Transforming growth factor beta 1 (TGFβ1)-induced epithelial-mesenchymal transition (EMT) in kidney epithelial cells plays a key role in renal tubulointerstitial fibrosis in chronic kidney diseases. As hypoxia-inducible factor (HIF)-1α is found to mediate TGFβ1 signaling pathway, we tested the hypothesis that HIF-1α and its upstream regulator prolyl hydroxylase domain-containing proteins (PHDs) are involved in TGFβ1-induced EMT in renal tubular cells. Our results showed that TGFβ1 treatment for 48 h stimulated EMT in cultured renal tubular cells as indicated by the decrease in epithelial marker P-cadherin from 1.0 ± 0.02 to 0.40 ± 0.05 (
P
< 0.05), and the increase in mesenchymal markers α-smooth muscle actin (2.14 ± 0.32 fold,
P
< 0.05) and fibroblast-specific protein (2.0 ± 0.17 fold,
P
< 0.05) as shown in Western blot assay. Meanwhile, TGFβ1 time-dependently increased HIF-1α, which reached its maximum value (2.36 ± 0.2 fold,
P
< 0.05) at 24 h, and that HIF-1α siRNA significantly inhibited TGFβ1-induced EMT, suggesting that HIF-1α mediated TGFβ1 induced-EMT. Real-time PCR showed that PHD1 and PHD2, rather than PHD3, could be detected, with PHD2 as the predominant form of PHDs (PHD1 : PHD2 = 0.21:1.0). Importantly, TGFβ1 time-dependently decreased PHD2 mRNA and protein level, which reached their maximum value from 1.0 ± 0.15 to 0.45 ± 0.08 (
P
< 0.05) for mRNA at 16 h and from 1.0 ± 0.08 to 0.26 ± 0.08 (
P
< 0.05) for protein at 24 h, respectively. In contrast, TGFβ1 had no effect on PHD1 mRNA and protein levels. Furthermore, over-expression of PHD2 transgene almost fully prevented TGFβ1-induced HIF-1α accumulation and EMT marker changes, indicating that PHD2 is involved in TGFβ1-induced EMT. Finally, Smad2 inhibitor SB431542 prevented TGFβ1-induced PHD2 decrease, suggesting that Smad2 may mediate TGFβ1-induced EMT through PHD2/HIF-1α. It is concluded that TGFβ1 decreased PHD2 expression via a Smad2-dependent signaling pathway, thereby leading to HIF-1α accumulation and EMT in renal tubular cells. The present study suggests that PHD2/HIF-1α is a novel signaling pathway mediating the fibrogenic effect of TGFβ1 and that manipulating PHD2/HIF-1α pathway may be used as a therapeutic strategy in chronic kidney diseases. (support: NIH grant HL89563 and HL106042)
Hypoxia-inducible factor prolyl-hydroxylase-2 mediates transforming growth factor beta 1-induced epithelial–mesenchymal transition in renal tubular cells
