Michael G. Borland

Ligand Activation of Peroxisome Proliferator-Activated Receptor-β/δ Inhibits Cell Proliferation in Human HaCaT Keratinocytes

Although there is strong evidence that ligand activation of peroxisome proliferator-activated receptor (PPAR)-β/δ induces terminal differentiation and attenuates cell growth, some studies suggest that PPARβ/δ actually enhances cell proliferation. For example, it was suggested recently that retinoic acid (RA) is a ligand for PPARβ/δ and potentiates cell proliferation by activating PPARβ/δ. The present study examined the effect of ligand activation of PPARβ/δ on cell proliferation, cell cycle kinetics, and target gene expression in human HaCaT keratinocytes using two highly specific PPARβ/δ ligands [4-[[[2-[3-fluoro-4-(trifluoromethyl)phenyl]-4-methyl-5-thiazolyl]methyl]thio]-2-methylphenoxy acetic acid (GW0742) and 2-methyl-4-((4-methyl-2-(4-trifluoromethylphenyl)-1,3-thiazol-5-yl)-methylsulfanyl)phenoxy-acetic acid (GW501516)] and RA. Both PPARβ/δ ligands and RA inhibited cell proliferation of HaCaT keratinocytes. GW0742 and GW501516 increased expression of known PPARβ/δ target genes, whereas RA did not; RA increased the expression of known retinoic acid receptor/retinoid X receptor target genes, whereas GW0742 did not affect these genes. GW0742, GW501516, and RA did not modulate the expression of 3-phosphoinositide-dependent protein kinase or alter protein kinase B phosphorylation. GW0742 and RA increased annexin V staining as quantitatively determined by flow cytometry. The effects of GW0742 and RA were also examined in wild-type and PPARβ/δ-null primary mouse keratinocytes to determine the specific role of PPARβ/δ in modulating cell growth. Although inhibition of keratinocyte proliferation by GW0742 was PPARβ/δ-dependent, inhibition of cell proliferation by RA occurred in both genotypes. Results from these studies demonstrate that ligand activation of PPARβ/δ inhibits keratinocyte proliferation through PPARβ/δ-dependent mechanisms. In contrast, the observed inhibition of cell proliferation in mouse and human keratinocytes by RA is mediated by PPARβ/δ-independent mechanisms and is inconsistent with the notion that RA potentiates cell proliferation by activating PPARβ/δ.

Cellular and Pharmacological Selectivity of the Peroxisome Proliferator-Activated Receptor-β/δ Antagonist GSK3787

The availability of high-affinity agonists for peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) has led to significant advances in our understanding of the functional role of PPARβ/δ. In this study, a new PPARβ/δ antagonist, 4-chloro-N-(2-{[5-trifluoromethyl)-2-pyridyl]sulfonyl}ethyl)benzamide (GSK3787), was characterized using in vivo and in vitro models. Orally administered GSK3787 caused antagonism of 4-[2-(3-fluoro-4-trifluoromethyl-phenyl)-4-methyl-thiazol-5-ylmethylsulfanyl]-2-methyl-phenoxy}-acetic acid (GW0742)-induced up-regulation of Angptl4 and Adrp mRNA expression in wild-type mouse colon but not in Pparβ/δ-null mouse colon. Chromatin immunoprecipitation (ChIP) analysis indicates that this correlated with reduced promoter occupancy of PPARβ/δ on the Angptl4 and Adrp genes. Reporter assays demonstrated antagonism of PPARβ/δ activity and weak antagonism and agonism of PPARγ activity but no effect on PPARα activity. Time-resolved fluorescence resonance energy transfer assays confirmed the ability of GSK3787 to modulate the association of both PPARβ/δ and PPARγ coregulator peptides in response to ligand activation, consistent with reporter assays. In vivo and in vitro analysis indicates that the efficacy of GSK3787 to modulate PPARγ activity is markedly lower than the efficacy of GSK3787 to act as a PPARβ/δ antagonist. GSK3787 antagonized GW0742-induced expression of Angptl4 in mouse fibroblasts, mouse keratinocytes, and human cancer cell lines. Cell proliferation was unchanged in response to either GW0742 or GSK3787 in human cancer cell lines. Results from these studies demonstrate that GSK3787 can antagonize PPARβ/δ in vivo, thus providing a new strategy to delineate the functional role of a receptor with great potential as a therapeutic target for the treatment and prevention of disease.

PPARβ/δ Activation Induces Enteroendocrine L Cell GLP-1 Production

BACKGROUND & AIMS Glucagon-like peptide (GLP)-1, an intestinal incretin produced by L cells through proglucagon processing, is secreted after nutrient ingestion and acts on endocrine pancreas beta cells to enhance insulin secretion. Peroxisome proliferator-activated receptor (PPAR) β/δ is a nuclear receptor that improves glucose homeostasis and pancreas islet function in diabetic animal models. Here, we investigated whether PPARβ/δ activation regulates L cell GLP-1 production. METHODS Proglucagon regulation and GLP-1 release were evaluated in murine GLUTag and human NCI-H716 L cells and in vivo using wild-type, PPARβ/δ-null, and ob/ob C57Bl/6 mice treated with the PPARβ/δ synthetic agonists GW501516 or GW0742. RESULTS PPARβ/δ activation increased proglucagon expression and enhanced glucose- and bile acid-induced GLP-1 release by intestinal L cells in vitro and ex vivo in human jejunum. In vivo treatment with GW0742 increased proglucagon messenger RNA levels in the small intestine in wild-type but not in PPARβ/δ-deficient mice. Treatment of wild-type and ob/ob mice with GW501516 enhanced the increase in plasma GLP-1 level after an oral glucose load and improved glucose tolerance. Concomitantly, proglucagon and GLP-1 receptor messenger RNA levels increased in the small intestine and pancreas, respectively. Finally, PPARβ/δ agonists activate the proglucagon gene transcription by interfering with the β-catenin/TCF-4 pathway. CONCLUSIONS Our data show that PPARβ/δ activation potentiates GLP-1 production by the small intestine. Pharmacologic targeting of PPARβ/δ is a promising approach in the treatment of patients with type 2 diabetes mellitus, especially in combination with dipeptidyl peptidase IV inhibitors.

Analysis of the peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) cistrome reveals novel co-regulatory role of ATF4

BackgroundThe present study coupled expression profiling with chromatin immunoprecipitation sequencing (ChIP-seq) to examine peroxisome proliferator-activated receptor-β/δ (PPARβ/δ)-dependent regulation of gene expression in mouse keratinocytes, a cell type that expresses PPARβ/δ in high concentration.ResultsMicroarray analysis elucidated eight different types of regulation that modulated PPARβ/δ-dependent gene expression of 612 genes ranging from repression or activation without an exogenous ligand, repression or activation with an exogenous ligand, or a combination of these effects. Bioinformatic analysis of ChIP-seq data demonstrated promoter occupancy of PPARβ/δ for some of these genes, and also identified the presence of other transcription factor binding sites in close proximity to PPARβ/δ bound to chromatin. For some types of regulation, ATF4 is required for ligand-dependent induction of PPARβ/δ target genes.ConclusionsPPARβ/δ regulates constitutive expression of genes in keratinocytes, thus suggesting the presence of one or more endogenous ligands. The diversity in the types of gene regulation carried out by PPARβ/δ is consistent with dynamic binding and interactions with chromatin and indicates the presence of complex regulatory networks in cells expressing high levels of this nuclear receptor such as keratinocytes. Results from these studies are the first to demonstrate that differences in DNA binding of other transcription factors can directly influence the transcriptional activity of PPARβ/δ.

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

Peroxisome proliferator-activated receptor-β/δ (PPARβ/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 PPARβ/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 PPARβ/δ or PPARγ. Over-expression of PPARβ/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 PPARβ/δ. While PPARβ/δ-dependent attenuation of staurosporine- or UVB-induced poly (ADP-ribose) polymerase (PARP) cleavage was not observed, PPARβ/δ- and PPARγ-dependent repression of UVB-induced expression and secretion of inflammatory cytokines was found in HaCaT keratinocytes over-expressing PPARβ/δ or PPARγ. These studies suggest that FABP5 does not transport atRA or GW0742 to PPARβ/δ and promote anti-apoptotic activity by increasing expression of PDPK1, or that PPARβ/δ interferes with PPARγ transcriptional activity. However, these studies demonstrate that stable over-expression of PPARβ/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.

Effect of ligand activation of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) in human lung cancer cell lines

There is compelling evidence that peroxisome proliferator-activated receptor-beta/delta (PPARbeta/delta) mediates terminal differentiation and is associated with inhibition of cell growth. However, it was recently suggested that growth of two human lung cancer cell lines is enhanced by PPARbeta/delta. The goal of the present study was to provide insight in resolving this controversy. Therefore, the effect of ligand activation of PPARbeta/delta in A549 and H1838 human lung cancer cell lines was examined using two high affinity PPARbeta/delta ligands. Ligand activation of PPARbeta/delta caused up-regulation of a known PPARbeta/delta target gene, angiopoietin-like 4 (Angptl4) but did not influence expression of phosphatase and tensin homolog (PTEN) or phosphorylation of protein kinase B (Akt), and did not affect cell growth. Results from this study demonstrate that two human lung cancer cell lines respond to ligand activation of PPARbeta/delta by modulation of target gene expression (Angptl4), but fail to exhibit significant modulation of cell proliferation.

Functional characterization of peroxisome proliferator‐activated receptor‐β/δ expression in colon cancer

This study critically examined the role of PPARβ/δ in colon cancer models. Expression of PPARβ/δ mRNA and protein was lower and expression of CYCLIN D1 protein higher in human colon adenocarcinomas compared to matched non‐transformed tissue. Similar results were observed in colon tumors from Apc+/Min‐FCCC mice compared to control tissue. Dietary administration of sulindac to Apc+/Min‐FCCC mice had no influence on expression of PPARβ/δ in normal colon tissue or colon tumors. Cleaved poly (ADP‐ribose) polymerase (PARP) was either increased or unchanged, while expression of 14‐3‐3ε was not influenced in human colon cancer cell lines cultured with the PPARβ/δ ligand GW0742 under conditions known to increase apoptosis. While DLD1 cells exhibited fewer early apoptotic cells after ligand activation of PPARβ/δ following treatment with hydrogen peroxide, this change was associated with an increase in late apoptotic/necrotic cells, but not an increase in viable cells. Stable over‐expression of PPARβ/δ in human colon cancer cell lines enhanced ligand activation of PPARβ/δ and inhibition of clonogenicity in HT29 cells. These studies are the most quantitative to date to demonstrate that expression of PPARβ/δ is lower in human and Apc+/Min‐FCCC mouse colon tumors than in corresponding normal tissue, consistent with the finding that increasing expression and activation of PPARβ/δ in human colon cancer cell lines inhibits clonogenicity. Because ligand‐induced attenuation of early apoptosis can be associated with more late, apoptotic/necrotic cells, but not more viable cells, these studies illustrate why more comprehensive analysis of PPARβ/δ‐dependent modulation of apoptosis is required in the future.

Modulation of aryl hydrocarbon receptor (AHR)-dependent signaling by peroxisome proliferator-activated receptor β/δ (PPARβ/δ) in keratinocytes

Whether peroxisome proliferator-activated receptor β/δ (PPARβ/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 Pparβ/δ-null skin and keratinocytes. Pparβ/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 PPARβ/δ promotes demethylation of the Cyp1a1 promoter. Experiments with human HaCaT keratinocytes stably expressing shRNA against PPARβ/δ also support this conclusion. Consistent with the lower AHR-dependent activities in Pparβ/δ-null mice compared with wild-type mice, 7,12-dimethylbenz[a]anthracene (DMBA)-induced skin tumorigenesis was inhibited in Pparβ/δ-null mice compared with wild-type. Results from these studies demonstrate that PPARβ/δ is required to mediate complete carcinogenesis by DMBA. The mechanisms underlying this PPARβ/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 PPARβ/δ-dependent demethylation of AHR target gene promoters including Cyp1a1 that reduces AHR accessibility as shown by reduced promoter occupancy. This PPARβ/δ/AHR crosstalk is unique to keratinocytes and conserved between mice and humans.

Editor’s Highlight: PPARβ/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-β/δ (PPARβ/δ) and PPARγ on cell proliferation, anchorage-dependent clonogenicity, and ectopic xenografts in the UACC903 human melanoma cell line. Stable overexpression of either PPARβ/δ or PPARγ enhanced ligand-induced expression of a PPARβ/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 PPARβ/δ, or vice versa. Stable overexpression of either PPARβ/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 PPARβ/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 PPARβ/δ or PPARγ and/or ligand activation of either PPARβ/δ or PPARγ inhibited cell proliferation, and anchorage-dependent clonogenicity of UACC903 cell lines as compared with controls. Further, overexpression of either PPARβ/δ or PPARγ and/or ligand activation of either PPARβ/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 PPARβ/δ and PPARγ and suggest that targeting these receptors may be useful for primary or secondary melanoma chemoprevention.

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-β/δ (PPARβ/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 PPARβ/δ or PPARγ in A431 cells expressing these receptors. PPARβ/δ expression blocked the cell cycle at the G2/M phase, and this effect was increased by ligand activation. Ligand activation of PPARβ/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 PPARβ/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 PPARβ/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, PPARβ/δ or PPARγ could be therapeutically targeted for the treatment of squamous cell carcinomas.