THE MECHANISM OF PPARN3 MEDIATED DOWN REGULATION OF SODIUM HYDROGEN EXCHANGER 1 (NHE1) GENE EPXRESSION AND ITS INHIBITION BY ESTROGEN RECEPTOR n1 6

4. DISCUSSION 4.1 ESTABLISHING THE RELATIONSHIP BETWEEN PPARγ ACTIVATION AND NHE1 EXPRESSION The present study provides evidence supporting a relationship between PPARγ activation and NHE1 regulation. Firstly, treatment with PPARγ agonists downregulated NHE1 protein and mRNA expression in human breast cancer cells overexpressing PPARγ. Furthermore, a functional PPRE sequence was identified in the promoter region of NHE1 gene. The repressive effect of PPARγ ligands on NHE1 was abrogated in the presence of PPARγ antagonist GW9662, further confirming the inhibitory effect of PPARγ agnists on NHE1 expression is mediated through PPARγ receptor. 4.1.1 Identification of NHE1 gene as a transcriptional target of PPARγ. The peroxisome proliferator-activator receptors (PPARs) are transcription factors that can be ligand-activated and they belong to the nuclear hormone receptor family. Upon ligand activation, PPARγ heterodimerizes with a retinoid X receptor (RXR) and subsequently binds to specific peroxisome proliferator response elements (PPREs) in promoter regions of regulated target genes. In the past 20 years since the discovery of PPAR, more than 70 PPAR target genes have been identified to contain functional PPREs. However, majority of the genes are clustered in two functional pathways of adipocyte differentiation and fatty acid metabolism. Among the PPAR target genes listed in table 1, Acy-CoA oxidase, Fatty acid transport protein, HMG-CoA synthase, Malic enzyme and 169 Phosphoenolpyruvate carboxykinase are known to be invoved in fatty acid metabolism. These genes share common DR1 motif of classical PPRE consensus: ATGGTCA N AGGTCA, with slight difference in one or two nucleotides. As the first step to establish NHE1 as a PPARγ target, we located a putative PPRE situated between nucleotide (nt) –977 to –990 relative to the TATA box in the human NHE1 promoter (Accession number: L25272). However, the putative PPRE identified contained DR2 repeats instead of the DR1 motif found in classical PPRE-regulated genes. Interestingly, evidence from litreature has demonstrated binding of PPARγ to DR2 element (Fontaine et al., 2003; Kumar et al., 2004). The PPRE found in NHE1 promoter is located in Alu receptor response element (AluRRE) (Jurka and Milosavljevic, 1991). Sequence alignment of AluRRE of the human NHE1 with human myeloperoxidase (MPO) promoter revealed extensive sequence identity (Figure 1B). Incidentally, PPARγ was reported to bind to and regulate MPO gene (Kumar et al., 2004). Hence, the presence of similar PPRE in NHE1 promoter may also allow PPARγ regulation in the same manner. It is noteworthy that DR2 element can also be recognized by retinoic acid recptors by functioning as retinoic acid response element (Laperriere et al., 2007), suggesting the possibility of the same promoter region in NHE1 being regulated by retinoic acids. To validate the identified PPRE on NHE1 promoter as a functional PPARγbinding element, we performed Noshift Transcritption Factor assay as well as chromatin immunoprecipitation (ChIP). Noshift Transcription Factor assay was used to assess direct interaction between protein and DNA oligomers in vitro, 170 while the ChIP assay was for detecting in vivo interaction between protein of interst and DNAs of known sequences. In Noshift Trancription Factor assay, we confirmed that PPARγ binds to the DR2 of putative PPRE in NHE1 promoter (Figure 8B). The ability of competitive non-biotinylated oligonucleotides to dimish the ELISA signal further confirmed the specificity of the protein-DNA interaction. The assay was also validated using PTEN promoter sequence (Figure 8B), which was reported to be bound by activated PPARγ (Patel et al., 2001). One limitation of the assay is that it only assesses the binding of protein with naked DNA that is artificially synthesized and exogenously introduced, thus it does not reflect the binding between protein and chromatin in their native cellular environment. Hence, ChIP assay was also performed to confirm the in vivo binding of activated PPARγ to putative PPRE on NHE1 promoter. Consistent with data from the Noshift Transcription Factor assay, PPARγ was shown to bind to the PTEN promoter in vivo (Figure 8C). Interestingly, the binding capacity of PPARγ to NHE1 promoter appeared to be weaker than that to PTEN promoter in both assays. As PTEN contains classical DR1 motif of PPRE, we surmise the binding of PPARγ to DR2 in NHE1 promoter is not optimal compared to DR1. Furthermore, the binding is depedent on PPARγ activation, as the amount of PPARγ increased at NHE1 promoter with higher concentration of the ligand (Figure 8C). Although the binding pattern of auxiliary proteins such as RXR was not demonstrated, the results from these two assays sufficiently confirmed that PPARγ can bind to the PPRE identified at the promoter of NHE1. 4.1.2 The mechanism of PPARγ-mediated repression of NHE1 gene. 171 After confirming the prescence of functional PPRE on NHE1 promoter, we next investigated how PPARγ agonists regulate NHE1 expression in breast cancer cell lines. To this end, MCF-7, MDA-MB-231 and T47D cells were treated with different doses of synthetic ligands as well as natural ligand of PPARγ, and the protein expression and mRNA transcript level of NHE1 were assessed. 15d-PGJ2 is a metabolite of eicosanoid prostaglandin J2, and was reported to be the most potent natural agonist for PPARγ with Kds varying from 325nM to 2.5µM. In all cell lines tested, 15d-PGJ2 induced a concentration-dependent down-regulation of NHE1 expression both at protein and transcriptional level (Figure 2, 3). Synthetic PPARγ ligands, which belong to the TZD family of drugs, ciglitazone, troglitazone and rosiglitazone, also induced similar inhibition on NHE1 mRNA in these cell lines (Figure 2). The extent of inhibition varied from ligand to ligand, this could be explained by the different binding affinities of these ligands to PPARγ receptor. Taken together, we surmise that PPARγ ligands induced downregulation of NHE1 expression is mediated through PPARγ receptor, and is not a drug-specific effect. Although repressive effect was seen in all three cell lines tested, the magnitude of inhibition varied from cell line to cell line. The most drastic inhibition of NHE1 expression by 15d-PGJ2 was observed in MDA-MB-231 cells and the least inhibition was found in T47D (Figure 2, 3). The extent of inhibition correlatively mirror-imaged the basal PPARγ receptor level present in different cell lines. T47D which expresses the lowest amount of PPARγ was minimally affected by treatment with 15d-PGJ2. Conversely, MDA-MB-231 with highest level of 172 PPARγ was the most susceptible to 15d-PGJ2-induced down-regulation of NHE1 expression. This result provides the first evidence that PPARγ ligands-regulated NHE1 repression is PPARγ-dependent. To further confirm the hypothesis, we overexpressed PPARγ in T47D cells, and checked whether the increased basal PPARγ level would lead to enhanced repression by PPARγ ligand. As expected, T47D transfected with PPARγ receptor showed augmented reduction in NHE1 protein and mRNA expression upon treatment with 15d-PGJ2 as compared to cells that were transfected with empty vector (Figure 4). On the other hand, overexpression of PPARγ defective in DNA binding domain abrogated the ligand-mediated down-regulation of NHE1 expression in MCF-7 cells (Figure 7B, C). We also silenced PPARγ in MDA-MB231 cells which express higher basal level of PPARγ and assessed whether the absence of PPARγ would lead to attenuated down-regualtion of NHE1 by PPARγ ligand. As expected, silencing of PPARγ produced a reverse effect as compared to overexpression of PPARγ. The initial 15d-PGJ2-mediated down-regualtion of NHE1 protein was abrogated upon PPARγ silencing (Figure 5). Furthermore, pharmocoligical PPARγ antagonist, GW9662 produced similar effect as PPARγ silencing in MDA-MB-231 cells. It was observed that inhibiting PPARγ activation by GW9662 blocked the down-regulation of NHE1 protein by PPARγ ligand (Figure 6). Together, these data confirmed our hypothesis that PPARγ ligands inhibit NHE1 expression through a PPARγ-dependent mechanism. The inhibition of NHE1 by PPARγ can be due to negative interference with activating transcription factors such as NF-κB and activator protein-1 (Delerive et 173 al., 1999; Rossi et al., 2000). Alternatively, the inhibitory effect can be caused by sequestration of limiting coactivator such as CBP (Li et al., 2000). Beside, the regulatory mechanism can also involve direct recruitment of corepressors such as NCoR and SMRT by PPARγ in a promoter-specific manner (Guan et al., 2005). Recently, it was also reported that sumolyated PPARγ inhibits the expression of inducible nitric oxide synthase gene in a ligand-dependent manner (Pascual et al., 2005). Given that PPARγ binds to PPRE on the NHE1 promoter and PPARγ defective in DNA-binding abrogated the PPARγ-mediated NHE1 repression, the mechanism underlying down-regulation of NHE1 expression by PPARγ ligands may involve direct binding of sumolyated PPARγ or its recruitment of corepressors. 4.1.3 Production of ROS/RNS by PPARγ ligands in breast cancer cells. Cyclopentenone prostaglandins including 15d-PGJ2 are reported to generate intracellular ROS/RNS in various cell lines (Li et al., 2001; Bureau et al., 2002; Lennon et al., 2002). Furthermore, studies have also shown that the TZD class of synthetic PPARγ ligands are capable of induce ROS/RNS by affecting mitochondria function (Brunmair et al., 2004; Perez-Ortiz et al., 2007). Corroborating with these reports, we also confirmed the production of ROS/RNS in breast cancer cells by 15d-PGJ2 and ciglitazone. Using redox sensitive DCFDA staining, we observed an increase in intracellular ROS/RNS induced by PPARγ ligands (Figure 9). 174 As the DCFDA dyes are sensitive to a variety of ROS/RNS species such as H2O2, ONOO–, HO• and ROO•, it is imperative to charactarize the specific oxygen derivatives induced by PPARγ ligands. A study by Perez-Ortiz et al. revealed that the PPARγ synthetic ligands, glitazones yield peroxynitrite from superoxide anion and NO in astroglioma cells (Perez-Ortiz et al., 2007). To investigate whether peroxynitrite is produced in our system, 5,10,15,20-Tetrakis(4- sulfonatophenyl)prophyrinato iron (III), chloride (FeTPPS) was used. The porphyrin complex catalytically isomerizes peroxynitrite to nitrate in vivo as well as in vitro (Misko et al., 1998). As expected, FeTPPS significantly inhibited the production of ROS/RNS which was detected by DCFDA staining (Figure 11A). Furthermore, the H2O2 scanvenger catalase powder failed to elicit any change in the amount of ROS/RNS produced by PPARγ ligand. This strongly suggests that the oxygen derivative induced by PPARγ ligand is peroxynitrite. It was also reported that PPARγ ligands 15d-PGJ2 and ciglitazone induce nitric oxide (NO) release from endothelial cells (Calnek et al., 2003). Given the fact that peroxynitrite produced can be a product from combining superoxide with nitric oxide, we investigated whether NO was produced by PPARγ ligand in our system. For this, NO spefic probe, DAF-FM dye was used. DAF-FM reacts with NO to form a fluorescent benzotrozol that can be captured by flow cytometry (Nakatsubo et al., 1998). The DAF staining showed significant production of NO in MCF-7 cells upon treatment with 15d-PGJ2 (Figure 10B). Moreover, the inhibitor for nitric oxide synthase, L-NG-monomethyl Arginine citrate (L- 175 NMMA) was able to decrease the amount of NO induced by 15d-PGJ2 (Figure 11B). Based on the above experimental evidence, it is demonstrated that the ROS/RNS species generated by 15d-PGJ2 is peroxynitrite. Given that peroxynitrite can be formed by NO and superoxide anion at a rapid rate in a nonenzymatic reaction, the production of NO and superoxide are highly likely in our system. Although we have convincingly showed the production of NO by 15d-PGJ2, the exact source of production remains to be further elucidated. Study has shown that 15dPGJ2 and ciglitazone stimulate NO release from endothelial cells, through a transcriptional mechanism (Calnek et al., 2003). However, this mechanism induced NO release only after 24h of treatment with 15d-PGJ2, and may not be responsible for the rapid and immediate production of NO seen in our system. A direct activation of cytosolic or mitochondrial NO synthase (NOS) isoforms by PPARγ ligand can be a plausible mechanism. The other component of peroxynitrite, suproxide anion, on the other hand can be produced from mitochondria. It was reported that 15d-PGJ2 strongly induced ROS/RNS production by inhibiting the mitochondrial complex I activity in MCF-7 cells, and the generation of oxidative stress by 15d-PGJ2 was abolished by complex I inhibitor, rotenone (Martinez et al., 2005). It was also demonstrated that ciglitazone resulted in mitochondrial depolarization by opening the mitochondrial permeability transition pore, thus enhancing ROS/RNS levels through interfering with the electron transport chain (Masubuchi et al., 2006; Perez-Ortiz et al., 2007). From these published results, we postulate the source of superoxide anion 176 to be mitochondria. However, further investigation is needed to confirm the origins of nitric oxide and superoxide in breast cancer cells treated with PPARγ ligands. 4.1.4 The mechanism of ROS/RNS-mediated repression of NHE1 gene. It is not novel that intracellular ROS/RNS can regulate transcription of various target genes. The human cytochrome P450 1A1 (CYP1A1) was shown to be reduced at the mRNA level by oxidative stress and glutathione depletion in human HepG2 or rat H4 hepatoma cells (Morel and Barouki, 1998). Our group has previously demonstrated that NHE1 is a redox-regulated gene, as exposure of cells to H2O2 inhibited the NHE1 promtoer activity as well as gene expression (Akram et al., 2006). Recently, the study has been furthered to demonstrate that the sustained repression of NHE1 by H2O2 is dependent on iron as well as peroxynitrite. We have previously shown that PPARγ ligands induce production of peroxynitrite and concurrently repress NHE1 gene expression, hence we investigated whether the generation of peroxynitrite contributes to the inhibitory effect of PPARγ ligand on NHE1 expression. To this end, N-acetyl cysteine (NAC), a general antioxidant, was added to MCF-7 cells before exposure to 15dPGJ2. NAC functions by reducing the overall oxidation state in the system partly through formation of glutathione. DCFDA staining revealed that the production of ROS/RNS was completely blocked in the presence of NAC (Figure 11A). This result confirms the previous report that treatement with NAC prevents the production of ROS/RNS in SH-SY5Y cells and human hepatic fibroblasts (Li et al., 2001). After confirming the effectiveness of NAC in scavenging the 177 ROS/RNS produced by 15d-PGJ2 in MCF-7 cells, we then assessed its functional consequence on NHE1 expression at both mRNA and protein level. Surprisingly, large disparity exists in the extents of resue by NAC between mRNA and protein expression. While NAC was able to induce an almost complete reversal of NHE1 protein down-regulation by 15d-PGJ2, its presence only rescued 20% of the reduction in NHE1 mRNA by the same PPARγ ligand (Figure 11B, C). This result suggests that other than regulating the factor controlling NHE1 transcription, NAC has altered the rate of NHE1 protein turnover. Indeed, NAC was shown to inhibit 26s of proteasome activity and increased the protein level of those molecules dependent on the ubiquitin/proteasome system for their degradation in EVC 304 bladder carcinoma cells (Pajonk et al., 2002). Hence, the observed rescue at protein level can be due to the slowed NHE1 protein degradation rather than the desuppressed NHE1 transciption. This was confirmed further by using FeTPPS and L-NMMA, which not affect the proteosomal degradation pathway, hence these two drugs only partially rescued the protein expression in NHE1 (Figure 11D, E). Notably, the rescue of NHE1 expresion by ROS/RNS scavengers was more prominent at 5µM than 3µM of 15d-PGJ2, suggesting a more important role of ROS/RNS in NHE1 regulation at higher concentration of PPARγ ligand. 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Three ERα binding sites were also included in the ChIP assay: ERA, ERB and ERC. 230 [...]... contributed by ROS/RNS generated by the PPARγ ligand (Figure 12 ) This agrees with the previous finding that the ROS scavenger NAC could only rescue 20% of the reduction in NHE1 mRNA brought about by 5µM of 15 d-PGJ2 (Figure 11 C) Taken together, we show that 15 d-PGJ2 -mediated NHE1 repression is largely dependent on PPAR receptor and minimally through ROS/RNS 4.2 ANTI-CANCER EFFECTS OF PPARγ LIGANDS As reviewed... recognized (Neri and Supuran, 2 011 ; Webb et al., 2 011 ) For this, two strategies have been proposed: direct pharmacological inhibitors of NHE1 activity or controlling the gene/ protein expression of NHE1 18 6 In this study, we have demonstrated that activation of the nuclear receptor PPARγ down- regualtes NHE1 transcription and thus its protein level The reduction in NHE1 expression by PPARγ ligands was shown... activity of PPARγ, and inhibits up -regulation of PTEN by activated PPARγ (Wang and Kilgore, 2002; Bonofiglio et al., 2005) In our case, we demonstrate that ERα is not only effective in blocking the transcriptional upregulation of PPARγ target genes, it is also capable of inhibiting transcriptional repression of NHE1 gene 4.3.2 Unravelling the mechanism of how ERα inhibits PPARγ -mediated down- regulation of. .. down- regulation by PPARγ ligand In both case, removal of ERα restored the ability of 15 d-PGJ2 to repress the expression of NHE1 both at protein and mRNA level (Figure 21) In regular serum condition, re-expression of ERα in ER negative MDA-MB-2 31 cells also 19 1 elevated the basal level of NHE1 mRNA, suggesting a constitutive repression of NHE1 by PPARγ in ER negative MDA-MB-2 31 cells and its alleviation upon... inhibits PPARγ ligand-induced reduction in NHE1 expression, we next investigated whether the effect of E2 is mediated through its receptor, ERα Like most of the natural ligands, E2 is reported to exert both ERα– 19 0 dependent and independent effects in cancer cells (Yager and Davidson, 20 06; Santen et al., 2009) To confirm that the inhibitory effect of E2 on NHE1 repression by PPARγ ligands is mediated. .. ERα receptor, we used ER negative breast caner cell line MDA-MB-2 31 and tested the effect of E2 on its NHE1 expression Due to the absence of ERα in these cells, E2 failed to elicit the inhibitory effect on PPARγ -mediated NHE1 down- regualtion in ER negative MDA-MB-2 31 cells (Figure 19 A) However, re-expression of ERα by stable transfection in the same cell line reinstated the effect of E2 on PPARγ -mediated. .. both AF1 and AF2 domains of ERα (Fawell et al., 19 90) Raloxifene acts as an estrogen antagonist in both breast and uterus (Mitlak and Cohen, 19 99) by blocking the assessbility of 19 2 AF2 domain in ERα receptor (Thiebaud and Secrest, 20 01) The antagonistic properties of the drugs were confirmed first using ERE luciferase reporter assay and Western blot of ER-response genes Both drugs were capable of reducing... effects of 17 β-estradiol on PPAR mediated metabolic effects as well as anti-cancer effects (Wang and Kilgore, 2002; Bonofiglio et al., 2005; Jeong and Yoon, 2 011 ) Having established NHE1 gene as a bona fide PPARγ target gene and an important therapeutic target, we next examined whether ERα interferes with PPARγ-dependent down- regulation of NHE1 gene expression 4.3 .1 ERα negatively interferes with PPARγ -mediated. .. association of PPARγ and ERα and shown that activated ERα is capable of modulating the binding efficiency of PPARγ to NHE1 and PTEN promoter, we next examined other PPRE present on the human chromosome 1 and assessed the inhibitory effect of ERα on the binding of PPARγ to these PPRE sequences At all PPARγ-binding sites on chromosome 1, E2 invariably abrogated the binding of activated PPARγ to these sites... shown the inhibitory effect of active ERα on 2 PPARγ target genes: PPARγ-indcued RhoB upregulation and PPARγ -mediated NHE1 down- regualtion are suppressed in the presence of E2-activated ERα receptor Given that ERα and PPARγ physically interact, and the ability of ERα to modulate the binding affinity of PPARγ to its response element, this led us to investigate the ability of PPARγ to interfere with the . reversal of NHE1 protein down- regulation by 15 d-PGJ 2 , its presence only rescued 20% of the reduction in NHE1 mRNA by the same PPARγ ligand (Figure 11 B, C). This result suggests that other than. MDA-MB-2 31 cells. It was observed that inhibiting PPARγ activation by GW 966 2 blocked the down- regulation of NHE1 protein by PPARγ ligand (Figure 6) . Together, these data confirmed our hypothesis. in the promoter region of NHE1 gene. The repressive effect of PPARγ ligands on NHE1 was abrogated in the presence of PPARγ antagonist GW 966 2, further confirming the inhibitory effect of PPARγ