THE MECHANISM OF PPARN3 MEDIATED DOWN REGULATION OF SODIUM HYDROGEN EXCHANGER 1 (NHE1) GENE EPXRESSION AND ITS INHIBITION BY ESTROGEN RECEPTOR n1 5

dependent reduction in NHE1 protein expression in ERα-silenced MCF-7 (Figure 21A). To further confirm that the active ERα present in regular serum blocked the down-regulation of NHE1 by PPARγ ligand at transcriptional level, cells were transfected with scrambled control siRNA or ERα siRNA and treated with 15dPGJ2 in regular serum condition for 40h. Following that, cells were harvested for RNA and subjected to mRNA analysis using real-time PCR. The mRNA data corroborated with the data on NHE1 protein expression. In regular serum, 5μM of 15d-PGJ2 failed to induce any changes to the NHE1 mRNA level. However, the same concentration of 15d-PGJ2 induced about 50% reduction in NHE1 mRNA expression when ERα was removed by siRNA (Figure 21C). To further rule out the possibility that the observed down-regulation of NHE1 by PPARγ ligand in ERα-silenced MCF-7 cells was due to unspecific gene silencing by ERα siRNA, ERα was pharmocolically degraded by high concentration of fulvestrat for 4h before the addition of 15d-PGJ2. Fulvestrant is a 7αalkylsulphinyl analogue of 17β-estradiol. It functions as an estrogen receptor antagonist devoid of agonist activities. It was reported that fulvestrant has higher affinity for estrogen receptors and thus competitively inhibits binding of estrogen to ERs. It was also shown to degrade estrogen receptors (McClelland et al., 1996). MCF-7 cells were kept in RPMI supplemented with 10% regular serum for 48h before 1μM of fulvestrant was introduced. After 4h, the cells were harvested for analysis of ERα protein expression by Western blot. Figure 23A shows ERα in MCF-7 were degraded by end of 4h after fulvestrant addition. After ensuring the 137 complete degradation of ERα by fulvestrant, we exposed the cells to 15d-PGJ2 for 48h. As shown in figure 23B, 15d-PGJ2 could not inhibit the NHE1 protein expression in regular serum, but the edogenous PPARγ ligand succeeded in repressing NHE1 when ERα was removed by fulvestrant (Figure 21B). Again real-time PCR was performed to assess the NHE1 mRNA level of the same experimental setting. Nontheless, it should be noted that cells were harvested for RNA 40h after 15d-PGJ2 introduction. The mRNA data were highly similar to that obtained from the ERα silencing experiment, degradation of ERα by fulvestrant significantly enhanced the inhibitory effect of 15d-PGJ2 on NHE1 mRNA expression (Figure 21D). Taken together, the above results demonstrate that the active ERα present in regular serum interferes with and blocks PPARγ-mediated down-regulation of NHE1 expression in ER positive MCF-7 cells. 138 139 Figure 21: Reduced ERα level enhances PPARγ-mediated down-regulation of NHE1 in regular serum conditon. (A) MCF-7 (1.5 X105 cells/6-well dishes) cells were transfected with ERα specific siRNA or negative siRNA as described in Materials and Methods. After 48h of transfection, ERα expression was determined by Western blot with β-actin as loading control. The transfected cells were exposed to 15d-PGJ2 for 48h in media containing regular serum, and the protein expression of NHE1 was analyzed by Western blot. NHE1 band intensity was normalized to β-actin. (B) MCF-7 (2 X105 cells/6-well dishes) cells were treated with 15d-PGJ2 for 48h with or without 4h pre-incubation of 1µM Fulvestrant, and the protein expression of NHE1 was analyzed by Western blot. After 4h of Fulvestrant treatment, ERα 140 expression was determined by Western blot. NHE1 band intensity was normalized to β-actin. (C) MCF-7 (1.5 X105 cells/6-well dishes) cells were transfected with ERα specific siRNA or negative siRNA as described in Materials and Methods. The transfected cells were exposed to 15d-PGJ2 for 40h in medium containing regular serum, and the fold change of NHE1 mRNA expression was determined by Taqman real-time PCR, normalized to the endogenous control: human 18s. Relative NHE1 mRNA expression is expressed as percentage of control. Results denote means +/-SD computed from two experiments done in duplicate. (D) MCF-7 (1.5 X10 cells/6-well dishes) cells were treated with 15d-PGJ2 for 40h with or without 4h pre-incubation of 1µM Fulvestrant, and the fold change of NHE1 mRNA expression was determined by Taqman real-time PCR, normalized to the endogenous control: human 18s. Relative NHE1 mRNA expression is expressed as percentage of control. Results denote means +/-SD computed from two experiments done in duplicate. *, p[...]... to PPARγ -mediated NHE1 down- regulation From the results shown above, we concluded further that the DNA binding ability of ERα is involved in inhibiting PPARγ -mediated NHE1 repression 15 1 15 2 Figure 24: Transfection of DNA-binding defective ERα enhances PPAR mediated down- regulation of NHE1 in regular serum condition (A) MCF-7 (7 .5 X 10 4 cells /12 -well dishes) cells were co-transfected with 4µg of plasmid... PPARγ -mediated down- regulation of NHE1 expression remains to be elucidated further 15 4 Figure 25: Identification of putative ERE on NHE1 promoter (A) The AluRRE of NHE1 promoter is aligned with AluRRE of myeloperoxidase (MPO) (Kumar et al., 2004) .The putative ERE and PPRE in 5 proximal promoter region of NHE1 is aligned with the consensus ERE (B) MCF-7 (1. 78 X 10 6 /10 0mm dish) cells were exposed to 1 M... activity and facilitate down- regulation of NHE1 expression by PPARγ ligand 14 7 Figure 23: ERα antagonists enhance PPARγ -mediated down- regulation of NHE1 in regular serum condition (A) MCF-7 (2 X10 5 cells/6-well dishes) cells were treated with 10 nM Fulvestrant or 10 0nM Raloxifene in media containing 10 % regular serum for 48h, and the protein expressions of c-Myc and progesterone receptor were analyzed by. .. transcripts from the untreated control (Figure 27C) On the contrary, presence of E2 inhibited PPARγ -mediated up -regulation of the gene (Figure 27C) Taken together, these data suggest that both positive and negative gene regulation of PPARγ target gene can be blocked by activated ERα 16 0 Figure 27: Transcriptional activity of PPARγ is blocked in the presence of estrogen (A) MCF-7 (7 .5 X 10 4 cells /12 -well dishes)... 3B.4 THE MECHANISM BY WHICH ERα BLOCKS THE EFFECT OF PPARγ ON NHE1 EXPRESSION 3B4 .1 ERα does not bind to the putative ERE on NHE1 promoter Since the DNA binding ability of ERα was found to be essential in its inhibitory effect on PPARγ -mediated down- regulation of NHE1, it was hyptotheiszed that ERα and PPARγ compete sterically for DNA binding site in close proximity, and activation of ERα by estrogen. .. X1 05 cells/6-well dishes) cells were exposed to increasing doses of 3μM 15 d-PGJ2 with or without 2h pre-incubation of 1 M E2 for 24h, and the protein expressions of RhoB was analyzed by Western blot, using β-actin as the loading control MCF-7 (3 X1 05 cells/6-well dishes) cells were exposed to increasing doses of 3μM 15 d-PGJ2 with or without 2h pre-incubation of 1 M E2 for 6h, and the fold changes of. .. binding enhances the effect of PPARγ ligand on NHE1 down- regulation Having demonstrated that transcriptionally inactivating ERα in regular serum can facilitate the down- regulation of NHE1 expression by 15 d-PGJ2, we next investigated whether the DNA binding ability of ERα was involved in preventing PPARγ -mediated reduction of NHE1 expression To this end, we overexpressed a dominant-negative form of ERα in... MCF-7 (2 X10 5 cells/6-well dishes) cells were treated with 15 d-PGJ2 with or without 6h pre-incubation of 10 0nM Raloxifene in media containing 10 % regular serum for 48h, and the protein expressions of NHE1 was analyzed by Western blot NHE1 band intensity was 14 8 normalized to β-actin (D) MCF-7 (2 X1 05 cells/6-well dishes) cells were treated with 15 d-PGJ2 with or without 6h pre-incubation of 10 nM Fulvestrant... However, the same concentration of 15 d-PGJ2 produced significant 47% and 61% reduction in colonogenic ability from the untreated control in the presence of 10 nM fulvestrant and 10 0nM raloxifene respectively (Figure 30) This finding has provided the clinical relevance of the study, suggesting the use of combining antiestrogen and PPARγ ligand for better efficacy in treating ER positive breast cancer 16 7...antagonists on PPARγ-regulated NHE1 expression was also analyzed at transcriptional level using real-time PCR In the absence of ERα antagonist, 40h incubation with 5 M of 15 d-PGJ2 failed to bring down the NHE1 mRNA expression However, the same concentration of 15 d-PGJ2 resulted in 20% and 30% reduction in NHE1 mRNA level when cells were pre-incubated with 10 nM fulvestrant and 10 0nM raloxifene respectively . reduction in NHE1 mRNA in ER positive MDA-MB-2 31 cells. It is interesting that in the absence of fulvestrant, 1 M of 15 d-PGJ 2 brought down NHE1 mRNA to 50 % of the control. 14 3 However,. alone. Together, these data confirm that re-expression of ERα in MDA-MB-2 31 and its activation by estrogen present in serum blocks PPARγ -mediated down- regulation of NHE1 expression. 14 4 . PCR. In the absence of ERα antagonist, 40h incubation with 5 M of 15 d-PGJ 2 failed to bring down the NHE1 mRNA expression. However, the same concentration of 15 d-PGJ 2 resulted in 20% and 30%