Regulation of Cell Proliferation and Differentiation by PPARbeta/delta.

Peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) is a ligand-activated transcription factor with essential functions in the regulation of lipid catabolism, glucose homeostasis, and inflammation, which makes it a potentially relevant drug target for the treatment of major human diseases. In addition, there is strong evidence that PPARbeta/delta modulates oncogenic signaling pathways and tumor growth. Consistent with these observations, numerous reports have clearly documented a role for PPARbeta/delta in cell cycle control, differentiation, and apoptosis. However, the precise role of PPARbeta/delta in tumorigenesis and cell proliferation remains controversial. This review summarizes our current knowledge and proposes a model corroborating the discrepant data in this area of research.

Ligand-mediated regulation of peroxisome proliferator-activated receptor (PPAR) beta/delta: a comparative analysis of PPAR-selective agonists and all-trans retinoic acid.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that modulate target gene expression in response to natural fatty acid ligands and synthetic agonists. It is noteworthy that all trans-retinoic acid (atRA) has recently been reported to act as a ligand for PPARbeta/delta, to activate its transcriptional activity, and, in contrast to the "classic" function of atRA, to stimulate cell proliferation (Schug et al., 2007). Here, we report that in contrast to synthetic PPARbeta/delta agonists, atRA failed to induce the transcriptional activity of PPARbeta/delta using different types of reporter gene assays. Likewise, atRA did not affect the expression of the bona fide PPARbeta/delta target genes ADRP and ANGPTL4 but strongly increased expression of the retinoic acid target gene CYP26A under the identical experimental conditions. Consistent with these observations, atRA did not compete with established PPARbeta/delta agonists in a ligand binding assay, and atRA did not enable the interaction of PPARbeta/delta with a coactivator peptide in a time-resolved fluorescence resonance energy transfer assay in vitro. These results are in sharp contrast to the effect of established PPARbeta/delta agonists in both in vitro assays. Taken as a whole, these data strongly suggest that atRA does not function as a ligand of PPARbeta/delta in any of the experimental systems tested and that the previously reported atRA effects are more likely to reflect an uncharacterized and less direct mechanism.

Expression level and agonist-binding affect the turnover, ubiquitination and complex formation of peroxisome proliferator activated receptor beta.

Peroxisome proliferator-activated receptors (PPARs) are members of the nuclear hormone receptor superfamily that modulate target gene expression in response to fatty acid ligands. Their regulation by post-translational modifications has been reported but is poorly understood. In the present study, we investigated whether ligand binding affects the turnover and ubiquitination of the PPARbeta subtype (also known as PPARdelta). Our data show that the ubiquitination and degradation of PPARbeta is not significantly influenced by the synthetic agonist GW501516 under conditions of moderate PPARbeta expression. By contrast, the overexpression of PPARbeta dramatically enhanced its degradation concomitant with its polyubiquitination and the formation of high molecular mass complexes containing multiple, presumably oligomerized PPARbeta molecules that lacked stoichiometical amounts of the obligatory PPARbeta dimerization partner, retinoid X receptor. The formation of these apparently aberrant complexes, as well as the ubiquitination and destabilization of PPARbeta, were strongly inhibited by GW501516. Our findings suggest that PPARbeta is subject to complex post-translational regulatory mechanisms that partly may serve to safeguard the cell against deregulated PPARbeta expression. Furthermore, our data have important implications regarding the widespread use of overexpression systems to evaluate the function and regulation of PPARs.

Three members of the human pyruvate dehydrogenase kinase gene family are direct targets of the peroxisome proliferator-activated receptor beta/delta.

The nuclear receptors peroxisome proliferator-activated receptors (PPARs) are known for their critical role in the metabolic syndrome. Here, we show that they are direct regulators of the family of pyruvate dehydrogenase kinase (PDK) genes, whose products act as metabolic homeostats in sensing hunger and satiety levels in key metabolic tissues by modulating the activity of the pyruvate dehydrogenase complex. Mis-regulation of this tightly controlled network may lead to hyperglycemia. In human embryonal kidney cells we found the mRNA expression of PDK2, PDK3 and PDK4 to be under direct primary control of PPAR ligands, and in normal mouse kidney tissue Pdk2 and Pdk4 are PPAR targets. Both, treatment of HEK cells with PPARbeta/delta-specific siRNA and the genetic disruption of the Pparbeta/delta gene in mouse fibroblasts resulted in reduced expression of Pdk genes and abolition of induction by PPARbeta/delta ligands. These findings suggest that PPARbeta/delta is a key regulator of PDK genes, in particular the PDK4/Pdk4 gene. In silico analysis of the human PDK genes revealed two candidate PPAR response elements in the PDK2 gene, five in the PDK3 gene and two in the PDK4 gene, but none in the PDK1 gene. For seven of these sites we could demonstrate both PPARbeta/delta ligand responsiveness in context of their chromatin region and simultaneous association of PPARbeta/delta with its functional partner proteins, such as retinoidXreceptor, co-activator and mediator proteins and phosphorylated RNA polymerase II. In conclusion, PDK2, PDK3 and PDK4 are primary PPARbeta/delta target genes in humans underlining the importance of the receptor in the control of metabolism.

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.

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.

Induction of PPARbeta and prostacyclin (PGI2) synthesis by Raf signaling: failure of PGI2 to activate PPARbeta.

A role for the nuclear receptor peroxisome proliferator-activated receptor-beta (PPARbeta) in oncogenesis has been suggested by a number of observations but its precise role remains elusive. Prostaglandin I2 (PGI2, prostacyclin), a major arachidonic acid (AA) derived cyclooxygenase (Cox) product, has been proposed as a PPARbeta agonist. Here, we show that the 4-hydroxytamoxifen (4-OHT) mediated activation of a C-Raf-estrogen receptor fusion protein leads to the induction of both the PPARbeta and Cox-2 genes, concomitant with a dramatic increase in PGI2 synthesis. Surprisingly, however, 4-OHT failed to activate PPARbeta transcriptional activity, indicating that PGI2 is insufficient for PPARbeta activation. In agreement with this conclusion, the overexpression of ectopic Cox-2 and PGI2 synthase (PGIS) resulted in massive PGI2 synthesis but did not activate the transcriptional activity of PPARbeta. Conversely, inhibition of PGIS blocked PGI2 synthesis but did not affect the AA mediated activation of PPARbeta. Our data obtained with four different cell types and different experimental strategies do not support the prevailing opinion that PGI2 plays a significant role in the regulation of PPARbeta.