THE MECHANISM OF PPARN3 MEDIATED DOWN REGULATION OF SODIUM HYDROGEN EXCHANGER 1 (NHE1) GENE EPXRESSION AND ITS INHIBITION BY ESTROGEN RECEPTOR n1 4

washed and reseeded at a density of 1.0 X 104cells/100mm dish. After 14 days of incubation, cells were stained with crystal violet. Graphical representation of colony counts from colony forming assays. Results denote means +/-SD computed from three experiments. *, p[...]... described earlier However, the presence of E2 succefully inhibited the down- regulation of NHE1 mRNA induced by all three PPARγ ligands tested (Figure 18 C) In this section, we have further confirmed that PPARγ -mediated repression of NHE1 protein and mRNA are inhibited by estrogen 12 7 Figure 18 : Estrogen blocks PPARγ -mediated down- regulation of NHE1 expression 12 8 MCF-7 (3 X105 cells/6-well dishes) cells... down- regulation of NHE1 expression induced by PPARγ lgiands, we next investigated the role of estrogen receptor in PPARγ -mediated inhibition of NHE1 3B2 .1 Re-expression of ERα in ER negative MDA-MB-2 31 cells restores its response to E2 on inhibiting PPARγ -mediated down- regulation of NHE1 The status of ERα is a determining factor in the biological behaviors of breast cancers and is closely associated with their... amount of NHE1 mRNA in both MDA-MB-2 31 and MDA-MB-2 31 ER+ cells after treatment of 15 d-PGJ2 in the presence or absence of E2 Consistent with the data on NHE1 protein expression, 3μM of 15 d-PGJ2 reduced NHE1 mRNA to about 60% of the control in normal MDA-MB-2 31 cells Moreover, pre-incubation with estrogen failed to rescue the down- regulation of NHE1 mRNA by active PPARγ in this cell line On the other hand,... estrogen recptor alpha 13 1 Figure 19 : Re-expression of ERα blocks PPARγ -mediated down- regulation of NHE1 expression in MDA-MB-2 31 cells (A) MDA-MB-2 31 (2X105 cells/6-well dishes), and (B) MDA-MB-2 31 ERα+ (2X10 5 cells/6-well dishes) cells were treated with 15 d-PGJ2 for 24h with or without 2h pre-incubation of 1 M E2, and the protein expression of NHE1 was 13 2 analyzed by Western blot NHE1 band intensity was... that the higher amount of E2 blocked the down- regulation of NHE1 expression by PPARγ ligand in regular serum condition To assess whether estrogen was responsible for the inhibitory effect on PPARγ -mediated reduction of NHE1 expression in regular FBS, we re-introduced 17 β-estradiol to serumdeprived MCF-7 before addition of 15 d-PGJ2 Interestingly, the down- regulation 12 6 of NHE1 protein expression by 15 d-PGJ2... gene and tested the effect of E2 on NHE1 expression in this ERα positive MDA-MB-2 31 cells We hypothesized that ERα receptor was responsible for the observed E2 -mediated reversal of NHE1 down- regulation induced by PPARγ ligands, hence re-expression of ERα receptor in MDA-MB2 31 cells would restore the modulation of E2 on PPARγ -mediated reduction in NHE1 expression In accordance with our hypothesis, 15 d-PGJ2... other hand, the same 3μM of 15 d-PGJ2 only decreased the NHE1 mRNA to about 75% of the control in MDA-MB-2 31 ER+ cells More prominently, E2 was able to reverse 20% reduction in NHE1 mRNA brought about by 3μM of 15 dPGJ2 in this cell line (Figure 19 C) Taken together, these findings support that the inhibitory effect of estrogen on PPARγ-induced repression of NHE1 protein and mRNA is mediated by estrogen. .. NHE1 protein in ER+ MDA-MB-2 31 cells in the absence of E2 However, when these cells were pre-incubated with 1 M of E2 for 2h, the down- regualtion of NHE1 by PPARγ ligand was much attenuated (Figure 19 B) To further confirm that re-expression of ERα receptor in MDA-MB-2 31 cells restores the modulation of E2 on PPARγ -mediated reduction in NHE1 13 0 transcription, real-time PCR was used to quantify the. .. Transient silencing of ERα in ER positive MCF-7 cells abrogates the inhibitory effect of E2 on PPARγ -mediated down- regulation of NHE1 Since re-expression of ERα and its activation by E2 in ER negative MDA-MB2 31 cells rescued the inhibition of NHE1 expression by PPARγ ligands, it was of interest to investigate if silencing ERα in ER positive MCF-7 would enhance the effect of PPARγ ligands on NHE1 expression... examine whether E2 rescue the PPARγ -mediated suppression of NHE1 at transcriptional level To this end, MCF-7 cells were pre-incubated with 1 M of E2 before they were exposed to 3μM of 15 d-PGJ2 or 10 μM of ciglitazone or 15 μM of troglitazone, and the cells were next harvested and subjected to NHE1 mRNA analysis by real-time PCR In cells without E2 treatment, PPARγ ligands brought down the NHE1 mRNA level . EFFECT OF PPARγ ON NHE1 Given that the presence of estrogen blocked the down- regulation of NHE1 expression induced by PPARγ lgiands, we next investigated the role of estrogen receptor in PPARγ -mediated. rescue the down- regulation of NHE1 mRNA by active PPARγ in this cell line. On the other hand, the same 3μM of 15 d-PGJ 2 only decreased the NHE1 mRNA to about 75% of the control in MDA-MB-2 31 ER+. onto 10 0mm dish after 16 h exposure to 15 d-PGJ 2 . The selection of NHE1-overexpressing cells was then achieved by including 12 1 hygromycin in the culturing medium for 14 days, before the