Recent studies show that the ex pression of PPAR�� is also regulated by DNA methylation and histone deacetylation. Furthermore, renal megalin expression is known to be regulated by PPARs. We therefore tested whether epigenetic regulation of PPARs mediated the effects sellckchem of 5Aza and TSA on cubilin and megalin expression. Computerized transcription fac tor binding site analysis identified three PPAR response elements conserved in human and mouse cubilin pro moters. Analysis of cubilin promoter activity in cells transfected with a cubilin promoter luciferase re porter found that cubilin transcription was increased by transfection of cells with PPAR and expression con structs. Furthermore, endogenous cubilin mRNA expression in NRK cells was blocked by the PPAR and antagonists, GW6471 and GW9662.
To further evaluate the role of PPARs in regulating Inhibitors,Modulators,Libraries cubilin expression, we utilized PRTCs. PPAR agonist, Wy14643, treatment of PRTCs resulted in a significant increase in cubilin protein levels over a range of agonist concentrations. Cubilin mRNA levels were also Inhibitors,Modulators,Libraries significantly increased by PPAR agonist treatment at 100 uM concentration. By contrast, PPAR antagonist, GW6471, significantly decreased endogenous cubilin mRNA ex pression and also inhibited the increased cubilin expres sion achieved by agonist treatment. PPAR agonist and antagonist treatments resulted in similar re ciprocal changes in the mRNA levels of Acadl, a known PPAR responsive gene. Treat ment of PRTCs with the PPAR�� agonist also resulted in a significant increase in cubilin protein levels.
Rosiglitazone at 50 nM concentration also elicited a modest, but significant increase in cubilin mRNA expression. Furthermore, the PPAR�� antagonist, GW9662, inhibited the increased cubilin ex pression achieved by Rosiglitazone treatment. Together, these findings indicate that, similar to megalin, cubilin is a gene regulated by both PPAR and. We next evaluated the effects Inhibitors,Modulators,Libraries of 5Aza and TSA on the expression of PPAR and in NRK cells. As shown in Figure 9A, 5Aza treatment elicited a dose dependent in crease in PPAR mRNA expression. 5Aza also augmented PPAR�� expression at low concentrations but not at the highest concentration tested. Similarly, TSA treatment of NRK cells also produced a concentration dependent increase in PPAR mRNA levels. TSA also increased PPAR�� expression at the highest con centration tested.
The observations that TSA and 5Aza Inhibitors,Modulators,Libraries increased PPAR and mRNA levels suggested the possibility that cubilin upregulation by TSA and Inhibitors,Modulators,Libraries 5Aza resulted from increased expression of PPAR. We therefore eval uated the selleck chemical effects of PPAR and antagonists on TSA and 5Aza induction of cubilin expression. As shown in Figure 9E, the upregulation of cubilin by 5Aza was inhibited by PPAR�� antagonist as well as combined PPAR and antagonist treatments. Acadl, a known PPAR responsive gene, was also increased by 5Aza treatment and the increase was inhibited by PPAR antagonist treatment.