1887

Abstract

Calreticulin is a molecular chaperone responsible for proper protein folding in the endoplasmic reticulum (ER), and supports assembly of protein oligomers. Calreticulin also plays a role in quality control of protein expression in the ER. Calreticulin is a ubiquitous protein that shows high levels of expression in fetal cardiomyocytes. Both knockout and over-expression of Calreticulin in the mouse embryos is lethal arguing for an important role for this protein in survival and importantly for tight regulation of its expression in that regard to support physiological functions. Interestingly, over-expression of constitutively active Calreticulin only in the hearts of Calreticulin-null mice, rescues the lethal phenotype. Collectively these results argue for an important role for Calreticulin in the development and differentiation of cardiac myocytes. The exact mechanism by which Calreticulin affects cardiac development remains unclear. Previous studies on Calreticulin knockout cells showed inhibition of IP3– dependent Ca2 release, suggesting a role for Calreticulin in Ca2 homeostasis. Ca2 is a ubiquitous second messenger that can be either release from intracellular Ca2 stores or flows into the cell from the extracellular space through multiple Ca2 influx pathways at the cell membrane. Ca2 release occurs through either the IP3-receptor (IP3R) or the ryanodine receptor depending on the cell type. An important Ca2 influx pathway at the cell membrane is the store-operated Ca2 entry pathway (SOCE). SOCE is activated in response to the depletion of intracellular Ca2 store following Ca2 release. SOCE is mediated by two primary molecules, stim1 and Orai1. stim1 is an ER resident membrane protein with lumenal EF-hands that allows it to sense ER Ca2 levels. Upon store depletion stim1 clusters and moves close to the cell membrane where it binds to and activates Orai1. Orai1 is a four trans-membrane pass Ca2 channel that is gated by direct coupling to stim1. Both Orai1 and Stim1 were shown to be expressed in cardiomyocytes and required for cardiomyocyte SOCE. SOCE is important in cardiomyocyte biology and was shown to regulate normal and hypertrophic postnatal cardiac growth. Moreover, studies in zebra fish suggest an important role of Orai1 in regulating cardiac function, linking Orai1-mediated calcium signaling to cardiomyocyte function. Ca2 influx through SOCE has been shown to activate the transcription factor, nuclear factor of activated T-cells (NFAT), by enhancing its translocation from the cytoplasm to the nucleus, leading to specific induction of gene expression. NFAT activation occurs downstream of the activation of the Ca2-dependent phosphatase calcineurin. This nuclear import of NFAT was shown to be impaired in Calreticulin knockout cells. This supports a role for Calreticulin in the Ca2/calcineurin/NF-AT transcription pathway. Here we use Calreticulin knockout MEF cells to study the effect of the absence of Calreticulin on SOCE. We specifically focus on Orai1, and study its subcellular localization and function in Calreticulin knockout MEFs as compared to wild type MEFs. We used specific Ca2 imaging assays to asses SOCE function. Our Calcium imaging data show no difference in SOCE between wild-type and Calreticulin knockout MEF cells. We further studied the membrane trafficking and plasma membrane localization of YFP-tagged Orai1 in knockout MEF cells. Calreticulin knockout cells show higher proliferation rate than wildtype cells in culture, which will be further investigated by MTT and CFSE labeling. Our results suggest that the lethal phenotype in Calreticulin-null mice is not due to improper folding of Orai1, but can be related to the change of calcium homeostasis in the absence of Calreticulin.

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/content/papers/10.5339/qfarc.2016.HBPP1846
2016-03-21
2020-02-25
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http://instance.metastore.ingenta.com/content/papers/10.5339/qfarc.2016.HBPP1846
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