[PP.07.10] THE TRUNCATED STIM1 CAUSED IMPAIRED STORE-OPERATED CALCIUM ENTRY IN ASTROCYTES OF SHRSP

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Abstract

Objective:

We previously identified a nonsense mutation in the stromal interaction molecule 1 (Stim1) gene of SHRSP, which was a candidate genetic variation responsible for the phenotype of SHRSP. As STIM1 was an important regulator of the store-operated Ca2+ entry (SOCE), in this study, we evaluated effects of the truncated STIM1 on SOCE in cultured astrocytes.

Design and method:

Astrocytes were isolated from two strains with the truncated STIM1 [SHRSP and a congenic strain, SHRSPwch1.71], and three with the wild-type [WKY, SHR and the other congenic strain, SHRSPwch1.72]. In the two congenic strains, small chromosomal fragments of WKY with or without the Stim1 locus was transferred to the SHRSP background. Changes in the intracellular calcium level was monitored with Fluo-8. Gene expression was quantified by RT- PCR. Transfection of the wild-type and the truncated Stim1 was performed using fibroblasts obtained from a Stim1-knockout mouse embryo.

Results:

Ca2+-imaging experiments showed that SOCE induced by thapsigargin was significantly reduced in astrocytes with the truncated STIM1 when compared with those with the wild-type. Expression of cyclooxygenase-2 (Cox-2) was significantly lower in the cells with the truncated STIM1 as well. BTP-2 (a SOCE inhibitor) and cyclosporine A (a calcineurin inhibitor) suppressed the Cox-2 expression, indicating that it was downstream of SOCE and the Ca2+/calcineurin pathway. Although transient expression of the truncated STIM1 restored the SOCE activity in fibroblasts obtained from Stim1-knockout mice, the activation seemed lower when compared with the response in cells transfected with the wild-type STIM1.

Conclusions:

Our results indicated that the truncated STIM1 gave impaired SOCE activity, which might be responsible for pathological phenotypes in SHRSP.

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