Antiapoptotic Role of PPARβ in Keratinocytes via Transcriptional Control of the Akt1 Signaling Pathway

Molecular Cell, Vol. 10, 721–733, October, 2002, Copyright 2002 by Cell Press

Antiapoptotic Role of PPAR␤ in Keratinocytes via Transcriptional Control of the Akt1 Signaling Pathway Nicolas Di-Poı¨,2 Nguan Soon Tan,2 Liliane Michalik, Walter Wahli, and Be´atrice Desvergne1 Institut de Biologie Animale Universite´ de Lausanne CH-1015 Lausanne Switzerland

Summary Apoptosis, differentiation, and proliferation are cellular responses which play a pivotal role in wound healing. During this process PPAR␤ translates inflammatory signals into prompt keratinocyte responses. We show herein that PPAR␤ modulates Akt1 activation via transcriptional upregulation of ILK and PDK1, revealing a mechanism for the control of Akt1 signaling. The resulting higher Akt1 activity leads to increased keratinocyte survival following growth factor deprivation or anoikis. PPAR␤ also potentiates NF-␬B activity and MMP-9 production, which can regulate keratinocyte migration. Together, these results provide a molecular mechanism by which PPAR␤ protects keratinocytes against apoptosis and may contribute to the process of skin wound closure. Introduction Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that belong to the nuclear hormone receptor family. Three isotypes (␣, NR1C1; ␤/␦, NR1C2; and ␥, NR1C3) which have distinct tissue distributions and functions have been found in vertebrates (Desvergne and Wahli, 1999). Important roles of PPAR␣ and PPAR␥ in lipid homeostasis and in inflammation have been unveiled. Little is known about the exact function of PPAR␤, although it has been implicated in colon tumorigenesis (Park et al., 2001), skin wound healing (Michalik et al., 2001), and embryonic development (Barak et al., 2002). In adult epidermis, PPAR␤ is undetectable in interfollicular keratinocytes (Michalik et al., 2001), which is consistent with the observation that PPAR␤ null (PPAR␤⫺/⫺) mice exhibit no apparent skin defects (Peters et al., 2000). However, the expression of PPAR␤ is reactivated upon proliferative stimuli such as cutaneous injury, suggesting a role of PPAR␤ in regulating keratinocyte proliferation/differentiation processes (Michalik et al., 2001; Tan et al., 2001). In accordance, wound repair is delayed in PPAR␤ mutant mice due to impaired adhesion/migration of the mutant keratinocytes (Michalik et al., 2001). Recently, we have also shown that cultured keratinocytes isolated from the PPAR␤⫺/⫺ mice display increased apoptosis in

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Correspondence: [email protected] These authors contributed equally to this work.

response to TNF-␣. Increased keratinocyte death was also observed in vivo, indicating an antiapoptotic role of PPAR␤ (Tan et al., 2001). Apoptosis, differentiation, and proliferation are complex biological processes, in which Akt1, also known as protein kinase B-␣ (PKB␣), plays a pivotal role. Akt1 is a major downstream effector of the phosphatidylinositol-3-kinase (PI3K) signaling, which is involved in the response to various stimuli including TNF-␣ and deprivation of growth factors (GF) (Nicholson and Anderson, 2002). Akt1 also inhibits anoikis, an apoptosis that is induced by bringing cells into suspension (Khwaja et al., 1997). It exerts antiapoptotic effects either by inhibiting proapoptotic proteins, such as Bad and the forkhead family of transcription factors (FKHR) via phosphorylation, or by inducing antiapoptotic signals via the nuclear factor-␬B (NF-␬B) (Nicholson and Anderson, 2002). Maximal Akt1 activity requires phosphorylation of threonine 308 (T308) by the 3-phosphoinositide-dependent kinase-1 (PDK1) and of serine 473 (S473) by the integrinlinked kinase (ILK) (Nicholson and Anderson, 2002) or other kinases. Both PDK1 and ILK are ubiquitously expressed. The overexpression of ILK in epithelial cells leads to the stimulation of anchorage-independent cell cycle progression, the suppression of anoikis, and increased tumorigenicity in nude mice (Wu and Dedhar, 2001). PDK1 is a key enzyme in transducing signals to multiple effector pathways and has been implicated in the control of apoptosis (Flynn et al., 2000). Importantly, activation of ILK and PDK1, which in turn activates Akt1, requires their binding to phosphatidylinositol-3,4,5-triphosphate (PIP3) (Nicholson and Anderson, 2002; Wu and Dedhar, 2001). The increase and decrease of the cellular PIP3 levels are governed by PI3K and phosphatase and tensin homolog deleted on chromosome 10 (PTEN), respectively (Nicholson and Anderson, 2002). Herein, we decipher the mechanism by which PPAR␤ exerts its antiapoptotic effects via the Akt1 pathway. We show that both ILK and PDK1 are target genes of PPAR␤, revealing transcriptional regulation as an important level of control in this pathway. The upregulation of these genes together with the downregulation of PTEN leads to an increase of Akt1 activity in keratinocytes, which is sufficient to suppress apoptosis induced by GF deprivation or anoikis in cell culture. Consistent with a critical role of PPAR␤ during wound healing, we also show that, in response to TNF-␣, PPAR␤ potentiates NF-␬B activity and matrix metalloproteinase-9 (MMP-9) secretion via the PI3K cascade. Such insights into the roles of PPAR␤ are crucial to the understanding of the healing process. Results PPAR␤ Regulates Genes Involved in the Akt1 Signaling Pathway PPAR␤ is a key regulator of timely wound repair (Michalik et al., 2001) and plays a role in the control of keratinocyte apoptosis (Tan et al., 2001). The pivotal

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Figure 1. PPAR␤ Regulates the Expression of Components of the Akt1 Signaling Pathway (A) PPAR␤ modulates the expression of PDK1, ILK, and PTEN. RPA was carried out using total RNA extracted from primary keratinocytes derived from PPAR␤⫹/⫹ and ⫺/⫺ mice, treated (LD) or not (⫺) for 12 and 24 hr with 1 ␮M of PPAR␤ ligand L-165041, in the absence or presence of 5 ␮g/ml or 10 ␮g/ml of cycloheximide (CH; right panel). A representative experiment is shown. Fold changes, indicated below each band, represent the mean of three experiments, after normalization to the wt or untreated keratinocytes. L27 ribosomal protein (L27) mRNA was used as internal control.

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Figure 2. PPAR␤ Ligand Induces the Expression of ILK and PDK1 in Primary Keratinocytes PPAR␤⫹/⫹ keratinocytes were plated on chamber slides (Lab-Tek) and treated for 24 hr with vehicle or 1 ␮M PPAR␤ ligand L-165041 (LD). Immunofluorescence was performed with polyclonal anti-ILK or anti-PDK1 antibodies (1:50, Santa Cruz Biotechnology) and FITC-conjugated IgG (1:100). Arrows indicate the absence of staining in the nucleus. Magnification bars, 25 ␮m.

role of Akt1 signaling in apoptotic regulation pointed to a possible link between PPAR␤ and the Akt1 pathway. Thus, we assessed the expression of various actors of this pathway (see Figure 7). No difference between keratinocytes derived from wild-type (wt) (PPAR␤⫹/⫹) or mutant PPAR␤⫺/⫺ mouse skin was observed in the expression levels of Akt1 and focal adhesion kinase (FAK) (Figure 1A, left panel). FAK is a tyrosine kinase that is involved at an early step in the Akt1 signaling cascade by promoting anchorage-dependent cell survival (Sonoda et al., 2000). Interestingly, the expression of ILK and PDK1 was reduced in the PPAR␤⫺/⫺ cells as compared to their wt counterparts whereas the expression of PTEN was 2-fold higher in PPAR␤⫺/⫺ cells (Figure 1A). In agreement with these observations, treatment of wt keratinocytes for 12 or 24 hr with the synthetic PPAR␤ ligand L-165041 (LD) resulted in an increase of both PDK1 and ILK and a decrease in PTEN expression. The effect of LD on the expression of PDK1 and ILK is PPAR␤ dependent (Figure 1A, middle panels) and direct, as it was unaffected by protein synthesis inhibitor cycloheximide (CH) (Figure 1A, right panel). In contrast, the PPAR␤-mediated downregulation of PTEN was sensitive to CH, indicating an indirect expression control by

PPAR␤. The modulation of expression of these genes was confirmed at the protein levels (Figure 1B, left panel) and by immunohistochemistry using antibodies against ILK and PDK1 performed on keratinocytes treated with the PPAR␤ agonist (Figure 2). In addition, the reduced phosphorylation of Akt1 at T308 and S473 in PPAR␤⫺/⫺ cells is consistent with the decreased PDK1/ILK protein levels, while transfection of PPAR␤ in these cells restored the levels of both kinases and phosphorylated Akt1 (Figure 1B, left panels). PPAR␤-dependent modulation of Akt1 and PTEN activity was further confirmed by direct determination of their activity using an in vitro kinase assay and by measuring PIP3 production, respectively (Figure 1C). Ultimately, the reduced Akt1 activity in PPAR␤⫺/⫺ cells was reflected in lower phosphorylated Bad and FKHR levels, without changes in their total protein expression level (Figure 1B, right panel). Bad and FKHR are direct targets of Akt1 and are involved in its antiapoptotic activity. These results conclusively show that, in keratinocytes, transcriptional upregulation of ILK and PDK1 and repression of PTEN by PPAR␤ is important because it strongly affects the Akt1 signaling cascade (Akt1, Bad, and FKHR).

(B) PPAR␤ regulates the Akt1 pathway at the protein level. Western blot assays were performed using equal amounts of cell extracts (20 ␮g) from PPAR␤⫹/⫹ and ⫺/⫺ mouse skin tissues, or from PPAR␤⫺/⫺ keratinocytes control (⫺) or transfected with mPPAR␤ (PPAR␤), and were treated for 6 hr with 1 ␮M PPAR␤ ligand (left panel, right column). All primary antibodies specific for total or phosphorylated proteins were used at a dilution of 1:1000, except for anti-ILK (1:2500). Fold changes, indicated below each band, were normalized to total Akt1 protein and represent the mean of at least two independent experiments. ␤-tubulin was used as internal control. The apparent molecular weight is indicated for each protein. (C) PPAR␤ modulates Akt1 activity and PIP3/PIP2 ratio. Left panel: After immunoprecipitation from PPAR␤⫹/⫹ and ⫺/⫺ keratinocytes treated (LD) or not (⫺) for 6 hr with LD, the Akt1 kinase activity was assessed in vitro using as a substrate a GSK-3␣/␤ peptide fused to a GST tag. Levels of phosphorylated GSK-3␣/␤ were measured by Western blot using an anti-phospho-GSK-3 antibody (GSK-3␣/␤ S9/21-P), and equal substrate loading of the gels was assessed by silver staining (substrate). Right panel: PTEN activity was evaluated by measurement of the PIP3/PIP2 ratio in PPAR␤⫹/⫹ and ⫺/⫺ cells or in PPAR␤⫹/⫹ treated for 3 hr with LD (LD) or not (⫺) and was labeled with 32P orthophosphate. The first lane shows the migration of control PIP2 and PIP3 (Std). A representative thin layer chromatography is shown. Relative Akt1 activity or PIP3/PIP2 ratios, respectively, are indicated below each band and represent the means of at least two independent experiments.

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Figure 3. ILK and PDK1 Are Two Direct Target Genes of PPAR␤ (A) PPAR␤ transactivates the ILK and PDK1 genes. Relative positions with respect to the assigned transcription start site (⫹1) of putative PPREs in the ILK gene (ILK PPRE1 and PPRE2) and the PDK1 promoter region (PDK1 PPRE1) are given. Different luciferase reporter constructs used for transient transfection in HaCaT keratinocytes are as indicated. Transfected cells were treated for 24 hr in serum-free medium with either DMSO (vehicle) or 1 ␮M L-165041 (LD). Results were normalized with ␤-galactosidase activity and expressed in arbitrary units, with

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ILK and PDK1 Are Two Direct Target Genes of PPAR␤ The above data suggest that ILK and PDK1 are direct targets of PPAR␤. Thus, functional PPAR response elements (PPREs) should reside in the regulatory region of these genes. The human ILK gene (accession number AJ404847) is under the control of a short 280 bp promoter (Figure 3A) containing putative binding sites for AP-1 and SP-1, as identified by MatInspector (Wingender et al., 2000) (data not shown). This analysis also revealed two putative PPREs, named herein ILK PPRE1 (nucleotides ⫹1535 to ⫹1547) and ILK PPRE2 (⫹4238 to ⫹4250) in a long intron 2 (Figures 3A and 3C, left panel). In keeping with the genomic organization of the ILK gene, the promoter region (pILK-Pr) and different fragments of intron 2 (pILK-In, pILK-⌬1, and pILK-⌬2) were subcloned upstream and downstream of a luciferase reporter gene, respectively. Transfection studies showed that the short promoter region was sufficient for basal promoter activity but was not responsive to LD (Figure 3A, top panel). In contrast, the constructs harboring intron 2 (pILK-In) or the 5⬘ intronic fragment with the ILK PPRE1 (pILK-⌬1) responded efficiently to LD, suggesting that the ILK PPRE1 is the functional PPRE in the ILK gene. In a similar approach, analysis of the human PDK1 promoter gene sequence (accession number AC092117) revealed a putative PPRE (PDK1 PPRE1) between nucleotides ⫺5077 and ⫺5065 (Figure 3A). Transfection of a SV40-luciferase reporter vector containing either a 1.5 kb fragment of the PDK1 promoter that includes the PDK1 PPRE1 (pPDK-Pr) or a shorter construct (pPDK⌬) showed that the PPAR␤-responsive region in PDK1 is located between ⫺5515 and ⫺4691, where the identified PPRE resides (Figure 3A, bottom panel). In order to determine whether PPAR␤ binds to the ILK and PDK1 genes in vivo, chromatin immunoprecipitation (chIP) was performed using a human HaCaT keratinocyte cell line treated or not with LD. ILK and PDK1 gene sequences spanning each putative PPRE and a random control sequence from the corresponding gene were analyzed by PCR in the immunoprecipitated chromatin. As shown in Figure 3B, in the presence of LD, the sequences spanning the ILK PPRE1 and PDK1 PPRE1 were significantly enriched in the immunoprecipitates obtained with PPAR␤ and acetylated histone H4 antibodies compared to the controls (preimmune serum and control DNA fragment). Finally, electrophoretic mobility shift assays (EMSA) demonstrated that PPAR␤/RXR␣ heterodimers were able to recognize these two newly discovered PPREs (Figure 3C, right panel). Together,

these results demonstrate that the ILK PPRE1 and PDK1 PPRE1 are functional and that ILK and PDK1 are authentic PPAR␤ target genes in keratinocytes. PPAR␤ Protects Keratinocytes from Anoikis and GF Deprivation-Induced Apoptosis Keratinocytes are anchorage-dependent cells that undergo apoptosis in the absence of interaction with the extracellular matrix (anoikis) (Frisch and Francis, 1994) or in the absence of soluble GF (Jost et al., 1999). The effect of PPAR␤ on the Akt1 pathway via ILK and PDK1 should have consequences with respect to the known role of Akt1 in suppressing apoptosis (Khwaja et al., 1997; Chen et al., 2001). To study the effects of this PPAR␤-dependent Akt1 activity in apoptosis, human HaCaT and mouse BALB/ MK keratinocyte cell lines were used. We first assessed that both cell lines respond to LD by an increased PDK1/ ILK expression (Figure 4A, left panel) and PIP3 production (Figure 4B, bottom panel), hence an increased Akt1 activity (Figure 4B, top panel, and supplemental data at http://www.molecule.org/cgi/content/full/10/4/721/ DC1). Interestingly and as expected, overexpression of PTEN, the negative regulator of the PI3K signaling that is inhibited by PPAR␤, did not affect the upregulation of PDK1 and ILK by LD but abrogated the activation of Akt1 by phosphorylation (Figure 4A, right panel). These results further demonstrate the concomitant requirement of increased ILK and PDK1 expression and efficient PIP3 production for PPAR␤-regulated Akt1 activation. When challenged with GF deprivation or anoikis, HaCaT cells underwent apoptosis, resulting at 24 hr in 40%–80% of cells positive for annexin-V (Figures 4C and 4D, left panel). Activation of PPAR␤ by LD resulted in a reduced apoptosis to only 10%–20% of apoptotic cells for all tested conditions (Figure 4D, left panel). Interestingly, LD treatment of cells in suspension led to a profound cellular rearrangement, with the formation of multicellular aggregates (Figure 4C, right inset). To assess the involvement of ILK and PDK1 in this PPAR␤-induced resistance to apoptosis, the same experiment was performed in cells overexpressing PTEN, the natural inhibitor of ILK and PDK1 activity. As shown in Figure 4D, PTEN largely abrogated the antiapoptotic effect of PPAR␤. The same results were obtained using the specific PI3K inhibitor LY294002 (data not shown). Nevertheless, overexpression of PTEN did not totally suppress the PPAR␤-mediated protection against apoptosis, suggesting a possible minor contribution of a

the activity of the promoterless vector (pluc) set at 1. Values represent means of at least three independent experiments. (B) PPAR␤ binds to ILK and PDK1 genes in vivo. Schematic representations of the ILK gene and PDK1 promoter region with identified PPREs are illustrated (left panel). Bars below each construct represent the relative positions and the length of the fragments that are amplified by PCR. Soluble chromatin from HaCaT cells treated with (⫹) or without (⫺) LD was immunoprecipitated with antibodies against PPAR␤ (antiPPAR␤, middle panel), acetylated histone H4 (anti-acH4, right panel), or preimmune serum (pre-imm.), and analyzed by PCR. Aliquots of the chromatin were also analyzed before immunoprecipitation (input). (C) PPAR␤/RXR␣ heterodimers bind to ILK and PDK1 PPRE1. Sequence of putative PPREs identified in ILK gene (ILK PPRE1 and PPRE2) and PDK1 promoter (PDK1 PPRE1) are aligned to consensus PPRE sequences and to the rat acyl CoA oxidase (ACoA) PPRE (Dreyer et al., 1993) (left panel). EMSA was done using in vitro translated PPAR␤ and/or RXR␣, and radiolabeled PPRE oligonucleotides (right panel). The first and second lanes represent probe alone or with unprogrammed TNT reticulocyte lysate, respectively. Specific competition was performed with the indicated molar excess of unlabeled ACoA probe. Arrows indicate the specific complexes between the PPAR␤/RXR␣ heterodimer and the probes.

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PI3K-independent survival pathway. Similar observations were also obtained using BALB/MK cells (see supplemental data at http://www.molecule.org/cgi/content/ full/10/4/721/DC1). Reciprocally, stable cell lines (three independent clones for each condition) expressing elevated levels of either PDK1, ILK, or constitutively active (ca) Akt1 revealed that increased PDK1, ILK expression, or Akt1 activity to levels comparable to those induced by LD is sufficient to protect the cells from apoptosis to a degree similar to that achieved by LD (Figure 4D, right panel). This result indicates that the effect of LD is dependent on increased levels of ILK and PDK1. Finally, the antiapoptotic effect of PPAR␤ agonist was evaluated by examining caspase-3 activation reflected by a decrease of the proenzymatic form (32 kDa) and an increase of the active cleaved form (17 kDa). Caspase-3 became activated after 6 hr of GF deprivation or anoikis while cells treated with LD displayed a reduced caspase-3 activation (Figure 4E). Thus, these results show a major antiapoptotic effect of the PPAR␤ ligand against anoikis and GF deprivation-induced apoptosis. Similar experiments using PPAR␤⫺/⫺ and PPAR␤⫹/⫹ primary keratinocytes further demonstrated the direct implication of PPAR␤ in the protection against anoikis and GF deprivation, as PPAR␤⫺/⫺ cells were more susceptible than PPAR␤⫹/⫹ cells to both kinds of apoptosis (Figure 5A). Importantly, PPAR␤⫺/⫺ cells that were transiently transfected with vectors expressing either dominant-negative (dn) PTEN (C124S; Wu et al., 1998), wtPDK1, wtILK, or caAkt1 (T308D/S473D) were rescued and became resistant to GF deprivation and anoikis. Conversely, transfection of PPAR␤⫹/⫹ cells with wtPTEN, kinase dead (kd) forms of PDK1 (K114A; Filippa et al., 2000), kdILK (E356K; Novak et al., 1998), or dnAkt1 (K179A; Welch et al., 1998) led to an increased apoptosis reaching levels observed in PPAR␤⫺/⫺ cells (Figure 5B). Interestingly, overexpression of caAkt1 in PPAR␤⫺/⫺ cells did not totally rescue cells from anoikis, suggesting a moderate involvement of an Akt1-independent pathway.

Together, these results demonstrate that the major antiapoptotic role of PPAR␤ against anoikis and GF deprivation occurs through the activation of the PI3K/Akt1 pathway via the upregulation of PDK1 and ILK and the downregulation of PTEN. This antiapoptotic effect requires an important contribution of the PI3K activity, primarily for the functional activation of PDK1 and ILK by PIP3. PPAR␤ Increases NF-␬B Activity and MMP-9 Production in Response to TNF-␣ Akt1 has been reported to modulate the activity of the transcription factor NF-␬B in response to TNF-␣ (Ozes et al., 1999). Since PPAR␤⫺/⫺ keratinocytes are more susceptible to TNF-␣-induced apoptosis (Tan et al., 2001), the possibility of an interplay between PPAR␤ and NF-␬B activity via the Akt1 signaling pathway was tested. NF-␬B activation involves its translocation to the nucleus, triggered by the degradation of its inhibitor I␬B␣, via the activation of the inhibitor-␬B kinase (IKK). Upon nuclear entry, NF-␬B activates gene transcription by binding to the NF-␬B response elements of its target genes. Thus, we performed transient transfection experiments of PPAR␤⫹/⫹ keratinocytes with a NF-␬B-responsive luciferase reporter construct. As expected, treatment of these cells with TNF-␣ increased NF-␬B dependent luciferase activity by 4- to 5-fold. Interestingly, cotreatment with LD significantly enhanced the TNF-␣-dependent expression of the NF-␬B reporter gene, and this activation was abolished by treatment with an IKK inhibitor, pyrrolidine dithiocarbamate (PDTC) (Schreck et al., 1992) (Figure 6A). The PI3K inhibitor LY also diminished the effect of the PPAR␤ agonist, indicating that PPAR␤ can modulate the NF-␬B activity triggered by TNF-␣ via the PI3K pathway. Conversely, the TNF-␣-induced NF-␬B activation was reduced in PPAR␤⫺/⫺ keratinocytes compared to

Figure 4. PPAR␤ Ligand Confers Resistance to Growth Factor Deprivation-Induced Apoptosis and Anoikis in HaCaT Cells (A) PPAR␤ activates the Akt1 pathway in the human keratinocyte HaCaT cell line. Left panel: Total cellular proteins (30 ␮g) from HaCaT cells treated for 12 hr with 1 ␮M of PPAR␤ ligand (LD) or vehicle (⫺) were used for Western blot analysis. LD increased PDK1 and ILK protein levels, which increased phosphorylation of Akt1 at T308 and S473. Right panel: Overexpression of HA-tagged mPTEN (HA-PTEN) into these cells abrogated the activation of Akt1 by phosphorylation induced by LD. In this panel, normalization takes into account the PTEN transfection efficiency using anti-HA antibodies. (B) PPAR␤ modulates Akt1 activity and PIP3 levels in HaCaT cells. In vitro kinase activity of Akt1 (top panel) and PIP3/PIP2 ratios (bottom panel) were measured as in Figure 1C using HaCaT cells treated for 6 hr with LD (LD) or untreated (⫺). (C) Activation of PPAR␤ induces cell survival following growth factor deprivation and anoikis. Apoptosis was induced in HaCaT cells treated with either vehicle (DMSO) or LD (1 ␮M) using serum-free medium (GF deprivation) or agarose-coated culture plates that prevent cell attachment (anoikis). Apoptotic cells were stained by annexin V-EGFP (green) and propidium iodide (PI, red) and examined by fluorescence microscopy. A representative picture of total cells superimposed with annexin V/PI staining after 24 hr of treatment is shown. Insets: High magnifications of typical apoptotic cells stained with annexin V/PI (left insert) and of cellular aggregation induced by LD (right insert). Magnification bars, 25 ␮m. (D) Antiapoptotic role of PPAR␤ requires PI3K activity. Left panel: HaCaT cells were transfected with plasmids expressing mPTEN (⫹PTEN) or empty (⫺PTEN). These plasmids also express the red fluorescent protein to allow identification of transfected cells (transfection efficiency ⬎75%). The transfected cells were allowed to recover for 24 hr and then treated with vehicle (open bars) or LD (green bars). After cell dispersion by trypsinisation, the number of annexin V-positive cells was quantified at indicated times by fluorescence microscopy. Values represent percentage of annexin V-positive cells among all cells expressing the red fluorescent protein (count ⬎150 cells). Right panel: Stable control cell line (Ctrl) and lines expressing ILK, PDK1, or caAkt1 were used for similar studies as in the left panel. Three isolated lines were selected for protein expression or active Akt1 levels comparable to that induced by LD (top of right panel). Values represent the means of three independent experiments each performed using the three independent clones. (E) Activated PPAR␤ reduces caspase-3 activation induced by growth factor deprivation and anoikis. Western blots of cell extracts from HaCaT cells subjected to GF deprivation or anoikis for 6, 12, or 24 hr, in the absence (⫺) or presence of LD (LD), using specific antibodies against both activated (cleaved caspase-3) and inactivated forms (procaspase-3) of caspase-3. Equal protein loading of the gels was confirmed by Coomassie staining of blots.

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Figure 5. ILK and PDK1 Are Responsible for the Antiapoptotic Role of PPAR␤ (A) PPAR␤⫺/⫺ keratinocytes are more susceptible to growth factor deprivation-induced apoptosis and anoikis. Apoptosis was induced in PPAR␤⫹/⫹ and ⫺/⫺ primary keratinocytes in culture and measured at indicated times as described in Figure 4C. (B) ILK and PDK1 rescue PPAR␤⫺/⫺ cells from induced apoptosis. PPAR␤⫹/⫹ and ⫺/⫺ keratinocytes were transiently transfected with pcDNAred encoding the indicated constructs. The transfected cells were allowed to recover for 24 hr, after which apoptosis was induced by a 12 hr GF deprivation (left panel) or by a 3 hr anoikis (right panel). The number of apoptotic cells was quantified by annexin V-EGFP staining. Values represent the percentage of both red fluorescent and annexin V-positive cells among all cells expressing the red fluorescent protein (⬎150 cells).

PPAR␤⫹/⫹ cells (Figure 6B, left panel). This decreased NF-␬B activity was due to a lower activation of the Akt1 pathway, since ectopic expression of caAkt1 or wtPDK1 in these cells increased the reporter activity by ⵑ6 fold. Reciprocally, overexpression of dnAkt1 or kdPDK1 in PPAR␤⫹/⫹ cells inhibited the PPAR␤ effect on NF-␬B activity (Figure 6B, left panel), while ILK had a weaker effect. Parallel experiments using stably transfected HaCaT cell lines showed that increased Akt1 activity to a level comparable to that induced by LD is sufficient to potentiate NF-␬B activity (Figure 6B, right panel). The functional activity of NF-␬B correlated to the corresponding changes in the NF-␬B DNA binding activity, as evaluated by EMSA analysis (Figure 6C). The increased NF-␬B activity induced by LD in the presence of TNF-␣ was reflected in a higher production of MMP-9 protease as measured by gelatin zymography. This effect was PPAR␤- and PI3K-dependent as suggested by parallel experiments using PPAR␤⫺/⫺ cells and PI3K inhibitor LY, respectively (Figure 6D). MMP-9 protease genes are known targets of NF-␬B, and their activities influence diverse physiological and pathologi-

cal processes including wound repair (Sternlicht and Werb, 2001). Altogether, these results indicate that the ability of PPAR␤ to potentiate NF-␬B activity via the PI3K/Akt1 pathway can lead to increased MMP-9 secretion. The reduced production of MMP-9 observed in PPAR␤⫺/⫺ cells in response to TNF-␣ is consistent with defective wound repair in PPAR␤ mutant mice (Michalik et al., 2001). In the context of skin wound healing, the above findings, together with those obtained recently (Michalik et al., 2001; Tan et al., 2001), show that PPAR␤ is important for keratinocyte survival and thus for a well-tuned balance between cell death and proliferation. Discussion The results presented here show that PPAR␤ directly controls apoptosis in keratinocytes via the Akt1 pathway. Two major biological consequences of PPAR␤mediated activation of the Akt1 pathway were examined. First, ligand-activated PPAR␤ increases keratinocyte survival upon exposure to stress, such as in GF depriva-

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tion and anoikis. This effect is mediated via a coordinated upregulation of ILK and PDK1 expression and a downregulation of PTEN expression, leading to Akt1 activation (Figure 7). Second, PPAR␤ potentiates the TNF-␣-mediated activity of NF-␬B in a PI3K-dependent manner, which may affect cell adhesion and migration via increased MMP-9 expression. Altogether, these results provide important insights into the role of PPAR␤ during wound repair and explain, at least in part, the delayed wound-healing phenotype observed in PPAR␤ mutant mice. PPAR␤ Activates the Akt1 Signaling Cascade via a Coordinated Regulation of Gene Expression The activation of Akt1 is dependent on PI3K activity, and signals that trigger PI3K also activate Akt1 (Nicholson and Anderson, 2002). Precisely, activation of PI3K leads to the production of PIP3 to which ILK and PDK1 must bind to be functional and to phosphorylate Akt1. Herein, we show that ILK and PDK1 are direct target genes of PPAR␤ in keratinocytes, and we have identified a functional PPRE in each gene. In addition, the downregulation of the expression of PTEN which dephosphorylates PIP3 to PIP2 (Nicholson and Anderson, 2002) results in higher PIP3 levels that contribute to the functional activation of ILK and PDK1. Intriguingly, PTEN was reported to be upregulated by the PPAR␥ agonist rosiglitazone in human primary macrophages, and two PPREs were identified in the promoter region of the gene (Patel et al., 2001). The opposite regulation of PTEN by PPAR␤ in keratinocytes and PPAR␥ in macrophages suggests a PPAR- and cell-specific regulation of PTEN, which remains to be elucidated. Antiapoptotic Role of PPAR␤ via Akt1 Activation Apoptosis is executed by two major cascades: the death receptor and the mitochondrial pathways. Death receptors are activated by the binding of specific ligands such as TNF-␣ or Fas-Ligand (Fas-L), which leads to the activation of the initiator caspase-8. The mitochondrial pathway involves the Bcl-2 family of cytoplasmic proteins that includes both antiapoptotic (Bcl-2, Bcl-XL) and proapoptotic members (Bax, Bad). The intracellular balance between these proteins determines the release of cytochrome c from mitochondria and subsequent activation of the initiator caspase-9. Activated initiator caspases in turn converge to the activation of effector caspases (caspase-3, -6, and -7) responsible for the general features of apoptosis. The Akt1 pathway has been shown to play a central role in protecting cells against apoptosis by modulating both major apoptotic cascades mentioned above. GF deprivation-induced apoptosis and anoikis are cytotoxic conditions known to invoke the mitochondrial apoptotic cascade. In this context, Akt1 phosphorylates Bad, thus sequestering it away from the mitochondria, which prevents cytochrome c release. Reduced levels of activated Akt1 in PPAR␤⫺/⫺ keratinocytes result in lower levels of phosphorylated Bad, hence increased sensitivity to apoptosis. In support of our earlier report (Tan et al., 2001), Akt1 can also prevent death receptormediated apoptosis, as invoked by TNF-␣, via phosphorylation of the transcription factor FKHR, which pre-

vents its nuclear translocation and hence its ability to induce Fas-L expression. Accordingly, we show herein that PPAR␤⫺/⫺ cells indeed exhibit lower levels of phosphorylated FKHR. Regardless of the origin of the apoptotic stimuli, the commitment to apoptosis occurs through the activation of effector caspases and finally the degradation of nuclear DNA into nucleosomal units. Interestingly, we recently reported that PPAR␤⫺/⫺ keratinocytes express lower levels of inhibitor of caspase-activated deoxyribonuclease (ICAD) (Tan et al., 2001). ICAD, which is degraded by caspase-3 during apoptosis, is involved in the inhibition of DNA fragmentation. The ability of PPAR␤ to influence both early apoptotic events via the ILK/PDK1/ Akt1 pathway, as well as genes involved in late events of apoptosis, such as ICAD, confirms the role of PPAR␤ as an important mediator of the survival of keratinocytes exposed to stress situations. PPAR␤ Modulates NF-␬B Activity in a PI3K-Dependent Manner Recent reports have pointed to a significant role of NF-␬B in vivo in determining the balance between keratinocyte growth and differentiation. Suppression of NF-␬B activity by skin-specific expression of an I␬B transgene caused epidermal hyperplasia, while NF-␬B overexpression resulted in epidermal hypoplasia (Gerondakis et al., 1999). Interestingly, the PPAR␤⫺/⫺ mice also exhibit a keratinocyte hyperproliferation in response to diverse stimuli (Peters et al., 2000; Michalik et al., 2001), and there is a delayed differentiation of these mutated cells in response to TNF-␣ (Tan et al., 2001). Consistent with these observations, we show here that a PPAR␤ agonist can potentiate the activation of the transcription factor NF-␬B in a PI3K-dependent manner. Whereas we cannot exclude that the increase in transactivation is also due to enhanced phosphorylation of nuclear NF-␬B (Silverman and Maniatis, 2001), we observed that PPAR␤⫺/⫺ keratinocytes, in response to TNF-␣, have reduced NF-␬B DNA binding and transactivation activities. As suggested by the phenotype of transgenic and knockout mice whose NF-␬B cascade is altered (Gerondakis et al., 1999), the observed decrease of NF-␬B activity in PPAR␤⫺/⫺ cells may be responsible for the delayed initiation of the differentiation process and cell cycle withdrawal. NF-␬B is also a critical transcription factor necessary for the expression of genes involved in inflammation and in cell migration, such as MMPs. MMPs are extracellular proteases whose activities influence diverse physiological and pathological processes, including wound repair (Sternlicht and Werb, 2001). In skin, MMP-9 is restricted to migrating keratinocytes at the margins of the healing wound. The reduced production of MMP-9 observed in PPAR␤⫺/⫺ cells might provoke a stalemate in cell migration during wound healing, which is consistent with the defective wound repair that we have characterized in PPAR␤ mutant mice (Michalik et al., 2001). Reduced Activation of the Akt1 Cascade Contributes to the Phenotype of PPAR␤ Mutant Keratinocytes For efficient wound healing, the balance between proliferation and apoptosis has to be tightly controlled.

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Figure 6. PPAR␤ Potentiates NF-␬B Activity in Response to TNF-␣ in Primary Keratinocytes (A) PPAR␤ potentiates NF-␬B transactivation in the presence of TNF-␣. PPAR␤⫹/⫹ keratinocytes were transiently transfected with a NF-␬B responsive luciferase reporter construct and treated with the indicated ligand and/or inhibitors (PPAR␤ ligand [LD, 1 ␮M], PI3K inhibitor LY294002 [LY, 50 ␮M], and NF-␬B inhibitor [PDTC, 300 ␮M]) for 12 hr prior to the exposure to 1 ng/ml TNF-␣ for 6 hr. The fold induction in treated versus untreated cells was calculated after normalization to ␤-galactosidase activity. Values represent means of at least three independent experiments. (B) PPAR␤ increases NF-␬B activity through an Akt1-dependent pathway. Left panel: PPAR␤⫹/⫹ and ⫺/⫺ keratinocytes were cotransfected with the NF-␬B reporter plasmid and indicated constructs. Twenty-four hours after transfection, cells were treated for 6 hr with 1 ng/ml TNF-␣.

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Figure 7. Model for the Antiapoptotic Role of PPAR␤ in Keratinocytes Following stimulation by TNF-␣, PPAR␤ directly upregulates ILK and PDK1 and downregulates PTEN, leading to the activation of Akt1 in a PI3K-dependent manner. In response to this activation, the activity of several of its targets, including Bad, FKHR, and NF-␬B, is modified, leading to the inhibition of apoptosis and changes in cell adhesion/ migration. Dotted lines represent a modification at the transcriptional level, and continuous lines represent a modification of the protein activity. Inhibitors of PI3K (LY294002) and NF-␬B (PDTC) are indicated.

Wound repair requires activation of keratinocytes. Triggered by cytokines released from injured cells and inflammatory cells, keratinocytes become migratory and hyperproliferative, finally covering the wound with a neoepithelium. We previously showed that inflammatory cytokines, such as TNF-␣, induce PPAR␤ expression (Tan et al., 2001). This induced PPAR␤ expression accelerates keratinocyte differentiation and protects the cells from cytokine-induced apoptosis during inflammation. In concordance, the delayed wound closure observed in PPAR␤⫹/⫺ mice was due to both defective migration of the activated keratinocytes and increased apoptosis in the PPAR␤⫹/⫺ skin (Tan et al., 2001). The present study shows that PPAR␤ mediates its antiapoptotic effect via down- and upregulation of PTEN and ILK/PDK1 expression, respectively, which modulate Akt1 activity. Thus,

this study provides a molecular explanation, at least valid in keratinocytes, for the contrasting effects of TNF-␣ that triggers both pro- and antiapoptotic responses. In addition to the reduced MMP-9 expression discussed above, it also provides an additional molecular mechanism for the delayed wound closure in PPAR␤ mutant mice (Michalik et al., 2001). TNF-␣ exposure can also trigger PPAR␤ ligand production, as we previously demonstrated (Tan et al., 2001), possibly through an increased expression of COX-2 via the activation of the NF-␬B pathway. Interestingly, COX-2 suppresses apoptosis in human cancer cells and, in view of our current result on the antiapoptotic role of PPAR␤, it is conceivable that some of the antiapoptotic roles of COX-2 may be mediated via PPAR␤. Our results showed that, in keratinocytes, PPAR␤ is

Right panel: HaCaT cells stably transfected with caAkt1 (see Figure 4D, top of right panel) were transfected with the NF-␬B reporter plasmid and treated as described above. caAkt1 expression mimics the results obtained with PPAR␤ activation. (C) PPAR␤ enhances NF-␬B DNA binding activity. NF-␬B DNA binding was assessed by EMSA using a NF-␬B response element and nuclear extracts prepared from PPAR␤⫹/⫹ or ⫺/⫺ cells treated for 6 hr with 1 ␮M of LD and/or 50 ␮M LY, prior to exposure to 1 ng/ml of TNF-␣ for 30 min. The first and second lanes contain free probe alone and extracts from cells not treated with TNF-␣ (control), respectively. Specificity was confirmed by competition with the indicated molar excess of unlabeled NF-␬B probe. Arrows indicate the specific complexes between NF-␬B and the labeled probe. (D) PPAR␤ increases MMP-9 production. PPAR␤⫹/⫹ and ⫺/⫺ cells were treated as indicated in (C), and conditioned media were analyzed by gelatin zymography. Loading was normalized to equal total cellular proteins. The clear band of apparent molecular mass ⵑ92 kDa against a Coomassie blue stained background indicates the presence of MMP-9 gelatinase activity.

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important for the proper activation of the Akt1 pathway. It is thus not surprising that PPAR␤⫺/⫺ and Akt1⫺/⫺ mice share many similar manifestations. Both mutant mice display embryonic lethality with partial penetrance and growth retardation, and exhibit no diabetic phenotype (Peters et al., 2000; Cho et al., 2001). Furthermore, both PPAR␤⫺/⫺ and Akt1⫺/⫺ cells are more susceptible to apoptosis induced by TNF-␣, GF deprivation, and anoikis (herein and Chen et al., 2001). Our data also highlight the possibility that aberrant PPAR␤ expression may promote cell growth and accelerate cancer metastasis, consistent with a recently identified role of Akt1 signaling in mouse skin tumorigenesis (Segrelles et al., 2002). Finally, our results show a close interplay between PPAR␤ and Akt1 activation in keratinocytes. This study may give some clues about the mechanisms underlying the fascinating paradoxes observed during wound repair in the PPAR␤⫺/⫺ mice, such as increased proliferation versus apoptosis and hyperproliferative cells versus delayed wound closure. Experimental Procedures Reagents The PI3K inhibitor LY294002 was from Cell Signaling. TNF-␣ was from CalBiochem. 32P orthophosphate was from Amersham, 3H-PIP2 from NEN, and Silica Gel 60 plates from MERCK. Cell culture media and supplies were obtained from Gibco, BRL. Fetal calf serum (FCS, ⬍10 EU/ml) and PDTC were from Sigma. All antibodies were from Cell Signaling, except anti-ILK, anti-PPAR␤ (sc-7197X), FITC-conjugated IgG secondary antibody (Santa Cruz Biotechnology), monoclonal anti-PDK1 (Transduction Laboratories), anti-HA-tag (Clontech), and anti-acetylated histone H4 (Upstate Biotechnology). Plasmid Constructs cDNAs encoding mouse PPAR␤ (mPPAR␤), human RXR␣ (hRXR␣), hAkt1, mPDK1, mILK, and mPTEN were subcloned into pSG5 (Stratagene) or a modified pcDNA3.1 (Invitrogen) expression vector. The plasmid pcDNAred contains the red fluorescent protein cDNA (pDsRed1; Clontech) driven by a CMV promoter subcloned into pcDNA3.1. mPTEN was also subcloned in fusion with an epitope tag into pCMV-HA (Clontech). The kinase-dead (kd) mILK (E356K), mPDK1 (K114A), dominant-negative (dn) mPTEN (C124S), dn hAkt1 (K179A), and constitutively active (ca) hAkt1 (T308D, S473D) constructs were created by using the QuikChange mutagenesis kit (Stratagene). The hILK promoter/intronic regions and the hPDK1 promoter were subcloned into pGL2 luciferase vector (Promega). NF-␬B responsive reporter construct was obtained by cloning six copies of NF-␬B elements into the pGL2-Promoter. Cell Culture and Transfection Assays Mouse primary keratinocytes were isolated and cultured as described in Tan et al. (2001). HaCaT cells were maintained in DMEM supplemented with 10% FCS and 5 ␮g/ml gentamycin. Stable HaCaT cell lines expressing ILK, PDK1, or caAkt1 into pcDNA3.1 vector were selected with 600 ␮g/ml and maintained with 500 ␮g/ml G418. Transient transfection assays were performed using Superfect reagent (Qiagen) and luciferase activity (Promega) measured according to the manufacturers’ instructions. ␤-galactosidase activity was used for normalization. RNase Protection Assay (RPA) Gene-specific probes for mFAK (nucleotides 2828–3339 from the start codon ATG), mPTEN (244–449), mPDK1 (1657–1871), mILK (1194–1424), mAkt1 (1125–1446), and mL27 (157–356) were subcloned into pGEM3Zf(⫹) (Promega). Antisense riboprobes were synthesized by in vitro transcription with either T7 or Sp6 RNA polymerase. A ratio of 1:1 ␣32-P-UTP to cold UTP was used for all riboprobes, except for L27 (1:20) and PTEN (1:0). RPA was carried out as described in Tan et al. (2001).

Western Blot and Immunofluorescence Assays Cells or tissues were lysed in ice-cold lysis buffer (20 mM Na2H2PO4, 250 mM NaCl, Triton-100 1%, SDS 0.1%) supplemented with Complete protease inhibitors (Roche). Equal amounts of protein extracts were resolved by SDS-PAGE and electrotransferred onto PVDF membranes. Membranes were processed as described by the manufacturer (Cell Signaling) and detected by chemiluminescence (Pierce). Equal loading/transfer was verified by Coomassie staining of blots. For immunofluorescence, keratinocytes were fixed with 100% methanol. Staining was performed as described by the manufacturer (Cell Signaling). Akt1 Kinase Assay and Measure of Cellular PIP3/PIP2 In vitro kinase assay was done as described by the manufacturer (Cell Signaling) except that 1 ⫻ 106 cells and 1 ␮g of GST fused to GSK-3␣/␤ were used as substrates. Bands were quantified by densitometry and normalized to substrate loading. Control PIP3 and PIP3/PIP2 levels in PPAR␤⫹/⫹ and ⫺/⫺ were done according to Ptasznik et al. (1996). Radioactive spots corresponding to PIP3 and PIP2 were isolated and counted. Chromatin Immunoprecipitation (chIP) chIP was performed as described by the manufacturer (Upstate Biotechnology) with some modifications. Cells were fixed with 1% formaldehyde at 37⬚C for 15 min. Crosslinked DNA was sonicated to fragments ranging from 200–500 bp in length. Reverse crosslink of DNA fragments was achieved at 65⬚C for 6 hr. The DNA was subsequently purified using Qiaquick column (Qiagen). PCR was performed using 4 ␮l of DNA for 20–23 cycles, depending on the product to be amplified. chIP was done using PPAR␤ and acetylated histone H4-specific antibodies. Electrophoretic Mobility Shift Assay (EMSA) The PPRE studies were done using in vitro transcribed and translated (TNT, Promega) receptors and 32P end-labeled oligonucleotides (20,000 cpm) in 20 ␮l of binding buffer (20 mM HEPES [pH 7.5], 50 mM KCl, 0.1 mM EDTA, 5 mM MgCl2, 5% glycerol, 1 mM DTT, and 250 ␮g/ml poly dIdC). NF-␬B studies were done using 10 ␮g of cell nuclear extract and 32P end-labeled oligonucleotides in 20 ␮l of binding buffer (10 mM HEPES [pH 7.9], 50 mM KCl, 0.1 mM EDTA, 10% glycerol, 2.5 mM DTT, 0.05% NP40, and 500 ␮g/ml poly dIdC). For competition, unlabeled ACoA PPRE or NF-␬B oligonucleotide was added at the indicated molar excess. Gelatin Zymography Conditioned media of cells treated under different indicated conditions were used for gelatin zymography. Samples were resolved on a 10% nonreducing SDS-PAGE containing 0.1% gelatin and subsequently developed as described in the Ready Gels applications guide (Bio-Rad). Loading was normalized to equal total cellular proteins and ␤-galactosidase. Anoikis and Apoptotic Assays Anoikis was induced according to Jost et al. (1999) except that serum-free MEM was used. Apoptotic cells were stained using ApoAlert Annexin V-EGFP apoptosis kit (Clontech) and monitored by fluorescence microscopy. Acknowledgments We thank Nathalie Deriaz for valuable technical help, Prof. J. Tschopp (University of Lausanne, Epalinges) and Prof. S. Werner (Institute of Cell Biology, ETH Zurich) for helpful discussions, and Prof. N. Noy (Cornell University, New York) for critical reading of this manuscript. We thank Prof. R. Parsons (Columbia University, New York) and J.R. Woodgett (Ontario Cancer Institute, Toronto) for the PTEN and the Akt1 cDNAs, respectively. This work was supported by grants from Swiss National Science Foundation, Etat de Vaud, and Human Frontier Science Program to B.D. and W.W. Received: January 28, 2002 Revised: August 21, 2002

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