Lysine methyltransferase SMYD2 promotes cyst growth in autosomal dominant polycystic kidney disease
Linda Xiaoyan Li, … , Julien Sage, Xiaogang Li
Linda Xiaoyan Li, … , Julien Sage, Xiaogang Li
Published June 12, 2017
Citation Information: J Clin Invest. 2017;127(7):2751-2764. https://doi.org/10.1172/JCI90921.
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Research Article Genetics Nephrology

Lysine methyltransferase SMYD2 promotes cyst growth in autosomal dominant polycystic kidney disease

Abstract

Autosomal dominant polycystic kidney disease (ADPKD) is driven by mutations in PKD1 and PKD2 genes. Recent work suggests that epigenetic modulation of gene expression and protein function may play a role in ADPKD pathogenesis. In this study, we identified SMYD2, a SET and MYND domain protein with lysine methyltransferase activity, as a regulator of renal cyst growth. SMYD2 was upregulated in renal epithelial cells and tissues from Pkd1-knockout mice as well as in ADPKD patients. SMYD2 deficiency delayed renal cyst growth in postnatal kidneys from Pkd1 mutant mice. Pkd1 and Smyd2 double-knockout mice lived longer than Pkd1-knockout mice. Targeting SMYD2 with its specific inhibitor, AZ505, delayed cyst growth in both early- and later-stage Pkd1 conditional knockout mouse models. SMYD2 carried out its function via methylation and activation of STAT3 and the p65 subunit of NF-κB, leading to increased cystic renal epithelial cell proliferation and survival. We further identified two positive feedback loops that integrate epigenetic regulation and renal inflammation in cyst development: SMYD2/IL-6/STAT3/SMYD2 and SMYD2/TNF-α/NF-κB/SMYD2. These pathways provide mechanisms by which SMYD2 might be induced by cyst fluid IL-6 and TNF-α in ADPKD kidneys. The SMYD2 transcriptional target gene Ptpn13 also linked SMYD2 to other PKD-associated signaling pathways, including ERK, mTOR, and Akt signaling, via PTPN13-mediated phosphorylation.

Authors

Linda Xiaoyan Li, Lucy X. Fan, Julie Xia Zhou, Jared J. Grantham, James P. Calvet, Julien Sage, Xiaogang Li

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Figure 5

SMYD2 regulates the phosphorylation of STAT3 and the p65 subunit of NF-κB.

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SMYD2 regulates the phosphorylation of STAT3 and the p65 subunit of NF-κ...
(A) Western blot analysis of p-STAT3 and p-p65 expression from whole cell lysates of Pkd1 WT MEK and Pkd1-null MEK cells. The phosphorylation of STAT3 and p65 was increased in Pkd1-null MEK versus WT MEK cells, whereas the expression of STAT3 and p65 was almost at the same level in these cells. (B) Western blot analysis of the expression of SMYD2, p-STAT3, STAT3, p-p65, and p65 in kidneys from Pkd1+/+:Smyd2+/+:Ksp-Cre, Pkd1fl/fl:Smyd2+/+:Ksp-Cre, and Pkd1fl/fl:Smyd2fl/fl:Ksp-Cre neonates at P7. The expression of SMYD2 and the phosphorylation of STAT3 and p65 were increased in kidneys from Pkd1fl/fl:Smyd2+/+:Ksp-Cre compared with Pkd1+/+:Smyd2+/+:Ksp-Cre mice, whereas the expression of these 3 proteins was decreased in kidneys from Pkd1fl/fl:Smyd2fl/fl:Ksp-Cre compared with Pkd1fl/fl:Smyd2+/+:Ksp-Cre neonates. (C) Interactions between SMYD2 and STAT3 (top panel) as well as SMYD2 and p65 (bottom panel) in Pkd1 WT and Pkd1-null MEK cells were detected with anti-SMYD2 antibody and then blotted with STAT3 antibody (top panel) and p65 (bottom panel) antibody, respectively. IgG was used as a negative control. (D) Methylation of STAT3 and p65 was increased in Pkd1 WT MEK cells compared with Pkd1-null MEK cells. These cells were immunoprecipitated with anti-STAT3 antibody (top panel) and anti-p65 antibody (bottom panel), and then blotted with STAT3, p65, and pan–methyl lysine antibody, respectively. IgG was used as a negative control. (E) The level of methylated STAT3 and p65 was decreased in Pkd1-null MEK cells treated with AZ505 (2 hours) compared with that in cells treated with DMSO as examined with anti–pan–methyl lysine antibody after these proteins were pulled down with STAT3 and p65 antibodies. (F) GFP-tagged SMYD2 together with Flag-tagged WT STAT3 or mutant STAT3 with lysine-to-arginine substitution were transfected into HEK293T cells. The effect of overexpression of SMYD2 on the methylation and phosphorylation of Flag-tagged STAT3 was examined by Western blot analysis. (G) GFP-tagged SMYD2 together with T7-tagged WT p65 (RelA) or mutant p65 (RelA) with lysine-to-arginine substitution were transfected into HEK293T cells. The effect of overexpression of SMYD2 on the methylation and phosphorylation of T7-tagged p65 (RelA) was examined by Western blot analysis.