Parathyroid hormone induces endothelial-to-adipocyte transition in endothelial cells by Wnt/β-catenin pathway

Abstract

Objective To investigate whether elevated parathyroid hormone (PTH) levels could induce endothelial-to-mesenchymal transition (EndMT) and adipocyte transition in endothelial cells (ECs), and to determine the possible underlying mechanism. Methods (1) A rat model of secondary hyperparathyroidism and chronic kidney disease (CKD) was established. The adiposity in bone marrow was detected by oil red O staining. Immunofluorescence staining was performed to detect the expression and localization of cluster of differentiation 31 (CD31) and fibroblast-specific protein 1 (FSP1). (2) The human umbilical vein ECs were cultured in vitro. Western blotting was performed to detect protein expressions of EndMT-related markers CD31, FSP1 and α-smooth muscle actin (α-SMA) in interference groups with different PTH concentrations (0, 10-11, 10-9, 10-7 mol/L PTH for 48 h) and times (0, 12, 24, 48 h, 10-7 mol/L PTH), as well as the expression of β-catenin in interference groups with different PTH concentrations. The localizations of CD31, FSP1 and β-catenin were observed by cell immunofluorescence. Protein expressions of adipocytes markers peroxisome proliferator-activated receptor-γ (PPAR-γ) and CCAAT/enhancer binding protein-α (C/EBP-α) by Western blotting and the degree of adipogenesis by oil red O staining were detected after transformed ECs were cultured in adipogenic culture medium for one week. Small interfering RNA (siRNA) was performed to silence β-catenin expression. ECs were divided into control siRNA group, β-catenin siRNA group, PTH+control siRNA group and PTH+β-catenin siRNA group. Protein expressions of CD31, FSP1 and PPAR-γ by Western blotting and the degree of adipogenesis by oil red O staining were determined. Results (1) In vivo, compared with the control, CKD rats had increased adipocytes in bone marrow (P<0.05), and the co-expression of CD31 and FSP1 in bone marrow ECs. (2) In vitro, PTH significantly inhibited the expression of endothelial marker CD31 and increased the expressions of mesenchymal markers FSP1 and α-SMA in concentration-and time-dependent manners. These indexes in 10-7 mol/L PTH group and 0 mol/L PTH group, in 48 h group and 0 h group showed statistical differences (all P<0.05). In PTH group ECs with 10-7 mol/L PTH for 48 h showed FSP1 accumulation in the cytoplasm and reduced expressions of CD31, and ECs had higher expressions of PPAR-γ and C/EBP-α as well as the degree of adipogenesis than those in control group (all P<0.05). Furthermore, PTH enhanced the nuclear β-catenin protein levels in ECs in concentration-dependent. The expressions of β-catenin in 10-7 mol/L PTH group and 0 mol/L PTH group showed statistical differences (P<0.05). β-catenin expressed in the cytoplasm in control group, while it enter into the nucleus in PTH group. Compared with those in PTH+control siRNA group, the expressions of CD31 and PPAR-γ as well as the degree of adipogenesis decreased in PTH+β-catenin siRNA group (all P<0.05), while the expression of FSP1 increased (P<0.05). Conclusions PTH induces ECs-to-adipocytes transition by the canonical Wnt/β-catenin signaling pathway, which might account for bone loss in CKD. Silenced β-catenin expression can inhibit PTH-induced EndMT and adipogenesis. Key words: Chronic kidney disease-mineral and bone disorder; Parathyroid hormone; Human umbilical vein endothelial cells; Adipocytes; Wnt signaling pathway; Endothelial-to-mesenchymal transition