expression

Reactive intermediates produced during oxidative stressful conditions cause the oxidation of
polyunsaturated fatty acids such as arachidonic, linolenic, and linoleic acids of membrane
lipid bilayers or low-density lipoprotein [156]
leading eventually to the formation of several aldehydes. Among the products of oxidative breakdown
of polyunsaturated fatty acid, 4-hydroxy-2,3-trans-alkenals have been proposed as ultimate messengers of lipid peroxidation-induced injury,
because they can diffuse from the site where they are produced and can reach
different intracellular and extracellular targets [157–159]. 4-hydroxynonenal (HNE), the aldehyde most represented in the 4-hydroxy-2,3-trans-alkenal
class, has long been investigated, since, at concentrations near to those
“physiologically” found in normal cells and plasma, it modulates cellular functions,
gene expression and biochemical pathways, without cytotoxic effects [160]. For this reason, HNE has been proposed by several
authors as an intracellular signalling mediator, rather than a toxic product of
lipid peroxidation [159, 161].
Previous results demonstrated the antiproliferative and differentiative effects
of HNE in leukemic cells [162, 163] and the antiproliferative and proapoptotic effects in a number of different
cell models [164, 165]. Deeper investigations into HL-60
cells showed that the proliferation block occurred at
the level of the G0/G1 stage of the cell cycle [163]. Further experiments
showed that the HNE effects depend on the inhibition of the cyclin expression,
and especially of cyclins D2, D1, and A [166]. The reduction of cyclin
expression can result in a reduced activity of cyclin/CDK complexes which
principally regulate the phosphorylation of the pRb. In highly proliferating
tumour cells, pRb is constantly in the hyperphosphorylated status. When
hyperphosphorylated, pRB cannot bind to E2F transcription factors that can
promote the G1/S cell cycle phase passage. After HNE treatment, pRb remains
hypophosphorylated, and E2F remains bound to pRb [167]. HNE not only reduces
the phosphorylation of pRb, but also decreases the amount of “free” E2F
bound to the P2 c-myc promoter. These effects can explain the blocking of c-myc
expression demonstrated in HNE-treated cells.The hypophosphorylation of pRb proteins may depend on the inhibition of cyclin expression, however, this effect may also be
related to the increase of the expression of p21, an inhibitor of the
cyclin/CDK complexes, induced by HNE treatment [167].
Another effect of HNE, also important for cell
multiplication, is that displayed on telomerase activity and hTERT expression.
The activity of telomerase and the expression of its catalytic subunit hTERT,
were inhibited by HNE in three different human leukemic cell lines, HL-60, U937
and ML-1 [168]. The binding studies of E-box in the hTERT promoter demonstrated that
in HNE-treated HL-60 cells there is a decrease in Myc binding complexes and an
increase in Mad-1 binding complexes which could contribute to the switch from c-Myc/Max to Mad1/Max with repressor
activity of the transcription.HNE is able to induce p53 expression in ML-1 cells, according to previous results
demonstrating the induction of p53 expression by HNE in the SK-N-BE human
neuroblastoma cell line [165]. Moreover, in SK-N-BE cells apoptosis was
substantially increased even with 1 μM HNE. At the same time, the
expression of the p53 family members, p63 and p73, was strongly increased as
well as the expression of the cyclin/CDK inhibitor p21 and the proapoptotic bax
gene. Since p21 and bax are the two main targets for the transcription factor
p53, these results indicate that HNE, by acting on p53 gene
expression, can regulate the p53 target genes.


## 6. Interaction between PPAR Ligands and Lipoperoxidation Products

The relationship between oxidative stress-related molecules and PPAR activation has not yet been
elucidated. Based upon their capacity to elicit cellular responses to a variety
of stimuli, PPARs may represent a class of molecules which allow the
biochemical adaptation to a diverse range of internal and external signals.
These include oxidised LDL [169] and
inflammatory agents as well as 15d-PG J2 [170]
and leukotriene B4 [65]. However, other molecules generated during inflammation
may be involved. In the cultured mesangial cells, PPARγ is activated by various
oxidative stress-related molecules such as TPA, TNF alpha, and H2O2 [171]. The physiological ligand of PPARγ, 15d-PG J2,
is a potential inducer of intracellular oxidative stress that mediates the cytotoxic
effects in human neuroblastoma cells [172]. On
the other hand, the activation of PPARα leads to increased oxidative stress in
liver cells [173]. On the basis of this link
between oxidative stress and PPAR activation and between oxidative stress and
lipoperoxidation induction, our research group investigated, for the first
time, the interaction between the major lipoperoxidation product, HNE, and PPAR
activation in HL-60 and U937 human leukemic cells [94]. We demonstrated that
HNE increases the monocytic differentiation induced by the PPARα ligand
ciprofibrate, and PPARγ ligands, troglitazone and 15d-PG J2, in HL-60 cells.
Whereas, neither PPARα nor PPARγ ligands induce U937 differentiation. Moreover,
in this cell line, only PPARγ ligands reduce cell growth. HNE also significantly inhibits cell growth when
given alone, and strengthens the growth inhibitory effect of a low dose of PPARγ ligands. HNE promotes at the same time a great
increase in the expression of PPARγ in both HL-60 and U937 cells, without any modification of the PPARα expression.
These results suggest a synergistic effect of HNE and PPARγ ligands in blocking
cell growth and in promoting the differentiation in HL-60 cells.More recently, we analysed the effects of PPARγ ligands (rosiglitazone and 15d-PG J2) and HNE, alone or in association,
on proliferation, apoptosis, differentiation, and growth-related and
apoptosis-related gene expressions in CaCo-2, colon cancer cells. Results
obtained indicate that, in this cell line, PPARγ ligands and HNE inhibited cell
growth and induced differentiation or apoptosis by different signalling pathways.
The common feature consisted of the inhibition of c-myc expression, whereas the
apoptosis was induced by 15d-PG J2 and HNE and, to a minor extent, by
rosiglitazone and the differentiation was induced by rosiglitazone and by
15d-PG J2, but not by HNE. Moreover, HNE induced p21 expression, while PPARγ
ligands did not. Bax expression was increased by HNE and 15d-PG J2, but not by
rosiglitazone. HNE did not induce an increase of PPARγ expression and did not display
synergism or antagonism towards PPARγ ligands.These various results, obtained in different cell models, strongly demonstrate that the gene expression control exerted
by PPAR ligands is dependent on the cell type examined.An interaction between HNE and PPARγ has also been demonstrated by Muzio et al. (2006) [174]. These
authors found that arachidonic acid induces suppression of
human lung tumor A549 cell growth, increases lipid peroxidation and decreases
aldehyde dehydrogenase 3A1 ALDH3A1, which may determine an accumulation of
endogenous HNE. These phenomena are associated with the increased expression of
PPARγ, suggesting a relationship between endogenous HNE levels and PPARγ
expression. Moreover, it has been postulated that HNE can represent an
endogenous modulator of PPARβ/δ activity, since HNE is an endogenous
ligand for PPARβ/δ and activates PPARβ/δ target genes [175]. This datum suggest that the binding between HNE and PPARβ/δ can modulate PPARβ/δ activity in all cell types, since PPARβ/δ is
ubiquitously expressed.The different interactions between HNE and PPAR are summarized in Figure2.Different interactions between HNE and PPAR. (a) HNE increases
PPARγ expression in leukemic cell lines; (b) HNE binds and activates PPARβ/δ.
(a)(b)These findings represent an
intriguing suggestion about the role played by the lipoperoxidation products in
controlling cellular PPAR-dependent responses, not only regarding cell
proliferation control but also in the regulation of different metabolic
pathways, and indicate that the interaction between oxidative stress products
and PPAR activity represents a new research field in expansion.



---

*Source: 524671-2008-07-06.xml*