Transcriptional Repression of Peroxisome Proliferator-activated Receptor β/δ in Murine Keratinocytes by CCAAT/Enhancer-binding Proteins*

The roles of peroxisome proliferator-activated receptors (PPARs) and CCAAT/enhancer-binding proteins (C/EBPs) in keratinocyte and sebocyte differentiation suggest that both families of transcription factors closely interact in the skin. Initial characterization of the mouse PPARβ promoter revealed an AP-1 site that is crucial for the regulation of PPARβ expression in response to inflammatory cytokines in the skin. We now present evidence for a novel regulatory mechanism of the expression of the PPARβ gene by which two members of the C/EBP family of transcription factors inhibit its basal promoter activity in mouse keratinocytes. We first demonstrate that C/EBPα and C/EBPβ, but not C/EBPδ, inhibit the expression of PPARβ through the recruitment of a transcriptional repressor complex containing HDAC-1 to a specific C/EBP binding site on the PPARβ promoter. Consistent with this repression, the expression patterns of PPARβ and C/EBPs are mutually exclusive in keratinocytes of the interfollicular epidermis and hair follicles in mouse developing skin. This work reveals the importance of the regulatory interplay between PPARβ and C/EBP transcription factors in the control of proliferation and differentiation in this organ. Such insights are crucial for the understanding of the molecular control regulating the balance between proliferation and differentiation in many cell types including keratinocytes.

The roles of peroxisome proliferator-activated receptors (PPARs) and CCAAT/enhancer-binding proteins (C/EBPs) in keratinocyte and sebocyte differentiation suggest that both families of transcription factors closely interact in the skin. Initial characterization of the mouse PPAR␤ promoter revealed an AP-1 site that is crucial for the regulation of PPAR␤ expression in response to inflammatory cytokines in the skin. We now present evidence for a novel regulatory mechanism of the expression of the PPAR␤ gene by which two members of the C/EBP family of transcription factors inhibit its basal promoter activity in mouse keratinocytes. We first demonstrate that C/EBP␣ and C/EBP␤, but not C/EBP␦, inhibit the expression of PPAR␤ through the recruitment of a transcriptional repressor complex containing HDAC-1 to a specific C/EBP binding site on the PPAR␤ promoter. Consistent with this repression, the expression patterns of PPAR␤ and C/EBPs are mutually exclusive in keratinocytes of the interfollicular epidermis and hair follicles in mouse developing skin. This work reveals the importance of the regulatory interplay between PPAR␤ and C/EBP transcription factors in the control of proliferation and differentiation in this organ. Such insights are crucial for the understanding of the molecular control regulating the balance between proliferation and differentiation in many cell types including keratinocytes.
Peroxisome proliferator-activated receptors (PPARs) 3 are ligand-activated transcription factors that belong to the nuclear hormone receptor family. Three isotypes encoded by separate genes have been identified in vertebrates, PPAR␣ (NR1C1), PPAR␤/␦ (NR1C2; called ␤ below), and PPAR␥ (NR1C3), which have a variety of functions (1). Most particularly, the importance of PPARs in regulating lipid metabolism (2) has led to investigate PPAR expression and function during the differentiation of the skin, a tissue with high rates of fatty acid and cholesterol metabolism. We previously showed that the three PPAR isotypes, and predominantly PPAR␤, are expressed in the interfollicular epidermis and hair follicles during embryonic mouse development (3).
In adult skin, PPAR␤ is very low in interfollicular keratinocytes, but its expression is reactivated upon proliferative stimuli such as cutaneous injury and hair plucking (4). Using specific PPAR agonists and in vivo gene disruption approaches in mice, we demonstrated the critical role of PPAR␤ in regulating the balance between proliferation and apoptosis in keratinocytes during skin wound healing (4,5), as well as during postnatal hair follicle development (6), through direct activation of the antiapoptotic phosphatidylinositol 3-kinase/Akt1 signaling pathway (7,8). Furthermore, PPAR␤ was reported to keratinocyte differentiation and epidermal permeability maturation under normal and inflammatory conditions (3,5).
CCAAT/enhancer-binding proteins (C/EBPs) are members of the basic leucine zipper family of transcription factors and also play pivotal roles in the regulation of human and mouse skin homeostasis. Genes coding for six C/EBP isotypes (C/EBP␣, C/EBP␤, C/EBP␦, C/EBP␥, C/EBP⑀, and C/EBP or CHOP-10) have been cloned and characterized in mammalian cells (9). All C/EBP⑀ consist of three structural domains: a C-terminal leucine zipper domain, a highly conserved canonical basic region, and an N-terminal domain, which contains both positive and negative regulatory regions (10). The basic region allows binding to specific palindromic CCAAT motifs located in the promoter of C/EBP target genes (11), whereas the leucine zipper motif is responsible for homo-and heterodimerization between C/EBP members, which is absolutely required for DNA binding as well as transcriptional activity. C/EBPs also interact with many other basic leucine zipper and nonbasic leucine zipper factors such as NF-B, p21, and activator protein-1 (AP-1) (9). They control the transcription of many key genes, either as transcriptional activators or repressors, demonstrating the importance of this family of transcription factors in the regulation of a number of cellular processes, including energy metabolism, inflammation, and liver regeneration (9,10). In addition, they are well known regulators of the balance between differentiation and proliferation in various cell types like adipocytes, keratinocytes, granulocytes, and hepatocytes (9,12). In the skin, C/EBP␣ and C/EBP␤ are the most abundantly expressed isotypes in keratinocytes from the interfollicular and follicular epidermis (13)(14)(15)(16). Prodifferentiative and antiproliferative functions of these two proteins have been characterized in keratinocytes, on the basis of their expression profiles and presence of binding sites in the promoter region of genes encoding early and late keratinocyte differentiation markers, such as keratin 10 (K10), K1, and involucrin (13)(14)(15)(16)(17)(18)(19). In accordance, analysis of C/EBP␤-null epidermis revealed a mild epidermal hyperplasia and slightly decreased expression of epidermal differentiation markers (15). Finally, it has been recently reported that C/EBP␣ and C/EBP␤ have a specific expression pattern during the mouse hair growth cycle, suggesting that C/EBP family members may also regulate gene expression during hair cycling (16) and sebocyte differentiation (20).
The important roles of PPARs and C/EBPs identified in skin strongly suggest that both families of transcription factors may closely interact to regulate the epidermal and/or hair follicle differentiation program. In favor of this hypothesis, an interplay between PPARs and C/EBPs was observed during the differentiation of adipocytes (21), sebocytes (20), and astrocytes (22). In this study, we provide compelling evidence for the transcriptional repression in mouse keratinocytes of PPAR␤ expression by C/EBP␣ and C/EBP␤, through a mechanism that requires both binding to DNA and histone deacetylation. Consistent with this, PPAR␤ and C/EBP expression are mutually exclusive in the interfollicular epidermis and hair follicles of mouse skin. Such interplay between PPAR␤ and C/EBP transcription factors is crucial in the molecular control of the balance between differentiation and proliferation in keratinocytes and many other cell types.
Cell Culture and Transient Transfections-Mouse BALB/MK keratinocytes were grown in Eagle's minimum essential medium (Joklik modification) containing 10% dialyzed fetal calf serum, 0.05 mM CaCl 2 , 10 ng/ml epidermal growth factor, and 5 g/ml gentamycin. Transient transfection assays were performed in 12-well plates using Superfect reagent (Qiagen), and luciferase activity was measured with the Promega dual reporter kit, according to the manufacturer's instructions. To reduce the background, all transfections were performed in the absence of fetal calf serum.
In Vivo Protein-Protein Cross-link and Chromatin Immunoprecipitation (ChIP)-Protein-protein cross-link and ChIP were performed as previously described (8) with some modifications. Cells were fixed with 1% formaldehyde at 37°C for 15 min before sonication in lysis buffer (10 mM EDTA, 1% SDS, 50 mM Tris-HCl, pH 8.1, protease inhibitor mixture (Roche Applied Science)) to obtain cross-linked DNA fragments of 200 -600 bp in length. The immunoprecipitates were reverse crosslinked for PCR or boiled for 5 min in SDS loading buffer for Western blot analysis. For double chromatin immunoprecipitation (re-ChIP) assays, complexes were eluted from the agarose beads by sequential incubation with one volume of Immunopure Gentle Ag/Ab elution buffer (Pierce), followed by one volume of the same buffer containing 0.1 mM dithiothreitol. The eluates were pooled, diluted 20-fold in re-ChIP dilution buffer (1 mM EDTA, 50 mM NaCl, 1% Triton X-100, 20 mM Tris-HCl, pH 8.1), and subjected to another ChIP procedure. ChIP and re-ChIP assays were done using anti-acetyl-histone H4, HA-probe, and/or HDAC-1 C-19 antibodies. PCR was performed using 25 cycles with primers flanking either the C/EBP response element (Ϫ567 to Ϫ284) or an unrelated control sequence (Ϫ1179 to Ϫ894) on the mouse PPAR␤ promoter.
Western Blot Assays-Western blots were performed according to standard procedures, as previously described (6). Primary antibodies against HDAC-1, HDAC-2, and ␤-tubulin were used at dilutions of 1:2000, and the anti-HA antibody was diluted 1:1000. Detection was performed using chemiluminescence (Pierce) with horseradish peroxidase.

PPAR␤, C/EBP␣, and C/EBP␤ Are Highly Expressed in the Developing
Mouse Skin-The important roles of PPAR and C/EBP transcription factors in regulating overlapping pathways in keratinocytes strongly suggest that both families of transcription factors closely interact to regulate the differentiation program of the skin. In favor of this hypothesis, similar expression patterns of PPARs and C/EBPs were previously reported during sebocyte differentiation in this organ (20). Therefore, we first performed a detailed analysis of the expression of PPAR␤ and of the two main C/EBP isotypes, C/EBP␣ and C/EBP␤, in the developing mouse skin from postnatal day 1 to 7 (P1-P7) by immunohistochemistry ( Fig. 1). As we previously described (6), the PPAR␤ protein is highly expressed in the basal layer keratinocytes of the interfollicular epidermis as well as in the proliferative epithelial compartments of developing hair follicles, including hair pegs and hair matrix (Fig. 1A). On the contrary, and consistent with previous reports (13,14), C/EBP␣ is mainly expressed in the nuclei of keratinocytes located in all of the suprabasal layers of the interfollicular epidermis, whereas C/EBP␤ is found in low amounts in some basal keratinocytes and strongly increases in the lower suprabasal layers ( Fig. 1B; P1). In hair follicles, PPAR␤ and C/EBPs strictly colocalize in the developing sebaceous glands ( Fig. 1; P4 -P7), consistent with previous observations made in adult skin (16) and cultured sebocytes (20). Intense C/EBP␣ expression was also specifically detected in the nuclei of dermal papilla fibroblasts, from early hair follicle stages (P1) to mature follicles (P7), whereas C/EBP␤ was found restricted to the outer root sheath keratinocytes. A weak staining of C/EBP␤ was also observed in hair shaft precursors (precortex region) of differentiating hair follicles ( Fig. 1B; P7). However, in contrast to PPAR␤ expression, neither C/EBP␣ nor C/EBP␤ expression was detectable in the highly proliferative epithelial compartments of developing hair follicles, including hair peg (P1) and hair matrix (P4 -P7) keratinocytes.
Altogether, these results indicate that the expression of PPAR␤ and C/EBPs are mutually exclusive in mouse keratinocytes in the interfollicular epidermis as well as in the differentiating hair follicles, suggesting a negative interplay in keratinocytes between the two families of transcription factors during skin development.
C/EBPs Modulate PPAR␤ Expression in Mouse Keratinocytes-To assess whether C/EBPs could directly modulate PPAR␤ expression in keratinocytes, mouse BALB/MK cells were transiently co-transfected with C/EBP-expressing vectors and a luciferase reporter construct containing the proximal promoter of the mouse PPAR␤ gene. This 1.88-kb PPAR␤ promoter region used herein was characterized in our laboratory. It contains sequences related to binding sites for AP-1, Ets, and C/EBP transcription factors (5).
Overexpression in BALB/MK cells of either C/EBP␣ or C/EBP␤ repressed basal PPAR␤ promoter activity by 2-3-fold in a dose-dependent manner, whereas expression of C/EBP␦ had no significant effect ( Fig. 2A). Consistent with these observations, transfection of C/EBP␣ and C/EBP␤ in mouse keratinocytes resulted in a decrease of endogenous PPAR␤ mRNA levels, as evaluated by real time PCR (Fig. 2B, left). In similar conditions, PPAR␥ expression was increased, with C/EBP␣ and C/EBP␦ having a greater effect than C/EBP␤ (Fig. 2B, right). As a control of the functionality of the transfected C/EBPs, we used the proximal murine promoter regions of the genes for keratin 10 (K10) and cyclooxygenase-2, which both contain multiple functional C/EBP-binding sites (13,25). As previously described in other cell types, a strong activation of the K10 promoter by both C/EBP␣ and C/EBP␤ was observed in BALB/MK cells (Fig. 2C, left) (13), whereas the cyclooxygenase-2 promoter activity, in agreement with a previous report, was only increased by C/EBP␦ (Fig. 2C, right) (25).
To further identify the region in the mouse PPAR␤ promoter that mediates the inhibition described above, the consequence of C/EBP␣ and C/EBP␤ overexpression on the activity of a series of truncations of In the developing hair follicles, specific expression of C/EBP␣ was observed in the dermal papilla fibroblasts (DP), whereas C/EBP␤ was mainly found in the outer root sheath (ORS) cells as well as in the precortex region (Px). In contrast to PPAR␤ expression, no C/EBP staining was observed in hair peg (HP) and hair matrix (HM) keratinocytes. Cell nuclei were counterstained with 4Ј,6Ј-diamidino-2-phenylindole (blue). The epidermal-dermal junction is indicated by dashed lines. Magnification bars, 50 m. the proximal PPAR␤ promoter was analyzed in BALB/MK cells. As shown in Fig. 3A, C/EBP␣ and C/EBP␤ still inhibited the activity of the promoter constructs PPAR␤(Ϫ846) and PPAR␤(Ϫ587). In contrast, the shorter promoter constructs PPAR␤(Ϫ445) and PPAR␤(Ϫ223) were not responsive to C/EBP␣ or C/EBP␤ overexpression, suggesting that the region mediating inhibition by C/EBPs is located between nucleotides Ϫ587 and Ϫ445 of the PPAR␤ promoter. Sequence analysis of this region revealed the presence of a putative C/EBP response element between nucleotides Ϫ494 and Ϫ485, as well as putative binding sites for Oct1 and AP-1 transcription factors (Fig. 3B). To determine whether C/EBP-mediated inhibition of PPAR␤ expression requires this newly identified C/EBP response element, we introduced mutations known to prevent recognition by C/EBP transcription factors (Fig. 3B) (11). Interestingly, disruption of the putative C/EBP response element in the PPAR␤(Ϫ587) reporter construct totally abrogated the inhibitory effect of both C/EBP␣ and C/EBP␤ (Fig. 3C), suggesting that the action of these factors on the activity of the PPAR␤ promoter depends on the occupancy of the C/EBP response element at position Ϫ494/Ϫ495. C/EBP␣ and C/EBP␤ Inhibit PPAR␤ Promoter Activity Independently of Their Repression Domains-C/EBP␣ and C/EBP␤ are known to inhibit the promoter activity of a number of genes in various cell types, although the molecular mechanisms of this repression have not been explored (26 -28). Intrinsic domains that negatively regulate transcrip-tional activity have been identified in C/EBPs (10), and the inhibitory functions of these domains have been attributed to a repression of DNA binding, transcriptional activation, and synergy with factors bound to adjacent promoter elements.
To determine the functional domains of C/EBP␣ and C/EBP␤ required for inhibition of the PPAR␤ promoter activity, several C/EBP mutant proteins tagged with hemagglutinin (HA) were transiently overexpressed in BALB/MK keratinocytes (Fig. 4A). These mutants were deleted of the repression domain and/or of the activation domains or carried single point mutations (KE) in the DNA-binding basic region (23) that prevent binding of C/EBPs to DNA, as confirmed using ChIP assays on the K10 promoter (Fig. S1A). All C/EBP mutants were expressed at comparable protein levels when transiently transfected in mouse keratinocytes (Fig. 4B). As expected, they all have lost their ability to transactivate the K10 promoter (supplemental Fig. S1B), which requires both binding to specific DNA response elements and transactivation activity (13). Interestingly, deletion that includes the repression domain of C/EBP␣ (C/EBP␣-⌬1) did not prevent its ability to inhibit PPAR␤ promoter activity (Fig. 4A), suggesting that C/EBP␣ uses an alternative mechanism to repress PPAR␤ transcription. Most importantly, further deletion of the region up to amino acids 170 -273 in the C/EBP␣ protein (C/EBP␣-⌬2) or deletion of the corresponding region  NOVEMBER 18, 2005 • VOLUME 280 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 38703 on C/EBP␤ protein (amino acids 115-211, C/EBP␤-⌬2) abrogated the inhibitory effects of C/EBPs on the PPAR␤ promoter (Fig. 4A). Similar results were obtained by mutating the basic domain of C/EBP␣ (C/EBP␣-KE) and C/EBP␤ (C/EBP␤-KE), suggesting that the inhibitory effect of C/EBPs on PPAR␤ promoter requires both an internal domain spanning residues 170 -273 for C/EBP␣ or residues 115-210 for C/EBP␤ as well as an intact DNA binding domain.

Interplay between PPAR␤/␦ and C/EBPs in Keratinocytes
To confirm that the inhibitory effect of C/EBP␣ and C/EBP␤ on the PPAR␤ promoter requires binding to DNA in vivo, chromatin immunoprecipitation assays were performed in BALB/MK cells transfected with the HA-tagged C/EBP␣ and C/EBP␤ mutants. DNA fragments encompassing the C/EBP response element or a random control sequence of the endogenous mouse PPAR␤ promoter were amplified by PCR in the immunoprecipitated chromatin with anti-HA antibodies. As shown in Fig. 5, the sequence spanning the C/EBP response element was significantly enriched compared with the control DNA fragment for all C/EBP mutants, except for the proteins with mutated DNA binding domain (KE). Together, these results demonstrate that C/EBP␣ and C/EBP␤ bind to the C/EBP response element identified at position Ϫ494/Ϫ495 in the PPAR␤ promoter and indicate that the internal domain of C/EBPs needed to repress the promoter activity of PPAR␤ (Fig. 4A) is not required for DNA binding.
C/EBP-mediated Inhibition of PPAR␤ Promoter Activity Involves Histone Deacetylation-Since histone acetylation/deacetylation is a key component in the regulation of gene expression, we next assessed the importance of this modification in the repression activity of C/EBPs on the PPAR␤ promoter. Transient transfections were performed in the presence of the most potent mammalian histone deacetylase inhibitor trichostatin A (29). As shown in Fig. 6A, the inhibitory effect of C/EBP␣ and C/EBP␤ on PPAR␤ promoter activity was largely abrogated in the presence of trichostatin A in a dose-dependent manner, suggesting that transcriptional repression by C/EBPs occurs via promoting histone deacetylation on the PPAR␤ promoter. In favor of this model, C/EBP␤ was recently reported to repress gene transcription in preadipocytes FIGURE 3. Identification of the PPAR␤ promoter region that mediates the C/EBP inhibitory response. A, mouse BALB/MK keratinocytes were co-transfected with a luciferase reporter construct containing or not containing (pGL-luc) various deletions of the proximal promoter of the PPAR␤ gene and with vectors expressing or not expressing (control) C/EBP␣ or C/EBP␤. Reporter plasmids (PPAR␤(Ϫ1880), PPAR␤(Ϫ846), PPAR␤(Ϫ587), PPAR␤(Ϫ445), and PPAR␤(Ϫ223)) are named according to the length of the promoter region they contain upstream of the transcription start site. The relative -fold induction was calculated after normalization to ␤-galactosidase activity, with the activity of the promoterless control construct (pGL-luc) set at 1. Values are means of three independent experiments. B, sequence analysis of the PPAR␤ promoter region between Ϫ587 and Ϫ445 revealed the presence of a putative C/EBP response element between nucleotides Ϫ494 and Ϫ485, as well as putative binding sites for Oct1 and AP-1 transcription factors. The sequence of the putative C/EBP response element identified in the PPAR␤ promoter (C/EBP) is aligned to the consensus C/EBP response element (consensus). Mutations introduced in this C/EBP response element are also given (C/EBP*). C, BALB/MK keratinocytes were co-transfected with the promoter construct PPAR␤(Ϫ587) containing unchanged (C/EBP) or mutated (C/EBP*) C/EBP response element and with vectors expressing or not expressing (control) C/EBP␣ or C/EBP␤. The relative -fold inductions were calculated after normalization to ␤-galactosidase activity, with the activity of the promoterless (pGL-luc) activity set at 1. Values are means of three independent experiments. through its direct association with a general transcription corepressor complex containing histone deacetylase-1 (HDAC-1) (30). To test whether C/EBPs and HDACs similarly interact with each other in keratinocytes in vivo, BALB/MK cells were transfected with the HA-tagged C/EBP mutants described above, and immunoprecipitations were carried out on cell extracts using anti-HA antibodies. The presence of class I HDACs interacting with the C/EBPs was detected by Western blot analysis (Fig. 6B). The results indicate that full-length C/EBP␣ and C/EBP␤ strongly interact with HDAC-1, but not HDAC-2, in mouse keratinocytes. Most importantly, C/EBP␣-⌬2 and C/EBP␤-⌬2 mutants, which failed to inhibit PPAR␤ promoter activity in transfection assays (Fig. 4A), also lost their interaction with HDAC-1, suggesting again that  NOVEMBER 18, 2005 • VOLUME 280 • NUMBER 46 JOURNAL OF BIOLOGICAL CHEMISTRY 38705 C/EBP-mediated inhibition of PPAR␤ expression most probably involves histone deacetylation.

Interplay between PPAR␤/␦ and C/EBPs in Keratinocytes
We next examined whether histone H4 is acetylated at the endogenous PPAR␤ promoter in keratinocytes transfected with the C/EBP mutants, using chromatin immunoprecipitation assays with antiacetyl-H4 antibodies. H4 acetylation was diminished in cells transfected with full-length C/EBP␣ and C/EBP␤, with C/EBP␣ having a greater effect than C/EBP␤, confirming that C/EBPs induce histone deacetylation, therefore decreasing PPAR␤ promoter activity (Fig. 7A). Most importantly, C/EBP mutants that lost their capacity to bind DNA (C/EBP-KE) or to interact with HDAC-1 (C/EBP-⌬2) also failed to decrease H4 acetylation. These results are consistent with transient transfection assays showing that the overexpression of these two mutants did not affect the basal PPAR␤ promoter activity (Fig. 4) and with the lack of interaction of this mutant with HDAC-1 (Fig. 6). To confirm that C/EBPs inhibit PPAR␤ promoter activity via recruitment of HDAC-1, we next performed two ChIP experiments (ChIP/re-ChIP) in mouse keratinocytes transfected with C/EBP␣ or C/EBP␤. As shown in Fig. 7B, PCR amplification of the PPAR␤ promoter region encompassing the C/EBP response element was observed following sequential immunoprecipitation with anti-HA and anti-HDAC-1 antibodies. No amplification was observed after a second immunoprecipitation with IgG immunoglobulins. Altogether, these results clearly demonstrate the formation of a transcriptional repressor complex between C/EBPs and HDAC-1 to a specific C/EBP binding site on mouse PPAR␤ promoter in keratinocytes.

DISCUSSION
The important roles of PPARs and C/EBPs in the regulation of similar pathways in the skin strongly suggest that both families of transcription factors closely interact to regulate keratinocyte proliferation and differentiation. We demonstrate here that C/EBP␣ and C/EBP␤ isotypes, but not C/EBP␦, inhibit the basal activity of the mouse PPAR␤ promoter in mouse keratinocytes through a mechanism that involves binding to a specific C/EBP response element and recruitment at this site of HDAC-1 that results in histone deacetylation. Most importantly, the expression of PPAR␤ and C/EBPs are mutually exclusive in keratinocytes from the interfollicular epidermis and hair follicles, suggesting an important role for C/EBP transcription factors in regulating the expression pattern of PPAR␤ during mouse skin development. Unveiling this interplay leads to a better understanding of the molecular control regulating the balance between differentiation and proliferation in kerati-nocytes, a balance that is impaired in pathologies such as psoriasis and skin cancers.
Molecular Mechanism for the C/EBP-mediated Inhibition of PPAR␤ Expression in Keratinocytes-Controlled transcriptional repression, like transcriptional activation, has emerged as a mechanism by which tissuespecific gene expression is regulated. Initial characterization of the mouse PPAR␤ promoter revealed the presence of an AP-1 site that is central for the regulation of PPAR␤ expression in response to inflammatory cytokines in mouse skin (5,24). In this study, we present evidence for a novel regulatory mechanism by which C/EBP␣ and C/EBP␤ inhibit basal PPAR␤ expression in keratinocytes. We have demonstrated that C/EBP␣ and C/EBP␤ bind to a C/EBP response element located in the proximal promoter of the mouse PPAR␤ gene and recruit a transcriptional repressor complex containing HDAC-1 but lacking HDAC-2. The inhibitory effect on PPAR␤ expression thus results from deacetylation of histone H4. Binding of C/EBP transcription factors to specific DNA response elements was already reported to be necessary for the inhibition of a number of other genes, including gonadotrophinreleasing hormone (31), liver X receptor-␣ (27), dentin sialophosphoprotein (32), inhibin ␣-subunit (33), and trefoil factor-1 (26). However, the underlying molecular mechanisms remained poorly characterized. The model reported here for the inhibition of PPAR␤ gene expression  NOVEMBER 18, 2005 • VOLUME 280 • NUMBER 46 through histone deacetylation might be a general mechanism by which C/EBPs inhibit gene transcription. In favor of this, C/EBP␤ was recently reported to inhibit gene transcription via a similar mode of action in preadipocytes (30).

Interplay between PPAR␤/␦ and C/EBPs in Keratinocytes
Many additional regulatory mechanisms may influence the effect of C/EBP␣ and C/EBP␤ on PPAR␤ promoter in vivo, including changes in the relative proportion of C/EBP homodimers and heterodimers. In this context, the C/EBP-homologous protein CHOP-10, also known as growth arrest and DNA damage-inducible gene 153, may be of interest. It is expressed in the interfollicular epidermis and hair follicles (13,16,34) and was shown to act as a dominant negative inhibitor of C/EBP␣ and C/EBP␤ by preventing their binding to DNA (35). Furthermore, post-translational modifications play a key role in the regulation of C/EBP functions in many cell types, by modulating their DNA binding activity, their activation or inhibition potential, and their intracellular localization (9). Finally, all three C/EBP␣, C/EBP␤, and HDAC-1 have the ability to participate in auto-and cross-regulation of their transcriptional expression (36 -38), and HDAC-1 may also regulate the transcriptional regulation activity of C/EBPs directly (30), thereby adding to the potential complexity of the regulation of the expression of PPAR␤ by C/EBPs.

PPAR␤ and C/EBP Expression Are Mutually Exclusive in Mouse
Keratinocytes-C/EBP␣ and C/EBP␤ are abundantly expressed in mouse and human skin in an organized manner along differentiation. Similar timing in the pattern of expression of C/EBPs is also seen during keratinocyte differentiation in culture. Indeed, C/EBP␤ is expressed during early keratinocyte differentiation in vitro (13,34) and appears in the lower differentiated layers of the epidermis. Accordingly, analysis of C/EBP␤-null epidermis revealed a mild epidermal hyperplasia and slight decreased expression of the early keratinocyte differentiation marker K10, without changes in the expression of late differentiation markers such as involucrin and loricrin (15). On the contrary, C/EBP␣ is expressed during late keratinocyte differentiation (13,34), and it appears in all of the suprabasal layers of the interfollicular epidermis in mouse skin. However, no phenotypic changes were reported in the skin of C/EBP␣-deficient mice, except for a substantial thinning of the subcutaneous fat layer (39). Interestingly, a similar differentiation program was observed in adipocytes, where high levels of C/EBP␤ are expressed in the early phase of preadipocyte differentiation, whereas C/EBP␣ maintains the adipocyte terminal differentiation program (21).
The expression patterns of PPAR␤ and C/EBPs are mutually exclusive in normal or pathologic conditions in skin. Indeed, PPAR␤ expression appears in the basal layer of the interfollicular epidermis in mouse skin (4, 6), whereas C/EBP␣ and C/EBP␤ were mainly found in the suprabasal layers. It is note worthy that ␣2and ␣5-integrins, whose expression is restricted to the basal cell layer of the epidermis, are also repressed by C/EBP␣ and C/EBP␤, C/EBP␣ being the more effective repressor in both cases (16). We observed a similar exclusion between PPAR␤ and C/EBPs in developing postnatal hair follicles, with high PPAR␤ expression levels in the proliferative follicular epithelial compartment (6), abundant C/EBP␣ in the dermal compartment, and some C/EBP␤ expression in the precortex region. Finally, the expression of C/EBP␣ was diminished in many cancers, including squamous cell carcinomas and colorectal cancers (12), whereas PPAR␤ expression levels are frequently increased in similar cancer models (40). Altogether, these data strongly suggest that PPAR␤ and C/EBPs may have antagonistic functions in keratinocytes and that C/EBPs participate or are responsible for establishing the PPAR expression pattern in mouse skin. or not expressing (ctr) the indicated C/EBP full-length or mutants were immunoprecipitated (ChIP) with either an anti-acetyl-histone H4 antibody (anti-AcH4) or control immunoglobulins (control IgG). Enrichment of the DNA fragment encompassing the C/EBP response element (Ϫ494/Ϫ485) on the endogenous mouse PPAR␤ promoter was evaluated by PCR. Aliquots of the chromatin were also analyzed before immunoprecipitation (input). B, complexes from BALB/MK cells transfected with vectors expressing or not expressing (ctr) C/EBP␣ (␣) or C/EBP␤ (␤) were first immunoprecipitated with an anti-HA tag antibody (anti-HA). A second immunoprecipitation step (re-ChIP) was then performed with either an anti-HDAC-1-specific antibody (H1) or control immunoglobulins (IgG). PCR amplification of the C/EBP response element on the PPAR␤ promoter is shown.
Importance of the Interplay between PPAR␤ and C/EBPs for the Differentiation of the Epidermis and Hair Follicles-Because less than 1% of C/EBP␣-null mice survive after birth, a detailed analysis of the skin phenotype of these mutant mice is difficult to perform (41). In addition, compensatory effects by other C/EBPs may mask hair follicle or other skin abnormalities in the C/EBP␣ knock-out mice. Indeed, an up-regulation in C/EBP␣ expression has been reported in keratinocytes of C/EBP␤-deficient mice. However, the absence of C/EBP␤ was not totally compensated, since these mice showed a skin phenotype characterized by a scruffy appearance of their coats (13). Detailed analysis of C/EBP␣ and C/EBP␤ expression performed in human and murine adult skin revealed that C/EBPs are expressed in a hair follicle cycling-dependent manner (16). Consistent with our results in the developing hair follicles, high levels of C/EBP␣ were observed in the fibroblasts of the dermal papilla in murine anagen hair follicle, whereas strong immunoreactivity of C/EBP␤ was detected in most outer root sheath cells in the distal hair follicle. The high expression of C/EBP␣ and C/EBP␤ in defined follicular compartments during hair follicle growth strongly suggests that the C/EBP family of transcription factors might be important regulators of hair follicle morphogenesis and cycling.
We previously demonstrated that PPAR␤ is required for normal postnatal hair follicle development, by regulating the balance between apoptosis and proliferation in follicular keratinocytes (6). The molecular mechanism involves direct activation of the antiapoptotic phosphatidylinositol 3-kinase/Akt1 signaling pathway. Interestingly, the same pathway was also reported to regulate the phosphorylation status of C/EBPs, hence their ability to regulate cell proliferation (12). Antimitotic activities of C/EBP␣ have been well documented in a variety of cell types and physiological situations (12), including in keratinocytes (15,19). Growth arrest induced by C/EBP␤ is highly cellular context-specific, but several observations indicate an antiproliferative function for this isotype in keratinocytes. It was shown that forced expression of C/EBP␤ in BALB/MK keratinocytes inhibits their growth, and mice lacking C/EBP␤ exhibit mild epidermal hyperplasia. C/EBP␤-deficient keratinocytes also display resistance to calcium-induced growth arrest, and C/EBP␤ expression is diminished in squamous cell carcinomas (14,15). In addition, C/EBP␤ was reported to regulate keratinocyte survival, which is required to complete the differentiation program in the skin (42). Thus, the negative interplay between PPAR␤ and C/EBPs presented in this study may explain, at least partially, the slight decrease in proliferation observed in PPAR␤-deficient follicular keratinocytes of developing hair follicles (6). Another possible explanation for the reduced proliferation of PPAR␤-mutant keratinocytes might come from the ability of C/EBP members to modulate the expression of the paracrine factor hepatocyte growth factor in mouse fibroblasts (43). Like C/EBP␣, hepatocyte growth factor is specifically expressed in the fibroblasts of the dermal papilla in the developing hair follicle (44), and we already demonstrated that hepatocyte growth factor stimulates PPAR␤ activity in mouse keratinocytes (6). Thus, it is important to note that, in addition to their ability to regulate the expression level of PPAR␤, C/EBPs might also indirectly modulate PPAR␤ activity in keratinocytes through the regulation of hepatocyte growth factor production within the follicular mesenchyme, allowing for normal hair follicle development.
In summary, this work demonstrates the importance of the regulatory interplay between PPAR␤ and C/EBP transcription factors in the molecular control of proliferation and differentiation in mouse keratinocytes. Furthermore, the ability of C/EBP␣ and C/EBP␤ to modulate PPAR␤ expression may have significant impact on the differentiation program in other cell types and subsequently on other processes where C/EBP transcription factors have been involved, including the regulation of liver and lung homeostasis.