Clausmarin A, Potential Immunosuppressant Revealed by Yeast-Based Assay and Interleukin-2 Production Assay in Jurkat T Cells

<div><p>Small-molecule inhibitors of Ca²⁺-signaling pathways are of medicinal importance, as exemplified by the immunosuppressants FK506 and cyclosporin A. Using a yeast-based assay devised for the specific detection of Ca²⁺-signaling inhibitors, clausmarin A, a previously reported terpenoid coumarin, was identified as an active substance. Here, we investigated the likely mechanism of clausmarin A action in yeast and Jurkat T-cells. In the presence of 100 mM CaCl₂ in the growth medium of Ca²⁺-sensitive Δ<i>zds1</i> strain yeast, clausmarin A exhibited a dose-dependent alleviation of various defects due to hyperactivation of Ca²⁺ signaling, such as growth inhibition, polarized bud growth and G2 phase cell-cycle arrest. Furthermore, clausmarin A inhibited the growth of Δ<i>mpk1</i> (lacking the Mpk1 MAP kinase pathway) but not Δ<i>cnb1</i> (lacking the calcineurin pathway) strain, suggesting that clausmarin A inhibited the calcineurin pathway as presumed from the synthetic lethality of these pathways. Furthermore, clausmarin A alleviated the serious defects of a strain expressing a constitutively active form of calcineurin. In the human Jurkat T-cell line, clausmarin A exhibited a dose-dependent inhibition of IL-2 production and IL-2 gene transcription, as well as an inhibition of NFAT dephosphorylation. The effects of clausmarin A observed in both yeast and Jurkat cells are basically similar to those of FK506. Our study revealed that clausmarin A is an inhibitor of the calcineurin pathway, and that this is probably mediated via inhibition of calcineurin phosphatase activity. As such, clausmarin A is a potential immunosuppressant.</p></div

Clausmarin A inhibits IL-2 and IL-2 mRNA production in Jurkat cells.

<p><b>(A)</b> Jurkat cells were treated with various concentrations of clausmarin A or with 100 nM FK506 for 30 min, then stimulated with 25 ng/mL PMA and 1 μg/mL Io at 37°C for 24 h. The treatments were: (1) none, (2) 0.5% (v/v) DMSO, (3–7) clausmarin A at (3) 0.25 μM, (4) 1 μM, (5) 2.5 μM, (6) 5 μM and (7) 25 μM, (8) 100 nM FK506 and (9) unstimulated (no PMA/Io treatment). (<b>B)</b> Effect of clausmarin A on IL-2 mRNA expression. Jurkat cells were treated with various concentrations of clausmarin A or with 100 nM FK506 for 30 min prior to stimulation with PMA/Io as described in <b>(A</b>). The various treatments were: (1) none, (2–4) clausmarin A at (2) 5 μM, (3) 25 μM and (4) 50 μM, (5) 100 nM FK506 and (6) unstimulated (no PMA/Io treatment). RNA was extracted and subjected to two-stage gene specific sqRT-PCR. (<b>C</b>) Corrected IL-2 expression levels relative to that of β-actin. Data are shown as the mean ± 1 SD, derived from three replicated. Means that are significantly different from the untreated cells at <i>p</i> ≤ 0.001 are indicated by *.</p

Yeast strains used in this study.

<p>Yeast strains used in this study.</p

Clausmarin A inhibits the calcineurin but not the Mpk1 pathway.

<p>The effect of clausmarin A on the growth of the loss-of-function mutations in the (<b>A</b>) calcineurin (Δ<i>cnb1</i>) or (<b>B</b>) the Mpk1 MAP kinase (Δ<i>mpk1</i>) Ca²⁺-signaling pathways. The experimental procedures were similar to that described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136804#pone.0136804.g001" target="_blank">Fig 1</a>, except no CaCl₂ was added. Legend: no addition (○); 250 μM clausmarin A (▲); 250 nM FK506 (△). Data shown are from one trial and are representative of those seen from three independent trials. *Statistically different with <i>p</i>-value <0.0001.</p

Clausmarin A alleviates the growth inhibition of a constitutively active calcineurin.

<p><b>(A)</b> The effect of clausmarin A on the YRC1 yeast strain expressing an inducible constitutively active form of calcineurin. The experimental procedures were similar to those described in the legend to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136804#pone.0136804.g001" target="_blank">Fig 1</a>, except the YRC1 strain with a chromosomally integrated construct for the galactose-inducible constitutively active form of the calcineurin catalytic subunit (<i>CMP2</i>ΔC) and the appropriate medium (SC) without CaCl₂ addition for induction of <i>GAL1</i> promoter was used. Legend: SC medium with no addition (□); 250 μM clausmarin A (▲); 250 nM FK506 (△). The <i>GAL1</i> promoter was induced by the addition of 2% (w/v) galactose and incubated at 30°C with shaking for the indicated period of time. *Statistically different with <i>p</i>-value <0.001. <b>(B)</b> The samples obtained after 12 h of incubation were observed under phase-contrast microscopy (left), fluorescence microscopy of Hoechst 33342-stained cells (middle) or flow cytometric analysis of Hoechst 33342-stained cells (right). Data shown are from one trial and are representative of those seen from three independent trials.</p

Clausmarin A alleviates the Ca²⁺-dependent growth inhibition of the YNS17 (Δ<i>zds1</i>) yeast strain.

<p>YNS17 cells (5 x 10⁶ cells/mL) were incubated for 30 min in YPD medium containing the indicated concentration of clausmarin A before the addition of CaCl₂ to 100 mM final concentration and incubating at 30°C with shaking for the indicated time. Legend: 100 mM CaCl₂ (■);100 mM CaCl₂ + 125 μM clausmarin A (□<b>)</b>;100 mM CaCl₂ + 250 μM clausmarin A (▲);100 mM CaCl₂ + 250 nM FK506 (△); none (○) and 250 μM clausmarin A (●). Data shown are from one trial and are representative of those seen from three independent trials. *Statistically different with <i>p</i>-value <0.0001.</p