Bokai Zhu

Quantitative expression patterns of peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) protein in mice

The expression patterns of PPARbeta/delta have been described, but the majority of these data are based on mRNA data. To date, there are no reports that have quantitatively examined the expression of PPARbeta/delta protein in mouse tissues. In the present study, a highly specific PPARbeta/delta antibody was developed, characterized, and used to examine tissue expression patterns of PPARbeta/delta. As compared to commercially available anti-PPARbeta/delta antibodies, one of six polyclonal anti-PPARbeta/delta antibodies developed was significantly more effective for immunoprecipitation of in vitro-translated PPARbeta/delta. This antibody was used for quantitative Western blot analysis using radioactive detection methods. Expression of PPARbeta/delta was highest in colon, small intestine, liver, and keratinocytes as compared to other tissues including heart, spleen, skeletal muscle, lung, brain, and thymus. Interestingly, PPARbeta/delta expression was localized in the nucleus and RXRalpha can be co-immunoprecipitated with nuclear PPARbeta/delta. Results from these studies demonstrate that PPARbeta/delta expression is highest in intestinal epithelium, liver, and keratinocytes, consistent with significant biological roles in these tissues.

Activation of Peroxisome Proliferator-Activated Receptor-β/δ (PPAR-β/δ) Inhibits Human Breast Cancer Cell Line Tumorigenicity

The effect of activation and overexpression of the nuclear receptor PPAR-β/δ in human MDA-MB-231 (estrogen receptor–negative; ER−) and MCF7 (estrogen-receptor-positive; ER+) breast cancer cell lines was examined. Target gene induction by ligand activation of PPAR-β/δ was increased by overexpression of PPAR-β/δ compared with controls. Overexpression of PPAR-β/δ caused a decrease in cell proliferation in MCF7 and MDA-MB-231 cells compared with controls, whereas ligand activation of PPAR-β/δ further inhibited proliferation of MCF7 but not MDA-MB-231 cells. Overexpression and/or ligand activation of PPAR-β/δ in MDA-MB-231 or MCF7 cells had no effect on experimental apoptosis. Decreased clonogenicity was observed in both MDA-MB-231 and MCF7 overexpressing PPAR-β/δ in response to ligand activation of PPAR-β/δ as compared with controls. Ectopic xenografts developed from MDA-MB-231 and MCF7 cells overexpressing PPAR-β/δ were significantly smaller, and ligand activation of PPAR-β/δ caused an even greater reduction in tumor volume as compared with controls. Interestingly, the decrease in MDA-MB-231 tumor size after overexpressing PPAR-β/δ and ligand activation of PPAR-β/δ correlated with increased necrosis. These data show that ligand activation and/or overexpression of PPAR-β/δ in two human breast cancer cell lines inhibits relative breast cancer tumorigenicity and provide further support for the development of ligands for PPAR-β/δ to specifically inhibit breast carcinogenesis. These new cell-based models will be invaluable tools for delineating the role of PPAR-β/δ in breast cancer and evaluating the effects of PPAR-β/δ agonists. Mol Cancer Ther; 13(4); 1008–17. ©2014 AACR.

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.

Aryl Hydrocarbon Receptor Antagonism Attenuates Growth Factor Expression, Proliferation, and Migration in Fibroblast-Like Synoviocytes from Patients with Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic autoimmune disease with high morbidity and mortality. Within the inflammatory milieu, resident fibroblast-like synoviocytes (FLS) in the synovial tissue undergo hyperplasia, which leads to joint destruction. Epidemiologic studies and our previous research suggest that activation of the aryl hydrocarbon receptor (AHR) pathway plays an instrumental role in the inflammatory and destructive RA phenotype. In addition, our recent studies implicate the AHR in the regulation of the expression of several growth factors in established tumor cell lines. Thus, under inflammatory conditions, we hypothesized that the AHR is involved in the constitutive and inducible expression of several growth factors, FLS proliferation and migration, along with protease-dependent invasion in FLS from patients with RA (RA-FLS). Treatment with the AHR antagonist GNF351 inhibits cytokine-induced expression of vascular endothelial growth factor-A (VEGF-A), epiregulin, amphiregulin, and basic fibroblast growth factor mRNA through an AHR-dependent mechanism in both RA-FLS and FLS. Secretion of VEGF-A and epiregulin from RA-FLS was also inhibited upon GNF351 treatment. RA-FLS cell migration, along with cytokine-induced RA-FLS cell proliferation, was significantly attenuated by GNF351 exposure. Treatment of RA-FLS with GNF351 mitigated cytokine-mediated expression of matrix metalloproteinase-2 and -9 mRNA and diminished the RA-FLS invasive phenotype. These findings indicate that inhibition of AHR activity may be a viable therapeutic target in amelioration of disease progression in RA by attenuating growth factor release; FLS proliferation, migration, and invasion; and inflammatory activity.

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.

The Nuclear Receptor Peroxisome Proliferator-activated Receptor-β/δ (PPARβ/δ) Promotes Oncogene-induced Cellular Senescence through Repression of Endoplasmic Reticulum Stress

Background: It is unclear whether ER stress and associated unfolded protein response (UPR) can influence oncogene-induced senescence. Results: ER stress attenuated senescence by modulating kinases, and a positive feed forward loop was delineated where ER stress caused loss of senescence and promotion of tumorigenesis. Conclusion: A new role for ER stress and UPR that attenuates H-RAS-induced senescence was discovered. Significance: PPARβ/δ may suppress RAS-dependent tumorigenesis. Endoplasmic reticulum (ER) stress and ER stress-associated unfolded protein response (UPR) can promote cancer cell survival, but it remains unclear whether they can influence oncogene-induced senescence. The present study examined the role of ER stress in senescence using oncogene-dependent models. Increased ER stress attenuated senescence in part by up-regulating phosphorylated protein kinase B (p-AKT) and decreasing phosphorylated extracellular signal-regulated kinase (p-ERK). A positive feed forward loop between p-AKT, ER stress, and UPR was discovered whereby a transient increase of ER stress caused reduced senescence and promotion of tumorigenesis. Decreased ER stress was further correlated with increased senescence in both mouse and human tumors. Interestingly, H-RAS-expressing Pparβ/δ null cells and tumors having increased cell proliferation exhibited enhanced ER stress, decreased cellular senescence, and/or enhanced tumorigenicity. Collectively, these results demonstrate a new role for ER stress and UPR that attenuates H-RAS-induced senescence and suggest that PPARβ/δ can repress this oncogene-induced ER stress to promote senescence in accordance with its role as a tumor modifier that suppresses carcinogenesis.

PPARβ/δ promotes HRAS-induced senescence and tumor suppression by potentiating p-ERK and repressing p-AKT signaling.

Peroxisome proliferator-activated receptor-β/δ (PPARβ/δ) inhibits skin tumorigenesis through mechanisms that may be dependent on HRAS signaling. The present study examined the hypothesis that PPARβ/δ promotes HRAS-induced senescence resulting in suppression of tumorigenesis. PPARβ/δ expression increased p-ERK and decreased p-AKT activity. Increased p-ERK activity results from the dampened HRAS-induced negative feedback response mediated in part through transcriptional upregulation of RAS guanyl-releasing protein 1 (RASGRP1) by PPARβ/δ. Decreased p-AKT activity results from repression of integrin-linked kinase (ILK) and phosphoinositide-dependent protein kinase-1 (PDPK1) expression. Decreased p-AKT activity in turn promotes cellular senescence through upregulation of p53 and p27 expression. Both over-expression of RASGRP1 and shRNA-mediated knockdown of ILK partially restore cellular senescence in Pparβ/δ-null cells. Higher PPARβ/δ expression is also correlated with increased senescence observed in human benign neurofibromas and colon adenoma lesions in vivo. These results demonstrate that PPARβ/δ promotes senescence to inhibit tumorigenesis and provide new mechanistic insights into HRAS-induced cellular senescence.

Chemoprevention of Chemically Induced Skin Tumorigenesis by Ligand Activation of Peroxisome Proliferator–Activated Receptor-β/δ and Inhibition of Cyclooxygenase 2

Ligand activation of peroxisome proliferator–activated receptor-β/δ (PPARβ/δ) and inhibition of cyclooxygenase-2 (COX2) activity by nonsteroidal anti-inflammatory drugs (NSAID) can both attenuate skin tumorigenesis. The present study examined the hypothesis that combining ligand activation of PPARβ/δ with inhibition of COX2 activity will increase the efficacy of chemoprevention of chemically induced skin tumorigenesis over that observed with either approach alone. To test this hypothesis, wild-type and Pparβ/δ-null mice were initiated with 7,12-dimethylbenz[a]anthracene (DMBA), topically treated with 12-O-tetradecanoylphorbol-13-acetate to promote tumorigenesis, and then immediately treated with topical application of the PPARβ/δ ligand GW0742, dietary administration of the COX2 inhibitor nimesulide, or both GW0742 and nimesulide. Ligand activation of PPARβ/δ with GW0742 caused a PPARβ/δ-dependent delay in the onset of tumor formation. Nimesulide also delayed the onset of tumor formation and caused inhibition of tumor multiplicity (46%) in wild-type mice but not in Pparβ/δ-null mice. Combining ligand activation of PPARβ/δ with dietary nimesulide resulted in a further decrease of tumor multiplicity (58%) in wild-type mice but not in Pparβ/δ-null mice. Biochemical and molecular analysis of skin and tumor samples show that these effects were due to the modulation of terminal differentiation, attenuation of inflammatory signaling, and induction of apoptosis through both PPARβ/δ-dependent and PPARβ/δ-independent mechanisms. Increased levels and activity of PPARβ/δ by nimesulide were also observed. These studies support the hypothesis that combining ligand activation of PPARβ/δ with inhibition of COX2 activity increases the efficacy of preventing chemically induced skin tumorigenesis as compared with either approach alone. Mol Cancer Ther; 9(12); 3267–77. ©2010 AACR.