Reverse crosstalk of TGFß and PPARß/δ signaling identified by transcriptional profiling.

Previous work has provided strong evidence for a role of peroxisome proliferator-activated receptor ß/δ (PPARß/δ) and transforming growth factor-ß (TGFß) in inflammation and tumor stroma function, raising the possibility that both signaling pathways are interconnected. We have addressed this hypothesis by microarray analyses of human diploid fibroblasts induced to myofibroblastic differentiation, which revealed a substantial, mostly reverse crosstalk of both pathways and identified distinct classes of genes. A major class encompasses classical PPAR target genes, including ANGPTL4, CPT1A, ADRP and PDK4. These genes are repressed by TGFß, which is counteracted by PPARß/δ activation. This is mediated, at least in part, by the TGFß-induced recruitment of the corepressor SMRT to PPAR response elements, and its release by PPARß/δ ligands, indicating that TGFß and PPARß/δ signals are integrated by chromatin-associated complexes. A second class represents TGFß-induced genes that are downregulated by PPARß/δ agonists, exemplified by CD274 and IL6, which is consistent with the anti-inflammatory properties of PPARß/δ ligands. Finally, cooperative regulation by both ligands was observed for a minor group of genes, including several regulators of cell proliferation. These observations indicate that PPARß/δ is able to influence the expression of distinct sets of both TGFß-repressed and TGFß-activated genes in both directions.

High-affinity peroxisome proliferator-activated receptor ß/δ-specific ligands with pure antagonistic or inverse agonistic properties.

Peroxisome proliferator-activated receptor ß/δ (PPARß/δ) is a ligand-regulated nuclear receptor with essential functions in metabolism and inflammation. We have synthesized a new derivative [methyl 3-(N-(4-(hexylamino)-2-methoxyphenyl)sulfamoyl)thiophene-2-carboxylate (ST247) structurally related to the published PPARß/δ inhibitory ligand methyl 3-(N-(2-methoxy-4-(phenylamino)phenyl)sulfamoyl)thiophene-2-carboxylate (GSK0660). ST247 has a higher affinity to PPARß/δ than GSK0660, and at equimolar concentrations, it more efficiently 1) induces the interaction with corepressors both in vitro and in vivo, 2) inhibits the agonist-induced transcriptional activity of PPARß/δ, and 3) down-regulates basal level expression of the peroxisome proliferator responsive element-driven PPARß/δ target gene ANGPTL4. Methyl 3-(N-(4-(tert-butylamino)-2-methoxyphenyl)sulfamoyl)thiophene-2-carboxylate (PT-S58), another high-affinity derivative from our series, also efficiently inhibits agonist-induced transcriptional activation, but in contrast to ST247, it does not enhance the interaction of PPARß/δ with corepressors. PT-S58 rather prevents corepressor recruitment triggered by the inverse agonist ST247. These findings classify ST247 as an inverse agonist, whereas PT-S58 is the first pure PPARß/δ antagonist described to date. It is noteworthy that ST247 and PT-S58 are also effective on PPRE-independent functions of PPARß/δ: in monocytic cells, both ligands modulate expression of the activation marker CCL2 in the opposite direction as an established PPARß/δ agonist. The possibility to differentially modulate specific functions of PPARß/δ makes these novel compounds invaluable tools to advance our understanding of PPARß/δ biology.

Transcriptional profiling identifies functional interactions of TGF ß and PPAR ß/δ signaling: synergistic induction of ANGPTL4 transcription.

Peroxisome proliferator-activated receptors (PPARs) not only play a key role in regulating metabolic pathways but also modulate inflammatory processes, pointing to a functional interaction between PPAR and cytokine signaling pathways. In this study, we show by genome-wide transcriptional profiling that PPARß/δ and transforming growth factor-ß (TGFß) pathways functionally interact in human myofibroblasts and that a subset of these genes is cooperatively activated by TGFß and PPARß/δ. Using the angiopoietin-like 4 (ANGPTL4) gene as a model, we demonstrate that two enhancer regions cooperate to mediate the observed synergistic response. A TGFß-responsive enhancer located ∼8 kb upstream of the transcriptional start site is regulated by a mechanism involving SMAD3, ETS1, RUNX, and AP-1 transcription factors that interact with multiple contiguous binding sites. A second enhancer (PPAR-E) consisting of three juxtaposed PPAR response elements is located in the third intron ∼3.5 kb downstream of the transcriptional start site. The PPAR-E is strongly activated by all three PPAR subtypes, with a novel type of PPAR response element motif playing a central role. Although the PPAR-E is not regulated by TGFß, it interacts with SMAD3, ETS1, RUNX2, and AP-1 in vivo, providing a possible mechanistic explanation for the observed synergism.

15-hydroxyeicosatetraenoic acid is a preferential peroxisome proliferator-activated receptor beta/delta agonist.

Peroxisome proliferator-activated receptor (PPARs) modulate target gene expression in response to unsaturated fatty acid ligands, such as arachidonic acid (AA). Here, we report that the AA metabolite 15-hydroxyeicosatetraenoic acid (15-HETE) activates the ligand-dependent activation domain (AF2) of PPARbeta/delta in vivo, competes with synthetic agonists in a PPARbeta/delta ligand binding assay in vitro, and triggers the interaction of PPARbeta/delta with coactivator peptides. These agonistic effects were also seen with PPARalpha and PPARgamma, but to a significantly weaker extent. We further show that 15-HETE strongly induces the expression of the bona fide PPAR target gene Angptl4 in a PPARbeta/delta-dependent manner and, conversely, that inhibition of 15-HETE synthesis reduces PPARbeta/delta transcriptional activity. Consistent with its function as an agonistic ligand, 15-HETE triggers profound changes in chromatin-associated PPARbeta/delta complexes in vivo, including the recruitment of the coactivator cAMP response element-binding protein binding protein. Both 15R-HETE and 15S-HETE are similarly potent at inducing PPARbeta/delta coactivator binding and transcriptional activation, indicating that 15-HETE enantiomers generated by different pathways function as PPARbeta/delta agonists.

Specific components of prostanoid-signaling pathways are present in non-small cell lung cancer cells.

In the present study, we measured prostanoid synthesis and the expression of genes associated with prostanoid signaling in human non-small cell lung carcinoma (NSCLC) cell lines and in primary human tumors. Consistent with the proposed growth promoting role of PGE2, we found that NSCLC cell lines frequently co-expressed the genes encoding cyclooxygenase-2 and the prostaglandin E2 (PGE2) receptors EP1, 2 and 4 concomitant with the synthesis of PGE2. In contrast, NSCLC cells did not synthesize appreciable amounts of prostaglandin I2 (PGI2, prostacyclin), lacked PGI2 synthase (PGIS) and did not express the gene coding for the PGI2 receptor IP at detectable levels. In agreement with this finding, PGIS mRNA levels were dramatically diminished in primary human tumor samples as compared to matched normal lung tissue. Finally, thromboxane A2 (TxA2) was synthesized in NSCLC cell lines, but transcription of the gene coding for the TxA2 receptor TP was not observed in these cells. In marked contrast, lung fibroblasts synthesized all three prostanoids and their receptors at high levels. While the observed expression patterns were consistent with the existence of autocrine/paracrine PGE2 signaling loops in NSCLC cells, PGI2- and TxA2-mediated signals may play a role in tumor stroma cells.

Regulation of TAK1/TAB1-mediated IL-1ß signaling by cytoplasmic PPARß/δ.

The peroxisome proliferator-activated receptor subtypes PPARα, PPARß/δ, PPARγ are members of the steroid hormone receptor superfamily with well-established functions in transcriptional regulation. Here, we describe an unexpected cytoplasmic function of PPARß/δ. Silencing of PPARß/δ expression interferes with the expression of a large subset of interleukin-1ß (IL-1ß)-induced target genes in HeLa cells, which is preceded by an inhibition of the IL-1ß-induced phosphorylation of TAK1 and its downstream effectors, including the NFκBα inhibitor IκBα (NFKBIA) and the NFκBα subunit p65 (RELA). PPARß/δ enhances the interaction between TAK1 and the small heat-shock protein HSP27, a known positive modulator of TAK1-mediated IL-1ß signaling. Consistent with these findings, PPARß/δ physically interacts with both the endogenous cytoplasmic TAK1/TAB1 complex and HSP27, and PPARß/δ overexpression increases the TAK1-induced transcriptional activity of NFκB. These observations suggest that PPARß/δ plays a role in the assembly of a cytoplasmic multi-protein complex containing TAK1, TAB1, HSP27 and PPARß/δ, and thereby participates in the NFκB response to IL-1ß.

Genomewide analyses define different modes of transcriptional regulation by peroxisome proliferator-activated receptor-ß/δ (PPARß/δ).

Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors with essential functions in lipid, glucose and energy homeostasis, cell differentiation, inflammation and metabolic disorders, and represent important drug targets. PPARs heterodimerize with retinoid X receptors (RXRs) and can form transcriptional activator or repressor complexes at specific DNA elements (PPREs). It is believed that the decision between repression and activation is generally governed by a ligand-mediated switch. We have performed genomewide analyses of agonist-treated and PPARß/δ-depleted human myofibroblasts to test this hypothesis and to identify global principles of PPARß/δ-mediated gene regulation. Chromatin immunoprecipitation sequencing (ChIP-Seq) of PPARß/δ, H3K4me3 and RNA polymerase II enrichment sites combined with transcriptional profiling enabled the definition of 112 bona fide PPARß/δ target genes showing either of three distinct types of transcriptional response: (I) ligand-independent repression by PPARß/δ; (II) ligand-induced activation and/or derepression by PPARß/δ; and (III) ligand-independent activation by PPARß/δ. These data identify PPRE-mediated repression as a major mechanism of transcriptional regulation by PPARß/δ, but, unexpectedly, also show that only a subset of repressed genes are activated by a ligand-mediated switch. Our results also suggest that the type of transcriptional response by a given target gene is connected to the structure of its associated PPRE(s) and the biological function of its encoded protein. These observations have important implications for understanding the regulatory PPAR network and PPARß/δ ligand-based drugs.

Proteomic profile of mouse fibroblasts with a targeted disruption of the peroxisome proliferator activated receptor-beta/delta gene.

The peroxisome proliferator activated receptor-beta (PPARbeta) plays an essential role in lipid metabolism, immune modulation, differentiation and cell proliferation. There is also strong evidence for a function in oncogenesis and tumor vascularization, but the underlying molecular mechanisms remain elusive. In the present study, we have used fibroblasts derived from Pparb wild-type and null mice to determine by 2-DE and PMF analysis the contribution of PPARbeta to the protein profile of fibroblasts. Thirty-one differentially expressed proteins of different functional categories were identified. For at least two proteins a role in tumorigenesis and/or tumor vascularization has previously been reported: while the calcium intracellular channel-4 (CLIC4) was expressed at lower levels in Pparb null cells, expression of the cellular retinol binding protein 1 (CRBP1) was enhanced. Clic4 and Crbp1 gene expression patterns observed in different experimental settings in vitro and in vivo confirmed the proteomics data. Our findings indicate that the expression of a defined set of proteins is altered in fibroblasts and endothelial cells from Pparb null mice, that this is due to aberrant gene regulation, and that the altered expression of these proteins is consistent with the tumor vascularization phenotype of Pparb null mice.

Deregulation of tumor angiogenesis and blockade of tumor growth in PPARbeta-deficient mice.

The peroxisome proliferator-activated receptor-beta (PPARbeta) has been implicated in tumorigenesis, but its precise role remains unclear. Here, we show that the growth of syngeneic Pparb wild-type tumors is impaired in Pparb(-/-) mice, concomitant with a diminished blood flow and an abundance of hyperplastic microvascular structures. Matrigel plugs containing pro-angiogenic growth factors harbor increased numbers of morphologically immature, proliferating endothelial cells in Pparb(-/-) mice, and retroviral transduction of Pparb triggers microvessel maturation. We have identified the Cdkn1c gene encoding the cell cycle inhibitor p57(Kip2) as a PPARbeta target gene and a mediator of the PPARbeta-mediated inhibition of cell proliferation, which provides a possible mechanistic explanation for the observed tumor endothelial hyperplasia and deregulation of tumor angiogenesis in Pparb(-/-) mice. Our data point to an unexpected essential role for PPARbeta in constraining tumor endothelial cell proliferation to allow for the formation of functional tumor microvessels.