Parathyroid hormone induces epithelial-to-mesenchymal transition via the Wnt/β-catenin signaling pathway in human renal proximal tubular cells

Abstract

Epithelial-to-mesenchymal transition (EMT) has been shown to play an important role in renal fibrogenesis. Recent studies suggested parathyroid hormone (PTH) could accelerate EMT and subsequent organ fibrosis. However, the precise molecular mechanisms underlying PTH-induced EMT remain unknown. The present study was to investigate whether Wnt/β-catenin signaling pathway is involved in PTH-induced EMT in human renal proximal tubular cells (HK-2 cells) and to determine the profile of gene expression associated with PTH-induced EMT. PTH could induce morphological changes and gene expression characteristic of EMT in cultured HK-2 cells. Suppressing β-catenin expression or DKK1 limited gene expression characteristic of PTH-induced EMT. Based on the PCR array analysis, PTH treatment resulted in the up-regulation of 18 genes and down-regulation of 9 genes compared with the control. The results were further supported by a western blot analysis, which showed the increased Wnt4 protein expression. Wnt4 overexpression also promotes PTH-induced EMT in HK-2 cells. The findings demonstrated that PTH-induced EMT in HK-2 cells is mediated by Wnt/β-catenin signal pathway, and Wnt4 might be a key gene during PTH-induced EMT.

Keywords: Epithelial-to-mesenchymal transition; Wnt/β-catenin signal pathway; parathyroid hormone; renal tubular epithelial cell.

Figure 1

EMT was induced by PTH. (A) HK-2 cells were cultured in medium with or without 10^-10 M PTH for 48 h. Cellular morphology was photographed by phase contrast microscope. (B) HK-2 cells were exposed to 10^-10 M PTH for 12, 24, 36, 48 and 72 h. The mRNA expression of E-cadherin and α-SMA were quantified by qRT-PCR. GAPDH was used as an internal control. Results are expression as the mean ± SE (*P < 0.05, **P < 0.01 compared with control). (C) HK-2 cells were treated as (B). Cell lysates were prepared, and the protein expression of E-cadherin and α-SMA was detected using western blotting with anti-E-cadherin antibody and anti-α-SMA antibody. GAPDH was used as an internal control. (D) The loss of expression of E-cadherin and the increase of expression of α-SMA were confirmed by immunofluorescence. (E, F) HK-2 cells were treated as B. The protein expression of TGF-β1 was detected by western blotting. GAPDH was used as an internal control. Results are expression as the mean ± SE (*P < 0.05, **P < 0.01 compared with control).

Figure 2

Wnt/β-catenin pathway is required for PTH-induced EMT in HK-2 cells. A, B. HK-2 cells were transfected with β-catenin siRNA or negative control siRNA (NC siRNA). After transfection for 48 h, cells were collected and β-catenin expression was revealed by western blot. GAPDH from the same loading was used as control. Data shown are means ± SEM from three independent cell preparations, **P < 0.01. C, D. Control siRNA and β-catenin siRNA cells were treated without or with PTH for 48 h. Blots were probed with antibodies for E-cadherin, α-SMA, and β-catenin. GAPDH from the same loading was used as control. Results are expression as the mean ± SE. E, F. After treatment for 48 h with PTH with or without DKK1, cells were collected and cell lysates were prepared. The protein expression of E-cadherin and α-SMA was detected using western blotting with anti-E-cadherin antibody and anti-α-SMA antibody. GAPDH was used as an internal control. Results are expression as the mean ± SE.

Figure 3

Overexpression of WNT4 promote PTH-induced EMT in HK-2 cell. Cells transfected with pcDNA 3.1 vector or pcDNA 3.1-Wnt4 were treated without or with PTH for 48 h. Blots were probed with antibodies for E-cadherin, α-SMA, and Wnt4. GAPDH from the same loading was used as control. Results are expression as the mean ± SE.