Modulation of aryl hydrocarbon receptor (AHR)-dependent signaling by peroxisome proliferator-activated receptor ß/δ (PPARß/δ) in keratinocytes.

Whether peroxisome proliferator-activated receptor ß/δ (PPARß/δ) reduces skin tumorigenesis by altering aryl hydrocarbon receptor (AHR)-dependent activities was examined. Polycyclic aromatic hydrocarbons (PAH) increased expression of cytochrome P4501A1 (CYP1A1), CYP1B1 and phase II xenobiotic metabolizing enzymes in wild-type skin and keratinocytes. Surprisingly, this effect was not found in Pparß/δ-null skin and keratinocytes. Pparß/δ-null keratinocytes exhibited decreased AHR occupancy and histone acetylation on the Cyp1a1 promoter in response to a PAH compared with wild-type keratinocytes. Bisulfite sequencing of the Cyp1a1 promoter and studies using a DNA methylation inhibitor suggest that PPARß/δ promotes demethylation of the Cyp1a1 promoter. Experiments with human HaCaT keratinocytes stably expressing shRNA against PPARß/δ also support this conclusion. Consistent with the lower AHR-dependent activities in Pparß/δ-null mice compared with wild-type mice, 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin tumorigenesis was inhibited in Pparß/δ-null mice compared with wild-type. Results from these studies demonstrate that PPARß/δ is required to mediate complete carcinogenesis by DMBA. The mechanisms underlying this PPARß/δ-dependent reduction of AHR signaling by PAH are not due to alterations in the expression of AHR auxiliary proteins, ligand binding or AHR nuclear translocation between genotypes, but are likely influenced by PPARß/δ-dependent demethylation of AHR target gene promoters including Cyp1a1 that reduces AHR accessibility as shown by reduced promoter occupancy. This PPARß/δ/AHR crosstalk is unique to keratinocytes and conserved between mice and humans.

Inhibition of tumorigenesis by peroxisome proliferator-activated receptor (PPAR)-dependent cell cycle blocks in human skin carcinoma cells.

To examine the functional role of peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) and PPARγ in skin cancer, stable cell lines were created in the A431 human squamous cell carcinoma cell line. Expression of PPAR target genes was greatly enhanced in response to ligand activation of PPARß/δ or PPARγ in A431 cells expressing these receptors. PPARß/δ expression blocked the cell cycle at the G2/M phase, and this effect was increased by ligand activation. Ligand activation of PPARß/δ markedly inhibited clonogenicity as compared to vehicle-treated controls. Similarly, ligand activation of PPARγ in A431 cells expressing PPARγ resulted in reduced clonogenicity. Expression of either PPARß/δ or PPARγ markedly reduced tumor volume in ectopic xenografts, while ligand activation of these receptors had little further influence on tumor volume. Collectively, these studies demonstrate that stable expression and activation of PPARß/δ or PPARγ in A431 cells led to reduced tumorigenicity. Importantly, PPAR expression or ligand activation had major impacts on clonogenicity and/or tumor volume. Thus, PPARß/δ or PPARγ could be therapeutically targeted for the treatment of squamous cell carcinomas.

Editor's Highlight: PPARß/δ and PPARγ Inhibit Melanoma Tumorigenicity by Modulating Inflammation and Apoptosis.

Skin tumorigenesis results from DNA damage, increased inflammation, and evasion of apoptosis. The peroxisome proliferator-activated receptors (PPARs) can modulate these mechanisms in non-melanoma skin cancer. However, limited data exists regarding the role of PPARs in melanoma. This study examined the effect of proliferator-activated receptor-ß/δ (PPARß/δ) and PPARγ on cell proliferation, anchorage-dependent clonogenicity, and ectopic xenografts in the UACC903 human melanoma cell line. Stable overexpression of either PPARß/δ or PPARγ enhanced ligand-induced expression of a PPARß/δ/PPARγ target gene in UACC903 cell lines as compared with controls. The induction of target gene expression by ligand activation of PPARγ was not altered by overexpression of PPARß/δ, or vice versa. Stable overexpression of either PPARß/δ or PPARγ reduced the percentage of cells in the G1 and S phase of the cell cycle, and increased the percentage of cells in the G2/M phase of the cell cycle in UACC903 cell lines as compared with controls. Ligand activation of PPARß/δ did not further alter the distribution of cells within each phase of the cell cycle. By contrast, ligand activation of PPARγ enhanced these changes in stable UACC903 cells overexpressing PPARγ compared with controls. Stable overexpression of either PPARß/δ or PPARγ and/or ligand activation of either PPARß/δ or PPARγ inhibited cell proliferation, and anchorage-dependent clonogenicity of UACC903 cell lines as compared with controls. Further, overexpression of either PPARß/δ or PPARγ and/or ligand activation of either PPARß/δ or PPARγ inhibited ectopic xenograft tumorigenicity derived from UACC903 melanoma cells as compared with controls, and this was likely due in part to induction of apoptosis. Results from these studies demonstrate the antitumorigenic effects of both PPARß/δ and PPARγ and suggest that targeting these receptors may be useful for primary or secondary melanoma chemoprevention.

A species difference in the peroxisome proliferator-activated receptor α-dependent response to the developmental effects of perfluorooctanoic acid.

This study examined the effect of prenatal perfluorooctanoic acid (PFOA) administration on pre- and postnatal development using peroxisome proliferator-activated receptor α (PPARα)-humanized mice to determine if species differences in receptor activity might influence the developmental effects induced by PFOA. Pregnant mice were treated daily with water or PFOA (3mg/kg) by po gavage from gestation day 1 (GD1) until GD17 and then either euthanized on GD18 or allowed to give birth and then euthanized on postnatal day 20 (PND20). No changes in average fetal weight, crown-to-rump length, or placental weight were observed on GD18. Expression of mRNA encoding the PPARα target genes acyl CoA oxidase (Acox1) and cytochrome P450 4a10 (Cyp4a10) in maternal and fetal liver was increased on GD18 in wild-type and PPARα-humanized mice but not in Pparα-null mice. On PND20, relative liver weight was higher in wild-type mice but not in Pparα-null mice or PPARα-humanized mice. Hepatic expression of Acox1 and Cyp4a10 mRNA was higher in wild-type mice but not in Pparα-null mice or PPARα-humanized mice on PND20. The percentage of mice surviving postnatally was lower in wild-type litters but not in litters from Pparα-null mice or PPARα-humanized mice. No changes in pup weight gain, onset of eye opening, or mammary gland development were found in any genotype. Results from these studies demonstrate that the developmental/postnatal effects resulting from prenatal PFOA exposure in mice are differentially mediated by mouse and human PPARα.

Stable over-expression of PPARß/δ and PPARγ to examine receptor signaling in human HaCaT keratinocytes.

Peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) function and receptor cross-talk with other nuclear receptors, including PPARγ and retinoic acid receptors (RARs), was examined using stable human HaCaT keratinocyte cell lines over-expressing PPARß/δ or PPARγ. Enhanced ligand-induced expression of two known PPAR target genes, adipocyte differentiation-related protein (ADRP) and angiopoietin-like protein 4 (ANGPTL4), was found in HaCaT keratinocytes over-expressing PPARß/δ or PPARγ. Over-expression of PPARß/δ did not modulate the effect of a PPARγ agonist on up-regulation of ADRP or ANGPTL4 mRNA in HaCaT keratinocytes. All-trans retinoic acid (atRA) increased expression of a known RAR target gene, yet despite a high ratio of fatty acid binding protein 5 (FABP5) to cellular retinoic acid binding protein II, did not increase expression of ANGPTL4 or 3-phosphoinositide-dependent-protein kinase 1 (PDPK1), even in HaCaT keratinocytes expressing markedly higher levels of PPARß/δ. While PPARß/δ-dependent attenuation of staurosporine- or UVB-induced poly (ADP-ribose) polymerase (PARP) cleavage was not observed, PPARß/δ- and PPARγ-dependent repression of UVB-induced expression and secretion of inflammatory cytokines was found in HaCaT keratinocytes over-expressing PPARß/δ or PPARγ. These studies suggest that FABP5 does not transport atRA or GW0742 to PPARß/δ and promote anti-apoptotic activity by increasing expression of PDPK1, or that PPARß/δ interferes with PPARγ transcriptional activity. However, these studies demonstrate that stable over-expression of PPARß/δ or PPARγ significantly increases the efficacy of ligand activation and represses UVB-induced expression of tumor necrosis factor α (TNFα), interleukin 6 (IL6), or IL8 in HaCaT keratinocytes, thereby establishing an excellent model to study the functional role of these receptors in human keratinocytes.