Abstract

Calcium concentration within the endoplasmic reticulum (ER) plays an essential role in cell physiology. We have investigated the effects of basiliolides, a novel class of C19 dilactones isolated from Thapsia garganica, on Ca²⁺ mobilization in T cells. Basiliolide A1 induced a rapid mobilization of intracellular Ca²⁺ in the leukemia T-cell line Jurkat. First, a rapid calcium peak was observed and inhibited by 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester. This initial calcium mobilization was followed by a sustained elevation, mediated by the entry of extracellular calcium through store-operated calcium release-activated Ca²⁺ (CRAC) channels and sensitive to inhibition by EGTA, and by the CRAC channel inhibitor N-{4-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]phenyl}-4-methyl-1,2,3-thiadiazole-5-carboxamide (BTP-2). Basiliolide A1 mobilized Ca²⁺ from ER stores, but in contrast to thapsigargin, it did not induce apoptosis. Basiliolide A1 induced nuclear factor of activated T cells 1 dephosphorylation and activation that was inhibited by BTP-2 and cyclosporine A. In addition, we found that basiliolide A1 alone did not mediate IκBα degradation or RelA phosphorylation (ser536), but it synergized with phorbol 12-myristate 13-acetate to induce a complete degradation of the nuclear factor-κB inhibitory protein and to activate the c-Jun NH₂-terminal kinase. Moreover, basiliolide A1 regulated both interleukin-2 and tumor necrosis factor-α gene expression at the transcriptional level. In basiliolide B, oxidation of one of the two geminal methyls to a carboxymethyl group retained most of the activity of basiliolide A1. In contrast, basiliolide C, where the 15-carbon is oxidized to an acetoxymethine, was much less active. These findings qualify these compounds as new probes to investigate intracellular calcium homeostasis.