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

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 contro

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 15d-PGJ2 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 7α-alkylsulphinyl 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

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

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

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 X105 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<0.05, **, p< 0.01, treated versus untreated control

3B2.4 Re-expression of ERα in ER negative MDA-MB-231 cells blocks PPARγ-mediated down-regulationof NHE1

From the experiemnts shown in section 2.2, we have demonstrated that in ER positive MCF-7 cells, high estrogen content in regular serum was able to block the PPARγ-mediated down-regulation of NHE1 At the same time, removing ERα

in these cells restored their response to PPARγ ligand on NHE1 Hence, we asked whether ER-expressing MDA-MB-231 cells would behave similarly as ER positive MCF-7 cells in regular serum condition

The endogenous level of NHE1 and ERα were first assessed in normal MB-231 cells and MDA-MB-231 cells stably transfected with ERα (MDA-MB-

MDA-231 ER positive cells) using Western blot The successful re-expression of ERα in MDA-MB-231 ER positive cells was shown in figure 24A It is noteworthy that

when the cells were kept in RPMI supplemented with regular serum condition, the basal level of NHE1 mRNA was significantly higher in ER positive MDA-MB-

231 cells compared to that in ER negative MDA-MB-231 cells (Figure 22A) MDA-MB-231 cells expressing ERα showed 40% more basal NHE1 mRNA level compared to ER negative MDA-MB-231 cells (Figure 22A) To reaffirm the role

of active ERα receptor, ERα activity was blocked by the ER antagonist fulvestrant Breifly, MDA-MB-231 cells stably transfected with ERα were incubatd with 10nM of fulvestrant for 2h, before 3µM of 15d-PGJ2 was introduced The cells were harvested after 18h and 24h for mRNA and protein expression respectively Western blot analysis of the NHE1 protein content shows

ER antagonist itself could significantly bring down NHE1 protein level in regular serum condition (Figure 22B) This result suggests that the both ERα and PPARγ were active in regular serum condition, probably due to the high concentration of estrogen and prostaglandins present Upon inhibition of ERα by its antagonist, the activated PPARγ resulted in spontaneous reduction in NHE1 protein level It is also noted that addition of 3µM of 15d-PGJ2 after 2h pre-incubation with 10nM fulvestrant further enhanced the magnitude of NHE1 down-regulation, demonstrating that inhibiting ERα could potentiate PPARγ ligand’s effect on NHE1 expression (Figure 22B) In figure 22C, NHE1 mRNA analysis by real-time PCR also revealved similar trend of regulation by ERα antagonist and PPARγ ligand 10nM of fulvestrant resulted in 30% reduction in NHE1 mRNA in

ER positive MDA-MB-231 cells It is interesting that in the absence of fulvestrant, 1µM of 15d-PGJ2 brought down NHE1 mRNA to 50% of the control

However, after intitial suppression of NHE1 mRNA by fulvestrant, the same concentration of 15d-PGJ2 was only able to reduce NHE1 mRNA to 60% of the control (Figure 22C) This observation suggests that when both PPARγ and ERα were activated in regular serum, ERα exerted a more dominant effect on PPARγ-regulated NHE1 expression The avaiblabiltiy of PPARγ ligand played a less important role as further addition of PPARγ agonist resulted in a smaller reduction in NHE1 expression than that with fulvestrant alone

Together, these data confirm that re-expression of ERα in MDA-MB-231 and its activation by estrogen present in serum blocks PPARγ-mediated down-regulation

of NHE1 expression

Figure 22: Re-expression of ERα blocks PPARγ-mediated down-regulation of NHE1in MDA-MB-231 cells kept in regular serum condition

(A) Endogenous NHE1 mRNA expressions in MDA-MB-231 and MDA-MB-231 ERα+ were 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 (B) MDA-MB-231 ERα+ (1.5 X105 cells/6-well dishes) cells were treated with 15d-PGJ2 for 48h with or without 2h pre-incubation of 10nM Fulvestrant in medium containing 10% regular serum, and the protein expressions

of NHE1 was analyzed by Western blot NHE1 band intensity was normalized to β-actin (C) MDA-MB-231 ERα+ (1.5 X105 cells/6-well dishes) cells were treated with 15d-PGJ2 for 40h with or without 2h pre-incubation of 10nM Fulvestrant in media containing 10% 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 *, p< 0.05, treated versus untreated control

3B.3 TRANSCRIPTIONALLY ACTIVE ERα BLOCKS THE EFFECT

OF PPARγ ON NHE1 EXPRESSION

The results so far from silencing ERα in MCF-7 cells and reexpression ERα in MDA-MB-231 cells have confirmed the role of ERα in PPARγ-mediated down-regulation in breast cancer cells Other than its classical mechanism of transcriptional action, ERα was reported to elicit non-genomic signaling pathways

by activating MAPK (Mitogen-Activated Protein Kinase) and PKA and PKC (Protein kinases) (Simoncini et al., 2003) Thus, we wanted to confirm whether the inhibitory effect of ERα on PPARγ-mediated reduction of NHE1 expression involves the transcriptional activity of estrogen receptor

3B3.1 ERα antagonist enhances the PPARγ-mediated NHE1 repression

Two ERα antagonists fulvestrant and raloxifene were utilized to inhibit the transcriptional activity of ERα and to examine their subsequent effects on PPARγ-regulated NHE1 expression Fulvestrant was shown to bind to ERα, rendering it incapable of dimerization and transcriptionally inactive by disabling both AF1 and AF2 domains of ERα (Fawell et al., 1990) Raloxifene acts as an estrogen antagonist in both breast and uterus (Mitlak and Cohen, 1999) by blocking the accessibility of AF2 domain in ERα receptor (Thiebaud and Secrest, 2001)

To ascertain that both fulvestrant and raloxifene acted as ERα antagonist in our system, the protein expression of two ERα target genes, progesterone receptor and

c-Myc were determined in MCF-7 cells exposed to 10nM fulvestrant and 100nM raloxifene As shown in figure 23A, both drugs induced a reduction in expressions

of c-Myc and progesterone receptor after 48h in regular serum condition The suppression of the two ERα target genes by fulvestrant and raloxifene demonstrates that the two antagonists successful inhibited the transcriptional activity of ERα Moreover, the transcriptional activity of ERα was assessed using ERE luciferase reporter assay Luciferase reporter assay was then performed on MCF-7 cells exposed to 10nM fulvestrant and 100nM raloxifene in RMPI supplemented with regular FBS The ERE reporter activity was measured after 6h When ERα receptors were activated by estrogen present in regular serum, both drugs induced a significant down-regulation of transcriptional activity at ERE (Figure 23B) This result corroborated with that in figure 24A, confirming that both fulvestrant and raloxifene were capable of inhibiting the transcriptional activity of ERα

Next we investigated how inhibition of ERα’s transcriptional activity by ERα antagonists would affect its ability to regulate PPARγ-mediated decrement of NHE1 expression in regular serum condition ER positive MCF-7 cells were pre-incubated with 10nM fulvestrant for 6h, before PPARγ ligand 15d-PGJ2 was introduced The cells were harvested for Western blot analysis after 48h The extent of NHE1 protein down-regualtion by PPARγ ligand was highly enhanced when ERα was inhibited by fulvestrant (Figure 23C) The same experimental set

up was repeated with 100nM raloxifene, and it was found that raloxifene similarly augmented PPARγ-mediated down-regulation in MCF-7 cells The effect of ERα

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 5μM of 15d-PGJ2 failed to bring down the NHE1 mRNA expression However, the same concentration of 15d-PGJ2 resulted in 20% and 30% reduction in NHE1 mRNA level when cells were pre-incubated with 10nM fulvestrant and 100nM raloxifene respectively (Figure 23D)

In this section, we demonstrated that ERα antagonists can inhibit ERα transcriptional activity and facilitate down-regulation of NHE1 expression by PPARγ ligand

Figure 23: ERα antagonists enhance PPARγ-mediated down-regulation of NHE1 in regular serum condition

(A) MCF-7 (2 X105 cells/6-well dishes) cells were treated with 10nM Fulvestrant

or 100nM Raloxifene in media containing 10% regular serum for 48h, and the protein expressions of c-Myc and progesterone receptor were analyzed by Western blot, using β-actin as the loading control (B) MCF-7 (7.5 X 104 cells/12-well dishes) cells were co-transfected with 3µg of reporter plasmid 3X ERE-luc and 0.3µg of renilla as described in Materials and Methods 48h after transfection, the activity of ERα was then determined using luciferase assay and the result was calculated as luciferase RLU/renilla/µg total Data represents the average +/- SD

of two experiments done in duplicate (C) MCF-7 (2 X105 cells/6-well dishes) cells were treated with 15d-PGJ2 with or without 6h pre-incubation of 100nM Raloxifene in media containing 10% regular serum for 48h, and the protein expressions of NHE1 was analyzed by Western blot NHE1 band intensity was

normalized to β-actin (D) MCF-7 (2 X10 cells/6-well dishes) cells were treated with 15d-PGJ2 with or without 6h pre-incubation of 10nM Fulvestrant or 100nM Raloxifene in medium containing 10% regular serum for 40h, 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<0.05, **, p<0.01, treaed versus untreated control

3B3.2 ERα defective in DNA 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 15d-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 cells The DNA binding domain of dominant-negative ERα contains mutations that rendered it defective in binding to DNA As a result, this form of ERα competes with wild type ERα for ligand and reduces the transcriptional activity of wild type ERα Luciferase reporter assay on ERE was performed to validate the loss of transcriptional activity in cells overexpressed with dominant-negative ERα MCF-7 cells were transfected with

plasmid encoding ERα defective in DNA-binding, 3X ERE-Luc and Renilla

plasmid 48h after transfection, the cells were subjected to luciferase reporter assay to check for transcriptional activity of ERα As expected, the high ERE luciferase reporter activity in regular serum was significantly abrogated in cells transfected with dominant-negative ERα (Figure 24A) Meanwhile, the cells were

subjected to Western blot analysis for ERα and its target gene c-Myc content The successful overexpression of dominant negative ERα was shown in figure 28B The PPARγ receptor level was not altered by overexpression of dominant negative ERα However, the expression of ERα response gene c-Myc was reduced

in cells transfected with dominant negative ERα, indicating the attenuated transcriptional activity of ERα in these cells (Figure 24B)

Next we went on to test the effect of dominant-negative ERα on PPARγ-mediated regulation of NHE1 expression in MCF-7 cells MCF-7 cells were transfected with plasmid encoding dominant negative ERα or parent empty vector as described in Materials and Methods 48h after transfection, the cells were treated with 15d-PGJ2 in RPMI supplemented with regular serum The protein level of NHE1 was analyzed by Western blot after 48h of drug treatment It was observed that in cells transfected with the empty vector, 15d-PGJ2 up to 5μM failed to induce significant reduction in NHE1 protein expression in regular serum condition (Figure 24C) This result is in agreement with our previous data in figure 23C, demonstrating that the activated ERα by serum prevented down-regulation of NHE1 by PPARγ ligand However, overexpression of dominant negative ERα was able to restore the sensitivity of NHE1 expression to PPARγ ligand In MCF-7 cells transfected with plasmid encoding DNA-binding defective ERα receptor, 15d-PGJ2 resulted in dose-dependent reduction in NHE1 protein expression (Figure 24C) This result further validated the data we obtained from experiment of silencing ERα in MCF-7 (Figure 24B): removing ERα completely

by ERα specific siRNA transfection or depleting them from DNA by

overexpressing dominant negative ERα both resulted in restored sensitivity 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