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. 2018 May;31:217-225.
doi: 10.1016/j.ebiom.2018.04.023. Epub 2018 May 11.

Runt-Related Transcription Factor 1 (RUNX1) Promotes TGF-β-Induced Renal Tubular Epithelial-to-Mesenchymal Transition (EMT) and Renal Fibrosis Through the PI3K Subunit p110δ

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Free PMC article

Runt-Related Transcription Factor 1 (RUNX1) Promotes TGF-β-Induced Renal Tubular Epithelial-to-Mesenchymal Transition (EMT) and Renal Fibrosis Through the PI3K Subunit p110δ

Tong Zhou et al. EBioMedicine. .
Free PMC article

Abstract

Renal fibrosis is widely considered a common mechanism leading to end-stage renal failure. Epithelial-to-mesenchymal transition (EMT) plays important roles in the pathogenesis of renal fibrosis. Runt-related transcription factor 1(RUNX1) plays a vital role in hematopoiesis via Endothelial-to-Hematopoietic Transition (EHT), a process that is conceptually similar to EMT, but its role in EMT and renal fibrosis is unclear. Here, we demonstrate that RUNX1 is overexpressed in the processes of TGF-β-induced partial EMT and renal fibrosis and that the expression level of RUNX1 is SMAD3-dependent. Knockdown of RUNX1 attenuated both TGF-β-induced phenotypic changes and the expression levels of EMT marker genes in renal tubular epithelial cells (RTECs). In addition, overexpression of RUNX1 promoted the expression of EMT marker genes in renal tubular epithelial cells. Moreover, RUNX1 promoted TGF-β-induced partial EMT by increasing transcription of the PI3K subunit p110δ, which mediated Akt activation. Specific deletion of Runx1 in mouse RTECs attenuated renal fibrosis, which was induced by both unilateral ureteral obstruction (UUO) and folic acid (FA) treatment. These findings suggest that RUNX1 is a potential target for preventing renal fibrosis.

Keywords: EMT; PI3K; RUNX1; Renal fibrosis; p110δ.

Figures

Fig. 1
Fig. 1
RUNX1 expression levels are increased in TGF-β-induced EMT and renal fibrosis. (a) HK-2 cells were stimulated with 5 ng/ml TGF-β for 24 h. RUNX1, RUNX2 and RUNX3 mRNA levels were detected by RT-PCR. Data are shown as the means ± SEM of three independent experiments. (b) HK-2 cells were stimulated with 5 ng/ml TGF-β for the indicated durations. Data are shown as the means ± SD of three independent experiments. (c) HK-2 cells were stimulated with 5 ng/ml TGF-β for the indicated durations to detect RUNX1, N-cadherin, SNAI1 and SLUG protein levels by immunoblotting. (d) HK-2 cells or RPTEC/TERT1 cells were transfected with SMAD3 siRNA (named siSD3) and nonspecific siRNA (named NC) or pretreated with the SMAD3 inhibitor SIS3 and control DMSO, followed by TGF-β stimulation to detect RUNX1 expression by immunoblotting. (e-g) Immunohistochemical and immunoblotting analysis of RUNX1 expression on day 7 after UUO, and Runx1, Tgf-β, Snai1 and Col1a1 mRNA levels by RT-qPCR in kidneys of UUO-induced and sham-control mice (n = 4). Data are shown as the mean ± SD of a representative of three independent experiments. *P < 0.05, **P < 0.01, *** P < 0.001.
Fig. 2
Fig. 2
RUNX1 is required for TGF-β-induced renal tubular EMT. (A, B) HK-2 cells were transfected with RUNX1 siRNA or control siRNA followed by 5 ng/ml TGF-β stimulation for 24 h. Morphological changes of HK-2 cells (a) and RUNX1, N-cadherin, SNAI1 and SLUG expression levels were detected by immunoblotting or RT-qPCR (b). Data are shown as the means ± SEM of three independent experiments. (c, d) HK-2 cells (c) or NRK-52E cells (d) were stably transfected to overexpress RUNX1 or GFP as a control and were then stimulated with 5 ng/ml TGF-β for 24 h. RUNX1, N-cadherin and SNAI1 expression were detected at the protein level by immunoblotting, or Slug at the mRNA level by RT-qPCR. Data are shown as the means ± SEM of three independent experiments. (e) RPTEC/TERT1 cells were transfected with RUNX1 siRNA or control siRNA followed by 5 ng/ml TGF-β stimulation for 24 h. RUNX1 and N-cadherin protein or Slug mRNA expression levels were detected. Data are shown as the mean ± SD of a representative of three independent experiments. *P < 0.05, **P < 0.01.
Fig. 3
Fig. 3
RUNX1 promotes EMT via p110δ-mediated Akt activation. (a, left panel) Plasmids for expressing GFP or RUNX1, together with plasmids for expressing SMAD3/4, were transfected into 293T cells in the presence of the CAGA luciferase reporter plasmid and the Renilla luciferase plasmid. After 24 h, cell lysates were prepared and analyzed by a dual luciferase reporter assay. Data are shown as the mean ± SD of a representative of three independent experiments. (a, right panel) NRK-52E cells were transfected with plasmids expressing RUNX1 or GFP, then stimulated with 20 ng/ml TGF-β for 30 min. RUNX1 and p-SMAD3 expression levels were detected by immunoblotting. (b) HK-2 cell lysates were immunoprecipitated with anti-SNAI1, SLUG or TWIST1 antibodies to detect endogenous interactions with RUNX1 by immunoblotting using anti-RUNX1 antibodies. (c-f) HK-2 cells were transfected with control siRNA, RUNX1 siRNA, or ATP1B1 siRNA as indicated, followed by stimulation with 5 ng/ml TGF-β for 24 h. Expression levels of N-cadherin and ATP1B1 (c) or HIF-1α and SMAD4 (d) or p110δ and phosphorylated AKT (e) or p110δ and phosphorylated ERK/p38 (f) were detected by immunoblotting. (g) RPTEC/TERT1 cells were transfected with RUNX1 siRNA or control siRNA, then stimulated with 5 ng/ml TGF-β for 24 h. p110δ, RUNX1 and Akt phosphorylation levels were detected by immunoblotting. (h) RUNX1 was knocked down or overexpressed in HK-2 cells to measure PIK3CD mRNA levels by RT-qPCR. Data are shown as the means ± SEM of three independent experiments. (i) HK-2 cells were stimulated with 5 ng/ml TGF-β for 24 h in the absence or presence of the PI3K inhibitor LY294002 (10 μM, named LY) or the p110δ inhibitor CAL-101 (1 μM, named CA). N-cadherin expression was detected by immunoblotting. (j, k) HK-2 or RPTEC/TERT1 cells were transfected with the control siRNA (named NC), RUNX1 siRNA (named siR), PTEN siRNA (named siP), then stimulated by 5 ng/ml TGF-β for 24 h. The mRNA levels of PTEN were detected by RT-qPCR (j). N-cadherin, RUNX1 and SNAI1 protein levels were detected by immunoblotting (k). Data are shown as the mean ± SD from three independent experiments. *P < 0.05, **P < 0.01.
Fig. 4
Fig. 4
Tubule-specific deletion of Runx1 ameliorates UUO-induced renal fibrosis. (a) Morphological changes of kidneys in control or UUO-induced Runx1cKO and WT mice. (b-e) mRNA levels of Runx1, Col1a1, Col3a1, Fibronectin and Pai-1 (b) or Runx2 (c) or Slc22a6 (d), or Il-6 (e), or Pik3cd (f) were detected by RT-qPCR in kidneys of control and UUO-induced Runx1cKO and WT mice (n = 4). (g) Immunoblotting analysis showed expression levels of SNAI1, SLUG, VIMETIN and α-SMA in kidneys of control or UUO-induced Runx1cKO and WT mice. (h) Representative images of MTS staining of kidneys from the indicated groups. Black bar: 50 μm. Data are shown as mean ± SD of a representative of three independent experiments. *P < 0.05, **P < 0.01, NS, not significant.
Fig. 5
Fig. 5
Tubule-specific deletion of Runx1 ameliorates FA-induced renal fibrosis. (a) Morphological changes of kidneys in the control or FA-treated Runx1cKO and WT mice. (b, c). The mRNA levels of Runx1, Col1a1, Col3a1, Pai-1, Il-6 and Pik3cd (b), or Slc22a6 (c) in kidneys from the control (n = 2) or FA-treated Runx1cKO (n = 6) and WT mice (n = 9). (d) Blood urea nitrogen (BUN) levels were detected in the control or FA-treated Runx1cKO and WT mice. (e) Representative images of MTS staining of kidneys from the indicated groups. Black bar: 50 μm. Data are shown as mean ± SD of a representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001.

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