Stromal interaction molecule 1 (STIM1)-mediated store-operated Ca2+ entry (SOCE) is now recognized as the main mechanism of the majority of nonexcitable
cell calcium influx. Calcium overload is a primary mechanism of endothelial cell injury
during systemic inflammatory response and sepsis. Whether STIM1-mediated SOCE plays
a role in calcium overload in vascular endothelial cell injury remains unclear.
Materials and methods
To explore the role of STIM1-gated SOCE in vascular endothelial cell calcium overload
and inflammation, we established a human septic serum or lipopolysaccharide (LPS)-induced
human umbilical vein endothelial cell (HUVEC) experimental system and derived ribonucleic
acid interference (RNAi)-mediated STIM1, ORAI1 (orai gene [HGNC: 25896 Entrez Gene:
84876] coding protein, ORAI Calcium Release-Activated Calcium Modulator 1), and transient
receptor potential channel 1 (TRPC1) (core components of store-operated Ca2+[SOC]) downregulated HUVECs, as well as STIM1 overinduced HUVECs.
Our results show that sepsis serum or LPS stimulation increased STIM1 in HUVECs and
increased all cytokines except for VEGF and the inflammatory mediators tumor necrosis
factor, intercellular cell adhesion molecule-1, and endothelin-1 in a time-dependent
manner. RNAi-mediated knockdown of STIM1 significantly inhibited serum or LPS-induced
inflammatory cytokine expression, and STIM1 overexpression in HUVECs promoted LPS-mediated
induction of these cytokines. Meanwhile, similar to the blocking effect of the specific
SOC inhibitors Gd3+ and La3+ on LPS-induced calcium influx, RNAi-mediated depletion of STIM1 or the SOC proteins
TRPC1 and ORAI1 could significantly inhibit serum or LPS-induced extracellular calcium
influx, as well as the expression of the inflammatory cytokines tumor necrosis factor,
intercellular cell adhesion molecule-1, and endothelin-1. Simultaneous downregulation
of the SOCE core units TRPC1 and ORAI1 inhibited LPS-induced calcium influx and cytokine
expression, which could not be restored by inducing STIM1. Forced expression of nuclear
factor-κB (NF-κB) in HUVECs significantly induced STIM1 expression, whereas RNAi-mediated
depletion of NF-κB significantly inhibited STIM1 mRNA levels and significantly reduced
the thapsigargin-mediated SOCE calcium influx, which was similar to results with the
NF-κB inhibitor wogonin.
Septic serum stimulates the expression of STIM1, cytokines, and inflammatory mediators
in HUVECs. STIM1-mediated SOCE is required for Ca2+ influx induced by LPS or septic serum and contributes cytokines and inflammatory
mediators in septic serum–stimulated HUVECs. In addition, STIM1-mediated SOCE on Ca2+ influx by septic serum or LPS involves NF-κB signaling.