Angiotensin II Type-2 receptors modulate inflammation through signal transducer and activator of transcription proteins 3 phosphorylation and TNFα production

Abstract

Angiotensin subtype-1 receptor (AT(1)R) influences inflammatory processes through enhancing signal transducer and activator of transcription proteins 3 (STAT3) signal transduction, resulting in increased tumor necrosis factor-α (TNF-α) production. Although angiotensin subtype-2 receptor (AT(2)R), in general, antagonizes AT(1)R-stimulated activity, it is not known if AT(2)R has any anti-inflammatory effects. In this study, we tested the hypothesis that AT(2)R activation plays an anti-inflammatory role by reducing STAT3 phosphorylation and TNF-α production. Changes in AT(2)R expression, TNF-α production, and STAT3 phosphorylation were quantified by Western blotting, Bio-Plex cytokine, and phosphoprotein cellular signaling assays in PC12W cells that express AT(2)R but not AT(1)R, in response to the AT(2)R agonist, CGP-42112 (CGP, 100 nm), or AT(2)R antagonist PD-123319 (PD, 1 μm). A 100% increase in AT(2)R expression in response to stimulation with its agonist CGP was observed. Further, AT(2)R activation reduced TNF-α production by 39% and STAT3 phosphorylation by 83%. In contrast, PD decreased AT(2)R expression by 76%, increased TNF-α production by 84%, and increased STAT3 phosphorylation by 67%. These findings suggest that increased AT(2)R expression may play a role in the observed decrease in inflammatory pathway activation through decreased TNF-α production and STAT3 signaling. Restoration of AT(2)R expression and/or its activation constitute a potentially novel therapeutic target for the management of inflammatory processes.

FIG. 1.

Laser scanning confocal images of AT₂R in PC12W cells, (A) and (B) 100 × magnification. Green fluorescent labeling for the AT₂R was detected on the cell membrane and in the nuclear and perinuclear region of the PC12W cells. AT₂R, angiotensin subtype-2 receptor.

FIG. 2.

Immunoblot quantification (A) of AT₂R expression in response to AT₂R agonist CGP-42112A (CGP, 100 nmol, □) or AT₂R antagonist PD-123319 (PD, 1 μmol, ▪) for 24 h using immunoblot detection with anti-AT₂R in PC12W cells. Bar graphs (B) represent the means of the band densities expressed as percentage change from control. Blots are representative of 3 experiments.

FIG. 3.

(A) Change in STAT3 phosphorylation in response to treatment with the AT₂R agonist CGP42112A (CGP, 100 nmol) or AT₂R antagonist PD123319 (PD, 1 μmol) for 24 h. Data acquired using the Bio-Plex suspension array system. (B) Change in the production of TNF-α in PC12W cells in response to AT₂R agonist CGP-42112A (CGP, 100 nmol) or AT₂R antagonist PD-123319 (PD, 1 μmol) for 24 h. Data shown are average of 3 experiments. STAT3, signal transducer and activator of transcription proteins 3; TNF-α, tumor necrosis factor-α.