# A Novel Mechanism of PPARγ Regulation of

Authors: Chang Ho Lee; Hyung Don Kim; Sang Mi Shin; Sang Geon Kim
Journal: PPAR Research
 (2008)

---

## Abstract

Peroxisome proliferator-activated receptor-γ (PPARγ) and retinoic acid X-receptor (RXR) heterodimer, which regulates cell growth and differentiation, represses the TGFβ1 gene that encodes for the protein involved in cancer biology. This review will introduce the novel mechanism associated with the inhibition of the TGFβ1 gene by PPARγ activation, which regulates the dephosphorylation of Zf9 transcription factor. Pharmacological manipulation of TGFβ1 by PPARγ activators can be applied for treating TGFβ1-induced pathophysiologic disorders such as cancer metastasis and fibrosis. In this article, we will discuss the opposing effects of TGFβ  on tumor growth and metastasis, and address the signaling pathways regulated by PPARγ for tumor progression and suppression.


---

## Body

## 1. Introduction

Peroxisome proliferator-activated receptor-γ (PPARγ) as a
ligand-activated transcription factor belongs to the members of nuclear hormone
receptor superfamily. PPARγ is implicated in a wide
variety of cellular functions, regulating the expression of gene networks required
for cell proliferation, differentiation, morphogenesis, and metabolic homeostasis. The
transforming growth factor isoforms (TGFβ1, β2, and β3)
as the members of the TGFβ superfamily are ubiquitously expressed
cytokines [1, 2]. TGFβ
exerts multiple functions with differential expression pattern in organs: each form
of TGFβ has similar biological activities [3]. Among the TGFβ
forms, it is recognized that TGFβ1 plays a major role in the regulation of cell proliferation and differentiation.
In this review paper, we will discuss the role of PPARγ on TGFβ gene expression.Accumulating evidences suggest that the interplay of PPARγ and TGFβ
contributes to the regulation of cell proliferation, differentiation, and their
associated cellular functions. For instance, the interaction of PPARγ
signaling with the proteins affected by the activation of TGFβ
receptor determines the outcome of the breast tumor progression [4]. Many  studies have shown that agonist-induced activation
of PPARγ interferes with TGFβ/Smad-dependent or Smad-independent signaling in different cell
types [5–12]. The crosstalk between PPARγ
and TGFβ can be achieved not only by PPARγ-dependent modulation of the propagation of TGFβ/TGFβ
receptor-mediated signaling pathways, but also by the regulation of TGFβ1
expression itself and TGFβ1-inducible target genes. Hence, suppression of TGFβ signaling by PPARγ could be counteracted by the inhibitory action of TGFβ on the PPARγ-mediated signaling [13–15].The TGFβ1 expression is regulated at multiple levels. Diverse transcription factors are
involved in the transcriptional regulation of TGFβ gene expression and post-translational modification makes precursors bound with
TGFβ1 binding proteins mature to TGFβ molecule [16, 17]. The role of PPARγ activation in TGFβ1
gene repression has been examined by the experiments using thiazolidinedione
PPARγ agonists [18, 19]. These studies on the
regulation of the TGFβ1 gene and the molecular interaction of ligand-activated nuclear receptors for the
activation of responsible transcription factor(s) brought insights into the
transcriptional control mechanism. The research results showed that PPARγ
activation might transrepress the TGFβ gene, interfering with TGFβ
signaling and thereby altering the expression of TGFβ-inducible
target genes [18], substantiating the fact that ligand activation of PPARγ
modulates TGFβ receptor-mediated gene regulation.


## 2. TGFβ and Cancer Cell Biology

TGFβ1 exerts its diverse biological
effects by acting on distinct combinations of type I and type II receptors and
recruiting downstream signal transducers including Smads, consequently
regulating a group of target gene expression responsible for a specific
biological activity. Smad proteins are
classified into R-Smads (receptor-regulated Smads: Smads 1, 2, 3, 5, and 8), Co-Smads
(common mediator Smad: Smad 4), and I-Smads (inhibitory Smads: Smad 6 and 7), and
these play roles as the transcriptional regulators for the superfamily of TGFβ1-inducible
target genes [1, 2, 20–22]. Smad 2 and Smad 3 are the specific mediators of TGFβ1, whereas Smad 1, Smad 5, and MADH6/Smad 9 are crucial
for bone morphogenic protein signaling [22]. In particular, Smad 3 is involved in
the TGFβ1 gene regulation, which
is crucial for the autocrine function of TGFβ1 [23].Following the activation of the TGFβ1 receptor by TGFβ1, TGFβ1-induced receptor kinase activation rapidly
phosphorylates Smads proteins and initiates formation of functional oligomeric
complexes. The resultant oligomeric complex translocates to the nucleus to
regulate target gene expression. Briefly, the type I TGFβ1 receptor kinase phosphorylates serine residues at the
C-terminal SSXS motif in the MH2 domain of Smad 3 (or Smad 2) [24]. Phosphorylated
Smad 3 (or Smad 2) forms an
oligomeric complex with Smad 4, which is crucial for the maximal transcription
of diverse TGFβ1-inducible target genes [25, 26]. The oligomeric complexes
of Smad 3 (or Smad 2) and Smad 4 recognize DNA binding element tetranucleotide
(CAGA) or GC-rich sequences, and several copies of which are present in the
promoter regions of many TGFβ1-responsive genes such
as plasminogen activator inhibitor-1 (PAI-1), α2(I) procollagen, and type VII collagen [25, 27]. It is
well known that the protein products encoded from these genes promote the accumulation
of extracellular matrix and that abnormal accumulation of the proteins may lead
to fibrosis, which represents a form of the epithelial to mesenchymal transition (EMT).Moreover, TGFβ1-activated kinase-1, a member of MAPK kinase kinase
family, activates its MAP kinase pathways [28, 29]. It is accepted that TGFβ1-activated ERK pathway synergistically enhances Smad
signaling of the TGFβ1 receptor due to the positive cross talk between the ERK
and Smad pathways [22, 30]. Serine phosphorylation of Smad 3/2, but not
phosphorylation of the C-terminal motif, was decreased by MEK-ERK inhibitors [31].
Smad 3/2 are differentially activated by TGFβ1 in hepatic stellate cells as a result of the differential
phosphorylations of the Smads. Smad 3 plays a key role in TGFβ signaling, which is strengthened by the observation that
the loss of Smad 3 interfered with TGFβ1-mediated
induction of target genes [32, 33]. In addition, activation
of CCAAT/enhancer binding protein (C/EBP) β is also involved in
the inhibition of TGFβ1 expression [34].During the process of carcinogenesis, TGFβ
action can be either tumor suppressive or tumor promoting, depending on the stage
of tumor development [35–37]. In an experimental
cell model, TGFβ could induce cell growth arrest and promote apoptosis of
carcinoma cells [1]. The antiproliferative action of TGFβ in epithelial cells, for example, is essentially
attributed to the cell cycle arrest and the apoptosis concomitantly induced. It
is well known that cell cycle arrest induced by TGFβ occurs at G1 phase through enhancing transcription of cyclin-dependent
kinase inhibitors, p21Cip1/WAF and p15Ink4b, while suppressing
the induction of c-Myc, a progrowth transcription factor, and of Id1–3,
the inhibitors of differentiation [38–43]. In a model of gastric adenocarinoma,
TGFβ-mediated apoptosis contributed to tumor suppression,
which resulted from TGFβ-induced caspase-8 activation [44]. Moreover, it has been
shown that TGFβ reduced the expression of antiapoptotic Bcl-2 family
members in prostate cancer cells [45].By contrast, TGFβ may also lead to tumor cell proliferation as a
consequence of EMT process [46–48], which is a cellular phenomenon
characterized by a loss of polarized epithelial phenotype with transition to a
mesenchymal or more migratory phenotype. Studies have shown that diverse
signaling pathways are involved in the TGFβ-dependent EMT process. Initiation of EMT by TGFβ receptor activation is mediated by either Smad-dependent
or Smad-independent pathway  [1, 49, 50]. Downstream of the TGFβ receptor
activation, the Smads activated by the TGFβ receptor kinase promote transcription
of the genes, which eventually play crucial roles in the process of EMT. The
responsible transcription factors primarily include Snail, Slug, and LEF-1 [1].
In addition, TGFβ also activates the non-Smad pathways, which include Ras,
phosphatidylinositol 3-kinase (PI3K), and Par 6. These molecules regulate the
expression of Snail and the activities of glycogen synthase kinase 3β (GSK3β) and RhoA, respectively [51, 52], thereby enhancing the process
of EMT. It is now accepted that the EMT phenomenon of primary cancer cells promoted
by the action of TGFβ may increase cancer metastasis.TGFβ acts on tumor cells directly, playing a role in cancer cell
migration and invasion. Diverse TGFβ-mediated signaling pathways are responsible for this
process. In glioblastoma cells, siRNA knockdowns of TGFβ1 and TGFβ2 resulted in the inhibition of cell motility or invasiveness
[53]. As a same token, TGFβ released from tumor tissues
might facilitate glioma cell migration and invasion via an autocrine signaling [54]. Several lines of evidence also support the
concept that TGFβ-induced
Smad signaling is responsible for the invasiveness of cancer cells [55–58]. This is explained in part by the TGFβ-dependent induction of matrix metalloproteases, which are known to be responsible for cell migration and
invasion [55, 59–62]. Activation of ERK
and JNK by TGFβ and their association with focal complexes may
also contribute to cell migration, as shown in the case of breast carcinoma [63]. Moreover, it has been shown that the activation of p38 MAPK pathway by
TGFβ facilitated invasion of head and neck squamous epithelial cells [61].In addition to the double-edged effects of TGFβ on cancer cells, TGFβ may
alter cancer growth by suppressing the growth of multiple immune cells, which compromises
the overall immune functions. Studies have shown that the proliferation and activity
of T cells are suppressed by the TGFβ blockade of IL-2 production and expression of T cell effector molecules [64–68].
Also, TGFβ attenuates the activity of natural killer (NK) cells by inhibiting NK
production of interferon-γ (IFN-γ) [69, 70].
Another study showed that TGFβ inhibited
the antigen presentation function of dendritic cells through suppressing the expression
of MHC class II and costimulatory molecules [71]. All of these results support
the alterations by TGFβ in immune
functions, which would impair immune surveillance or attack against cancer
cells.In summary, action of TGFβ1 on cancer cells switches from tumor suppression to tumor promotion, depending on the stage of tumor
progression. For instance, during the early phase of breast tumorigenesis, the
TGFβ signal inhibits primary tumor growth
via cell growth arresting and promoting apoptosis.
However, at later stage, cancer cells acquire a capacity to escape from the
tumor suppressive effects of TGFβ1 via induction of EMT. Interestingly, the aforementioned conflicting functions
of TGFβ might go through the same TGFβ receptor
complex and the associated signaling pathways involving Smad transcription
factors [1]. Probably, there should be certain stage-dependent
modifications in cellular signaling system including changes in receptor
function and downstream Smad signaling cascades.
Taken together, it is concluded that TGFβ may not
only induce growth arrest of cancer cells, but also increase cancer dissemination
[1], supporting the concept that the cytokine serves a dual function in tumor development
and  progression (Figure 1).Figure 1
A scheme showing the opposing effects of TGFβ on tumor growth and metastasis.


## 3. PPARγ and Cancer Biology

PPARγ has been extensively studied as an anticancer target in preclinical and
clinical settings [72]. The anticancer effects appeared to be cancer cell-specific.
A knock-out or loss of function mutation in PPARγ can
be an important risk factor for the incidence of cancer [73–75]. In this sense,
PPARγ has been considered as a novel target for designing new anticancer drugs for
chemotherapy. This is further supported by the finding that PPARγ activators
exert a potent tumor-suppressing activity against various human cancer cells [76–78]. As a matter of fact, PPARγ
activators such as troglitazone and ciglitazone exert antiproliferative
activities in epithelial cancer cell lines or animal models, which presumably
results from the activation of PPARγ receptor and the PPARγ
receptor-dependent pathways [76, 79–83]. Nevertheless, other anticancer pathways
have also been recognized in association with PPARγ, which might be PPARγ
receptor-independent [84, 85]. Multiple PPARγ-independent
anticancer targets of PPARγ
agonists have been suggested in several cancer cell types. The mechanisms may comprise
a variety of pathways such as the blockade of G1-S phase transition
by inhibiting translation initiation [86], activation of
JNK-dependent cell death pathway [87], induction of the early growth
response-1 (Egr-1) gene [88], inhibition of Bcl-xL and Bcl-2
function [85], counteracting TGFβ
release by tumor cells [54], and induction
of cyclin-dependent kinase inhibitor
p21WAF1/CIP1 [89]. However, the precise antiproliferative
mechanisms of the PPARγ agonists remain to be further studied. On the contrary, there are also other reports
available on the opposite effects showing that PPARγ signaling promoted
carcinogenesis [90, 91].It should be noted that the antitumor effects of PPARγ may be explained at least in two different ways. One mechanism involves cell growth
regulation [4], which should be further clarified, whereas the other mechanism includes
cancer chemopreventive effects mediated by the induction of antioxidant enzymes
[92]. It is well recognized that PPARγ
affects cell survival, growth, and differentiation by acting on the peroxisomal
proliferator-response element (PPRE), thereby modulating an expression of a
group of genes controlling cell growth and differentiation pathways [93, 94]. The
PPARγ homodimer and PPARγ-retinoic acid X receptor (RXR) α heterodimer have the specificities of DNA-binding
with preferential binding of the latter to DR1, which is a PPRE DNA binding site. SRC-1 is a coactivator of
PPARγ [95].
Binding of the ligand-activated PPARγ-RXRα heterodimer to its DNA binding
sites stimulates the interaction between PPARγ-RXRα and p160/SRC-1 [95].A number of studies support the concept that cancer chemoprevention is accomplished by
the induction of antioxidant enzymes. The results from our laboratories indicated
that oltipraz and flavonoids as potential cancer chemopreventive agents
activate C/EBPβ in the antioxidant genes such as glutathione S-transferase (GST) A2 [96, 97]. In addition, treatments of cells with PPARγ activators
induced the nuclear translocation of NF-E2-related factor 2 (Nrf2) and C/EBPβ, and activating Nrf2 and C/EBPβ bindings to the antioxidant
response element (ARE) and C/EBP response elements, respectively [92].
Moreover, the Nrf2 and C/EBPβ genes contain PPRE sites,
which account for the induction of the target antioxidant proteins by PPARγ activators. Both
the ARE and the C/EBP binding site have crucial roles in transactivating the GSTA2 gene by PPARγ and RXR ligands [92].
Therefore, Nrf2 and/or C/EBPβ inductions(s) via the PPARγ and RXRα heterodimer binding to the
PPREs in the promoter regions of the target genes contribute(s) to the antioxidant
capacity of cells (e.g., GSTA2).A result of our
previous study indicated that specific mutations of these nuclear binding sites in the GSTA2 promoter, which are present as a
three-PPRE cluster, caused the complete loss of its responsiveness to PPARγ activators [92]. All of the putative PPRE sites
comprising DR1 were functionally active. Therefore, the binding of the
activating PPARγ-RXR
heterodimer to all of the PPRE sites appeared to be crucial for the inducible
gene activation, showing that the PPAR binding site cluster is the functionally
active PPRE-responsive enhancer module (PPREM) [92].
This study on the regulation of gene expression by the PPARγ-RXR
heterodimer at the promoter containing multiple DR1 elements brought additional
insight into the transcriptional control mechanism of the antioxidant enzymes.
The identified molecular mechanism would shed light on the contribution of cell
viability and cancer chemoprevention as a consequence of the induction of antioxidant
targets genes by PPARγ activators.


## 4. TGFβ Regulation by PPARγ-RXR and Cell Signaling

Activation of the PPARγ-RXR heterodimer represses the TGFβ1 gene through
dephosphorylation of a transcription factor called zinc finger transcription
factor-9 (Zf9), which has been shown to be induced by phosphatase and tensin homolog deleted on chromosome (PTEN)-mediated p70 ribosomal S6 kinase-1 (S6K1) inhibition [18]. Because RXRs
are modular proteins with a highly conserved central DNA binding domain and a
less conserved ligand binding domain [98], activation of the PPARγ and RXR heterodimer
contributes to the gene regulation. The role of PPARγ in repression of the TGFβ1 gene
was further evidenced by the effects of thiazolidinediones, and also by the
reversal of TGFβ1 repression by the dominant negative mutants, supporting to the novel aspect
that PPARγ activation contributes to TGFβ1 gene repression
and that RXRα is necessary for the full responsiveness in the gene repression. In
fact, the inhibition of TGFβ1 gene by the PPARγ
and RXR heterodimer might account for either tumor suppression or tumor
promotion [18]. Also, as an effort to identify the molecular
basis of TGFβ1 repression by PPARγ activators, the effects of PPARγ and RXR activation on
the TGFβ1 gene transactivation, that is regulated by the proximal DNA response elements, have
been examined [18]. The potential regulatory sites responsible for the TGFβ1 gene expression have been explored by
using the luciferase reporter gene assays, which identified the putative PPREs
located at the multiple sites upstream from −453 bp of the
promoter region [18]. Promoter deletion analyses indicate that neither the
putative PPREs nor the activator protein-1 (AP-1) binding sites are directly
regulated by PPARγ activators forthe gene repression.S6K1, a ubiquitous serine/threonine kinase, controls the translational efficiency by phosphorylating
ribosomal S6 protein [99].
S6K1 functions as a multifunctional kinase for
the phosphorylation of ribosomal S6 protein [99], CREM [100], BAD [101], and the eukaryotic
elongation factor 2 kinase [102]. Rapamycin, a well-known mammalian target of
rapamycin (mTOR) inhibitor, inhibited liver fibrosis and TGFβ1 expression in rats bile duct-ligated or
challenged with toxicants [103, 104], with a concomitant decrease in S6K1
activity. It is well recognized that rapamycin
inhibits S6K1 activity via mTOR inhibition [105]. Yet, other pharmacological agents that modulate S6K1
activity have not been reported. The mechanism of PPARγ-RXR
heterodimer-mediated repression of the TGFβ1 gene has been
elucidated in terms of the modulation of S6K1 activity (Figure 2).Figure 2
A schematic presentation of the multiple pathways regulated by PPARγ for
tumor suppression, progression, inhibition of metastasis, and cancer
chemoprevention.The PI3K-mTOR pathway regulates S6K1 for the regulation of transcription factors
involved in the TGFβ1 gene transactivation. A study identified the
inhibition of S6K1 activity by the PPARγ-RXR, which contributes to TGFβ1 gene
repression [18].
Another signaling molecule, PTEN, antagonizes the PI3-kinase-mTOR-S6K1-mediated signaling
cascade [106, 107]. Thus, it has been elucidated that PPARγ activators
upregulate PTEN, which leads to the S6K1 inhibition, consequently causing TGFβ1
repression [18].


## 5. Transcription Factors Responsible for TGFβ Repression by PPARγ-RXR

In the promoter region of the TGFβ1 gene
(Figure 3), the putative binding sites for PPARγ-RXR seemed to be neither active nor
responsible for the gene repression by the activated PPARγ and RXR heterodimer.
It has been claimed that the effects of PPARγ or retinoid ligands on TGFβ1 gene
expression might be mediated in part by AP-1 inhibition [108, 109]. Nevertheless, such a
result that deletion of the DNA region containing both AP-1 sites still had the
capability to repress the gene by PPARγ activator suggests that the AP-1
binding sites might not be a major regulatory target in the TGFβ1 gene
repression. Rather, the target molecule altered by PPARγ-RXRα-activated cell signal may be
involved in the interaction with the protein recruited on the AP-1 DNA complex.
It appeared that the TGFβ1 gene
repression may have not resulted from the direct inhibition of AP-1, but other
mechanistic basis [18].Figure 3
The human TGFβ1 promoter region.Another study showed that the mechanism associated with the inhibition of TGFβ1
by PPARγ activators involves the regulation of c-Fos [108]. In the study, thiazolidinediones inhibit
high-glucose-induced TGFβ1 promoter activity. A suggested mechanism was raised based on the observation that treatments of thiazolidinediones
reduced high-glucose-induced, activated PKC and c-Fos-mediated TGFβ1 gene expression in mesangial
cells [108].Zf9 as an immediate early gene reduces cell
proliferation with the induction of p21cip1 and the enhancement of
c-Jun degradation [110, 111], thus functioning as a potential tumor suppressor
gene. The transcription factors that interact
with the known DNA binding sites on the region downstream within the −323 bp of the TGFβ1 gene include Zf9, NF1, and SP1. It is noteworthy that Zf9 activation
induces TGFβ1 during the activation of hepatic
stellate cells [112]. Also, Zf9 regulates TGFβ receptors and collagen α1(I), promoting accumulation of extracellular
matrix [113]. Studies have shown that Zf9
phosphorylation enhances its nuclear localization and transcriptional activity [111].
Zf9 as a transcription factor plays a crucial role for the induction of TGFβ1 [113]. Thus, phosphorylation status of Zf9 may contribute
to the promotion of its target gene expression
[114]. Identification
of the partners of Zf9 or phosphorylated Zf9 for the TGFβ1 gene
regulation and their molecular interactions would be interesting to pursue. The constitutive Zf9 phosphorylation by S6K1 strengthened the important role of S6K1
as a multifunctional kinase for the transcription
factor regulation of target genes [100–102].The TGFβ1 gene contains the DNA
response element interacting with Zf9 [16] that regulates multiple genes
involved in tissue differentiation. Activation of Zf9 includes its
phosphorylation at serine (or tyrosine) residues [114]. Thus, phosphorylation
of Zf9 leads to transcription of its target genes [111, 114]. Although the
kinase catalyzing Zf9 phosphorylation has not been completely identified, the
inhibition of Zf9 phosphorylation by rapamycin that inhibits S6K1 activity via
mTOR inhibition supports the role of S6K1 in Zf9 phosphorylation [18]. More
importantly, the role of S6K1 in regulating TGFβ1 gene and the associated molecular mechanistic basis have
been clarified in terms of Zf9 dephosphorylation [18]. In view of the previous
observations that Zf9 is crucial as a transcription factor for TGFβ1 induction in hepatic stellate cells [113] and
that a phosphorylated form of Zf9 plays a role in the transactivation of the
target gene promoter [114], the potential ability of PPARγ activators to inhibit serine phosphorylation
of the transcription factor has also been investigated. Thus, it has been demonstrated
that the inhibition of the TGFβ1 gene by the activation of PPARγ-RXR includes Zf9
dephosphorylation [18]. Therefore, TGFβ1 gene repression by PPARγ activators appears to be related with
dephosphorylation of Zf9, supporting the conclusion
that the PPARγ-RXR heterodimer causes TGFβ1 repression via S6K1 inhibition,
and that the inhibition of S6K1 activity provides a central mechanism, by which PPARγ-RXR regulates
Zf9-dependent TGFβ1 gene expression (Figure 2).Moreover, it has been shown that PPARγ activation induces PTEN, which serves as a
PI(3,4,5)P3 lipid phosphatase and antagonizes
PI3-kinase-mediated cell signaling [106]. Functional PPREs located in the PTEN
promoter have been recognized [115]. The induction of PTEN by PPARγ activators may result
in TGFβ1 gene repression following S6K1 inhibition. Furthermore, PPARγ
activators inhibited phosphorylations of Akt, ERK1/2, p90 ribosomal S6 kinase-1
(RSK1), and mTOR, downstream of PTEN, indicating that PTEN induction by PPARγ
activators leads to S6K1 inhibition via the pathways of ERK1/2-RSK1 as well as
Akt-mTOR. In conclusion, the result showing that PPARγ activation upregulates PTEN,
which has also been implicated in tumor-inhibitory or anti-inflammatory actions
of PPARγ [106, 115], gives credence to
the concept that PPARγ activators induce PTEN during
S6K1 inhibition, and consequently causes TGFβ1 repression. Therefore, the inhibition of tumor proliferation by PPARγ
activators may be explained in part by PPARγ-dependent TGFβ1 repression
(Figure 2), supporting the concept that the PPARγ activators may be applied for
controlling TGFβ1-induced cancer metastasis and fibrosis.



---

*Source: 762398-2008-07-03.xml*