Abstract 5785: A Novel Function of Regulator of G-protein Signaling 2: Stabilization of Signal Transducer and Activator of Transcription 3 Phosphorylation
Background: Regulator of G protein signaling proteins (RGS) are known to regulate G protein coupled receptor (GPCR) signaling at the plasma membrane through their GTPase-activating protein activity. We have previously identified RGS2 as a downstream target of unphosphorylated Signal Transducer and Activator of Transcription 3 (U-STAT3). U-STAT3 mediated Angiotensin II (AngII) type 1 receptor (AT1R), a GPCR, induced cardiac hypertrophy but the mechanism is unclear.
Hypothesis: RGS2 is the mediator of U-STAT3 regulated Ang II/AT1R signaling.
Methods and Results: In AT1R overexpressing HEK293 cells and HL-1 cells (a mouse cardiomyocyte cell line), Ang II stimulation induced RGS2 expression in parallel with U-STAT3. The localization, distribution and expression of RGS2 were changed upon Ang II stimulation. RGS2 was mainly localized in the nucleus, which was also confirmed in human vascular smooth muscle cells. Gene transfection of RGS2 dose-dependently increased phosphorylated STAT3 (P-STAT3), and maintained a high level of P-STAT3 up to 24 hrs. Co-immunoprecipitation assay identified a complex formed between RGS2 and P-STAT3, suggesting that RGS2 plays a role in maintaining P-STAT3 stability. Treatment with AT1R antagonist, losartan, decreased RGS2/P-STAT3 interaction, suggesting that this interaction is dependent on AT1R activation. On the other hand, in cells overexpressing AT1R and Y705F mutant STAT3, which cannot be phosphorylated on residue 705 (U-STAT3), chromatin immunoprecipitation assay demonstrated that Y705F mutant STAT3 binds to a consensus GAS motif located in the RGS2 promoter, providing direct evidence that RGS2 is a downstream target of U-STAT3. siRNA-mediated gene knockdown of STAT3 consistently caused decreased RGS2 expression in AT1R overexpressing cells.
Conclusions: Chronic activation of AT1R induced U-STAT3 overexpression and subsequently induced overexpression of RGS2, which interacted with and stabilized P-STAT3. P-STAT3 further increases the transcription of stat3 gene, forming a positive feedback loop. We reported for the first time a novel function of RGS2, stabilization of P-STAT3, in mediating U-STAT3 regulated Ang II/AT1R signaling. Further studies are now underway utilizing engineered mice model.
This research has received full or partial funding support from the American Heart Association, Great Rivers Affiliate (Delaware, Kentucky, Ohio, Pennsylvania & West Virginia).