(W1030-04-28) Differential Modulation of Endoplasmic Reticulum Calcium Pumps SERCA2 and SERCA3 in T Lymphocytes by Small Molecule Allosteric SERCA Activator CDN1163

Purpose: T lymphocyte Ca²⁺ signals control activation, proliferation and differentiation, and therefore represent essential signaling mediators underlying proper immune system function. Ca²⁺ mobilization from the intracellular storage organelle the Endoplasmic Reticulum (ER) is a key component of the second messenger system leading to T cell activation. ER Ca²⁺ homeostasis is maintained tightly by Sarco/Endoplasmic Reticulum Ca²⁺-ATPases (SERCA), ATP-driven ion pumps, which limit and shape Ca²⁺ signaling patterns by transporting released Ca²⁺ back into the ER from the cytoplasm. SERCA2 and SERCA3-type calcium pumps are simultaneously expressed in T lymphocytes and play an important role during T cell activation and differentiation. Altered SERCA function leads to dysregulation of T cells and inappropriate immune responses. CDN1163, a quinoline derivative, has been reported to be a SERCA-activating compound in muscle tissue expressing SERCA1 and SERCA2 isoforms. In this study, we evaluated the effects of CDN1163 in T lymphocytes expressing SERCA2 and SERCA3 isoforms.

Methods: Intact Cell Calcium Assay: Jurkat T cells were loaded with fura-2/AM. Changes in cytosolic Ca²⁺ were measured in cell population experiments using a fluorescence spectrophotometer, permitting continuous stirring of samples. For cells loaded with fura 2/AM, excitation of the dye was achieved by rapidly alternating monochromator settings between 340 and 380 nm, with fluorescence emission measured at 510 nm. Changes in cytosolic Ca²⁺ concentrations are reported as the fluorescence ratio values for fura-2 measured at 340 and 380 nm. Permeabilized Cell Assay: For permeabilization studies cells were suspended in an intracellular-like medium. Saponin (20 µg/ml) was added, and the cell suspension was incubated for 5 min at 37º C to complete permeabilization. An ATP-regenerating system consisting of creatine kinase (40 units/ml) and phosphocreatine (20 mM) was added. Oligomycin (10 µg/ml) was also included to inhibit the mitochondrial ATPase. Following cell permeabilization, Fluo-3 (0.5 µM), was added to the cuvette. Subsequent addition of ATP (1 mM) resulted in a decrease in the fluorescence, indicating Ca²⁺ uptake by the intracellular stores. Ca²⁺ release from intracellular stores upon addition of drugs was measured using a fluorescence spectrophotometer equipped with a thermostatically controlled sample compartment, permitting continuous stirring of samples with wavelength settings of 503 nm (excitation) and 530 nm (emission).

Results: CDN1163 appears to exert an immediate action, within 1 minute, in perturbing T cell Ca²⁺ stores controlled by both SERCA2 and SERCA3. Using intact T cell Ca²⁺ assays we find that T cell receptor (TCR) activation via Phytohemagglutinin (PHA) treatment as well as application of SERCA blockers specific for SERCA 2b and SERCA 3 (low dose thapsigargin and tBHQ) produce reduced Ca²⁺ release responses in CDN1163 treated Jurkat lymphocytes as compared to untreated control cells. Permeabilized cell assays reveal that this early effect of CDN1163 likely arises due to the ability of the compound to inhibit SERCA-mediated Ca²⁺ uptake and to induce Ca²⁺ release by itself. Intriguingly, longer exposure to CDN1163 (greater than 12 hours) results in increased Ca²⁺ loading in T cell Ca²⁺ stores but in a SERCA specific manner. Ca²⁺ release induced by low dose TG (specific for SERCA 2b) is augmented by long-term CDN1163 treatment, whereas, Ca²⁺ release induced by low dose tBHQ (SERCA 3 specific) is reduced by CDN1163 extended exposure.

Conclusion: We report novel effects of the compound CDN1163 which is used increasingly as an important activator of SERCA ion pumps. Following an initial period of perturbation of SERCA regulated Ca²⁺ stores in T cells, our experiments show that increased Ca²⁺ loading occurs likely due to SERCA activation; yet, surprisingly CDN1163 appears to specifically stimulate SERCA 2b Ca²⁺ loading while inhibiting SERCA 3 dependent Ca²⁺ pools.

References: 1). Lewis R. S. (2001). Calcium signaling mechanisms in T lymphocytes. Annual review of immunology, 19, 497–521. https://doi.org/10.1146/annurev.immunol.19.1.497.
2). Kang, S., Dahl, R., Hsieh, W., Shin, A., Zsebo, K. M., Buettner, C., Hajjar, R. J., & Lebeche, D. (2016). Small Molecular Allosteric Activator of the Sarco/Endoplasmic Reticulum Ca2+-ATPase (SERCA) Attenuates Diabetes and Metabolic Disorders. The Journal of biological chemistry, 291(10), 5185–5198. https://doi.org/10.1074/jbc.M115.705012.

Acknowledgements:The project was funded in part by a Seed Grant from the Jie Du Center, University of the Pacific.

Fig 1: Presence of SERCA2 and SERCA3 sensitive Calcium pools in intact cells (A) and in permeabilized cells (B). Thapsigargin (TG) is a specific inhibitor of SERCA2 and 2,5-di-(t-butyl)-1,4-hydroquinone (tBHQ) is a specific inhibitor of SERCA3 at low doses.

Fig 2: Effect of short-term CDN treatment on PHA 10ug/ml (A), TG 1nM (B), and tBHQ 2µM (C) mediated Calcium release in intact cells, and on IP3 1µM (D), TG 1.5 nM (E), and tBHQ 1µM (F) mediated Calcium release in permeabilized cells.

Fig 3: Effect of short-term and long-term CDN treatment on Calcium uptake by SERCA upon addition of ATP (A & B respectively) in permeabilized cells, long-term CDN treatment on TG 1nM (C), and tBHQ 2µM (D) mediated Calcium release in intact cells, and on TG 1.5 nM (E), and tBHQ 1µM (F) mediated Calcium release in permeabilized cells.