Regulation of (pro)renin receptor expression by glucose-induced mitogen-activated protein kinase, nuclear factor-kappaB, and activator protein-1 signaling pathways

Abstract

Renal (pro)renin receptor (PRR) expression is increased in diabetes. The exact mechanisms involved in this process are not well established. We hypothesized that high glucose up-regulates PRR through protein kinase C (PKC)-Raf-ERK and PKC-c-Jun N-terminal kinase (JNK)-c-Jun signaling pathways. Rat mesangial cells exposed to 30 mm d-glucose demonstrated significant increase in PRR mRNA and protein expression, intracellular phosphorylation of Raf-1 (Y340/341), ERK, JNK, nuclear factor-kappaB (NF-kappaB) p65 (S536) and c-Jun (S63). By chromatin immunoprecipitation assay and EMSA, high glucose induced more functional NF-kappaB and activator protein (AP)-1 dimers bound to corresponding cis-regulatory elements in the predicted PRR promoter to up-regulate PRR transcription. Conventional and novel PKC inhibitors Chelerythrine and Rottlerin, Raf-1 inhibitor GW5074, MEK1/2 inhibitor U0126, JNK inhibitor SP600125, NF-kappaB inhibitor Quinazoline, and AP-1 inhibitor Curcumin, respectively, attenuated glucose-induced PRR up-regulation. Chelerythrine and Rottlerin also inhibited glucose-induced phosphorylation of Raf-1 (Y340/341), ERK1/2, JNK, NF-kappaB p65 (S536), and c-Jun (S63). GW5074 and U0126 inhibited the phosphorylation of ERK1/2 and NF-kappaB p65 (S536). SP600125 inhibited phosphorylation of NF-kappaB p65 (S536) and c-Jun (S63). We conclude that high glucose up-regulates the expression of PRR through mechanisms dependent on both PKC-Raf-ERK and PKC-JNK-c-Jun signaling pathways. NF-kappaB and AP-1 are involved in high-glucose-induced PRR up-regulation in rat mesangial cells.

Figure 1

PRR promoter prediction, mapping of NF-κB and AP-1 regulatory elements, and designs of ChIP primers and EMSA probes. Sp1, AP-1, and NF-κB elements in PRR promotor are designated with large capital letters A–G. EMSA probes corresponding to A–G elements were labeled as NF-κB01-02, AP-1.1-1.4, and Sp1, respectively. Position and orientation of ChIP primers corresponding to different A–G elements were labeled with black arrows and dashed lines. F, Forward primer; R, reverse primer.

Figure 2

Effect of glucose and different intracellular signaling pathways on PRR expression. A, Inhibition of PKC with Chelerythrine (Che; 5 μm) and Rottlerin (Ro; 5 μm). B, Inhibition of MAPKs with U0126 (10 μm) and SP600125 (20 μm). C, Inhibition of Raf-1 with GW5074 (10 nm). D, Inhibition of AP-1 with Curcumin (100 μm). E, Inhibition of NF-κB with Quinazoline (NF-κBi) (10 μm). Control, 5 mm d-glucose+25 mm l-glucose; glucose, 30 mm d-glucose. All the results represent the average of three independent experiments and each experiment was repeated at least three times.

Figure 3

PKC isomer proteins and their translocation, phosphorylation of intracellular signal proteins in response to high glucose alone, and combined with different kinases inhibitors in RMCs. Panel A, PKC isomers in membranous (M), cytosolic (C), and nuclear (N) compartments. Loading control (LC), β-Actin for membranous and cytosolic fractions and TATA binding protein (TBP) for nuclear fraction. Panels B–D, Phosphorylation of intracellular signal proteins in response to high glucose alone and combined with different kinases inhibitors. After 2 wk of glucose exposure and 12 h of serum starvation, cells were exposed to inhibitors for 15, 30, and 60 min. Panel B, PKC inhibition and phosphorylation of Raf-1, ERK1/2, JNK, c-Jun, and NF-κB p65. Panel C, Raf-1 inhibition and phosphorylation of ERK1/2, JNK, c-Jun, and NF-κB p65. Panel D, MEK1/2 and JNK inhibition and phosphorylation of NF-κB p65 and c-Jun. Control, 5 mm d-glucose+25 mm l-glucose; glucose, 30 mm d-glucose. The results are representative of three independent experiments.

Figure 4

Effect of glucose on binding of AP-1 regulatory elements in predicted PRR promoter. Panels A–D, In vivo mapping of AP-1 by ChIP. Panel E, EMSA and competitive EMSA. Oligo, AP-1 probes to AP-1 binding elements (Panels C–F as shown in Fig. 1). NE, Nuclear extract. Glucose (−), Control (5 mm d-glucose+25 mm l-glucose); glucose (+), 30 mm d-glucose. ChIP assay is based on three independent experiments and EMSA is a representative of three experiments.

Figure 5

Effect of glucose on binding of NF-κB and Sp1/Sp3 regulatory elements in predicted PRR promoter. Panels A and B, In vivo mapping of NF-κB by ChIP. Panel C, NF-κB EMSA and competitive EMSA. Panels D and E, In vivo mapping of Sp1 and Sp3 by ChIP. F, Sp1/Sp3 EMSA and competitive EMSA. Oligo, NF-κB probes to NF-κB binding elements A and B and Sp1 probes to Sp1 or Sp3 binding elements G as shown in Fig. 1. NE, Nuclear extract. Glucose (−), Control (5 mm d-glucose + 25 mm l-glucose); glucose (+), 30 mm d-glucose. ChIP assay is based on three independent experiments and EMSA is a representative of three experiments.

Figure 6

Working model of AP-1, NF-κB, and Sp1/Sp3 in predicted PRR promoter. Large open and solid black arrows refer to constitutive and regulated expression, respectively.