# Multiple Interactions between Peroxisome   Proliferators-Activated Receptors   and the Ubiquitin-Proteasome System  and Implications for Cancer Pathogenesis

Authors: Davide Genini; Giuseppina M. Carbone; Carlo V. Catapano
Journal: PPAR Research
 (2008)

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## Abstract

The peroxisome proliferator-activated receptors (PPAR)α,β/δ, and γ are ligand-activated nuclear receptors involved in a number of physiological processes, including lipid and glucose homeostasis, inflammation, cell growth, differentiation, and death. PPAR agonists are used in the treatment of human diseases, like type 2 diabetes and dyslipidemia, and PPARs appear as promising therapeutic targets in other conditions, including cancer. A better understanding of the functions and regulation of PPARs in normal and pathological processes is of primary importance to devise appropriate therapeutic strategies. The ubiquitin-proteasome system (UPS) plays an important role in controlling level and activity of many nuclear receptors and transcription factors. PPARs are subjected to UPS-dependent regulation. Interestingly, the three PPAR isotypes are differentially regulated by the UPS in response to ligand-dependent activation, a phenomenon that may be intrinsically connected to their distinct cellular functions and behaviors. In addition to their effects ongene expression, PPARs appear to affect protein levels and downstream pathways also by modulating the activity of the UPS in target-specific manners. Here we review the current knowledge of the interactions between the UPS and PPARs in light of the potential implications for their effects on cell fate and tumorigenesis.


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## Body

## 1. Introduction

Despite the everyday progress  in
understanding the genetic and molecular bases of cancer, this disease still
strikes millions of people worldwide. The quest for new targets and more
effective therapeutics is currently a major driving force in cancer research.
Multiple mutations that affect critical cellular pathways lead to uncontrolled
proliferation, increased survival, and block of differentiation in cancer cells
[1]. Several cellular pathways (e.g.,
cell surface receptors, signal transduction pathways, apoptosis,
angiogenesis, transcription, chromatin regulation, and
proteasome-mediated degradation) have provided relevant targets and
opportunities for development of clinically useful therapeutics [1]. Unfortunately, targeting each of
these major pathways individually may not be sufficient. Extensive cross-talks
occur between regulatory pathways and it is not unlikely that the same proteins
play multiple roles in different processes. The peroxisome
proliferator-activated receptor (PPAR) subfamily of nuclear receptors may
represent a prime example of proteins interacting with multiple cellular
pathways and exerting diverse and sometime apparently contrasting
functions. Here, we review how PPARs
interact with the ubiquitin-proteasome system (UPS), which is the major
cellular system responsible for protein turnover, and how these two systems
might reciprocally affect each other activity and functions.


## 2. The Ubiquitin-Proteasome System

Ubiquitin
is a 76-amino acid polypeptide that is post-transcriptionally linked to
proteins via a covalent linkage to one or multiple lysine residues [2]. Several proteins
including cell surface receptors, cell cycle regulators, and transcription
factors are ubiquitinated and protein ubiquitination affects many cellular
processes including proliferation, cell cycle progression, DNA damage repair,
and cell death [2]. Ubiquitination is a
regulatory signal that affects the fate and function of proteins.
Ubiquitination regulates mainly protein turnover directing ubiquitinated
proteins to proteasome-mediated proteolysis. Other nonproteolytic functions, like control of protein-protein
interactions, cellular localization, and catalytic activity, are emerging [2]. The proteasome is a
multicatalytic complex that comprises a 20S core with proteolytic activity and
a 19S subunit that recognizes poly-ubiquitinated proteins, unfolds them, and
passes into the 20S catalytic core for degradation. Ubiquitination is catalyzed
by three types of enzymes, called E1, E2, and E3 [2, 3]. Ubiquitin is first
activated by an E1 ubiquitin-activating enzyme in an ATP-dependent reaction.
The activated ubiquitin is then transferred to an E2 ubiquitin-conjugating
protein (UBC). Finally, E3 ubiquitin-ligases, which are the most critical
enzymes in the process, catalyze the transfer and covalent attachments of the
activated ubiquitin to the target protein. In human cells, a single E1 and
about 60 E2 enzymes have been identified, while there are approximately a
thousand E3 enzymes, which ensure a high degree of substrate specificity to the
system [2, 3]. E3 enzymes are split in two major
subfamilies: the Ring-H2 and the HECT domain proteins. The human genome
contains also more than 70 deubiquitinating enzymes (DUBs) that remove
ubiquitin chains from ubiquitinated proteins and can rescue them from
proteasomal degradation [4].Protein
ubiquitination is a highly dynamic process and ubiquitination-deubiquitination
cycles can serve to rapidly modulate protein level and function [4]. Ubiquitin and
proteasomal components play an important role in transcription [5, 6]. Ubiquitin ligases and proteasomal
subunits are present as integral components of transcription regulatory
complexes [5, 6]. Histones, the main component of
chromatin, are ubiquitinated and the process affects chromatin remodeling and
transcription [6, 7]. RNA polymerase II is also directly
regulated by ubiquitination [6, 8]. Moreover, the UPS regulates the
abundance, activity, and subcellular localization of many transcription factors
[5, 6]. Transcription factors are
ubiquitinated and degraded by the proteasome and,
paradoxically, the process is often essential for their transactivating ability
[6]. In fact, transcription
activation and degradation domains of transcription factors often overlap [6]. In addition, mono-ubiquitination (i.e.,
addition of single ubiquitin tag to a protein) can act as a post-translational
modification that modulates activity of transcription factors and regulates
transcription efficiency by nonproteolytic mechanisms [6]. Degradation of inhibitors of
transcription factors is also often required to release active transcription
factors. For example, activation of the transcription factor NF-κB is controlled by a signaling cascade based on
multiple ubiquitination and proteasome-dependent events [6].Alterations of the UPS are frequent in
cancer. They are mainly due to loss or gain of function of specific components
of the UPS and alterations of UPS substrates, like oncogene and tumor
suppressor gene products, which become less or more susceptible to
proteasomal-dependent degradation [9]. Tumor suppressor proteins are
often the targets of UPS alterations. The human papillomavirus (HPV), a cause
of cervical cancer, encodes two oncogenic proteins, E6 and E7. These viral proteins promote degradation of
the tumor suppressor p53 via ubiquitination by the E6-associated protein
(E6-AP) E3 ubiquitin ligase [10]. HDM2 is another E3 ubiquitin
ligase that targets p53 to proteosomal degradation [11]. Aberrant expression of HDM2 is
found in many human cancers [12]. Single nucleotide polymorphism in
the HDM2 promoter leading to HDM2 overexpression has been recently associated
to the development of sporadic and hereditary cancers [13]. The E3 ubiquitin ligase Skp2 is responsible for ubiquitination of the
cell cycle inhibitor and tumor suppressor p27 [14]. Skp2 overexpression is observed in
cancer cells leading to degradation and inactivation of this tumor suppressor
protein [15]. Oncogenic proteins are also
affected by alterations of UPS components. The E3 ubiquitin ligase encoded by
the von Hippel-Lindau gene (pVHL) mediates the ubiquitination and degradation
of the hypoxia-inducible transcription factor HIF-1α [16, 17]. Mutations in pVHL gene predispose
patients to renal cell carcinoma and other cancers. In these tumors, the level of HIF-1α is increased resulting in a potent oncogenic
and angiogenic stimulus.Due to the unique mechanism of cleavage at
the proteolytic active sites, selective proteasome inhibitors have been
synthesized and some, like bortezomib (Velcalde, PS341), have undergone
clinical evaluation as anticancer agents [18]. Bortezomib is a peptide boronate proteasome inhibitor that blocks the
chymotryptic activity of the 26S proteasome [18]. The anticancer effect of
bortezomib is likely to be achieved through its inhibitory effects on protein
degradation and modulation of important cellular pathways, including inhibition
of the NF-κB pathway [18]. Bortezomib is currently approved for clinical use for treatment of multiple
myeloma. Clinical trials with bortezomib and second generation proteasome
inhibitors as single agents or in combination with other chemotherapeutic
agents are ongoing in various tumor types [18].


## 3. Peroxisome Proliferator-Activated Receptors

PPARs emerged in the nineties as nuclear
receptors regulating transcription of genes involved in metabolic processes
like lipid and glucose homeostasis [19, 20]. Later, PPARs have found to be implicated
in many physiological and pathological processes [20]. PPARs belong to the nuclear
hormone receptor super-family,
which is one of the largest families of transcriptional regulators in the human
genome with more than 40 distinct nuclear receptors [21]. Nuclear receptors bind small
lipophilic molecules, such as steroid hormones, vitamins, and fatty acid
derivatives, and function as ligand-activated transcription factors,
interacting with specific DNA sequences (i.e., hormone response elements, HRE)
in target genes and stimulating their transcription [21]. Thus, nuclear receptors provide a
direct link between small lipophilic signaling molecules present in the cells
or their environment and the cellular transcriptional machinery, turning on
specific subsets of genes containing the appropriate HRE and inducing complex
cellular responses [21, 22]. The nuclear receptor super-family includes the
steroid hormone receptors (i.e., estrogen, progesterone, androgen, and
glucocorticoid receptors) and receptors for nonsteroidal hormones [21–23]. The latter include the PPARs, vitamin D (VDR), and retinoic acid (RAR)
receptors [21, 23]. The ligands of most nonsteroidal
receptors are dietary fatty acids or generated locally by lipid metabolism
within the target cell or tissue, while steroid and thyroid hormones are
produced by distant endocrine organs and released in the blood [21, 22].Nuclear receptors
exhibit a characteristic modular structure comprising an N-terminal domain with
the ligand-independent activation function domain (AF-1), a DNA binding domain
(DBD), and a C-terminal domain containing the ligand binding (LBD), and the
ligand-dependent transactivation (AF-2) domain [23]. The DBD contains two zinc finger
modules and determines the DNA binding specificity of the receptors. The LBD is
involved in homo- and heterodimerization and interaction with cofactors [23, 24]. The structure of the LBD is highly
conserved among nuclear receptors, comprising a large hydrophobic cavity that
accommodates the ligand. Variations of the size and shape of the ligand binding
pocket ensure ligand specificity among receptors [23, 24]. Ligand-binding induces a
conformational remodeling of the LBD that exposes surfaces required for
interaction with coactivators and affects the affinity of the receptors for
corepressors [23, 24]. The nonsteroidal receptors are
found primarily in the cell nucleus and are bound to HRE as heterodimers with
the retinoic X receptor (RXR) [19, 23]. These receptors can affect both
positively and negatively transcription of target genes with the LBD mediating
alternatively transcriptional activation or repression, although the mechanisms
of transrepression by PPARs are still poorly understood [25]. Transcriptional repression is due
to recruitment of corepressors, like NCoR/SMART, by the unliganded and
DNA-bound receptor and formation of multiprotein complexes containing histone
deacetylases and other chromatin remodeling enzymes [23, 25]. In the presence of ligands,
corepressor complexes are released and replaced by coactivators, like SRC1 and
CBP-p300, thus switching on transcription [23, 25]. Transcriptional activation is
associated with histone modifications, chromatin remodeling, and assembly of
the transcription initiation complex. Thus, transcriptional activation and
repression by nuclear receptors are very dynamic processes involving the
formation of protein complexes in which multiple coactivators and corepressors
need to be rapidly exchanged [25–27]. The UPS is perhaps the major
system controlling the assembly and turnover of these regulatory complexes ensuring
their timely interaction with the transcriptional machinery [26]. Ubiquitin and proteasome
components are associated with corepressor and coactivator complexes recruited
by nuclear receptors [25, 26]. Most nuclear receptors, including
thyroid hormone, estrogen, glucocorticoids receptor, RAR, and RXR receptors, as
well as coactivators, corepressors, and general components of the transcription
machinery are ubiquitinated and degraded by the proteasome [26, 28].PPARs have the
typical modular structure of the nuclear hormone receptors with a poorly
characterized N-terminal domain with putative ligand-independent AF-1 function,
a central DNA-binding domain (DBD), and a C-terminal ligand binding (LBD) and
ligand-dependent AF-2 domain (Figure1) [19, 23]. However, despite the high sequence
and structural homology, the three PPAR isotypes have distinct ligand
specificity, functions, and behaviors [19, 20]. PPARα is a key regulator of energy homeostasis and
plays a major role in lipid metabolism and glucogenesis. PPARα is expressed in tissues with significant fatty
acid and cholesterol catabolism, like brown adipose tissue, liver, kidney,
intestine, heart, and skeletal muscle [29]. PPARγ exists in two isoforms (γ1 and γ2) that differ only at the N-terminus. PPARγ2 is present at high levels in adipose tissue,
whereas PPARγ1 expression is broader and is present in gut,
brain, vascular cells, immune cells, and retina [30]. PPARγ plays a role in adipocyte differentiation,
glucose metabolism, and lipid homeostasis, and participates in
monocyte/macrophage differentiation [30]. Moreover, PPARγ influences fatty acid storage in the adipose
tissue and is implicated in insulin resistance and atherosclerosis [30]. PPARδ is ubiquitously expressed with high levels in
colon, skin, and brain [20]. PPARδ also functions in processes linked to lipid
metabolism, like fatty acid catabolism, cholesterol efflux, lipid uptake in
macrophages, and preadipocyte differentiation [31]. This nuclear receptor plays also a
role in placental and gut development, embryo implantation, tissue injury, and
wound healing [20, 32].Figure 1
Domain structure of PPARδ and truncated forms of the receptor. AF-1, N-terminal ligand-independent activation function 1 (aa 1–70). DBD,
DNA binding domain (aa 71–136). Hinge, flexible hinge region (aa 137–167). LBD,
ligand-binding domain (aa 168–431). AF-2, C-terminal ligand-dependent activation
function-2 (aa 432–441). The position of the mutations (K204R, K224R, and K229R)
introduced in the region 204–235 is shown.PPARs possess a
broad ligand-binding cavity that allows binding of a wide range of synthetic
and natural lipophilic compounds [19]. Medium- and long-chain unsaturated fatty acids (e.g., linoleic acid),
conjugated and oxidized fatty acids (e.g., phytanic acid), and eicosanoids bind
to PPARα [19]. Fibrates, like bezafibrate, fenofibrate, and clofibrate, which are
used for the treatment of dislipidemias and cardiovascular diseases, are
selective PPARα agonists [29]. PPARγ binds to long-chain fatty acids, prostaglandin
J2 (PG J2), and other eicosanoids [19]. Synthetic PPARγ agonists, such as pioglitazone and
rosiglitazone, are insulin sensitizers used to treat type 2 diabetes [30]. PPARδ has high affinity for prostaglandin I2 (PGI2), fatty acids, and synthetic compounds [19, 31].Beside their
metabolic functions, PPARs have an important role in inflammation. PPARα and PPARγ agonists can ameliorate chronic inflammatory
conditions, such as atherosclerosis, arthritis, and inflammatory bowel disease [20, 29, 30]. PPARs repress genes of the
inflammatory response pathway, such as cytokines (TNFα, IL-1β, IL-6), cell adhesion molecules (MMPs), and
other proinflammatory molecules (iNOS) [25]. These effects are mediated in large part by the ability of PPARs to
antagonize other transcription factors, like AP-1, STAT1, and NF-κB, which have proinflammatory functions [25]. Different mechanisms have been
proposed to explain the phenomenon of transrepression by PPARs, including
sequestration of limiting cofactors, direct physical interaction, and
antagonism between PPARs and other transcription factors, and promoter-specific
block of corepressor/coactivator exchange by PPARs in selected target genes [24, 25]. The latter involves a block of the
ubiquitin and proteasome-dependent processing of corepressor complexes as in
the case of PPARγ-mediated repression of proinflammatory NF-κB target genes [25]. PPARγ and PPARα can also interact physically with NF-κB and c-Jun blocking transcriptional activation
[33, 34]. Reciprocally, NF-κB and c-Jun can repress PPARγ and PPARα-induced transcription, respectively, by
inhibiting the binding to PPRE in target genes [33–35]. Also PPARδ has a role in inflammation controlling
expression of proinflammatory genes in macrophages in a ligand-dependent manner
[31]. Unliganded PPARδ binds to corepressor molecules including Bcl-6,
which is a repressor of inflammatory gene expression [31, 36]. Ligand binding releases the
corepressor complexes resulting in transcription of PPARδ target genes. At the same time, PPARδ-bound Bcl-6 is also released and is free to
repress its own target genes suppressing the inflammatory response [31, 36]. Paradoxically, PPARδ knockout has the same effects of the agonists
on the expression of Bcl-6 target genes since it also leads to release the
transcriptional repressor [36].The involvement of
PPARs in carcinogenesis has been widely discussed, although it is still
controversial whether the different isotypes either favor or inhibit
tumorigenesis [37, 38]. This may still represent a major
concern for developing PPAR-targeted therapeutics for clinical applications because
of the potential risk of promoting tumorigenesis as indicated by studies in
rodents [39]. PPARs are expressed in several
human cancers and PPAR ligands have been shown to modulate tumor growth [37, 38]. Inactivating mutations, deletions
and chromosomal translocations of PPARγ have been found in various cancers pointing to
a tumor suppressor role of this nuclear receptor [40–42]. PPARγ ligands promote differentiation, growth arrest,
and death of cancer cells in vitro [38]. PPARγ ligands reduce growth of human tumor
xenografts and spontaneous and carcinogen-induced tumors in rodents [38]. PPARα is also expressed in various tumors and cancer
cell lines [43, 44]. Activation of PPARα in cancer cells inhibits proliferation and
suppresses metastatic potential [45–47]. PPARα ligands have shown antitumor activity also in
murine models [46, 48, 49]. PPARδ participates in a number of important pathways
controlling adhesion, proliferation, differentiation, and survival [37]. Unlike the other isotypes, PPARδ has been shown to prevent apoptosis and induce
cell growth in normal cell types, like primary mouse keratinocytes,
preadipocytes, vascular smooth muscle cells, hepatic stellate cells [37]. Consistent with an antiapoptotic role, PPARδ increases the expression of antiapoptotic
genes and activates prosurvival signaling pathways in keratinocytes [50]. PPARδ agonists stimulate proliferation and survival
of cancer cells in vitro and
promote tumor growth in mice [51–57]. PPARδ is a downstream target of β-catenin/T cell factor-4, which is central in
colon cancer pathogenesis and regulates other cancer-promoting genes like c-myc
and cyclin D1 [58]. Cyclooxygenase-2 (Cox-2) modulates
PPARδ activity and nonsteroidal anti-inflammatory drugs that
have chemopreventive effects
in colon cancer inhibit PPARδ activity and expression [58–60]. Cox-2 is frequently upregulated in
cancer and preneoplastic lesions, and Cox-2 products like PGI2 act
as selective agonists of PPARδ [58–60]. To further support a protumorigenic
role of PPARδ, PPARδ expression is elevated in cancers, like
colorectal, endometrial, and head and neck cancers [58, 59, 61]. Additional evidence pointing to a
tumor promoting function of PPARδ comes from experiments in mice where
disruption of PPARδ decreased tumorigenicity of cancer cells in
nude mice and PPARδ activation increased tumor growth [55, 57, 62].Despite this large
body of evidence, some controversial results in animal experiments cast doubts both
on the anti- and protumorigenic activities of PPARs [37, 38]. Experiments in rodents have shown
increased frequency and enhanced tumor growth by PPARγ agonists [38, 63, 64]. Similar contradictory data have
been reported for PPARα, whereby prolonged administration of PPARα agonists caused hepatocarcinogenesis in rats
and mice [65]. The frequency of intestinal tumors
also increased in PPARδ knockout mice [66, 67] or decreased upon treatment of the
animals with PPARδ ligands [68]. These contradictory results
between cellular and animal models and different animal models suggest that the
function of these nuclear receptors is more complex than that has been assumed so far and may depend
heavily on cell and tissue context, cross-talks with multiple signaling
pathways and noncell autonomous mechanisms. A hint to this complexity is given
by recent studies of the role of PPARs in tumor angiogenesis. In addition to
cancer cell-autonomous effects, PPARs affect strongly tumor angiogenesis and
inflammation, two processes that have a critical role in tumor pathogenesis and
progression. PPARγ and PPARα agonists have anti-inflammatory properties,
which may contribute greatly to their in vivo antitumor activity under certain
circumstances. PPARγ ligands are also potent angiogenic inhibitors [69, 70] and PPARα agonists suppress VEGF production, endothelial
cell proliferation, and tumor growth in mice [48, 49]. PPARδ activation stimulates VEGF production in mice,
which at least in part had an autocrine prosurvival effect on cancer cells [71]. PPARδ has been recently identified as a critical
node in a tumor angiogenic network linking angiogenesis to inflammation and
carcinogenesis [72]. Knockout of PPARδ in host tissues but not in tumor cells reduced
tumor growth by impairing angiogenesis [72]. Interestingly, the in vivo
antitumor activity of PPARα agonists also depended heavily on the effects of
host endothelial and stromal cells rather than cancer cells blocking angiogenesis
and inflammation [48, 49]. Paradoxically, PPARα knockout impaired tumor growth in mice,
because it resulted in a strong inflammatory response and production of anti-angiogenic factors, like
TSP-1 and endostatin [73]. This paradoxical response is
similar to the effects of PPARδ on inflammatory gene expression in macrophages,
where both receptor activation and knockout suppressed expression of a subset
of target genes [31, 36]. This dual mode of regulation of
gene expression, whereby ligand- dependent and independent mechanisms lead to
transrepression, derepression, or trans-activation of distinct subsets of genes,
seems a common theme for these nuclear receptors and needs to be taken into
account when examining their functions in physiological and pathological
processes.


## 4. The Ubiquitin-Proteasome System and Peroxisome Proliferator-Activated Receptors

### 4.1. UPS and Control of PPAR Turnover

Important factors
to consider when studying the multiple and complex functions of PPARs are their
connections with other cellular systems and how these interactions reciprocally
impact on each system activity. Recent reports suggest that the activity of
PPARs is linked in many ways to the UPS [28]. All three PPARs are short-lived
proteins that undergo ubiquitination and proteosomal degradation and the UPS is
mainly responsible for the turnover of these nuclear receptors [28]. However, the three PPAR isotypes
have different behaviors with respect to ligand-dependent receptor turnover.
PPARγ undergoes negative autoregulation upon agonist
binding. PPARγ is ubiquitinated and degraded by the
proteasome in a negative feedback loop that probably serves to attenuate
receptor-mediated gene transactivation [74]. PPARα turnover is controlled by ligands in a
slightly different manner. Instead of enhancing ubiquitination and degradation,
PPARα ligands prevent ubiquitination and lead to
increased stability of the receptor [75].
The protective effect of the ligand,
however, is maximal during the first 3 hours of exposure to the ligand and the receptor is then rapidly degraded [75].We have recently examined the
ligand-dependent turnover of PPARδ and the role of the UPS in this process [76]. Our study revealed interesting
differences between PPARδ and other PPAR isotypes with respect to ligand-dependent
receptor turnover and interaction with the UPS. We found that PPARδ, like other nuclear receptors, is
ubiquitinated and rapidly degraded by the proteasome [76]. Brief incubation of cells expressing
both endogenous and recombinant PPARδ with proteasome inhibitors led to rapid
accumulation of the receptor in cell nuclei. Interestingly, in the presence of
proteasome inhibitors, PPARδ was transcriptionally competent as shown by
luciferase reporter assays and assessment of endogenous target genes by RT-PCR [76]. Thus, PPARδ was different from other nuclear receptors,
including the estrogen, androgen, thyroid hormone, and retinoic acid receptors,
whose transcriptional activity is reduced by proteasome inhibitors [26]. Furthermore, while in the absence
of ligands PPARδ had a very short half life (~30 minutes), the
addition of ligand increased considerably the receptor half life [76]. The effects of the synthetic and
natural ligands were rapid with an increase of PPARδ protein level within 4 hours upon addition to
the cell culture medium. The receptor level remained high as long as the
ligands were present [76]. Removal of the ligands was
followed by rapid reversal with return to the baseline level within few hours.
Once again, PPARδ behavior was unique among nuclear receptors,
whose turnover is generally accelerated by their own ligands [26, 77]. The progesterone receptor, thyroid
receptor, estrogen receptor, RAR, and RXR all show ligand-dependent increase of degradation associated with transcriptional
activation [26, 77]. The direct consequence of these
events is a rapid decrease of the receptor half life and switching-off the
transcriptional response. Only vitamin D3 receptor is known to be stabilized by
the ligand with a similar kinetics [78]. As mentioned above, PPARγ is also rapidly degraded upon exposure to
ligands [74] and PPARα is stabilized only transiently by ligands [75]. Further work demonstrated that
ligand-induced stabilization of PPARδ was due to a selective block of receptor
ubiquitination [76]. This ubiquitination block depended
on the continuous presence of the ligand, was rapidly reversed after removal of
the ligand, and was due to the direct interaction of the ligand with the
receptor [76]. Disruption of the LBD in PPARδ/Tr1-299 abolished the effect of the ligand on
ubiquitination and proteolysis, although the truncated form of the receptor was
still ubiquitinated and degraded by the proteasome [76]. Thus, binding of the ligand to the
LBD induced a conformational change that, in addition to allowing receptor
trans-activation, blocked the interaction of PPARδ with an ubiquitin ligase or, alternatively,
promoted binding of a deubiquitinating enzyme.Using site-directed
mutagenesis, we investigated further the role of distinct PPARδ  domains  in  the  ligand-dependent regulation of
receptor turnover [76]. This analysis revealed additional differences between PPARδ and other PPAR isotypes. Mutations in the DBD of PPARδ reduced the effect of ligands on receptor
ubiquitination [76]. This suggested that the ligand
acted preferentially on the DNA-bound receptor preventing its ubiquitination.
Interestingly, mutations in DBD of PPARγ did not affect ligand-dependent turnover,
indicating that DNA binding was not a prerequisite for ligand-induced
degradation of this receptor [74]. On the other hand, we showed that the AF-2 domain of PPARδ was not required for ligand-induced block of
ubiquitination, indicating that the effect was independent of coactivator
binding [76]. For most nuclear receptors, the
transactivating function is linked to proteolytic degradation and mutations in
the transactivating domain affect also receptor ubiquitination and proteolysis [77]. The AF-2 domain of PPARγ has a similar role and mediates ligand-induced
degradation of the receptor [74]. For PPARγ and other nuclear receptors, conformational
changes induced by the ligands may favor the concomitant interaction with
coactivators and components of the UPS. Overexpression of transcriptional coactivators led also to a decrease of
PPARα level in the presence of ligand, showing that
the interaction with coactivators via the AF-2 domain promoted proteolysis of
the α isotype [79]. Thus, for PPARα the initial stabilization is probably followed
by the recruitment of coactivators along with other factors that trigger
proteolysis of the receptor. In contrast, in the case of PPARδ we showed that transactivation and receptor
ubiquitination are functionally separated [76]. The absence of a link between these two processes allows independent
control of receptor transactivation and ubiquitination upon ligand binding and
may be a prerequisite to avoid rapid degradation and sustain its transcriptional
activity once it is engaged in transcriptional activation complexes. Further
analysis of PPARδ mutants indicates that the region between
amino acid 204 and 235 may play a role in controlling ubiquitination and proteolytic
degradation of the receptor (Figure 1). This region has a poor secondary
structure, forms a loop exposed to the surface, and may be in an environment
prone to ubiquitination [80, 81]. In addition, the region is quite
diverse between the PPAR isotypes, possibly explaining the divergent responses
in terms of ligand-dependent turnover. Pull-down experiments showed that the
truncated PPARδ/Tr1-235 was ubiquitinated, while the shorter PPARδ/Tr1-204 was not (Figure 2(a)). Different scenarios
can explain these results and are under consideration. The region between amino
acid 204 and 235 may contain lysine residues that are the major sites of
ubiquitination of PPARδ. However, mutations of the three lysines
present in this region (K204R, K224R and K229R) did not affect ubiquitination
of the PPARδ/Tr1-235 (Figure 2(b)). Thus, alternatively the
region 204–235 may be needed for the binding of an ubiquitin ligase or
cofactors that mediate the interaction of the receptor with the UPS.Ubiquitination of truncated and
mutated forms of PPARδ. (a) U2OS cells
were transfected with HA-ubiquitin expressing vector along with wild type
His-PPARδ or truncated forms of the receptor (PPARδ/Tr1-204, Tr1-235 and Tr1-299). After 24 hours,
cells were incubated overnight with vehicle or the PPARδ ligand GW501516 (5 μM) and subsequently all samples were incubated with
10 μM the proteasome inhibitor PS341 for 4 hours.
His-tagged wild type and truncated PPARδ were pulled-down with nickel affinity gel
under denaturing conditions. PPARδ was detected in pull-down fractions using an
anti-His antibody and ubiquitinated proteins with an anti-HA antibody. (b) U2OS
cells were transfected with HA-ubiquitin vector along with the indicated PPARδ expressing vectors. PPARδ/Tr1-235 had wild type sequence or the
indicated double or triple mutations (K204R, K224R and K229R). Cells were
treated and analyzed as above.
(a)(b)Thus, even if the
PPAR isotypes are structurally very similar, binding to specific ligands
induces divergent responses as far as receptor turnover. PPARγ upon ligand binding becomes ubiquitinated and
prone to degradation, whereas ligands prevent or delay ubiquitination and
degradation of PPARδ and PPARα. Most nuclear receptors exhibit negative
autoregulation upon interaction with the respective ligands [26, 77]. Ligand-induced stabilization is a
less common and has been observed only for very few nuclear receptors. The system in place for PPARδ may be geared to prevent both accumulation of
high levels of the receptor and its prolonged activation [76]. Overactivity of PPARδ may be detrimental to cells, perhaps due to
its antiapoptotic and potentially tumorigenic activity [32, 37]. The level of PPARδ is low and constantly controlled via
UPS-dependent proteolysis, which may affect greatly the ligand-independent
functions of the receptor like transrepression of other transcription factor
target genes. Under physiological conditions, the low abundance and short half life
of natural PPARδ ligands, like PGI2, would
contribute to keep the receptor in the unbound state [32]. In the presence of high concentrations
of ligands, the DNA-bound and liganded PPARδ is protected from proteasomal degradation by the
inhibition of its ubiquitination [76]. The stabilized DNA-bound receptor
would be able to transactivate target genes as long as enough ligand is
present. This would be consistent with the fact that in processes, such as
wound healing, inflammation, and cancer, PPARδ levels seem to increase concomitantly with
upregulation of cyclooxygenase-2 and other enzymes for the production of lipid
metabolites capable of stabilizing and activating PPARδ [32, 37, 55, 58, 59]. In the absence of
this coordinated increase of ligand and receptor levels, PPARδ might not be able to act as antiapoptotic and
growth-promoting factor. How ligand-induced stabilization of PPARδ affects ligand-dependent interactions with
other transcription factors leading to transrepression or derepression of gene
expression is still unknown.


### 4.2. UPS, PPARs, and Interactions with Other Signaling
Pathways

In addition to
ligand-dependent receptor turnover, the UPS is an important way to control PPAR
activity in response to upstream signal transduction pathways (Figure3). Receptor
phosphorylation by cellular kinases can regulate both basal and ligand-induced
activity of PPARs as well as modulate their protein level by indirectly controlling
proteasome-dependent degradation [82]. In colorectal cancer cells, the
polypeptide hormone gastrin promotes cell proliferation and the effect is
associated with decreased PPARγ level. This was mediated by phosphorylation of
PPARγ involving the epidermal growth factor receptor
and ERK1/2 kinase leading to increased PPARγ proteasome-mediated degradation [83]. In fat cells IFN-γ treatment induces a rapid reduction of PPARγ protein level, which is blocked by proteasome
inhibitors [84]. On the other hand, there are instances in which PPARs enhance
stabilization or degradation of proteins by affecting their susceptibility to
UPS-mediated degradation. Perhaps the best example of a signaling pathway in
which both PPARs and the UPS are implicated is the Wnt pathway. Suppression of
the canonical Wnt signaling is required for differentiation of preadipocytes
into adipocytes. The process is in part
mediated by PPARγ-induced degradation of β-catenin, which is a central element in the Wnt
pathway. Activation of PPARγ promotes degradation of β-catenin in glycogen synthase kinase 3β (GSK3B)-dependent or independent manner [85, 86]. β-catenin mutations that inhibit degradation
block expression of a subset of adipogenic and PPARγ target genes [85]. PPARγ-dependent degradation of β-catenin requires an active APC-containing
destruction-complex. Mutations of the T cell factor/lymphocyte enhancer factor
(TCF/LEF) binding domain of β-catenin or of a catenin-binding domain (CBD) within
PPARγ block proteasomal degradation of β-catenin [87]. The interaction between β-catenin and PPARγ affect their respective oncogenic and tumor
suppressor function [87]. A functional APC was found to be required
also for PPARγ-mediated suppression of colon carcinogenesis [88]. Activation of PPARγ induces degradation of cyclin D1, which has a
critical role in cell cycle regulation, along with β-catenin in hepatocytes [89]. Reduced cyclin D1 protein level
was observed also in breast cancer cells upon PPARγ activation by selective ligands and cyclin D1
downregulation was blocked by inhibition of the proteasome [90]. However, the ability of
thiazolidinedione ligands to reduce β-catenin and cyclin D1 levels might be in part
PPARγ-independent and determined by direct effects of
these compounds on protein degradation [91, 92]. Beside the induction of proteosomal
degradation, activation of PPARγ has been shown to increase the level of proteins
by blocking their proteolysis. Activation of PPARγ in human hepatocarcinoma cells inhibits
proteosomal degradation of p27, a cyclin-dependent kinase inhibitor, with
consequent inhibition of cell proliferation [93]. Similarly, PPARγ inhibits claudin 4 degradation resulting in
urothelial cell differentiation [94]. In both cases, the increased
protein level is probably due to reduced ubiquitination. Interestingly, transcriptome analysis of
ovarian cancer cells exposed to a PPARγ agonist revealed that PPARγ activation resulted in upregulation of several
genes involved in protein modification and ubiquitination, including many
ubiquitin ligases and ubiquitin-conjugating enzymes [95]. This finding may provide a plausible
explanation for the broad effects that PPAR-γ agonists have on protein ubiquitination and
turnover and clearly deserves further investigation [95].Figure 3
Interactions
between PPARs and the ubiquitin-proteasome system (UPS) affect multiple
cellular pathways. The UPS regulates activity of PPARs by controlling
receptor turnover in ligand dependent and independent manners and affecting the
ability of PPARs to regulate target gene transcription. Signaling pathways can
modulate PPAR activity by affecting UPS-mediated turnover (e.g., increased PPARγ degradation in response to growth factors or
hormones). PPAR can also affect biological pathways and cellular responses by
increasing or decreasing susceptibility of proteins to proteasomal degradation
(e.g., enhanced degradation of β-catenin and suppression of the Wnt pathway by
PPARγ).PPARα agonists also enhance protein degradation. In
LPS-treated macrophages PPARα agonists enhance degradation of inducible
nitric oxide synthase (iNOS), reducing nitric
oxide (NO) production, which is an important mediator in inflammatory
processes. PPARα agonists did not affect iNOS expression and proteasome
inhibitors reversed the effect on iNOS protein levels, indicating that PPARα agonists enhanced degradation of this protein by
the proteasome [96]. PPARδ has been found to regulate ubiquitin C
expression and this has been linked to the modulation of protein kinase Cα (PKCα) and attenuation of cell proliferation in the
skin. The level of PKCα was lower in the skin of PPARδ wild-type mice treated with TPA compared to
the skin of PPARδ-null mice [97]. On the
other hand, the amount of ubiquitinated PKCα was lower in skin of TPA-treated PPARδ-null mice compared to wild-type mice and inhibition
of the proteasome prevented TPA-induced downregulation of PKCα. Thus, the effects of PPARδ on cell proliferation in the skin could be due
to ubiquitin-dependent turnover of PKCα that in turn modulated the activity of the PKCα-dependent pathways [97].Finally, the UPS is
involved in the reciprocal regulation of PPARs and other transcription factors.
Activation of NF-κB is achieved when the inhibitor IκB, which normally holds NF-κB in the cytoplasm, is phosphorylated and
recognized by the E3-β-transducin repeat containing protein (β-TRCP). Ubiquitinated IκB is degraded by the proteasome, allowing NF-κB to translocate to the nucleus and induce gene
transcription [98]. NF-κB has a critical role in inflammation. In
experimental rat models of autoimmune myocarditis stabilization and
translocation of NF-κB were inhibited by PPARγ-dependent expression of IκB [99]. Likewise, PPARα activation induced IκB in aortic smooth muscle cells and in human
hepatocytes [100]. The transcription factor AP-1,
which is another key player in inflammation, interacts with the PPARs and may
be regulated in a similar combinatorial manner by PPARs and the UPS [33, 34].


## 4.1. UPS and Control of PPAR Turnover

Important factors
to consider when studying the multiple and complex functions of PPARs are their
connections with other cellular systems and how these interactions reciprocally
impact on each system activity. Recent reports suggest that the activity of
PPARs is linked in many ways to the UPS [28]. All three PPARs are short-lived
proteins that undergo ubiquitination and proteosomal degradation and the UPS is
mainly responsible for the turnover of these nuclear receptors [28]. However, the three PPAR isotypes
have different behaviors with respect to ligand-dependent receptor turnover.
PPARγ undergoes negative autoregulation upon agonist
binding. PPARγ is ubiquitinated and degraded by the
proteasome in a negative feedback loop that probably serves to attenuate
receptor-mediated gene transactivation [74]. PPARα turnover is controlled by ligands in a
slightly different manner. Instead of enhancing ubiquitination and degradation,
PPARα ligands prevent ubiquitination and lead to
increased stability of the receptor [75].
The protective effect of the ligand,
however, is maximal during the first 3 hours of exposure to the ligand and the receptor is then rapidly degraded [75].We have recently examined the
ligand-dependent turnover of PPARδ and the role of the UPS in this process [76]. Our study revealed interesting
differences between PPARδ and other PPAR isotypes with respect to ligand-dependent
receptor turnover and interaction with the UPS. We found that PPARδ, like other nuclear receptors, is
ubiquitinated and rapidly degraded by the proteasome [76]. Brief incubation of cells expressing
both endogenous and recombinant PPARδ with proteasome inhibitors led to rapid
accumulation of the receptor in cell nuclei. Interestingly, in the presence of
proteasome inhibitors, PPARδ was transcriptionally competent as shown by
luciferase reporter assays and assessment of endogenous target genes by RT-PCR [76]. Thus, PPARδ was different from other nuclear receptors,
including the estrogen, androgen, thyroid hormone, and retinoic acid receptors,
whose transcriptional activity is reduced by proteasome inhibitors [26]. Furthermore, while in the absence
of ligands PPARδ had a very short half life (~30 minutes), the
addition of ligand increased considerably the receptor half life [76]. The effects of the synthetic and
natural ligands were rapid with an increase of PPARδ protein level within 4 hours upon addition to
the cell culture medium. The receptor level remained high as long as the
ligands were present [76]. Removal of the ligands was
followed by rapid reversal with return to the baseline level within few hours.
Once again, PPARδ behavior was unique among nuclear receptors,
whose turnover is generally accelerated by their own ligands [26, 77]. The progesterone receptor, thyroid
receptor, estrogen receptor, RAR, and RXR all show ligand-dependent increase of degradation associated with transcriptional
activation [26, 77]. The direct consequence of these
events is a rapid decrease of the receptor half life and switching-off the
transcriptional response. Only vitamin D3 receptor is known to be stabilized by
the ligand with a similar kinetics [78]. As mentioned above, PPARγ is also rapidly degraded upon exposure to
ligands [74] and PPARα is stabilized only transiently by ligands [75]. Further work demonstrated that
ligand-induced stabilization of PPARδ was due to a selective block of receptor
ubiquitination [76]. This ubiquitination block depended
on the continuous presence of the ligand, was rapidly reversed after removal of
the ligand, and was due to the direct interaction of the ligand with the
receptor [76]. Disruption of the LBD in PPARδ/Tr1-299 abolished the effect of the ligand on
ubiquitination and proteolysis, although the truncated form of the receptor was
still ubiquitinated and degraded by the proteasome [76]. Thus, binding of the ligand to the
LBD induced a conformational change that, in addition to allowing receptor
trans-activation, blocked the interaction of PPARδ with an ubiquitin ligase or, alternatively,
promoted binding of a deubiquitinating enzyme.Using site-directed
mutagenesis, we investigated further the role of distinct PPARδ  domains  in  the  ligand-dependent regulation of
receptor turnover [76]. This analysis revealed additional differences between PPARδ and other PPAR isotypes. Mutations in the DBD of PPARδ reduced the effect of ligands on receptor
ubiquitination [76]. This suggested that the ligand
acted preferentially on the DNA-bound receptor preventing its ubiquitination.
Interestingly, mutations in DBD of PPARγ did not affect ligand-dependent turnover,
indicating that DNA binding was not a prerequisite for ligand-induced
degradation of this receptor [74]. On the other hand, we showed that the AF-2 domain of PPARδ was not required for ligand-induced block of
ubiquitination, indicating that the effect was independent of coactivator
binding [76]. For most nuclear receptors, the
transactivating function is linked to proteolytic degradation and mutations in
the transactivating domain affect also receptor ubiquitination and proteolysis [77]. The AF-2 domain of PPARγ has a similar role and mediates ligand-induced
degradation of the receptor [74]. For PPARγ and other nuclear receptors, conformational
changes induced by the ligands may favor the concomitant interaction with
coactivators and components of the UPS. Overexpression of transcriptional coactivators led also to a decrease of
PPARα level in the presence of ligand, showing that
the interaction with coactivators via the AF-2 domain promoted proteolysis of
the α isotype [79]. Thus, for PPARα the initial stabilization is probably followed
by the recruitment of coactivators along with other factors that trigger
proteolysis of the receptor. In contrast, in the case of PPARδ we showed that transactivation and receptor
ubiquitination are functionally separated [76]. The absence of a link between these two processes allows independent
control of receptor transactivation and ubiquitination upon ligand binding and
may be a prerequisite to avoid rapid degradation and sustain its transcriptional
activity once it is engaged in transcriptional activation complexes. Further
analysis of PPARδ mutants indicates that the region between
amino acid 204 and 235 may play a role in controlling ubiquitination and proteolytic
degradation of the receptor (Figure 1). This region has a poor secondary
structure, forms a loop exposed to the surface, and may be in an environment
prone to ubiquitination [80, 81]. In addition, the region is quite
diverse between the PPAR isotypes, possibly explaining the divergent responses
in terms of ligand-dependent turnover. Pull-down experiments showed that the
truncated PPARδ/Tr1-235 was ubiquitinated, while the shorter PPARδ/Tr1-204 was not (Figure 2(a)). Different scenarios
can explain these results and are under consideration. The region between amino
acid 204 and 235 may contain lysine residues that are the major sites of
ubiquitination of PPARδ. However, mutations of the three lysines
present in this region (K204R, K224R and K229R) did not affect ubiquitination
of the PPARδ/Tr1-235 (Figure 2(b)). Thus, alternatively the
region 204–235 may be needed for the binding of an ubiquitin ligase or
cofactors that mediate the interaction of the receptor with the UPS.Ubiquitination of truncated and
mutated forms of PPARδ. (a) U2OS cells
were transfected with HA-ubiquitin expressing vector along with wild type
His-PPARδ or truncated forms of the receptor (PPARδ/Tr1-204, Tr1-235 and Tr1-299). After 24 hours,
cells were incubated overnight with vehicle or the PPARδ ligand GW501516 (5 μM) and subsequently all samples were incubated with
10 μM the proteasome inhibitor PS341 for 4 hours.
His-tagged wild type and truncated PPARδ were pulled-down with nickel affinity gel
under denaturing conditions. PPARδ was detected in pull-down fractions using an
anti-His antibody and ubiquitinated proteins with an anti-HA antibody. (b) U2OS
cells were transfected with HA-ubiquitin vector along with the indicated PPARδ expressing vectors. PPARδ/Tr1-235 had wild type sequence or the
indicated double or triple mutations (K204R, K224R and K229R). Cells were
treated and analyzed as above.
(a)(b)Thus, even if the
PPAR isotypes are structurally very similar, binding to specific ligands
induces divergent responses as far as receptor turnover. PPARγ upon ligand binding becomes ubiquitinated and
prone to degradation, whereas ligands prevent or delay ubiquitination and
degradation of PPARδ and PPARα. Most nuclear receptors exhibit negative
autoregulation upon interaction with the respective ligands [26, 77]. Ligand-induced stabilization is a
less common and has been observed only for very few nuclear receptors. The system in place for PPARδ may be geared to prevent both accumulation of
high levels of the receptor and its prolonged activation [76]. Overactivity of PPARδ may be detrimental to cells, perhaps due to
its antiapoptotic and potentially tumorigenic activity [32, 37]. The level of PPARδ is low and constantly controlled via
UPS-dependent proteolysis, which may affect greatly the ligand-independent
functions of the receptor like transrepression of other transcription factor
target genes. Under physiological conditions, the low abundance and short half life
of natural PPARδ ligands, like PGI2, would
contribute to keep the receptor in the unbound state [32]. In the presence of high concentrations
of ligands, the DNA-bound and liganded PPARδ is protected from proteasomal degradation by the
inhibition of its ubiquitination [76]. The stabilized DNA-bound receptor
would be able to transactivate target genes as long as enough ligand is
present. This would be consistent with the fact that in processes, such as
wound healing, inflammation, and cancer, PPARδ levels seem to increase concomitantly with
upregulation of cyclooxygenase-2 and other enzymes for the production of lipid
metabolites capable of stabilizing and activating PPARδ [32, 37, 55, 58, 59]. In the absence of
this coordinated increase of ligand and receptor levels, PPARδ might not be able to act as antiapoptotic and
growth-promoting factor. How ligand-induced stabilization of PPARδ affects ligand-dependent interactions with
other transcription factors leading to transrepression or derepression of gene
expression is still unknown.


## 4.2. UPS, PPARs, and Interactions with Other Signaling
Pathways

In addition to
ligand-dependent receptor turnover, the UPS is an important way to control PPAR
activity in response to upstream signal transduction pathways (Figure3). Receptor
phosphorylation by cellular kinases can regulate both basal and ligand-induced
activity of PPARs as well as modulate their protein level by indirectly controlling
proteasome-dependent degradation [82]. In colorectal cancer cells, the
polypeptide hormone gastrin promotes cell proliferation and the effect is
associated with decreased PPARγ level. This was mediated by phosphorylation of
PPARγ involving the epidermal growth factor receptor
and ERK1/2 kinase leading to increased PPARγ proteasome-mediated degradation [83]. In fat cells IFN-γ treatment induces a rapid reduction of PPARγ protein level, which is blocked by proteasome
inhibitors [84]. On the other hand, there are instances in which PPARs enhance
stabilization or degradation of proteins by affecting their susceptibility to
UPS-mediated degradation. Perhaps the best example of a signaling pathway in
which both PPARs and the UPS are implicated is the Wnt pathway. Suppression of
the canonical Wnt signaling is required for differentiation of preadipocytes
into adipocytes. The process is in part
mediated by PPARγ-induced degradation of β-catenin, which is a central element in the Wnt
pathway. Activation of PPARγ promotes degradation of β-catenin in glycogen synthase kinase 3β (GSK3B)-dependent or independent manner [85, 86]. β-catenin mutations that inhibit degradation
block expression of a subset of adipogenic and PPARγ target genes [85]. PPARγ-dependent degradation of β-catenin requires an active APC-containing
destruction-complex. Mutations of the T cell factor/lymphocyte enhancer factor
(TCF/LEF) binding domain of β-catenin or of a catenin-binding domain (CBD) within
PPARγ block proteasomal degradation of β-catenin [87]. The interaction between β-catenin and PPARγ affect their respective oncogenic and tumor
suppressor function [87]. A functional APC was found to be required
also for PPARγ-mediated suppression of colon carcinogenesis [88]. Activation of PPARγ induces degradation of cyclin D1, which has a
critical role in cell cycle regulation, along with β-catenin in hepatocytes [89]. Reduced cyclin D1 protein level
was observed also in breast cancer cells upon PPARγ activation by selective ligands and cyclin D1
downregulation was blocked by inhibition of the proteasome [90]. However, the ability of
thiazolidinedione ligands to reduce β-catenin and cyclin D1 levels might be in part
PPARγ-independent and determined by direct effects of
these compounds on protein degradation [91, 92]. Beside the induction of proteosomal
degradation, activation of PPARγ has been shown to increase the level of proteins
by blocking their proteolysis. Activation of PPARγ in human hepatocarcinoma cells inhibits
proteosomal degradation of p27, a cyclin-dependent kinase inhibitor, with
consequent inhibition of cell proliferation [93]. Similarly, PPARγ inhibits claudin 4 degradation resulting in
urothelial cell differentiation [94]. In both cases, the increased
protein level is probably due to reduced ubiquitination. Interestingly, transcriptome analysis of
ovarian cancer cells exposed to a PPARγ agonist revealed that PPARγ activation resulted in upregulation of several
genes involved in protein modification and ubiquitination, including many
ubiquitin ligases and ubiquitin-conjugating enzymes [95]. This finding may provide a plausible
explanation for the broad effects that PPAR-γ agonists have on protein ubiquitination and
turnover and clearly deserves further investigation [95].Figure 3
Interactions
between PPARs and the ubiquitin-proteasome system (UPS) affect multiple
cellular pathways. The UPS regulates activity of PPARs by controlling
receptor turnover in ligand dependent and independent manners and affecting the
ability of PPARs to regulate target gene transcription. Signaling pathways can
modulate PPAR activity by affecting UPS-mediated turnover (e.g., increased PPARγ degradation in response to growth factors or
hormones). PPAR can also affect biological pathways and cellular responses by
increasing or decreasing susceptibility of proteins to proteasomal degradation
(e.g., enhanced degradation of β-catenin and suppression of the Wnt pathway by
PPARγ).PPARα agonists also enhance protein degradation. In
LPS-treated macrophages PPARα agonists enhance degradation of inducible
nitric oxide synthase (iNOS), reducing nitric
oxide (NO) production, which is an important mediator in inflammatory
processes. PPARα agonists did not affect iNOS expression and proteasome
inhibitors reversed the effect on iNOS protein levels, indicating that PPARα agonists enhanced degradation of this protein by
the proteasome [96]. PPARδ has been found to regulate ubiquitin C
expression and this has been linked to the modulation of protein kinase Cα (PKCα) and attenuation of cell proliferation in the
skin. The level of PKCα was lower in the skin of PPARδ wild-type mice treated with TPA compared to
the skin of PPARδ-null mice [97]. On the
other hand, the amount of ubiquitinated PKCα was lower in skin of TPA-treated PPARδ-null mice compared to wild-type mice and inhibition
of the proteasome prevented TPA-induced downregulation of PKCα. Thus, the effects of PPARδ on cell proliferation in the skin could be due
to ubiquitin-dependent turnover of PKCα that in turn modulated the activity of the PKCα-dependent pathways [97].Finally, the UPS is
involved in the reciprocal regulation of PPARs and other transcription factors.
Activation of NF-κB is achieved when the inhibitor IκB, which normally holds NF-κB in the cytoplasm, is phosphorylated and
recognized by the E3-β-transducin repeat containing protein (β-TRCP). Ubiquitinated IκB is degraded by the proteasome, allowing NF-κB to translocate to the nucleus and induce gene
transcription [98]. NF-κB has a critical role in inflammation. In
experimental rat models of autoimmune myocarditis stabilization and
translocation of NF-κB were inhibited by PPARγ-dependent expression of IκB [99]. Likewise, PPARα activation induced IκB in aortic smooth muscle cells and in human
hepatocytes [100]. The transcription factor AP-1,
which is another key player in inflammation, interacts with the PPARs and may
be regulated in a similar combinatorial manner by PPARs and the UPS [33, 34].


## 5. Conclusions

Here, we have
presented the current evidence linking PPARs and the UPS. Ubiquitination and proteasomal
degradation control the level and modulate the activity of PPARs in many ways. Ligand
binding and proteolytic degradation affect turnover and transcriptional
activity of the PPAR isotypes in distinct ways. PPARδ ubiquitination is selectively blocked by agonist
ligands ensuring the accumulation of DNA-bound receptor engaged in
transcriptional activation complexes. The opposite is true for the other PPAR isotypes. Distinct cellular
pathways can exploit the UPS to modulate PPAR turnover and activity affecting
their multiple functions. Furthermore, PPARs can control the level of specific proteins
by modulating the activity of the UPS. This could be mediated by their ability
to control the expression of components of the UPS, like ubiquitin ligases, or via
protein-protein interactions. Controlling turnover of the receptors, the UPS
can affect also the ligand-independent functions of PPARs. In this context, the control operated by the
UPS on nuclear receptor levels might affect their ability to modulate activity
of other transcriptional regulators. Increased proteolysis might reduce PPAR levels and produce apparently
paradoxical responses with derepression or transrepression of distinct subsets
of genes as seen in certain PPAR knockout experiments. The contribution of the
multiple interactions between PPARs and the UPS need to be taken in
consideration when examining the effects of PPAR overexpression, knock down or
ligand-dependent activation on complex biological processes, like inflammation,
angiogenesis, and tumorigenesis.



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*Source: 195065-2008-06-08.xml*