STIMs and Orai1 regulate cytokine production in spinal astrocytes

Abstract

Background: Our previous study demonstrated that a store-operated calcium channel (SOCC) inhibitor (YM-58483) has central analgesic effects. However, the cellular and molecular mechanisms of such effects remain to be determined. It is well-known that glial cells play important roles in central sensitization. SOC entry (SOCE) has been implicated in many cell types including cortical astrocytes. However, the role of the SOCC family in the function of astrocytes has not been determined. Here, we thoroughly investigated the expression and the functional significance of SOCCs in spinal astrocytes.

Methods: Primary cultured astrocytes were prepared from neonatal (P2-P3) CD1 mice. Expressions of mRNAs and proteins were respectively assessed by real-time PCR and Western blot analysis. SOCE was measured using a calcium imaging system. Live-cell STIM1 translocation was detected using a confocal microscope. Cytokine levels were measured by the enzyme-linked immunosorbent assay.

Results: We found that the SOCC family is expressed in spinal astrocytes and that depletion of calcium stores from the endoplasmic reticulum by cyclopiazonic acid (CPA) resulted in a large sustained calcium entry, which was blocked by SOCC inhibitors. Using the siRNA knockdown approach, we identified STIM1 and Orai1 as primary components of SOCCs in spinal astrocytes. We also observed thapsigargin (TG)- or CPA-induced puncta formation of STIM1 and Orai1. In addition, activation of SOCCs remarkably promoted TNF-α and IL-6 production in spinal astrocytes, which were greatly attenuated by knockdown of STIM1 or Orai1. Importantly, knockdown of STIM2 and Orai1 dramatically decreased lipopolysaccharide-induced TNF-α and IL-6 production without changing cell viability.

Conclusions: This study presents the first evidence that STIM1, STIM2, and Orai1 mediate SOCE and are involved in cytokine production in spinal astrocytes. Our findings provide the basis for future assessment of SOCCs in pain and other central nervous system disorders associated with abnormal astrocyte activities.

Keywords: Astrocytes; Cytokine; Orai1; STIM1; Store-operated calcium channels; The spinal cord.

Fig. 1

The SOCC family is expressed in spinal astrocytes. a Immunostaining of GFAP and DAPI in cultured astrocytes. b mRNA levels of STIM1, STIM2, Orai1, Orai2, and Orai3 in cultured astrocytes by RT-PCR (normalized to GAPDH). Values represent mean ± SEM, n = 4 samples each. c Protein expression of STIM1, STIM2, Orai1, Orai2, and Orai3 in astrocytes by Western blot analysis. Arrows indicate the corresponding molecular weights of Orai1 and STIM2 proteins

Fig. 2

Depletion of endoplasmic reticulum Ca²⁺ stores induces SOC Entry in cultured astrocytes. a Representative calcium imaging recordings. b Summary of effects of YM-58483 (YM, 3 μM) and 2-APB (30 μM) on TG-induced calcium influx. n = 14–22 astrocytes. c-e Effects of GdCl₃ (c), YM-58483 (d), and 2-APB (e) on CPA-induced calcium entry. Left panel, representatives; right panel, summary of effects of vehicle (control, n = 36), GdCl₃ (n = 19 cells), YM-58483 (n = 13–17 cells), and 2-APB (n = 12–22 cells) on CPA-induced calcium entry. Values represent mean ± SEM; *P < 0.05, compared with control by the one-way ANOVA

Fig. 3

Orai1, STIM1, and STIM2 are important for SOCE in spinal astrocytes. a Effects of specific siRNAs against STIM1 or STIM2 on protein levels of STIM1 and STIM2 (n = 4–5 samples). b Effects of specific siRNAs against STIMs on TG-induced SOCE. Left panel, representatives of TG-induced calcium responses recorded in astrocytes transfected with control siRNA or targeting siRNAs against STIMs; right panel, summary of effects of control siRNA (n = 78), STIM1 siRNA (n = 77), and STIM2 siRNA (n = 69). c Effects of specific siRNAs against Orai1, Orai2, or Orai3 on protein levels of Orai1, Orai2, and Orai3 (n = 4 samples each). The protein levels were normalized to control (non-target siRNA). d Effects of specific siRNAs against Orais on TG-induced SOCE. Left panel, representatives of TG-induced calcium responses recorded in astrocytes transfected with control siRNA or targeting siRNAs against Orais; right panel, summary of effects of control siRNA (n = 112), Orai1 siRNA (n = 76), Orai2 siRNA (n = 40), or Orai3 siRNA (n = 42). Values represent mean ± SEM, *P < 0.05, compared with control by Student’s t test or the one-way ANOVA

Fig. 4

Depletion of calcium stores from endoplasmic reticulum induces STIM1 puncta formation in spinal astrocytes. a Live-cell confocal time-lapse images of STIM1-transfected astrocytes treated with 1 μM thapsigargin (TG) or 30 μM cyclopiazonic acid (CPA) at 0, 4, and 8 min. b Average number of puncta per 100 μm² induced by respective treatments. Puncta were quantified as spots of high fluorescence intensity ranging from 0.4 to 2.0 μm in diameter size. c Confocal images of fixed spinal astrocytes containing endogenous STIM1 puncta after 8 min in the presence and absence of TG (1 μM)

Fig. 5

Depletion of calcium stores from endoplasmic reticulum induces STIM1-Orai1 puncta formation in spinal astrocytes. a Live-cell confocal images of astrocytes transfected with STIM1-YFP and Orai1-CFP taken before and after 10 min treatment of 1 μM thapsigargin (TG) or 30 μM cyclopiazonic acid (CPA). b Average number of STIM1 puncta (green) per 100 μm² induced by TG or CPA. c Average number of STIM1/Orai1 puncta (yellow) per 100 μm² induced by TG or CPA. Puncta were quantified as spots of high fluorescence intensity ranging from 0.4 to 2.0 μm in diameter size before and after 10 min TG and CPA treatments. Values represent mean ± SEM, *P < 0.05, compared with pretreatment by Student’s t test

Fig. 6

Depletion of Ca²⁺ store induces cytokine production in spinal astrocytes. a TG-induced TNF-α production. b TG-induced IL-6 production. c CPA-induced TNF-α and IL-6 production (n = 6–8). d Effects of YM-58483 (YM, 3 μM) and GdCl₃ (Gd³⁺, 0.3 µM) on TG-induced cytokine production and reduction of cell viability (n = 6–7). e Effects of knockdown STIM1 or Orai1 on TG-induced cytokine production and reduction of cell viability (n = 6). Values represent mean ± SEM, *P < 0.05, compared with control by Student’s t test or the one-way ANOVA

Fig. 7

LPS-induced cytokine production is inhibited by SOC inhibitors or knockdown of STIM2 or Orai1. a Effects of YM-58483 and 2-APB on LPS-induced TNF-α and IL-6 production. b Effects of knockdown STIM1, STIM2, or Orai1 on LPS-induced TNF-α and IL-6 production. n = 6. Values represent mean ± SEM, *P < 0.05, compared with control by the one-way ANOVA or Student’s t test