ABSTRACT
The Ca(2+) sensor stromal interacting molecule 1 (STIM1) and the Ca(2+) channel Orai1 mediate the ubiquitous store-operated Ca(2+) entry (SOCE) pathway activated by depletion of internal Ca(2+) stores and mediated through the highly Ca(2+)-selective, Ca(2+) release-activated Ca(2+) (CRAC) current. Furthermore, STIM1 and Orai1, along with Orai3, encode store-independent Ca(2+) currents regulated by either arachidonate or its metabolite, leukotriene C4. Orai channels are emerging as important contributors to numerous cell functions, including proliferation, migration, differentiation, and apoptosis. Recent studies suggest critical involvement of STIM/Orai proteins in controlling the development of several cancers, including malignancies of the breast, prostate, and cervix. Here, we quantitatively compared the magnitude of SOCE and the expression levels of STIM1 and Orai1 in non-malignant human primary astrocytes (HPA) and in primary human cell lines established from surgical samples of the brain tumor glioblastoma multiforme (GBM). Using Ca(2+) imaging, patch-clamp electrophysiology, pharmacological reagents, and gene silencing, we established that in GBM cells, SOCE and CRAC are mediated by STIM1 and Orai1. We further found that GBM cells show upregulation of SOCE and increased Orai1 levels compared to HPA. The functional significance of SOCE was evaluated by studying the effects of STIM1 and Orai1 knockdown on cell proliferation and invasion. Utilizing Matrigel assays, we demonstrated that in GBM, but not in HPA, downregulation of STIM1 and Orai1 caused a dramatic decrease in cell invasion. In contrast, the effects of STIM1 and Orai1 knockdown on GBM cell proliferation were marginal. Overall, these results demonstrate that STIM1 and Orai1 encode SOCE and CRAC currents and control invasion of GBM cells. Our work further supports the potential use of channels contributed by Orai isoforms as therapeutic targets in cancer.
Subject(s)
Brain Neoplasms/metabolism , Calcium Channels/metabolism , Calcium Signaling , Glioblastoma/metabolism , Membrane Proteins/metabolism , Neoplasm Proteins/metabolism , Action Potentials , Astrocytes/metabolism , Brain Neoplasms/pathology , Calcium/metabolism , Calcium Channels/genetics , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Glioblastoma/pathology , Humans , Membrane Proteins/genetics , Neoplasm Invasiveness , Neoplasm Proteins/genetics , ORAI1 Protein , Stromal Interaction Molecule 1 , Transcription, Genetic , Up-RegulationABSTRACT
Store-operated Ca²âº entry (SOCE) is a receptor-regulated Ca²âº entry pathway that is both ubiquitous and evolutionarily conserved. SOCE is activated by depletion of intracellular Ca²âº stores through receptor-mediated production of inositol 1,4,5-trisphosphate (IP3). The depletion of endoplasmic reticulum (ER) Ca²âº is sensed by stromal interaction molecule 1 (STIM1). On store depletion, STIM1 aggregates and moves to areas where the ER comes close to the plasma membrane (PM; within 25 nm) to interact with Orai1 channels and activate Ca²âº entry. Ca²âº entry through store-operated Ca²âº (SOC) channels, originally thought to mediate the replenishment of Ca²âº stores, participate in active downstream signaling by coupling to the activation of enzymes and transcription factors that control a wide variety of long-term cell functions such as proliferation, growth, and migration. SOCE has also been proposed to contribute to short-term cellular responses such as muscle contractility. While there are significant STIM1/Orai1 protein levels and SOCE activity in adult skeletal muscle, the precise role of SOCE in skeletal muscle contractility is not clear. The dependence on SOCE during cardiac and smooth muscle contractility is even less certain. Here, we will hypothesize on the contribution of SOCE in muscle and its potential role in contractility and signaling.
