SetD7-PKA Feedforward Signaling Enhances Cyst Growth in ADPKD

Background: Emerging evidence highlights a pivotal role of epigenetic mechanisms in ADPKD's pathophysiology and as drug targets. Our RNA-sequencing analysis showed that the histone methyltransferase SET domain containing 7 (SetD7) was upregulated in PKA activated Pkd1RC/RC kidneys and downregulated in PKA inhibited Pkd1RC/RC kidneys. Whether SetD7 contributes to ADPKD, and its relationship with cAMP-PKA remains unknown. Methods: To define the role of SetD7 and its cross talk with cAMP-PKA in ADPKD, we treated Pkd1 WT and null MEK cells as well as Pkd1 homozygous PN24 cells and heterozygous PH2 cells with SetD7 siRNA and inhibitor (PFI-2), adenylyl cyclase activator Forskolin (FSK) and PKA inhibitor, H-89, and expressed Myc–tagged SetD7 in these cells. We also treated Pkd1RC/RC and Pkd1nl/nl mice with PFI-2. The effects of these treatments in vitro and in vivo were evaluated by western blot, qRT-PCR, immunostaining as well as co-immunoprecipitation and ChIP assays. Results: We show that SetD7 is upregulated in 1) Pkd1 mutant MEK and PN24 cells compared to Pkd1 WT MEK and heterozygous PH2 cells, and 2) Pkd1mutant mouse kidneys, and human ADPKD kidneys compared to their respective normal control kidneys. Inhibition of SetD7 with PFI-2 delayed cyst growth and improved kidney function as seen by a decrease in cystic index, kidney weight (KW)/body weight (BW) ratios, and blood urea nitrogen (BUN) levels in Pkd1 mutant mice. Targeting SetD7 with PFI-2 and siRNA decreased the activation of PKD associated signaling pathways, including Stat3, p65 and ERK, in Pkd1 mutant cells and kidneys. We found that SetD7 methylates Stat3 to increase its activation. We also identified a crosstalk between SetD7 and the cAMP-PKA-CREB signaling in PKD. We found that 1) treatment with FSK increases and treatment with H-89 decreases SetD7 expression in PN24 cells, 2) SetD7 interacts with the catalytic subunit of PKA, leading to the activation of PKA, 3) CREB binds to the SetD7 promoter to increase its transcription, 4) SetD7 interacts with CREB to affect its activity, and 5) targeting SetD7 decreases the activation of PKA-CREB signaling pathway in Pkd1 mutant cells and kidneys Conclusion: This study reveals a novel PKA-Setd7-PKA positive feedback loop that promotes cystogenesis in ADPKD and targeting SetD7 with inhibitor may be a promising therapeutic strategy for ADPKD. Funding: NIDDK Support, Other U.S. Government Support