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

Suauam P, Yingyongnarongkul BE, Palaga T, Miyakawa T, Yompakdee C - PLoS ONE (2015)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Pathumwan, Bangkok, 10330, Thailand.

ABSTRACT
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 Δzds1 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 Δmpk1 (lacking the Mpk1 MAP kinase pathway) but not Δcnb1 (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.

No MeSH data available.


Related in: MedlinePlus

Clausmarin A alleviates the growth inhibition of a constitutively active calcineurin.(A) 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 Fig 1, except the YRC1 strain with a chromosomally integrated construct for the galactose-inducible constitutively active form of the calcineurin catalytic subunit (CMP2ΔC) and the appropriate medium (SC) without CaCl2 addition for induction of GAL1 promoter was used. Legend: SC medium with no addition (□); 250 μM clausmarin A (▲); 250 nM FK506 (△). The GAL1 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 p-value <0.001. (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.
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pone.0136804.g003: Clausmarin A alleviates the growth inhibition of a constitutively active calcineurin.(A) 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 Fig 1, except the YRC1 strain with a chromosomally integrated construct for the galactose-inducible constitutively active form of the calcineurin catalytic subunit (CMP2ΔC) and the appropriate medium (SC) without CaCl2 addition for induction of GAL1 promoter was used. Legend: SC medium with no addition (□); 250 μM clausmarin A (▲); 250 nM FK506 (△). The GAL1 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 p-value <0.001. (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.

Mentions: In the yeast Ca2+-signaling, the parallel calcineurin- and Mpk1-mediated pathways redundantly regulate some events essential for cell growth and morphogenesis [20]. Thus, a loss-of-function in either branch of the two parallel pathways is still viable (e.g., Δcnb1 single mutant lacking the calcineurin pathway or Δmpk1 single mutant lacking the Mpk1 MAP kinase pathway), but the simultaneous loss-of-function in both branches is lethal (e.g., Δcnb1 Δmpk1 double mutant). Therefore, to distinguish whether the calcineurin branch or the Mpk1 branch is likely to be inhibited by clausmarin A, the synthetic lethality due to clausmarin A was evaluated on each of the Δcnb1 and Δmpk1 single mutant yeast strains. The Δcnb1 and Δmpk1 cells were cultured in YPD medium in the presence of 250 μM clausmarin A or 250 nM FK506 and the cell growth was monitored (Fig 2). The growth of the Δcnb1 cells was unaffected by the inclusion of clausmarin A or FK506, indicating that the Mpk1 pathway is not likely to be inhibited by these drugs (Fig 2A). In contrast, the growth of the Δmpk1cells was severely inhibited (> 72%) by the inclusion of clausmarin A (250 μM) or FK506 (250 nM) to a similar extent (Fig 2B), implying that clausmarin A, similar to FK506, inhibited the calcineurin but not the Mpk1 pathway. If the target of clausmarin A is in the calcineurin branch, then it could be predicted that the drug will alleviate the deleterious effects caused by the expression of a constitutively active form of calcineurin, which otherwise leads to similar changes to that observed with the YNS17 cells treated with 100 mM CaCl2 [22]. The YRC1 strain (Δzds1 + [GAL1p-CMP2ΔC]), which over-expresses a constitutively active form of the catalytic subunit of calcineurin (Cmp2ΔC) upon galactose induction, was first treated with 250 μM clausmarin A (or 250 nM FK506) for 30 min and then 2% (w/v) galactose was added to induce the expression of the calcineurin gene (Fig 3A). As expected, a severe growth retardation (> 78%) resulted from the forced expression of the constitutively active form of calcineurin, but this was alleviated by the treatment with either 250 μM clausmarin A or 250 nM FK506 to a broadly similar extent, suggesting that calcineurin or a functionally closely related molecule is the target of clausmarin A. Hyper-activation of Ca2+ signals in Δzds1 cells (by externally supplemented CaCl2 or by CMP2ΔC overexpression) not only causes growth retardation, but also induces polarized bud growth and cell-cycle arrest in the G2 phase in affected cells [18]. Thus, whether clausmarin A could alleviate the altered morphology and the cell-cycle arrest caused by CMP2ΔC overexpression was also evaluated (Fig 3B). The abnormal budding morphology (polarized bud growth), as observed by a phase contrast microscopy, and the unequal nuclear division, as visualized in Hoechst 3342-stained cells using fluorescence microscopy, 6 h after CMP2ΔC induction, were both alleviated by treatment with 250 μM clausmarin A. The Ca2+-induced G2 arrest, as measured by FACS analysis, showed an increase in the 1C peak (from 23.4% to 29.1%) and a decrease in the 2C peak (from 76.6% to 70.9%), indicating that the G2 cell-cycle arrest was also alleviated (Fig 3B). These results demonstrated that each of the abnormalities caused by activated calcineurin was suppressed by clausmarin A, supporting the notion that the drug inhibited the calcineurin pathway in the yeast. Similar results were obtained with 250 nM FK506 (data not shown; [18], [23]).


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

Suauam P, Yingyongnarongkul BE, Palaga T, Miyakawa T, Yompakdee C - PLoS ONE (2015)

Clausmarin A alleviates the growth inhibition of a constitutively active calcineurin.(A) 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 Fig 1, except the YRC1 strain with a chromosomally integrated construct for the galactose-inducible constitutively active form of the calcineurin catalytic subunit (CMP2ΔC) and the appropriate medium (SC) without CaCl2 addition for induction of GAL1 promoter was used. Legend: SC medium with no addition (□); 250 μM clausmarin A (▲); 250 nM FK506 (△). The GAL1 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 p-value <0.001. (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.
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Related In: Results  -  Collection

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pone.0136804.g003: Clausmarin A alleviates the growth inhibition of a constitutively active calcineurin.(A) 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 Fig 1, except the YRC1 strain with a chromosomally integrated construct for the galactose-inducible constitutively active form of the calcineurin catalytic subunit (CMP2ΔC) and the appropriate medium (SC) without CaCl2 addition for induction of GAL1 promoter was used. Legend: SC medium with no addition (□); 250 μM clausmarin A (▲); 250 nM FK506 (△). The GAL1 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 p-value <0.001. (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.
Mentions: In the yeast Ca2+-signaling, the parallel calcineurin- and Mpk1-mediated pathways redundantly regulate some events essential for cell growth and morphogenesis [20]. Thus, a loss-of-function in either branch of the two parallel pathways is still viable (e.g., Δcnb1 single mutant lacking the calcineurin pathway or Δmpk1 single mutant lacking the Mpk1 MAP kinase pathway), but the simultaneous loss-of-function in both branches is lethal (e.g., Δcnb1 Δmpk1 double mutant). Therefore, to distinguish whether the calcineurin branch or the Mpk1 branch is likely to be inhibited by clausmarin A, the synthetic lethality due to clausmarin A was evaluated on each of the Δcnb1 and Δmpk1 single mutant yeast strains. The Δcnb1 and Δmpk1 cells were cultured in YPD medium in the presence of 250 μM clausmarin A or 250 nM FK506 and the cell growth was monitored (Fig 2). The growth of the Δcnb1 cells was unaffected by the inclusion of clausmarin A or FK506, indicating that the Mpk1 pathway is not likely to be inhibited by these drugs (Fig 2A). In contrast, the growth of the Δmpk1cells was severely inhibited (> 72%) by the inclusion of clausmarin A (250 μM) or FK506 (250 nM) to a similar extent (Fig 2B), implying that clausmarin A, similar to FK506, inhibited the calcineurin but not the Mpk1 pathway. If the target of clausmarin A is in the calcineurin branch, then it could be predicted that the drug will alleviate the deleterious effects caused by the expression of a constitutively active form of calcineurin, which otherwise leads to similar changes to that observed with the YNS17 cells treated with 100 mM CaCl2 [22]. The YRC1 strain (Δzds1 + [GAL1p-CMP2ΔC]), which over-expresses a constitutively active form of the catalytic subunit of calcineurin (Cmp2ΔC) upon galactose induction, was first treated with 250 μM clausmarin A (or 250 nM FK506) for 30 min and then 2% (w/v) galactose was added to induce the expression of the calcineurin gene (Fig 3A). As expected, a severe growth retardation (> 78%) resulted from the forced expression of the constitutively active form of calcineurin, but this was alleviated by the treatment with either 250 μM clausmarin A or 250 nM FK506 to a broadly similar extent, suggesting that calcineurin or a functionally closely related molecule is the target of clausmarin A. Hyper-activation of Ca2+ signals in Δzds1 cells (by externally supplemented CaCl2 or by CMP2ΔC overexpression) not only causes growth retardation, but also induces polarized bud growth and cell-cycle arrest in the G2 phase in affected cells [18]. Thus, whether clausmarin A could alleviate the altered morphology and the cell-cycle arrest caused by CMP2ΔC overexpression was also evaluated (Fig 3B). The abnormal budding morphology (polarized bud growth), as observed by a phase contrast microscopy, and the unequal nuclear division, as visualized in Hoechst 3342-stained cells using fluorescence microscopy, 6 h after CMP2ΔC induction, were both alleviated by treatment with 250 μM clausmarin A. The Ca2+-induced G2 arrest, as measured by FACS analysis, showed an increase in the 1C peak (from 23.4% to 29.1%) and a decrease in the 2C peak (from 76.6% to 70.9%), indicating that the G2 cell-cycle arrest was also alleviated (Fig 3B). These results demonstrated that each of the abnormalities caused by activated calcineurin was suppressed by clausmarin A, supporting the notion that the drug inhibited the calcineurin pathway in the yeast. Similar results were obtained with 250 nM FK506 (data not shown; [18], [23]).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, Faculty of Science, Chulalongkorn University, Phayathai Road, Pathumwan, Bangkok, 10330, Thailand.

ABSTRACT
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 Δzds1 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 Δmpk1 (lacking the Mpk1 MAP kinase pathway) but not Δcnb1 (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.

No MeSH data available.


Related in: MedlinePlus