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Sodium regulates PLC and IP₃ R-mediated calcium signaling in invasive breast cancer cells.

ABSTRACT: Intracellular Ca²⁺ signaling and Na⁺ homeostasis are inextricably linked via ion channels and co-transporters, with alterations in the concentration of one ion having profound effects on the other. Evidence indicates that intracellular Na⁺ concentration ([Na⁺ ]_i ) is elevated in breast tumors, and that aberrant Ca²⁺ signaling regulates numerous key cancer hallmark processes. The present study therefore aimed to determine the effects of Na⁺ depletion on intracellular Ca²⁺ handling in metastatic breast cancer cell lines. The relationship between Na⁺ and Ca²⁺ was probed using fura-2 and SBFI fluorescence imaging and replacement of extracellular Na⁺ with equimolar N-methyl-D-glucamine (0Na⁺ /NMDG) or choline chloride (0Na⁺ /ChoCl). In triple-negative MDA-MB-231 and MDA-MB-468 cells and Her2+ SKBR3 cells, but not ER+ MCF-7 cells, 0Na⁺ /NMDG and 0Na⁺ /ChoCl resulted in a slow, sustained depletion in [Na⁺ ]_i that was accompanied by a rapid and sustained increase in intracellular Ca²⁺ concentration ([Ca²⁺ ]_i ). Application of La³⁺ in nominal Ca²⁺ -free conditions had no effect on this response, ruling out reverse-mode NCX activity and Ca²⁺ entry channels. Moreover, the Na⁺ -linked [Ca²⁺ ]_i increase was independent of membrane potential hyperpolarization (NS-1619), but was inhibited by pharmacological blockade of IP₃ receptors (2-APB), phospholipase C (PLC, U73122) or following depletion of endoplasmic reticulum Ca²⁺ stores (cyclopiazonic acid). Thus, Na⁺ is linked to PLC/IP₃ -mediated activation of endoplasmic reticulum Ca²⁺ release in metastatic breast cancer cells and this may have an important role in breast tumors where [Na⁺ ]_i is perturbed.

SUBMITTER: James AD

PROVIDER: S-EPMC10074044 | biostudies-literature | 2023 Apr

REPOSITORIES: biostudies-literature

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Sodium regulates PLC and IP3 R-mediated calcium signaling in invasive breast cancer cells.

James Andrew D AD Unthank Katherine P KP Jones Isobel I Sajjaboontawee Nattanan N Sizer Rebecca E RE Chawla Sangeeta S Evans Gareth J O GJO Brackenbury William J WJ

Physiological reports 20230401 7

Intracellular Ca2+ signaling and Na+ homeostasis are inextricably linked via ion channels and co-transporters, with alterations in the concentration of one ion having profound effects on the other. Evidence indicates that intracellular Na+ concentration ([Na+ ]i ) is elevated in breast tumors, and that aberrant Ca2+ signaling regulates numerous key cancer hallmark processes. The present study therefore aimed to determine the effec ...[more]

PMID: 37017052

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Project description:Bok is a poorly characterized Bcl-2 protein family member with roles yet to be clearly defined. It is clear, however, that Bok binds strongly to inositol 1,4,5-trisphosphate (IP3) receptors (IP3Rs), which govern the mobilization of Ca2+ from the endoplasmic reticulum, a signaling pathway required for many cellular processes. Also known is that Bok has a highly conserved phosphorylation site for cAMP-dependent protein kinase at serine-8 (Ser-8). Whether Bok, or phosphorylated Bok, has any direct impact on the Ca2+ mobilizing function of IP3Rs remains to be established. Bok Ser-8 phosphorylation was characterized using purified proteins, G-protein coupled receptor agonists that increase cAMP levels in intact cells, mass spectrometry, and immunoreactivity changes. Also, using mammalian cells that exclusively or predominately express IP3R1, to which Bok binds strongly, and a fluorescent Ca2+-sensitive dye or a genetically-encoded Ca2+ sensor, we explored how endogenous and exogenous Bok controls the Ca2+ mobilizing function of IP3R1, and whether Bok phosphorylation at Ser-8, or replacement of Ser-8 with a phosphomimetic amino acid, is regulatory. Our results confirm that Ser-8 of Bok is phosphorylated by cAMP-dependent protein kinase, and remarkably that phosphorylation can be detected with Bok specific antibodies. Also, we find that Bok has suppressive effects on IP3R-mediated Ca2+ mobilization in a variety of cell types. Specifically, Bok accelerated the post-maximal decline in G-protein coupled receptor-induced cytosolic Ca2+ concentration, via a mechanism that involves suppression of IP3R-dependent Ca2+ release from the endoplasmic reticulum. These effects were dependent on the Bok-IP3R interaction, as they are only seen with IP3Rs that can bind Bok (e.g., IP3R1). Surprisingly, Bok phosphorylation at Ser-8 weakened the interaction between Bok and IP3R1 and reversed the ability of Bok to suppress IP3R1-mediated Ca2+ mobilization. For the first time, Bok was shown to directly suppress IP3R1 activity, which was reversed by Ser-8 phosphorylation. We hypothesize that this suppression of IP3R1 activity is due to Bok regulation of the conformational changes in IP3R1 that mediate channel opening. This study provides new insights on the role of Bok, its interaction with IP3Rs, and the impact it has on IP3R-mediated Ca2+ mobilization.

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Sodium regulates PLC and IP₃ R-mediated calcium signaling in invasive breast cancer cells.

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Sodium regulates PLC and IP<sub>3</sub> R-mediated calcium signaling in invasive breast cancer cells.

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Dataset Information

Sodium regulates PLC and IP3 R-mediated calcium signaling in invasive breast cancer cells.

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Sodium regulates PLC and IP&lt;sub&gt;3&lt;/sub&gt; R-mediated calcium signaling in invasive breast cancer cells.

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OmicsDI is part of the ELIXIR infrastructure

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Sodium regulates PLC and IP₃ R-mediated calcium signaling in invasive breast cancer cells.

Sodium regulates PLC and IP<sub>3</sub> R-mediated calcium signaling in invasive breast cancer cells.