Figure 7.

aPC regulates OTUB1 and YB-1 expression in tubular cells via PAR1 and EPCR. (A) PAR1, PAR2, PAR4, and EPCR are expressed in BUMPT cells. Representative images showing expression of PARs and EPCR in BUMPT cells and mouse kidney. (Kid, positive control; semi-quantitative RT-PCR, top, and immunoblot [IB] bottom). OTUB1 and YB-1 levels in HR-stressed BUMPT cells incubated with PAR (P1-P4) or EPCR (EP) blocking antibodies (Ab) before aPC treatment (B) or treated with PAR-agonist peptides (P1-P4 AP) (C). Blocking PAR1 or EPCR, but not PAR2 or PAR4, abolishes aPC’s effect in regard to OTUB1 and YB-1 expression. Activation of PAR1 by the aPC-specific agonist peptide (P1AP1: NPNDKYEPFWEDEEKNESGL), but not by the thrombin-specific agonist peptide (P1AP2: TFLLR) prevents the HR-induced loss of OTUB1 and YB1. Representative immunoblots showing OTUB1 [(B) and (C) top] and YB-1 [(B) and (C), bottom] expression in whole-cell lysates and bar graph summarizing results. Mean±SD value of at least three independent experiments [(B) and (C)]; *P<0.05; **P<0.01 (ANOVA).