New insights into the role of PPARs

Prostaglandins, Leukotrienes and Essential Fatty Acids 85 (2011) 235–243

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Review

New insights into the role of PPARs Alexandra Montagner, Gianpaolo Rando, Gwendoline Degueurce, Nicolas Leuenberger, Liliane Michalik n, Walter Wahli n Center for Integrative Genomics, National Research Center ‘‘Frontiers in Genetics’’, University of Lausanne, Genopode Building, CH-1015 Lausanne, Switzerland

abstract Peroxisome proliferator-activated receptors (PPARs) are fatty acid-activated transcription factors belonging to the nuclear hormone receptor family. While PPARs are best known as regulators of energy homeostasis, evidence also has accumulated recently for their involvement in basic cellular functions. We review novel insights into PPAR functions in skin wound healing and liver, with emphasis on PPARb/d and PPARa, respectively. Activation of PPARb/d expression in response to injury promotes keratinocyte survival, directional sensing, and migration over the wound bed. In addition, interleukin (IL)-1 produced by the keratinocytes activates PPARb/d expression in the underlying fibroblasts, which hinders the mitotic activity of keratinocytes via inhibition of IL-1 signaling. Initially, roles were identified for PPARa in fatty acid catabolism. However, PPARa is also involved in downregulating many genes in female mammals. We have elucidated the mechanism of this repression, which requires sumoylation of PPARa. Physiologically, this control confers protection against estrogen-induced intrahepatic cholestasis. & 2011 Elsevier Ltd. All rights reserved.

1. Introduction Metabolic regulation in complex organisms relies on (i) transcriptional regulation of genes encoding proteins involved in metabolic pathways, (ii) allosteric control of the activity of key enzymes, and (iii) various posttranslational modifications of key proteins, such as proteolytic cleavage, phosphorylation, glycosylation, sumoylation, and acetylation. Regulation at these different levels often operates in a strictly coordinated manner [1]. Among these regulatory pathways, transcriptional control depends on the transduction of signals to the cell nucleus for modulation of the activity of defined sets of genes. Many transcription factor families participate in metabolic regulation and contribute to the complex fine-tuning of gene activity required for the organism to adapt to changing conditions, such as availability of food, level of exercise, and circadian rhythms. The nuclear receptor family is among the most prominent transcription factor families involved in metabolism, and some of its members are known as ‘‘metabolic sensors’’. This group of regulators comprises the peroxisome proliferator-activated receptors (PPARs), liver X receptors, farnesol X receptor, hepatocyte nuclear factor 4, pregnane X receptor, and retinoid X receptors (RXRs). In addition, other factors outside of this family, such as

n

Corresponding authors. Tel.: þ 41 21 692 41 10 (lab); fax: þ 41 21 692 41 15. E-mail addresses: [email protected] (L. Michalik), [email protected] (W. Wahli). 0952-3278/$ - see front matter & 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2011.04.016

the sterol response element binding proteins, liver-enriched CAAT enhancer binding proteins, and transcriptional coactivators like PGC1a, are main actors in metabolic transcriptional control [1]. Here, we concentrate on PPARs that are lipid-activated transcription factors. They form a small subfamily consisting of three members, PPARa (NR1C1), PPARb/d (NR1C2; here referred to as PPARb), and PPARg (NR1C3) [2–5]. These proteins regulate gene expression as heterodimers with RXRs, binding to response elements in the regulatory regions of target genes. PPARs have been associated with many cellular and systemic functions ranging far beyond the processes that gave them their names; i.e., well beyond the activation of peroxisome proliferation in rodent liver (Fig. 1). PPARs present a broad but isotype-characteristic tissue distribution, which at least in part accounts for the variety of functions they regulate. PPARa is expressed in tissues with high fatty acid catabolism, such as brown adipose tissue, liver, heart, kidney, and intestine. PPARb is expressed ubiquitously; not only does it have metabolic functions in skeletal muscle and adipose tissue, but also it is involved in general fundamental cellular processes. PPARg is especially abundant in the white and brown adipose tissues, where it promotes lipid storage and adipocyte differentiation and maintenance [6–8]. As sensors, PPARs adapt gene expression to various lipid signals and other cues (Fig. 1). The great diversity of functions in which they are implicated parallels the large panel of ligands that can be accommodated in the PPAR ligand-binding pocket. The prevalent point of view is that PPARs act as sensors of fatty acids and fatty acid derivatives, translating modifications in the

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Nutrients Fatty acids Hormones Endocrine disruptors

Growth factors

CIRCADIAN REGULATION

Regulated gene activity by PPARα, β/δ, γ

LIVER BRAIN SKIN CELL SURVIVAL MUSCLE PROLIFERATION PANCREAS Cellular Systemic DIFFERENTIATION KIDNEY ADIPOSE TISSUE Development/growth Homeostasis (adult) Health Disease (cancer, inflammation) Aging/longevity

Metabolic Regulation

Fig. 1. PPARs regulate cellular and systemic metabolic pathways. Energy homeostasis depends on a complex web of interconnected pathways forming a coherent ensemble of integrated processes. PPARs are key regulators that are activated by fatty acids and fatty acid derivates and, alternatively, by endocrine disruptors (peroxisome proliferators). PPAR activity is also modulated by growth factors activating kinase signaling cascades.

PPAR ligandproducing enzymes

8/12/15-LO

PHYSIOLOGICAL CHANGES Feeding Starvation Exercise

Circulating and cellular fatty acids

Changes in free substrate levels

PATHOPHYSIOLOGICAL CONDITIONS Chronic inflammation Diabetes Atherosclerosis Cancer Injuries

Fatty acid derivatives agonists of PPARs 8/12/15-HETEs 9/13-HODEs Oxidized HETEs, HODEs, LDL

5-LO

LTB4

COX-1 COX-2

PGA1/2 15dPGJ2 PGI2

CYP450 epoxygenases

HETEs HEETs

Lipoprotein/ endothelial lipases

HODEs ?

FAS CEPT1

Non-enzymatic non-specific pathways

16:0/18:1 GPC

8-HEPE Components of oxLDL

Fig. 2. PPAR ligand production by endogenous enzymatic pathways. A list of enzymes that generate PPAR ligands is shown. Also, ligands can be generated by non-specific/ non-enzymatic pathways. The levels of substrates available for these enzymes and the expression levels of the enzymes themselves depend on the physiological and pathophysiological conditions of the organism.

intracellular levels of these natural compounds into changes in metabolic activities, and other processes. Several possible pathways may produce PPAR activators (Fig. 2). It is worth noting that some of the endogenous lipid mediators also signal through G-protein-linked receptors at the cell surface and therefore contribute to lipid signaling in a PPAR-independent manner [6]. The activity of the newly produced ligands depends on their PPAR binding specificity and affinity and additional cell-specific characteristics. The levels at which the regulatory action of the PPAR:RXR heterodimer can be exerted are many, including abundance of nuclear receptors and their corepressors and coactivators, activity of the pathways that produce PPAR and

RXR ligands, and physiological and pathophysiological conditions affecting lipid signals. The challenge that remains is to evaluate the contribution of each of these parameters to the biology of PPARs. Better-detailed knowledge of the spectrum of regulatory possibilities of PPAR activity will provide valuable keys for understanding their involvement in functions as diverse as energy homeostasis (lipid and carbohydrate metabolism), immune/ inflammatory responses, tissue repair, vascular biology, and cell differentiation and proliferation. The aim of this review is to illustrate novel insights into the roles of PPARs on two levels: first, by describing their actions at the level of the biology of the cell (proliferation, differentiation,

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and migration), and second, by addressing their systemic metabolic effects (lipid signaling, steroidogenesis, and circadian regulation; Fig. 1). In the first section of the paper, we review our work focused on the roles of PPARb in the skin, especially during wound healing. Then, we discuss regulatory functions associated with metabolic pathways based on results from studies of PPARa in the liver in the context of the circadian rhythm and ligandproducing enzyme activity. Finally, PPARa is described as a factor participating in hepatic sexual dimorphism.

2. PPAR in skin biology In the late stages of fetal development, the epidermis matures into a fully stratified and differentiated epithelium that is renewed continuously after birth. Undifferentiated keratinocytes from the basal layer of the epidermis undergo vectorial differentiation as they move from the basal to the uppermost epidermal layer, the stratum corneum. Homeostasis of the mature skin depends on tightly controlled coordination of keratinocyte proliferation, differentiation, and programmed death and is regulated by complex epithelial–mesenchymal interactions [9]. All three PPARs contribute to epidermis homeostasis in both rodents and humans. PPARb, which is the most abundantly expressed isotype, and PPARa both participate in keratinocyte differentiation and contribute to skin repair after an injury. The contribution of PPARg is less well known although it appears to be important for epidermal functions [10]. However, PPARg and PPARb are important for sebocyte differentiation and function [11,12]. Our work using the skin model has dealt mainly with PPARb. We therefore concentrate on the function of this receptor in the adult epidermis, more particularly during skin repair. 2.1. PPARb in skin wound healing Wound healing is a life-saving process during which the formation of a protective new epithelium that covers the wounded area is crucial. The occurrence of an injury triggers an immediate inflammatory response viewed as the initial stage of the repair process. Then follows re-epithelialization during which keratinocytes proliferate and migrate to cover the wound bed. The dermis also participates in the repair process via fibroblast proliferation, which plays a major role in wound closure, and the production of novel blood vessels that irrigate the repaired skin. While PPARb expression is easily detectable in fetal skin, its levels are extremely low in the interfollicular adult epidermis. Interestingly, PPARb expression is strongly reactivated in keratinocytes at the edges of wounds, and it accompanies the entire repair process [13]. The expression of PPARb is increased via the activation of the stress-associated protein kinase pathway in response to inflammatory cytokines, such as TNF-a, released after an injury [14] (Fig. 3). In addition to the stimulation of PPARb expression, the inflammatory response also induces the production of endogenous ligand(s), which is required for PPARb activation in the keratinocytes [14]. After the inflammatory phase, during which its expression is maximal, PPARb is progressively reduced in the epithelium under repair to finally reach levels observed in the unwounded skin. The mechanism of this downregulation is controlled by TGFb-1, which inhibits AP-1 binding to the PPARb promoter [15]. In genetically impaired PPARb-null mice, we observed that completion of skin repair is delayed by 2–3 days [13], demonstrating that PPARb is an important transcriptional regulator in the wounded epidermis. It promotes keratinocyte survival at the wound edges via activation of the PI3K/PKBa/Akt1 pathway [16] and regulates keratinocyte adhesion and migration, two key processes during re-epithelialization (Fig. 3).

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To better understand the link between PPARb and TGF-b1, we manipulated TGF-b1 levels using two in vivo models, the topical application of recombinant TGF-b1 (TGF-b1 gain of function) and genetic ablation of smad3 (TGF-b1 pathway loss of function) [17]. Manipulation of TGF-b1 activity changed PPARb expression in the epidermis of wounds, and we demonstrated that prolonged expression and activity of PPARb accelerated wound closure. These observations suggest that changes in the PPARb expression profile strongly affect healing efficiency. Additionally, such insights into how incoming extracellular signals regulate PPARb, which in turn coordinates their action on wound healing, may aid in the development of better treatments for chronic wound disorders. Keratinocyte proliferation also makes a major contribution to repair. In collaboration with NS Tan [18], we found that keratinocyte proliferation following wounding is fine tuned by a PPARb-dependent interaction between epidermal keratinocytes and dermal fibroblasts. We showed that Interleukin-1 (IL-1) produced by keratinocytes stimulates the activity of the AP-1 (Jun/Fos) transcription complex in dermal fibroblasts, resulting in an increased production of mitogenic cytokines that enhance keratinocyte proliferation (Fig. 3). In parallel, increased levels of PPARb in fibroblasts stimulate the production of the secreted IL-1 receptor antagonist (sIL-1ra), which results in an autocrine downregulation of the IL-1 signaling pathway. As a consequence, there is reduced production of secreted mitogenic factors by the IL-1-stimulated fibroblasts and thus reduced keratinocyte proliferation. Together, these findings show that the regulation of PPARb by IL-1 in fibroblasts contributes to the homeostatic control of keratinocyte proliferation. Interestingly, the ubiquitous expression of PPARb suggests that such epithelial–mesenchymal interactions might be regulated in a similar manner in other organs. 2.2. Comparison of gene expression profiles between PPARb wild-type and null keratinocytes As seen above, PPARb controls both keratinocyte-autonomous and non-autonomous functions. Keratinocyte differentiation, survival, and migration are governed by keratinocyte PPARb, while keratinocyte proliferation is controlled, at least in part, through PPARb in fibroblasts. Given the importance of these processes during wound healing, we analyzed the impact of genetic deletion of pparb in keratinocytes by comparing the expression profiles of wild-type and pparb-null primary keratinocytes obtained from newborn pups. Our functional analysis of a microarray experiment revealed that PPARb stimulates and represses approximately the same number of genes in keratinocytes. The genetic disruption of pparb caused changes in several important cellular functions, which are consistent with the above-described PPARb-null mouse phenotype and mechanisms we have previously observed (Fig. 4). Among these important functions, the three most significant are ‘‘cell cycle’’, ‘‘cell death’’, and ‘‘cellular growth and proliferation’’. Furthermore, the significantly represented functions of ‘‘cellular movement’’ and ‘‘cellular development’’ are in agreement with the in vivo results showing a role for PPARb in keratinocyte adhesion and migration. Among the top 10 genes exhibiting modified expression by pparb genetic ablation, we found akt1, cxcl12 (chemokine (C–X–C motif) ligand 12/stromal cell-derived factor 1 beta), and src, which were present in 18 of the 24 significantly represented functions. The potential involvement of these three genes in processes such as cell proliferation, angiogenesis, and tumor growth has been described previously [19–21]. Interestingly, Akt1, CXCL12, and Src are implicated in the PI3 kinase and MAP kinase pathways, both regulated by PPARb in keratinocytes (Fig. 3). In brief, the genetic deletion of pparb in keratinocytes generated many changes in gene expression that

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INJURY

RE-EPITHELIALIZATION

1. Inflammation

2. Survival

REMODELING

3. Proliferation

Epidermis

4. Adhesion and migration

1

2

TNF-α

4

PKBα/Akt1

AP-1 Endogenous ligands

GSK3β

REMODELING

Rho GTPases TGFβ-1

PDK PI3K

Smad3/Smad4

PPARβ/δ PPARβ expression

AP-1 Keratinocyte

AUTONOMOUS

NON-AUTONOMOUS

3

stress injury

IL-R2

R

KGF, GM-CSF, IL-6, IL-8, IL-10

IL-1 / sIL-1Ra

IL-R1 PPARβ/δ TAK1

AP-1

sIL-1Ra growth factors

Fibroblast

Fig. 3. Roles of PPARb in wound healing: keratinocyte-autonomous vs. non-autonomous functions. In inflamed keratinocytes, the expression of pparb is stimulated via binding of the AP-1 transcription factor complex to the promoter of the gene. AP-1 is activated by the stress-associated protein kinase pathway, which is enhanced by proinflammatory cytokines such as TNF-a (1). In parallel, pro-inflammatory cytokines also lead to the generation of PPARb ligands in keratinocytes at the wound edges (1). Increased levels of activated PPARb stimulate the expression of the genes coding for Integrin-linked kinase (ILK) and 3-phosphoinositide-dependent kinase-1 (PDK1). PDK1 activates the PKBa/Akt1-dependent cell survival pathway (2). PPARb also affects cell adhesion and migration via GSK3b and Rho GTPases (4). Once the novel epithelium is formed, TGFb-1 signaling inhibits AP-1 binding to the pparb promoter, leading to the downregulation of the gene. PPARb also regulates keratinocyte proliferation via a paracrine mechanism (3). IL-1 secreted by keratinocytes stimulates fibroblasts by activation of c-Jun, an obligate partner of the AP-1 transcription complex via TAK1. Consequently, the production of several mitogenic factors is increased. In parallel, activated PPARb enhances the production of sIL-1ra, whose gene is a direct PPARb target in fibroblasts, resulting in an attenuation of IL-1 signaling. The sIL-1ra binds with high affinity to IL-1R1 expressed by the fibroblasts, but has little affinity for IL-1R2, which is expressed in keratinocytes. Thus, sIL-1ra acts as an autocrine antagonist of IL-1 signaling in fibroblasts and consequently decreases the production of AP-1-mediated mitogenic factors, causing reduced keratinocyte proliferation.

appear to be responsible for the phenotypic changes we have observed in the mutated keratinocytes. 2.3. PPARb in hyperproliferative and inflammatory skin disorders As noted, PPARb expression is upregulated in mouse skin in response to inflammatory cytokines. Interestingly, PPARb is dramatically increased in the hyperproliferative lesional skin of patients with psoriasis, suggesting that this stimulation is most probably the result of pro-inflammatory signals [22]. In fact, numerous eicosanoids, such as lipoxygenase products, which are potent activators of PPARs in human keratinocytes, accumulate in psoriatic lesions [23]. So far, little is known about the consequences of PPARb activation in these lesions, but a recent study has shown that activation of PPARb causes a psoriasis-like skin disease in vivo [24]. In contrast, there is a known therapeutic benefit of PPARg agonists administered orally to patients suffering from psoriasis [25]. The fact that oral

administration rather than topical application is effective suggests that these therapeutic benefits are attributable to the general antiinflammatory functions of PPARg and its possible effects on the immune system. Whether PPARb stimulates or inhibits tumor development remains under debate, as does whether the response is context specific. A pilot clinical study, including 35 individuals and comprising an analysis of normal skin, actinic keratosis, and squamous cell carcinoma (SCC) showed that PPARb is upregulated in (pre)malignant skin lesions [26]. This finding is in line with a study of head and neck SCCs, suggesting a positive association between PPARb and vascular endothelial growth factor expression/microvessel density [27]. Mouse models have led to controversial results that have been recently extensively reviewed [28–30]. Of note, several papers have described the involvement of PPARb in the control of the expression of genes associated with tumorigenesis, such as tgfb1, twist1, or angptl-4 [31–33]. Although

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239

-log(p value)

Cellular functions

0

1

Cell cycle

52

Cell death

82

Cellular growth and proliferation

82

Cellular movement

53

Gene expression

61

Cellular development

63

DNA replication, recombination, and repair

21

Cellular assembly and organization

38

Cell morphology

37

Cell-to-cell signaling and interaction

35

Lipid metabolism

18

Carbohydrate metabolism

2

3

4

5

6

7

8

9

4

Molecular transport

17

Cell signaling

22

Inflammatory response

7

Protein trafficking

2

Nucleic acid metabolism

5

Energy production

4

Cellular function and maintenance

9

Amino acid metabolism

4

Free radical scavenging

8

Protein synthesis

6

Number of reprensented pathways

20

16

12

8

4

0

Akt1

Cxcl12

Src

Rassf1

Scarb1

Ptpre

Pim1

Gsk3b

Lpl

Ahr

Gene names

Fig. 4. Comparison of gene expression profiles between pparb wild-type and null keratinocytes. (A) Analysis of cellular function enrichment highlighted significantly changed functions identified using Ingenuity’s pathway analysis (IPA) v2.0 (Ingenuity Systems, Redwood City, CA). The data files containing the probe identifier (gene accession numbers) and the corresponding changes in expression values (fold change (FC) and p value) were uploaded into IPA. Probes from the microarray data set, which satisfied the cut-off criteria of 1.45 fold change (FC) in expression levels (up- or downregulated) and a p o 0.007, were considered for functional analyses that were performed using the right-tailed Fisher’s exact test (threshold set at 0.05). The numbers of genes belonging to the cellular function are written on the bars. (B) Heat map representation of the five most upregulated and the five most downregulated genes between PPARb-null and wild-type keratinocytes (use of linear models and empirical Bayes methods for assessing differential expression in microarray experiments) for the five most enriched cellular functions. Diagonal stripes(dark red in web version) represents the genes with FC between 2.5 and 3; light gray(light red in web version) represents genes with FC between 1.5 and 2.5; and dark gray(green in web version) indicates FC between  1.5 and  2. (C) Top 10 genes ranked by fold occurrence in significant PPARb-dependent cellular functions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

these studies were not performed on skin, it is reasonable to infer that such regulation also occurs in skin tumors. In the future, better models should help determine under which conditions PPARb has pro-cancer or anti-cancer activity. Defining these roles may lead to unexpected results, as illustrated by recent findings regarding the nature of PPARb ligands and the expression level of intracellular lipid transporters. Indeed, retinoic acid (RA), which

influences biological processes by activating the retinoic acid receptor (RAR), has been recently shown also to activate PPARb [12,34]. Remarkably, RA signaling through RAR or PPARb commits the cell to opposite fates, apoptosis or survival, respectively, by a mechanism that depends on the ratio of the proteins FABP5 and CRABP-II. When the FABP5 to CRABP-II ratio is high, RA serves as a physiological ligand for PPARb. Furthermore, FABP5 and PPARb

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are critical mediators of the ability of EGFR to enhance cell proliferation of MCF-7 mammary carcinoma cells, indicating that this transcriptional pathway may be key to EGFR-induced tumorigenesis [35]. Thus, PPARb is a tumor growth modifier via regulation of cell apoptosis/survival, proliferation, differentiation, and migration and through its action on the tumor cell environment, namely, angiogenesis, inflammation, and immune cell functions [29].

component, PER2, interacts with PPARa and REV-ERBa and serves as a coregulator of these receptors. Through this interaction, PER2 rhythmically binds to the promoters of nuclear receptor target genes in vivo, like Bmal1, Hnf1a, and glucose-6-phosphatase [50]. For instance, PPARa binds to a peroxisome proliferator response element (PPRE) located in the bmal1 promoter. Interestingly, BMAL1 is also an upstream regulator of PPARa gene expression. These results provide evidence for a regulatory feedback loop involving BMAL1 and PPARa in peripheral clock regulation [51].

3. PPAR and sexual dimorphism

3.3. Sexual dimorphism of PPARa functions

3.1. PPAR functions in liver

The liver is a sexually dimorphic organ. In mammals, more than 1600 hepatic genes are differentially expressed between males and females [52], contributing to sexual differences in energy homeostasis, lipid and steroid hormone metabolism, and degradation of xenobiotics. Among these genes, a few encode transcription factors. The best-characterized among them is the family of signal transducer and activators of transcription (STAT), which is differentially activated in the two sexes by a mechanism sensing differences in pituitary growth hormone secretion [53]. Additional transcription factors such as the GA-binding proteins (GABPs) also contribute to hepatic sexual dimorphism [54]. Different physiological phenotypes between females and males might reflect sexual dimorphism in transcription factor action, which can be demonstrated by a comparison of null animals of both sexes. For instance, in PPARa-null mice, severe hepatic abnormalities occur only in males, with triglycerides and cholesteryl-ester levels being 10-fold higher (males) and  3-fold higher (females) than in the wild-type counterparts, with a much more pronounced hepatic steatosis in males [55]. One study involving an acute blockade of hepatic fatty acid catabolism by a 5-day treatment with etomoxir, an inhibitor of long-chain fatty acid transport, resulted in the death of all PPARa-null males, while the female lethality was only 25% [56]. In brief, the action of PPARa presents a clear sexual dimorphism. This conclusion is in line with the lower PPARa expression in females [57] and with the finding that females are less responsive than males to the various effects of PPARa agonists, as seen with hepatic peroxisome proliferation [58] and PPRE-mediated luciferase expression in a transgenic model [59]. However, the idea that PPARa would be almost ‘silent’ in females because of its low expression is contradicted by years of efficacious fibrate treatments in both women and men. What, then, is different in the role of PPARa in the livers of females compared to males? To address this question, we compared mRNA expression profiles of male and female mice in the presence or absence of PPARa 2 h after dark, when the PPARa-stimulated genes are expected to reach maximum expression [60]. The microarray data were analyzed using a linear model with PPARa, sex, and their interaction as factors with a cut-off of 1.5 of fold change in expression levels (up- or downregulated) and a po0.05. Our results (Fig. 5A) showed that in liver, a similar number of genes (66 genes in females and 60 genes in males) were stimulated by PPARa in both sexes. Interestingly, only half of the genes (32, Fig. 5A) were commonly upregulated by PPARa, revealing a clear sexual dimorphism. The action of PPARa was not confined to stimulation. Of further note, the number of genes downregulated by PPARa in females was twice the number of genes repressed in the male liver (50 vs. 24). These results highlight the possible pitfalls of generalizing observations obtained from studies using animals of one sex only (often males). To better understand the nature of this repressive PPARa function in female liver, we looked for specific downregulated pathways. An Ingenuity pathway analysis identified several cellular functions preferentially repressed in females, mainly metabolic pathways

The liver performs many homeostatic functions, including (i) metabolism of carbohydrates, proteins, lipids, and hormones; (ii) production and secretion of bile; (iii) synthesis and secretion of most blood proteins; and (iv) detoxification of endogenous and exogenous compounds. The complexity of hepatic functions is primarily perceived at the level of the individual cell, as liver homeostasis depends on a variety of transcription factors that adapt gene expression to specific conditions. PPARa is particularly abundant in hepatocytes [36], PPARb is the prevalent isotype in the stellate cells [37], and PPARg expression increases in both parenchymal and stellate cells during pathologic lipid accumulation [38]. PPARa is a master hepatic energy sensor: it is maximally expressed during fasting and regulates lipid uptake and catabolism (e.g., enhancing b-oxidation, [39]), glucose metabolism (e.g., increasing glyconeogenesis, [40]), amino acid metabolism (e.g., inhibiting amino acid breakdown, [41]), and detoxification. Because we have reviewed the role of PPARa in liver metabolism recently [42], here we concentrate on its interaction with the circadian clock and involvement in hepatic sexual dimorphism. 3.2. Mutual interactions between PPARa and the circadian clock Because PPARa is a key regulator of the hepatic adaptation to fasting/feeding, one can speculate that inputs from the circadian clock might govern some of its actions. In fact, the expression of PPARa follows a diurnal rhythm, with the diurnal variation of glucocorticoid levels serving as an important stimulus of PPARa expression [43]. In rodents, PPARa expression is low in the morning and high in the evening. Oscillation in nuclear receptor expression in key metabolic tissues may contribute to coupling the circadian clock with nutrient and energy metabolism [44]. It is therefore not surprising that perturbations of the clock have been observed in hepatic diseases. In mice with hepatic fibrosis, for instance, several clock genes are silenced with concomitant attenuation of PPARa expression and abolition of its circadianity [45]. In addition, the three PAR-domain basic leucine zipper (PAR bZip) proteins DBP, TEF, and HLF, known as circadian transcription factors, are thought to connect peripheral oscillators to circadian gene expression. Gene expression profiling in the liver of triple PAR bZip-null mice has revealed many downregulated genes involved in lipid metabolism and xenobiotic catabolism, many of which were previously identified as targets of PPARa [46]. In response to nutritional cues, such as low-fat/high-glucose diets, several enzymes, like the fatty acid synthase FAS, might produce endogenous ligands for PPARa (Fig. 2) [47,48]. In collaboration with Schibler’s group, we observed that in triple PAR bZip-null mice, PPARa activity is impaired because of the alteration in the circadian transcription of genes specifying acyl-CoA thioesterases in charge of a cyclic release of fatty acids from thioesters [49]. Thus, PPARa contributes to the connection between the circadian clock and energy metabolism. This inference is further supported by the discovery that another clock

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Upregulated genes in KO

45 116 significantly deregulated genes in KO vs WT

5

34

19

32 28

84 significantly deregulated genes in KO vs WT

Downregulated genes in KO

59 28 65 34

21 7

29 14 12 2

9 6

Fig. 5. Sexual dimorphism in the gene expression profile regulated by PPARa in liver. (A) Venn diagram showing the number of genes significantly deregulated between PPARa-null vs. WT in males and females. (B) Heat map showing the expression (fold change, FC) and biological function (as identified by Ingenuity pathway analysis (IPA) v.2.0, Ingenuity Systems, Redwood City, CA) of the genes regulated by PPARa in both sexes. (C) Categories of biological functions (IPA) preferentially downregulated in females. The number of genes for each category is written in the bars, and p values o0.05 were considered significant (Fisher’s exact test). (D) Heat map showing the expression (FC) and biological function (IPA) of the genes differentially regulated by PPARa in the two sexes with respect to the ‘‘reproductive system’’ and ‘‘cholesterol biosynthesis’’.

(Fig. 5C). Furthermore, the analysis highlighted two major functions, ‘‘cholesterol biosynthesis’’ and ‘‘reproduction’’ (which includes genes involved in the synthesis of steroid hormones, such as Cyp7b1, Fig. 5D). In agreement with our previous results [60], this analysis revealed an important PPARa-dependent sexual dimorphism in hepatic gene expression, with a particularly marked repression function in females (Fig. 5B). 3.4. Downregulation of metabolic functions in females One of the most PPARa-repressed genes in female mice was the one encoding the steroid oxysterol 7a-hydroxylase cytochrome P4507b1 (Cyp7b1). Using this gene as a model, we analyzed the molecular mechanism of this sex-specific PPARa-dependent repression [60]. First, we found that PPARa was sumoylated in females but not in males. Modeling of the PPARa ligand-binding domain (LBD) revealed that the agonist-induced conformation of the LBD exposed the sumoylation-targeted Lys358 at the surface of the molecule, making it available for this modification (Fig. 6A). In fact, sumoylation of the PPARa LBD triggered the interaction of PPARa with the LXXLL motif of GABPa bound to the Cyp7b1 promoter. Furthermore, despite the ‘agonist-like’ conformation required for sumoylation,

the thus-modified PPARa recruited the corepressor NCoR [60,61]. DNA and histone methyltransferases were also recruited, and an Sp1-binding site close to the GABP sites and histones were methylated (Fig. 6B). These events resulted in loss of Sp1-stimulated expression and thus downregulation of Cyp7b1 [60]. Together, these results unveiled sumoylation as inducing a switch in PPARa activity: when sumoylated, PPARa functions as a repressor of specific promoters. The observation that sumoylated PPARa is more abundant in females compared to males prompted us to explore further the physiological significance of this sexual dimorphism. 3.5. Sumoylated PPARa protects against estrogen-mediated hepatotoxicity The CYP7b1 protein is a prominent member of the CYP family. It is significantly more expressed in male than in female liver and is involved in sexual hormone production and action. In females, CYP7b1 reduces the hepatic production of the male sexual hormone by hydroxylating dehydroepiandrosterone (DHEA), subtracting it from the testosterone synthesis pathway [60,62–64]. In contrast, it promotes estrogen receptor (ER) activity by catalyzing the clearance of 27-hydroxycholesterol, which acts as a competitive ER antagonist

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Lys 358

PPARα antagonist PPAR -null female Unstimulated male

PPARα agonist WT PPAR female Male treated with PPAR agonist NCoR Sumo PPARα

GABPα /β1 TTCC

Sp1 GGCGG

Cyp7b1 mRNA

H3-K9 ac

Active promoter

HDACs GABPα/β1 TTCC Histone Methyltransferase

Dnmt Methyl GGCGG

Cyp7b1 mRNA

H3-K9 trime

Repressed promoter

Fig. 6. Sex-dependent sumoylation-induced transrepression by PPARa. (A) Model of the ligand-binding domain (LBD) of mouse PPARa in the ligand-activated conformation (upper right panel). In Helix 7 of the LBD, Lysine 358 (Lys358) is sumoylated in female hepatocytes and appears at the surface of the LBD upon agonist binding. In contrast, Lys358 is masked in the presence of an antagonist (upper left panel). (B) Model of PPARa-induced repression of Cyp7b1 in female hepatocytes. Sumoylation of PPARa promotes interaction with GA-binding proteins (GABPs) and recruits NCoR, HDACs, histone, and DNA methyltransferases, which results in histone H3 deacetylation, tri-methylation, and DNA methylation at the Sp1-binding site. The displacement of Sp1 from its methylated binding site downregulates gene expression (right panel). Genetic removal of PPARa in female mice leads to stimulation of Cyp7b1 expression (left panel).

[64,65]. Activated ER indirectly stimulates the expression of CYP7b1, which participates in estrogen-induced hepatotoxicity and inflammation. Susceptible women using estrogen-containing oral contraceptives or postmenopausal hormone replacement therapy can suffer from estrogen-induced hepatotoxicity [66]. Moreover, high levels of estrogens cause the most common hepatic disease during pregnancy, intrahepatic cholestasis. This condition can result in intrauterine fetal death or spontaneous premature delivery [67]. The cross-talk between ER and PPARa has been previously documented in our laboratory [68], and its control over Cyp7b1 activity is an interesting facet of the overall picture. In fact, sumoylated PPARa mediates female-specific gene repression and protects the liver from estrogen-induced toxicity in mice, as documented by the observation that pretreatment with fibrate conferred on female mice protection against experimentally estrogen-induced intrahepatic cholestasis and inflammation [60]. In short, PPARa counteracts the negative effects of estrogen signaling in female liver, which needs to be optimally balanced, not least because of its action on lipid metabolism. For instance, when the synthesis of 17b-estradiol is ablated in mice (aromatase-null mice), the animals display hepatic steatosis [69]. A better understanding of the interconnection between lipid sensing by PPARs and estrogen signaling in females may lead to novel approaches in the treatment of energy dysfunction-related diseases in women.

4. Conclusions PPARs continue to be at the heart of many studies thanks to their multifaceted roles in several major cellular and physiological functions. At the molecular level, PPARs mainly act through ligand-dependent target gene stimulation, but as seen here, there is increasing evidence for alternative modes of action for PPAR-mediated signaling, such as transrepression. This diversity

of mechanisms is a central element in the broad spectrum of roles these receptors play, from the control of basic cellular processes to the regulation of complex metabolic functions in a sex-specific manner, especially in the liver. It can be speculated that this extremely developed multifaceted character derives from the key roles these proteins play in different aspects of energy balance that often show a cyclic pattern. With this in mind, we have discussed how PPARs occupy the crossroads of several pathways. In skin, we discussed the role of PPARb during cell proliferation/differentiation. In liver, we highlighted the role of PPARa during the fast-to-refeeding cycle and its interactions with the circadian clock. Finally, our results on sexual dimorphism allowed us to describe the involvement of these proteins in estrogen signaling. Not surprisingly, all of these processes require strict control of energy availability, in which PPARs as lipid sensors are the main regulators. Most likely, many facets of PPAR biology remain hidden, and new technologies allowing global analyses of the interactions among the components of biological systems within the body will contribute to unveiling these yet-to-be-identified functions.

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