The Protective Roles of Estrogen Receptor β in Renal Calcium Oxalate Crystal Formation via Reducing the Liver Oxalate Biosynthesis and Renal Oxidative Stress-Mediated Cell Injury

Figure 4

Mechanism dissection on how ERβ regulates NOX2 expression. (a) NOX2 mRNA levels were regulated by E2 or ERβ antagonists in HK-2 and HKC-8 cells. Cells were treated for 6 hr with vehicle (control), 0.75 mM oxalate, 0.75 mM oxalate + 10 nM E2, 0.75 mM oxalate + 10 nM E2 + 10 μM ICI, or 0.75 mM oxalate + 10 nM E2 + 10 μM PHTPP for q-PCR of NOX2 mRNA levels. Bonferroni’s test was applied for statistical analysis of results. (b) Predicted 3 potential EREs located in the 3 kb-NOX2 promoter region. (c) ChIP was performed in HK-2 cells, and only ERE I of the NOX2 promoter could be bound by ERβ. (d) ERE I wild-type or mutant of NOX2 promoter reporter constructs were cotransfected with pRL-TK at a ratio of 1000 : 1 into HK-2-shLuc and HK-2-shERβ cells. Luciferase reporter assay was performed after 48 hr incubation. For (a) and (d), data shown are . and . n.s. = not significant.

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