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A multiple-oscillator mechanism underlies antigen-induced Ca²⁺ oscillations in Jurkat T-cells.

ABSTRACT: T-cell receptor stimulation triggers cytosolic Ca²⁺ signaling by inositol-1,4,5-trisphosphate (IP₃)-mediated Ca²⁺ release from the endoplasmic reticulum (ER) and Ca²⁺ entry through Ca²⁺ release-activated Ca²⁺ (CRAC) channels gated by ER-located stromal-interacting molecules (STIM1/2). Physiologically, cytosolic Ca²⁺ signaling manifests as regenerative Ca²⁺ oscillations, which are critical for nuclear factor of activated T-cells-mediated transcription. In most cells, Ca²⁺ oscillations are thought to originate from IP₃ receptor-mediated Ca²⁺ release, with CRAC channels indirectly sustaining them through ER refilling. Here, experimental and computational evidence support a multiple-oscillator mechanism in Jurkat T-cells whereby both IP₃ receptor and CRAC channel activities oscillate and directly fuel antigen-evoked Ca²⁺ oscillations, with the CRAC channel being the major contributor. KO of either STIM1 or STIM2 significantly reduces CRAC channel activity. As such, STIM1 and STIM2 synergize for optimal Ca²⁺ oscillations and activation of nuclear factor of activated T-cells 1 and are essential for ER refilling. The loss of both STIM proteins abrogates CRAC channel activity, drastically reduces ER Ca²⁺ content, severely hampers cell proliferation and enhances cell death. These results clarify the mechanism and the contribution of STIM proteins to Ca²⁺ oscillations in T-cells.

SUBMITTER: Benson JC

PROVIDER: S-EPMC10641176 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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A multiple-oscillator mechanism underlies antigen-induced Ca2+ oscillations in Jurkat T-cells.

Benson J Cory JC Romito Olivier O Abdelnaby Ahmed Emam AE Xin Ping P Pathak Trayambak T Weir Sierra E SE Kirk Vivien V Castaneda Francisco F Yoast Ryan E RE Emrich Scott M SM Tang Priscilla W PW Yule David I DI Hempel Nadine N Potier-Cartereau Marie M Sneyd James J Trebak Mohamed M

The Journal of biological chemistry 20230929 11

T-cell receptor stimulation triggers cytosolic Ca2+ signaling by inositol-1,4,5-trisphosphate (IP3)-mediated Ca2+ release from the endoplasmic reticulum (ER) and Ca2+ entry through Ca2+ release-activated Ca2+ (CRAC) channels gated by ER-located stromal-interacting molecules (STIM1/2). Physiologically, cytosolic Ca2+ signaling manifests as regenerative Ca2+ oscillations, which are critical for nuclear factor o ...[more]

PMID: 37778728

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Project description:ObjectiveHepatic Ca2+ signaling has been identified as a crucial key factor in driving gluconeogenesis. The involvement of mitochondria in hormone-induced Ca2+ signaling and their contribution to metabolic activity remain, however, poorly understood. Moreover, the molecular mechanism governing the mitochondrial Ca2+ efflux signaling remains unresolved. This study investigates the role of the Na+/Ca2+ exchanger, NCLX, in modulating hepatic mitochondrial Ca2+ efflux, and examines its physiological significance in hormonal hepatic Ca2+ signaling, gluconeogenesis, and mitochondrial bioenergetics.MethodsPrimary mouse hepatocytes from both an AAV-mediated conditional hepatic-specific and a total mitochondrial Na+/Ca2+ exchanger, NCLX, knockout (KO) mouse models were employed for fluorescent monitoring of purinergic and glucagon/vasopressin-dependent mitochondrial and cytosolic hepatic Ca2+ responses in cultured hepatocytes. Isolated liver mitochondria and permeabilized primary hepatocytes were used to analyze the ion-dependence of Ca2+ efflux. Utilizing the conditional hepatic-specific NCLX KO model, the rate of gluconeogenesis was assessed by first monitoring glucose levels in fasted mice, and subsequently subjecting the mice to a pyruvate tolerance test while monitoring their blood glucose. Additionally, cultured primary hepatocytes from both genotypes were assessed in vitro for glucagon-dependent glucose production and cellular bioenergetics through glucose oxidase assay and Seahorse respirometry, respectively.ResultsAnalysis of Ca2+ responses in isolated liver mitochondria and cultured primary hepatocytes from NCLX KO versus WT mice showed that NCLX serves as the principal mechanism for mitochondrial calcium extrusion in hepatocytes. We then determined the role of NCLX in glucagon and vasopressin-induced Ca2+ oscillations. Consistent with previous studies, glucagon and vasopressin triggered Ca2+ oscillations in WT hepatocytes, however, the deletion of NCLX resulted in selective elimination of mitochondrial, but not cytosolic, Ca2+ oscillations, underscoring NCLX's pivotal role in mitochondrial Ca2+ regulation. Subsequent in vivo investigation for hepatic NCLX role in gluconeogenesis revealed that, as opposed to WT mice which maintained normoglycemic blood glucose levels when fasted, conditional hepatic-specific NCLX KO mice exhibited a faster drop in glucose levels, becoming hypoglycemic. Furthermore, KO mice showed deficient conversion of pyruvate to glucose when challenged under fasting conditions. Concurrent in vitro assessments showed impaired glucagon-dependent glucose production and compromised bioenergetics in KO hepatocytes, thereby underscoring NCLX's significant contribution to hepatic glucose metabolism.ConclusionsThe study findings demonstrate that NCLX acts as the primary Ca2+ efflux mechanism in hepatocytes. NCLX is indispensable for regulating hormone-induced mitochondrial Ca2+ oscillations, mitochondrial metabolism, and sustenance of hepatic gluconeogenesis.

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A multiple-oscillator mechanism underlies antigen-induced Ca²⁺ oscillations in Jurkat T-cells.

Publications

A multiple-oscillator mechanism underlies antigen-induced Ca<sup>2+</sup> oscillations in Jurkat T-cells.

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