Parathyroid Hormone Promotes Human Umbilical Vein Endothelial Cell Migration and Proliferation Through Orai1-Mediated Calcium Signaling

Abstract

Parathyroid hormone is the main endocrine regulator of extracellular calcium and phosphorus levels. Secondary hyperparathyroidism-induced endothelial dysfunction may be related to calcium homeostasis disorders. Here, we investigated the effects of parathyroid hormone on human umbilical vein endothelial cells (HUVECs) and characterized the involvement of store-operated Ca²⁺ entry (SOCE) and the nuclear factor of activated T cells (NFAT) signaling pathway. We used immunoblot experiments to find that parathyroid hormone significantly enhanced the expression of the Orai1 channel, a type of channel mediating SOCE, SOCE activity, and Orai1-mediated proliferation of HUVECs but did not increase Orai2 and Orai3. RNA-seq was utilized to identify 1,655 differentially expressed genes (823 upregulated and 832 downregulated) in parathyroid hormone-treated HUVECs as well as enhanced focal adhesion signaling and expression levels of two key genes, namely, COL1A1 and NFATC1. Increased protein and mRNA expression levels of COL1A1 and NFATC1 were confirmed by immunoblotting and quantitative RT-PCR, respectively. Cytosol and nuclei fractionation experiments and immunofluorescence methods were used to show that parathyroid hormone treatment increased NFATC1 nuclear translocation, which was inhibited by a calcineurin inhibitor (CsA), a selective calmodulin antagonist (W7), an Orai channel inhibitor (BTP2), or Orai1 small interfering RNA (siRNA) transfection. Parathyroid hormone also increased COL1A1 expression, cell migration, and proliferation of HUVECs. The PTH-induced increase in HUVEC migration and proliferation were inhibited by CsA, W7, BTP2, or COL1A1 siRNA transfection. These findings indicated that PTH increased Orai1 expression and Orai1-mediated SOCE, causing the nuclear translocation of NFATC1 to increase COL1A1 expression and COL1A1-mediated HUVEC migration and proliferation. These results suggest potential key therapeutic targets of Orai1 and the downstream calmodulin/calcineurin/NFATC1/COL1A1 signaling pathway in parathyroid hormone-induced endothelial dysfunction and shed light on underlying mechanisms that may be altered to prevent or treat secondary hyperparathyroidism-associated cardiovascular disease.

Keywords: COL1A1; NFAT; human umbilical vein endothelial cells; parathyroid hormone; secondary hyperparathyroidism; store-operated Ca2+ entry.

Figure 1

Effects of parathyroid hormone (PTH) on Orai expression and proliferation of human umbilical vein endothelial cells (HUVECs). (A–D) Representative Western blot images (A) and summary data (B–D) showing Orai1, Orai2, and Orai3 expression level changes in HUVECs after PTH treatment (1, 10, 100 pM) for 24 h compared with the vehicle control (Con). (E) Summary data showing viability of HUVECs treated with PTH (100 pM) for 24 h. (F,G) Representative Western blot images (F) and summary data (G) showing STIM1 expression level changes in HUVECs after PTH treatment (1, 10, 100 pM) for 24 h compared with the vehicle control (Con). Data are shown as the mean ± SEM; n = 3–5. *P < 0.05, **P < 0.01 vs. Con analyzed by one-way analysis of variance followed by Dunnett's multiple comparisons test.

Figure 2

Effect of parathyroid hormone (PTH) on store-operated Ca²⁺ entry (SOCE) in human umbilical vein endothelial cells (HUVECs). (A,B) Representative traces (A) and summary data (B) showing thapsigargin (TG)-evoked SOCE in HUVECs after treatment with vehicle control, PTH (100 pM) or PTH plus BTP2 (an Orai nonspecific inhibitor) for 24 h. After depletion of the intracellular Ca²⁺ stores by treatment with 2 μM TG for 10 min in a Ca²⁺-free medium, SOCE was evoked by 2 mM Ca²⁺ application to the medium. Data are shown as the mean ± SEM; n = 7. **P < 0.01, ***P < 0.001 vs. Con or PTH. (C–E) Representative traces (C,D) and summary data (E) showing TG-evoked SOCE in HUVECs transfected with scrambled siRNA or Orai1 siRNA and treated with vehicle control or PTH (100 pM) for 24 h. (F,G) Representative images (F) and summary data (G) showing the effect of scrambled siRNA or Orai1 siRNA transfection on Orai1 expression in HUVECs. GAPDH was used as a loading control. (H) Summary data showing viability of HUVECs transfected with scrambled siRNA or Orai1 siRNA and treated with vehicle control or PTH (100 pM) for 24 h. Data are shown as the mean ± SEM; n = 4–5. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Scrambled siRNA.

Figure 3

Effect of parathyroid hormone (PTH) on the transcriptome profile of human umbilical vein endothelial cells (HUVECs). (A,B) Volcano plot (A) and statistical graphs (B) of differentially expressed genes (DEGs). (C) Number of differentially expressed transcripts. (D) Heat map of the DEGs. Red represents highly expressed genes; blue, low expression. DEGs were identified with corrected P < 0.05 and absolute fold change ≥2. C1-4 represents the control group; P1-4, the PTH-treated (100 pM, 24 h) group.

Figure 4

Top 20 enriched Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway terms of differentially expressed genes (DEGs). Rich factor represents the percentage of DEGs in the KEGG relative to the identified genes in that classification. Circle size and color indicate the number of genes and P-value, respectively.

Figure 5

Effect of parathyroid hormone (PTH) on mRNA expressions of COL1A1, NFATC1, Orai1, Orai2, Orai3, STIM1 and STIM2 in human umbilical vein endothelial cells (HUVECs). (A–G) After the treatment with vehicle control or 100 pM PTH for 24 h, alterations in (A) COL1A1, (B) NFATC1, (C) Orai1, (D) Orai2, (E) Orai3, (F) STIM1, (G) STIM2 mRNA levels were detected by qPCR assays in HUVECs. Messenger RNA expression was normalized to that of GAPDH. Data are shown as the mean ± SEM; n = 5 – 6. *P < 0.05, **P < 0.01, ***P < 0.001 vs. Con.

Figure 6

Role of Orai1-mediated store-operated Ca²⁺ entry (SOCE) in parathyroid hormone (PTH)-induced NFAT nuclear translocation in human umbilical vein endothelial cells (HUVECs). (A–F) Representative confocal microscopy images and summary data (I) showing NFATC1 distribution in HUVECs treated for 24 h with (A) vehicle control, (B) 100 pM PTH, (C) PTH + CsA (calcineurin inhibitor), (D) PTH + W7 (calmodulin antagonist), (E) PTH + BTP2 (an Orai nonspecific inhibitor) or (F) PTH + Orai1 siRNA transfection. Green fluorescence indicates NFATC1; Blue, 4′,6-diamidino-2-phenylindole (DAPI) indicates nuclei. Data are shown as the mean ± SEM; n = 4. **P < 0.01, ***P < 0.001 vs. Con or PTH analyzed by one-way analysis of variance followed by Dunnett's multiple comparisons test. (G,H) Representative Western blot images showing fractionation assay results indicating the presence of p-NFATC1 in the cytoplasmic [Cyto, (H)] and NFATC1 in the nuclear [Nuc, (G)] extracts under the same treatment conditions as for confocal microscopy analyses. Lamin B1 is a nuclear marker; β-Tubulin is a cytoplasmic marker. (I) Summary data showing the ratio of green fluorescence intensity of NFATc-GFP in the nuclear (Nuc)/cytoplasmic (Cyto).

Figure 7

Effect of parathyroid hormone (PTH) on COL1A1 expression in human umbilical vein endothelial cells (HUVECs). (A) After HUVECs were treated with vehicle control, 100 pM PTH, PTH + BTP2 (an Orai nonspecific inhibitor), PTH + CsA (calcineurin inhibitor), or PTH + W7 (calmodulin antagonist) for 24 h, COL1A1 mRNA levels were analyzed by qPCR assays. Messenger RNA expression was normalized to that of GAPDH. (B,C) Representative images (B) and summary data (C) showing COL1A1 protein expression in HUVECs after treatment with vehicle control, 100 pM PTH, PTH + BTP2, PTH + CsA or PTH + W7 for 24 h. GAPDH expression was referenced as the loading control. Data are shown as the mean ± SEM; n = 5–6. *P < 0.05, ****P < 0.0001 vs. Con or PTH in (A) and (C) analyzed by one-way analysis of variance followed by Dunnett's multiple comparisons test.

Figure 8

Effect of parathyroid hormone (PTH) on migration of human umbilical vein endothelial cells (HUVECs) (A,B). (A) Representative images showing the migration of HUVECs treated with vehicle control (Con), 100 pM PTH, PTH + BTP2 (an Orai nonspecific inhibitor), PTH + CsA (calcineurin inhibitor), PTH + W7 (calmodulin antagonist), or PTH + COL1A1 siRNA transfection. (B) Summary data showing the percentage of cells that migrated during 24 h. (C) Summary data showing the viability of HUVECs treated with vehicle control, 100 pM PTH, PTH + BTP2, PTH + CsA, PTH + W7, or PTH + COL1A1 siRNA transfection for 24 h. Data are shown as the mean ± SEM; n = 4–5. *P < 0.05, **P < 0.01 vs. Con or PTH analyzed by one-way analysis of variance followed by Dunnett's multiple comparisons test. (D,E) Representative images and summary data showing the effect of scrambled siRNA or COL1A1 siRNA transfection on COL1A1 expression in HUVECs. GAPDH was used as a loading control. Data are shown as the mean ± SEM; n = 4–5. **P < 0.01 vs. Scrambled siRNA.

Figure 9

Schematic diagram of Orai1-mediated store-operated Ca²⁺ entry (SOCE) signal transduction pathway in human umbilical vein endothelial cells (HUVECs) after parathyroid hormone (PTH) stimulation. In this proposed mechanism, PTH acts on its receptor PTHR1, a G-protein coupled receptor, to produce phospholipase C (PLC) and then inositol triphosphate (IP₃), which stimulates its receptor to deplete Ca²⁺ stores in the endoplasmic reticulum (ER), leading to the opening of the Orai1 channel in HUVECs to induce extracellular Ca²⁺ influx via SOCE. The subsequent increase in intracellular Ca²⁺ ([Ca²⁺]_i) causes the nuclear translocation of NFATC1 through the Ca²⁺/calmodulin-dependent kinase 2 (CaMK-II)/calcineurin signaling pathway, and eventually induces COL1A1 overexpression and HUVEC migration and proliferation.