SARAF and Orai1 Contribute to Endothelial Cell Activation and Angiogenesis

Abstract

Angiogenesis is a multistep process that controls endothelial cells (ECs) functioning to form new blood vessels from preexisting vascular beds. This process is tightly regulated by pro-angiogenic factors, such as vascular endothelial growth factor (VEGF), which promote signaling pathways involving the increase in the intracellular Ca2+ concentration ([Ca2+]i). Recent evidence suggests that store-operated calcium entry (SOCE) might play a role in angiogenesis. However, little is known regarding the role of SARAF, SOCE-associated regulatory factor, and Orai1, the pore-forming subunit of the store-operated calcium channel (SOCC), in angiogenesis. Here, we show that SOCE inhibition with GSK-7975A blocks aorta sprouting, as well as human umbilical vein endothelial cell (HUVEC) tube formation and migration. The intraperitoneal injection of GSK-7975A also delays the development of retinal vasculature assessed at postnatal day 6 in mice, since it reduces vessel length and the number of junctions, while it increases lacunarity. Moreover, we find that SARAF and Orai1 are involved in VEGF-mediated [Ca2+]i increase, and their knockdown using siRNA impairs HUVEC tube formation, proliferation, and migration. Finally, immunostaining and in situ proximity ligation assays indicate that SARAF likely interacts with Orai1 in HUVECs. Therefore, these findings show for the first time a functional interaction between SARAF and Orai1 in ECs and highlight their essential role in different steps of the angiogenesis process.

Regulation of Store-Operated Ca2+ Entry by SARAF

“…This PM Ca 2+ flux was sensitive to changes in expression of either Orai1 or SARAF, suggesting that binding of SARAF to Orai1 stimulated channel activation [76]. In line with a stimulatory role for SARAF during SOCE activation, Ca 2+ imaging experiments in human endothelial cells showed that RNAi-mediated knockdown of Orai1 or SARAF expression attenuates VEGF-induced Ca 2+ entry [77]. Because Albarran et al had used cells with low expression levels of STIM1, their model suggested that SARAF contributes to Orai1 channel activation independently of STIM1 [76]; however, it is not clear that the contribution of SARAF to Ca 2+ entry in these cells was indeed STIM-independent as it may have involved STIM2, which also mediates SOCE in cells [12] and which is also regulated by SARAF [52].…”

“…Therefore, SARAF already physically engages the CRAC channel during channel activation and prior to induction of SCDI. While the functional significance of this early interaction is presently unclear, it may indicate an initial role for SARAF in channel activation [76,77] and raises a critical question regarding how the binding of SARAF to STIM1 ultimately promotes SCDI.…”

“…Cells 2021, 10, x FOR PEER REVIEW 10 of 19 that the dimeric state of SARAF accelerates CRAC channel deactivation; however, considering the effect of SARAF on channel activation [76,77], the results could alternatively imply that monomeric SARAF is more efficient in promoting CRAC channel activation than the SARAF dimer. Overall, while the luminal-facing domain of SARAF clearly affects CRAC channel function, remaining important questions are whether and how redox changes in the ER lumen induce oligomeric changes in SARAF and how such changes affect CRAC channel function.…”

“…It is possible that the differences in the experimental models employed in the above reports contributed to the different effects of SARAF on hypertrophy responses. Moreover, considering the tight relationship between angiogenesis and pathological cardiac hypertrophy, it is worth mentioning that SARAF was shown to contribute to different steps in the angiogenesis process [77]. Hence, since overexpression of SARAF was driven by cardiomyocyte-specific myosin light chain ventricular isoform (MLC-2v) promoter only in one of the two studies [120], cell-type specific functions of SARAF may have also contributed to the observed differences.…”

Store-Operated Calcium Channels Control Proliferation and Self-Renewal of Cancer Stem Cells from Glioblastoma

“…This decrease in stem cell population was observed with concentrations of SKF-96365 and YM-58483 that have been used to block the physiological effects of SOC [ 31 , 46 , 47 , 48 , 49 ]. As SKF-96365 and YM-58483 may also target other receptor channels (ROC), GSK-7975A, a more specific inhibitor of Orai-dependent SOCE was used [ 50 , 51 , 52 ]. The fact that GSK-7975A had also significant effects on GSC supports the idea that SOC play a key role in maintaining the GSC pool in GBM as they do in oral/oropharyngeal squamous cell carcinoma cancer stem cells and in liver cancer stem cells [ 53 , 54 ].…”