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.

Human Parathyroid Hormone (1-34) accelerates skin wound healing through inducing cell migration via up-regulating the expression of Rac1

“…The inhibition of Rac1 expression in epidermis or HaCaT cells with microRNA-200b led to damaged cell motility and eventually resulted in a substantially delayed wound-healing process [2]. PTH and its analogs have been recognized as an effective pro-migration agents in various types of cells which at least included HUVECs [25], BMSCs [26],…”

The Molecular Heterogeneity of Store-Operated Ca2+ Entry in Vascular Endothelial Cells: The Different roles of Orai1 and TRPC1/TRPC4 Channels in the Transition from Ca2+-Selective to Non-Selective Cation Currents

“…In the same endothelial cell type, genetic knockdown or pharmacological blockade with S66 or GSK abolished VEGF-induced extracellular Ca 2+ entry, proliferation, and tube formation [ 39 , 143 ]. In addition, genetic silencing of Orai1 interfered with parathyroid hormone-induced HUVEC proliferation and migration [ 156 ] and with VEGF-induced Ca 2+ entry and migration in human aortic endothelial cells (HAECs) [ 157 ]. In agreement with these in vitro findings, GSK inhibited aorta sprouting and the development of retinal vasculature in mice [ 143 ].…”

“…Intriguingly, RTK activation and PLCγ signalling were spatially restricted at the front of the migrating leader cell in HUVEC monolayers; herein, PLCγ promotes local InsP 3 -induced ER Ca 2+ pulses that recruit STIM1 and selectively activate SOCE to accelerate cell-matrix adhesion and stimulate forward migration [ 60 ]. In addition, Orai1-mediated SOCE could promote angiogenesis by stimulating the nuclear translocation of NFAT ( Table 3 ) [ 24 , 156 ].…”

Role of Orai‐family channels in the activation and regulation of transcriptional activity

“…The pivotal role of Orai1, but not Orai2 and Orai3, in the activation of NFAT2 (also known as NFATc, NFATc1) has been extensively demonstrated by using pharmacological or gene mutation approaches to inhibit channel function, or by gene silencing approaches to avoid protein expression (Carreras‐Sureda et al, 2021; Choi et al, 2018; Hwang & Putney, 2012; Jans et al, 2013; Wang et al, 2022; Zhou et al, 2014). Hence, specific siRNA against Orai1 or the overexpression of the dominant‐negative mutant Orai1R91W were used to demonstrate that Orai1‐mediated Ca 2+ entry promotes nuclear translocation and enhances transcriptional activity of NFAT2 after lysophosphatidic acid‐induced SOCE in keratinocytes (Jans et al, 2013).…”

“…In these cells, histamine treatment failed to promote SOCE activation and NFAT2 nuclear translocation, events required for interleukine‐8 (IL‐8) secretion and the inflammatory response (Zhou et al, 2014). Furthermore, the attenuation of Orai1 activity or expression in HUVEC cells, using the Orai1 channel inhibitor BTP2 or a specific siRNA against Orai1 respectively, also impaired NFAT2 nuclear translocation and transcriptional activity, compromising cell migration, and proliferation (Wang et al, 2022). The role of Orai1 in NFAT2 activation was also demonstrated by the generation of Orai1 knock‐out (Orai1KO) Jurkat cells using CRISPR‐mediated gene editing.…”