Elevated soluble (pro)renin receptor (s(P)RR) concentration in maternal blood is associated with gestational hypertension and preeclampsia. Placenta has abundant expression of (P)RR, and the binding of (P)RR with pyruvate dehydrogenase E1 beta subunit (PDHB) is reported to maintain oxidative metabolism. Thus, we hypothesized that placental hypoxia may increase (P)RR, and the increased (P)RR may preserve PDHB expression. Expression and functional analyses were performed using human placental trophoblast cells, mainly JAR cells. (P)RR co-immunoprecipitated and showed co-immunofluorescence with PDHB mainly in the mitochondria. Hypoxia treatment significantly increased intracellular s(P)RR protein expression, but secreted s(P)RR in the culture medium was decreased by hypoxia. Hypoxia treatment did not alter PDHB expression or activity in the basal condition, but when (P)RR was knocked down by siRNA, PDHB protein and activity were reduced by hypoxia. Acetyl-CoA, the product of PDH activity, was significantly reduced by hypoxia treatment with (P)RR siRNA. S(P)RR is generated from full-length PRR when cleaved by specific proteases. Protease inhibitor experiments suggested furin and site 1 protease as the enzymes generating s(P)RR in JAR cells, and only when treated by site 1 protease inhibitor, PF429242, PDHB protein showed a significant trend to decrease with hypoxia. In JAR cells, hypoxia increased intracellular s(P)RR, and (P)RR preserved the expression and function of PDHB during hypoxia. (P)RR may help maintain oxidative metabolism and efficient energy production during placental ischemia in hypertensive disorders of pregnancy.
Supplementary Figure 1. The level of intracellular s(P)RR protein was not altered by chamber treatment with normoxic air in JAR cells.
Supplementary Figure 2. (A) In JEG-3 cells, time course of 1% O2 treatment did not show any significant change in fl(P)RR, s(P)RR, or PDHB protein levels. (B) In JEG-3 cells, dose-response of different O2 levels for 24 hr did not show any significant change in fl(P)RR, s(P)RR, or PDHB protein levels.
Supplementary Figure 3. Chemical hypoxia using cobalt chloride (COCl2) time- and dose-dependently increased intracellular s(P)RR in JAR cells. (A) S(P)RR increased time-dependently with 500 µM CoCl2. (B) S(P)RR increased at CoCl2 concentrations of 500 µM or more with 16 hours of treatment.
Supplementary Table 1. Statistical results of the graphs shown in Figures 2 to 5.