STIM1 positively regulates the Ca2+ release activity of the inositol 1,4,5-trisphosphate receptor in bovine aortic endothelial cells

Abstract

The endothelium is actively involved in many functions of the cardiovascular system, such as the modulation of arterial pressure and the maintenance of blood flow. These functions require a great versatility of the intracellular Ca2+ signaling that resides in the fact that different signals can be encoded by varying the frequency and the amplitude of the Ca2+ response. Cells use both extracellular and intracellular Ca2+ pools to modulate the intracellular Ca2+ concentration. In non-excitable cells, the inositol 1,4,5-trisphosphate receptor (IP3R), located on the endoplasmic reticulum (ER), is responsible for the release of Ca2+ from the intracellular store. The proteins STIM1 and STIM2 are also located on the ER and they are involved in the activation of a store-operated Ca2+ entry (SOCE). Due to their Ca2+ sensor property and their close proximity with IP3Rs on the ER, STIMs could modulate the activity of IP3R. In this study, we showed that STIM1 and STIM2 are expressed in bovine aortic endothelial cells and they both interact with IP3R. While STIM2 appears to play a minor role, STIM1 plays an important role in the regulation of agonist-induced Ca2+ mobilization in BAECs by a positive effect on both the SOCE and the IP3R-dependent Ca2+ release.

Figure 1. STIM1 and STIM2 are expressed in BAECs and contribute to SOCE.

A) Cells were transfected with siCtrl, siSTIM1 or siSTIM2. After 48 h, cells were lysed and proteins were resolved by SDS-PAGE and identified by Western blot using selective antibodies against STIM1, STIM2 or actin (NT indicates non transfected cells). B) BAECs were loaded with fura-2/AM and imaged using an Olympus IX71 microscope (40× oil immersion objective) coupled to a MetaFluor imaging system for the recording of the intracellular Ca²⁺ concentration. In a nominally free Ca²⁺ medium, cells were treated with 1 µM TG to deplete their Ca²⁺ store and, once the Ca²⁺ concentration had stabilized, 1.8 mM Ca²⁺ was added to the medium to induce Ca²⁺ entry. The figure shows average traces from cells (>75 cells/condition) transfected with siCtrl (black line), siSTIM1 (dashed line) or siSTIM2 (gray line). C) Average Ca²⁺ increase (peak amplitude) after treatment with TG and subsequent Ca²⁺ entry (mean ± SD of results obtained from 3–4 independent experiments, *p<0.05). D) Total RNA was extracted from transfected cells (48 h post-transfection) and subjected to a qPCR analysis using specific primers for STIM1 and STIM2 to evaluate their relative level of encoding mRNAs. The results represent the mean ± SD of three independent experiments.

Figure 2. STIM1 and IP₃R-1 are widely distributed throughout the endoplasmic reticulum in BAECs.

A) BAECs were grown on cover glasses, fixed with methanol and incubated with mouse anti-STIM1 and rabbit anti-IP₃R-1 antibodies. Fluorescent staining was obtained with AlexaFluor 594-conjugated (STIM1, left panel) and AlexaFluor 488-conjugated (IP₃R-1, center panel) secondary antibodies. The right panel represents the overlay of these images. The results are representative of three independent experiments performed on different cells preparations.

Figure 3. STIM1 and STIM2 interact with IP₃R-1.

A) BAECs were solubilized in 1% Triton X-100 and the lysate was fractionated into samples that were immunoprecipitated with isoform-specific anti-STIM antibodies or, as control conditions, with IgG antibodies (IgG) or exclusively with protein-A/G agarose beads (A/G). The resulting immune complexes were separated by SDS-PAGE, transferred to PVDF membranes, and immunoblotted with an isoform-specific anti-IP₃R-1 antibody as indicated on the left side of the blot. B) BAECs lysate was immunoprecipitated with anti-IP₃R-1 antibody and the blot was revealed with an anti-STIM1 or anti-STIM2 antibodies as indicated. These results are representative of at least three independent experiments performed with different cells preparations.

Figure 4. The knockdown of STIM1 dampens the IP₃R-dependent agonist-induced intracellular Ca²⁺ release in BAECs.

BAECs were loaded with fura-2/AM and imaged using an Olympus IX71 microscope (40× oil immersion objective) coupled to a MetaFluor imaging system for the recording of intracellular Ca²⁺ concentration. (A and B) Average traces from cells (>75 cells/condition) transfected with siCtrl (black line), siSTIM1 (dashed line) or siSTIM2 (gray line) stimulated with 100 nM ATP (A) or 5 nM BK (B), in a nominally free Ca²⁺ medium. (C and D) Average Ca²⁺ releases (mean ± SD of results obtained from 4–6 independent experiments) induced by increasing concentrations of ATP (C) or BK (D). (E and F) Same data as in C and D expressed as the percentage of the maximal response under each condition. * indicates that the results are significantly different from those obtained with cells transfected with siCtrl.