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Selective role of mevalonate pathway in regulating perforin but not FasL and TNFalpha release in human Natural Killer cells.

Poggi A, Boero S, Musso A, Zocchi MR - PLoS ONE (2013)

Bottom Line: We have analyzed the effects of fluvastatin, an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase involved in mevalonate synthesis, on human NK cell-mediated anti-tumor cytolysis.Remarkably, fluvastatin did not affect the expression of the inhibiting receptors CD94, KIR2D and LAIR1.Altogether these findings suggest that interference with mevalonate synthesis impairs activation and assembly of cytoskeleton, degranulation and cytotoxic effect of perforins and granzyme but not FasL- and TNFα-mediated cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Angiogenesis Unit, IRCCS AOU San Martino - IST National Institute for Cancer Research, Genoa, Italy. alessandro.poggi@istge.it

ABSTRACT
We have analyzed the effects of fluvastatin, an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase involved in mevalonate synthesis, on human NK cell-mediated anti-tumor cytolysis. Fluvastatin inhibited the activation of the small guanosin triphosphate binding protein (GTP) RhoA and the consequent actin redistribution induced by ligation of LFA1 involved in NK-tumor target cell adhesion. Also, fluvastatin reduced ganglioside M1 rafts formation triggered through the engagement of NK cell activating receptors as FcγRIIIA (CD16), NKG2D and DNAM1. Cytolysis of tumor targets was inhibited up to 90% when NK cells were cultured with fluvastatin by affecting i) receptor-mediated increase of the intracellular free calcium concentration, ii) activation of akt1/PKB and iii) perforin and granzyme release. Fluvastatin displayed a stronger inhibiting effect on NKG2D, DNAM1, 2B4, NKp30, NKp44 and NKp46 than on CD16-mediated NK cell triggering. This was in line with the impairment of surface expression of all these receptors but not of CD16. Remarkably, fluvastatin did not affect the expression of the inhibiting receptors CD94, KIR2D and LAIR1. FasL release elicited by either NK-tumor cell interaction or CD16 or NKG2D engagement, as well as FasL-mediated killing, were not sensitive to fluvastatin. Moreover, TNFα secretion triggered in NK cells upon incubation with tumor target cells or engagement of NKG2D receptor was not impaired in fluvastatin-treated NK cells. Likewise, antibody dependent cellular cytotoxicity (ADCC) triggered through FcγRIIIA engagement with the humanized monoclonal antibody rituximab or trastuzumab was only marginally affected in fluvastatin-treated NK cells. Altogether these findings suggest that interference with mevalonate synthesis impairs activation and assembly of cytoskeleton, degranulation and cytotoxic effect of perforins and granzyme but not FasL- and TNFα-mediated cytotoxicity.

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Fluvastatin effects on intracellular calcium increase and akt1/PKB activation.(A–F) Fluvastatin inhibits intracellular free calcium increase [Ca2+]i induced upon engagement of NK cell activating receptors. (A). Effect of fluvastatin on calcium increase triggered by CD16 or NKG2D or DNAM1 or LFA1 with the specific mAbs followed by GAM; unmAb: unrelated mAb matched for isotype plus GAM. NK cells were cultured without drug (black), with fluvastatin (10 µM, red; 1 µM, blue; 0.1 µM, green; left panels) or in right subpanels with fluvastatin alone (red, 10 µM) or in combination with 1 mM of mevalonate (green) for 6d+IL2. Then cells were harvested, washed and labelled with the fluorescent calcium indicator Fura-2 at 37°C and [Ca2+]I analyzed on a spectrofluorimeter along time (800 sec). The arrows indicate the addition of GAM to achieve the optimal cross-linking for the indicated receptors. (B) Effect of fluvastatin on calcium increase triggered as in A. NK cells were cultured without drug (solvent), with fluvastatin alone (10 µM, fluva) or in combination with 1 mM of mevalonate (fluva+meva) or with mevalonate alone (meva) for 6d+IL2. Then cells were harvested, washed and labelled with the fluorescent calcium indicator Fluo-4 for 45 min at 37°C; intracellular free calcium increase was assessed by flowcytometry upon engagement of the indicated surface receptors after 600 sec of incubation. Results are shown as forward scatter (FSC, x-axis) vs Log green fluorescence intensity (Fluo-4, Y-axis). Each subpanel is divided in four quadrants: lower left: very small cells/debries; lower right: cells with FSC of living NK cells; upper left: small cells/debries labelled with Fluo-4; upper right: NK cells strongly labelled with Fluo-4. In each subpanel in the upper right quadrant is shown the % of NK cells with high Fluo-4 fluorescence. Evaluation of Fluo-4 staining was performed after 10 min upon engagement of each molecule. (C–F). Percentage of NK cells (% responding cells), cultured with different doses of fluvastatin (C,E) or different combination of drugs (D,F), with an increase of intracellular calcium concentration upon engagement of the indicated receptors. Results are expressed as mean of responding cells ±SD of 6 independent experiments. *p<0.001. (G). Evaluation of activation of akt1/PKB in NK cells cultured as in A–F. Results are expressed as % of akt1/PKB activation as ratio between pakt1 and total akt1×100 in each condition at 5 min after stimulation with mAb to the indicated receptors and GAM. Basal: basal level of akt1/PKB activation in cultured NK cells. LY294002: akt1/PKB activation in the presence of the upstream PI3K inhibitor LY294002. *p<0.001 vs solvent.
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pone-0062932-g002: Fluvastatin effects on intracellular calcium increase and akt1/PKB activation.(A–F) Fluvastatin inhibits intracellular free calcium increase [Ca2+]i induced upon engagement of NK cell activating receptors. (A). Effect of fluvastatin on calcium increase triggered by CD16 or NKG2D or DNAM1 or LFA1 with the specific mAbs followed by GAM; unmAb: unrelated mAb matched for isotype plus GAM. NK cells were cultured without drug (black), with fluvastatin (10 µM, red; 1 µM, blue; 0.1 µM, green; left panels) or in right subpanels with fluvastatin alone (red, 10 µM) or in combination with 1 mM of mevalonate (green) for 6d+IL2. Then cells were harvested, washed and labelled with the fluorescent calcium indicator Fura-2 at 37°C and [Ca2+]I analyzed on a spectrofluorimeter along time (800 sec). The arrows indicate the addition of GAM to achieve the optimal cross-linking for the indicated receptors. (B) Effect of fluvastatin on calcium increase triggered as in A. NK cells were cultured without drug (solvent), with fluvastatin alone (10 µM, fluva) or in combination with 1 mM of mevalonate (fluva+meva) or with mevalonate alone (meva) for 6d+IL2. Then cells were harvested, washed and labelled with the fluorescent calcium indicator Fluo-4 for 45 min at 37°C; intracellular free calcium increase was assessed by flowcytometry upon engagement of the indicated surface receptors after 600 sec of incubation. Results are shown as forward scatter (FSC, x-axis) vs Log green fluorescence intensity (Fluo-4, Y-axis). Each subpanel is divided in four quadrants: lower left: very small cells/debries; lower right: cells with FSC of living NK cells; upper left: small cells/debries labelled with Fluo-4; upper right: NK cells strongly labelled with Fluo-4. In each subpanel in the upper right quadrant is shown the % of NK cells with high Fluo-4 fluorescence. Evaluation of Fluo-4 staining was performed after 10 min upon engagement of each molecule. (C–F). Percentage of NK cells (% responding cells), cultured with different doses of fluvastatin (C,E) or different combination of drugs (D,F), with an increase of intracellular calcium concentration upon engagement of the indicated receptors. Results are expressed as mean of responding cells ±SD of 6 independent experiments. *p<0.001. (G). Evaluation of activation of akt1/PKB in NK cells cultured as in A–F. Results are expressed as % of akt1/PKB activation as ratio between pakt1 and total akt1×100 in each condition at 5 min after stimulation with mAb to the indicated receptors and GAM. Basal: basal level of akt1/PKB activation in cultured NK cells. LY294002: akt1/PKB activation in the presence of the upstream PI3K inhibitor LY294002. *p<0.001 vs solvent.

Mentions: As intracellular free calcium [Ca2+]i mobilization and phosphoinositide kinase 3 (PI3K) activation are crucial for the release of cytotoxic granules [8], [33], [36], we analyzed whether the inhibition of cytolytic activity mediated by fluvastatin was related to impaired [Ca2+]i increase and/or inhibition of PI3K-dependent activation of akt1/PKB in NK cells. To this aim, [Ca2+]i increase in NK cells cultured with IL2 for 6d with scalar doses of fluvastatin (10-1.0-0.1 µM, fig. 2A left subpanels) was analyzed on a spectrofluorimeter upon the engagement of CD16, or NKG2D or DNAM1 or LFA1 receptors after labelling of cells with Fura-2 calcium probe. We found that fluvastatin exerted a strong and dose dependent inhibiting effect (fig. 2A left panels) that was reverted by the addition during the culture of mevalonate (fig. 2A, right panels). Furthermore, to determine whether the observed inhibiting effect was dependent on the amount of responding cells, primary NK cells cultured with IL2 for 6d without or with fluvastatin, were washed and labelled with Fluo-4 calcium probe. The increase in the intensity of fluorescence of cell samples due to [Ca2+]i increase was analyzed by flowcytometry after 600 sec, either without or upon cross-linking of the same triggering receptors. The engagement of the activating receptors CD16, NKG2D, DNAM1 (fig. 2B–D) or NKp30, NKp44, NKp46 or 2B4 (fig. 2D,E) induced a strong [Ca2+]i increase while cross-linking of LFA1 was less efficient (fig. 2A–C). NK cells cultured with fluvastatin showed a lower [Ca2+]i rise (fig. 2B) and this effect was dose dependent (fig. 2C,D). We found a lower number of responding NK cells in cell cultures with 10 µM than with 1 µM fluvastatin (fig. 2C,E); at 0.1 µM fluvastatin no effect was observed (fig. 2C,E). The addition of mevalonate at the onset of cell culture completely restored the response to the triggering receptors in fluvastatin-treated NK cells (fig. 2D,F). Further, we found that the engagement of CD16 or NKG2D induced a marked activation of akt1/PKB; this activation was inhibited in NK cells treated with LY294002 indicating the involvement of the upstream PI3K (fig. 2G). Activating receptor-mediated triggering of akt1/PKB was impaired in NK cells incubated with fluvastatin; it is of note that CD16-mediated phosphorylation was less inhibited than that elicited upon NKG2D cross-linking (fig. 2G). Again, NK cells cultured with fluvastatin and mevalonate showed a response to these triggering receptors similar to untreated NK cells (fig. 2G).


Selective role of mevalonate pathway in regulating perforin but not FasL and TNFalpha release in human Natural Killer cells.

Poggi A, Boero S, Musso A, Zocchi MR - PLoS ONE (2013)

Fluvastatin effects on intracellular calcium increase and akt1/PKB activation.(A–F) Fluvastatin inhibits intracellular free calcium increase [Ca2+]i induced upon engagement of NK cell activating receptors. (A). Effect of fluvastatin on calcium increase triggered by CD16 or NKG2D or DNAM1 or LFA1 with the specific mAbs followed by GAM; unmAb: unrelated mAb matched for isotype plus GAM. NK cells were cultured without drug (black), with fluvastatin (10 µM, red; 1 µM, blue; 0.1 µM, green; left panels) or in right subpanels with fluvastatin alone (red, 10 µM) or in combination with 1 mM of mevalonate (green) for 6d+IL2. Then cells were harvested, washed and labelled with the fluorescent calcium indicator Fura-2 at 37°C and [Ca2+]I analyzed on a spectrofluorimeter along time (800 sec). The arrows indicate the addition of GAM to achieve the optimal cross-linking for the indicated receptors. (B) Effect of fluvastatin on calcium increase triggered as in A. NK cells were cultured without drug (solvent), with fluvastatin alone (10 µM, fluva) or in combination with 1 mM of mevalonate (fluva+meva) or with mevalonate alone (meva) for 6d+IL2. Then cells were harvested, washed and labelled with the fluorescent calcium indicator Fluo-4 for 45 min at 37°C; intracellular free calcium increase was assessed by flowcytometry upon engagement of the indicated surface receptors after 600 sec of incubation. Results are shown as forward scatter (FSC, x-axis) vs Log green fluorescence intensity (Fluo-4, Y-axis). Each subpanel is divided in four quadrants: lower left: very small cells/debries; lower right: cells with FSC of living NK cells; upper left: small cells/debries labelled with Fluo-4; upper right: NK cells strongly labelled with Fluo-4. In each subpanel in the upper right quadrant is shown the % of NK cells with high Fluo-4 fluorescence. Evaluation of Fluo-4 staining was performed after 10 min upon engagement of each molecule. (C–F). Percentage of NK cells (% responding cells), cultured with different doses of fluvastatin (C,E) or different combination of drugs (D,F), with an increase of intracellular calcium concentration upon engagement of the indicated receptors. Results are expressed as mean of responding cells ±SD of 6 independent experiments. *p<0.001. (G). Evaluation of activation of akt1/PKB in NK cells cultured as in A–F. Results are expressed as % of akt1/PKB activation as ratio between pakt1 and total akt1×100 in each condition at 5 min after stimulation with mAb to the indicated receptors and GAM. Basal: basal level of akt1/PKB activation in cultured NK cells. LY294002: akt1/PKB activation in the presence of the upstream PI3K inhibitor LY294002. *p<0.001 vs solvent.
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pone-0062932-g002: Fluvastatin effects on intracellular calcium increase and akt1/PKB activation.(A–F) Fluvastatin inhibits intracellular free calcium increase [Ca2+]i induced upon engagement of NK cell activating receptors. (A). Effect of fluvastatin on calcium increase triggered by CD16 or NKG2D or DNAM1 or LFA1 with the specific mAbs followed by GAM; unmAb: unrelated mAb matched for isotype plus GAM. NK cells were cultured without drug (black), with fluvastatin (10 µM, red; 1 µM, blue; 0.1 µM, green; left panels) or in right subpanels with fluvastatin alone (red, 10 µM) or in combination with 1 mM of mevalonate (green) for 6d+IL2. Then cells were harvested, washed and labelled with the fluorescent calcium indicator Fura-2 at 37°C and [Ca2+]I analyzed on a spectrofluorimeter along time (800 sec). The arrows indicate the addition of GAM to achieve the optimal cross-linking for the indicated receptors. (B) Effect of fluvastatin on calcium increase triggered as in A. NK cells were cultured without drug (solvent), with fluvastatin alone (10 µM, fluva) or in combination with 1 mM of mevalonate (fluva+meva) or with mevalonate alone (meva) for 6d+IL2. Then cells were harvested, washed and labelled with the fluorescent calcium indicator Fluo-4 for 45 min at 37°C; intracellular free calcium increase was assessed by flowcytometry upon engagement of the indicated surface receptors after 600 sec of incubation. Results are shown as forward scatter (FSC, x-axis) vs Log green fluorescence intensity (Fluo-4, Y-axis). Each subpanel is divided in four quadrants: lower left: very small cells/debries; lower right: cells with FSC of living NK cells; upper left: small cells/debries labelled with Fluo-4; upper right: NK cells strongly labelled with Fluo-4. In each subpanel in the upper right quadrant is shown the % of NK cells with high Fluo-4 fluorescence. Evaluation of Fluo-4 staining was performed after 10 min upon engagement of each molecule. (C–F). Percentage of NK cells (% responding cells), cultured with different doses of fluvastatin (C,E) or different combination of drugs (D,F), with an increase of intracellular calcium concentration upon engagement of the indicated receptors. Results are expressed as mean of responding cells ±SD of 6 independent experiments. *p<0.001. (G). Evaluation of activation of akt1/PKB in NK cells cultured as in A–F. Results are expressed as % of akt1/PKB activation as ratio between pakt1 and total akt1×100 in each condition at 5 min after stimulation with mAb to the indicated receptors and GAM. Basal: basal level of akt1/PKB activation in cultured NK cells. LY294002: akt1/PKB activation in the presence of the upstream PI3K inhibitor LY294002. *p<0.001 vs solvent.
Mentions: As intracellular free calcium [Ca2+]i mobilization and phosphoinositide kinase 3 (PI3K) activation are crucial for the release of cytotoxic granules [8], [33], [36], we analyzed whether the inhibition of cytolytic activity mediated by fluvastatin was related to impaired [Ca2+]i increase and/or inhibition of PI3K-dependent activation of akt1/PKB in NK cells. To this aim, [Ca2+]i increase in NK cells cultured with IL2 for 6d with scalar doses of fluvastatin (10-1.0-0.1 µM, fig. 2A left subpanels) was analyzed on a spectrofluorimeter upon the engagement of CD16, or NKG2D or DNAM1 or LFA1 receptors after labelling of cells with Fura-2 calcium probe. We found that fluvastatin exerted a strong and dose dependent inhibiting effect (fig. 2A left panels) that was reverted by the addition during the culture of mevalonate (fig. 2A, right panels). Furthermore, to determine whether the observed inhibiting effect was dependent on the amount of responding cells, primary NK cells cultured with IL2 for 6d without or with fluvastatin, were washed and labelled with Fluo-4 calcium probe. The increase in the intensity of fluorescence of cell samples due to [Ca2+]i increase was analyzed by flowcytometry after 600 sec, either without or upon cross-linking of the same triggering receptors. The engagement of the activating receptors CD16, NKG2D, DNAM1 (fig. 2B–D) or NKp30, NKp44, NKp46 or 2B4 (fig. 2D,E) induced a strong [Ca2+]i increase while cross-linking of LFA1 was less efficient (fig. 2A–C). NK cells cultured with fluvastatin showed a lower [Ca2+]i rise (fig. 2B) and this effect was dose dependent (fig. 2C,D). We found a lower number of responding NK cells in cell cultures with 10 µM than with 1 µM fluvastatin (fig. 2C,E); at 0.1 µM fluvastatin no effect was observed (fig. 2C,E). The addition of mevalonate at the onset of cell culture completely restored the response to the triggering receptors in fluvastatin-treated NK cells (fig. 2D,F). Further, we found that the engagement of CD16 or NKG2D induced a marked activation of akt1/PKB; this activation was inhibited in NK cells treated with LY294002 indicating the involvement of the upstream PI3K (fig. 2G). Activating receptor-mediated triggering of akt1/PKB was impaired in NK cells incubated with fluvastatin; it is of note that CD16-mediated phosphorylation was less inhibited than that elicited upon NKG2D cross-linking (fig. 2G). Again, NK cells cultured with fluvastatin and mevalonate showed a response to these triggering receptors similar to untreated NK cells (fig. 2G).

Bottom Line: We have analyzed the effects of fluvastatin, an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase involved in mevalonate synthesis, on human NK cell-mediated anti-tumor cytolysis.Remarkably, fluvastatin did not affect the expression of the inhibiting receptors CD94, KIR2D and LAIR1.Altogether these findings suggest that interference with mevalonate synthesis impairs activation and assembly of cytoskeleton, degranulation and cytotoxic effect of perforins and granzyme but not FasL- and TNFα-mediated cytotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Molecular Oncology and Angiogenesis Unit, IRCCS AOU San Martino - IST National Institute for Cancer Research, Genoa, Italy. alessandro.poggi@istge.it

ABSTRACT
We have analyzed the effects of fluvastatin, an inhibitor of the enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase involved in mevalonate synthesis, on human NK cell-mediated anti-tumor cytolysis. Fluvastatin inhibited the activation of the small guanosin triphosphate binding protein (GTP) RhoA and the consequent actin redistribution induced by ligation of LFA1 involved in NK-tumor target cell adhesion. Also, fluvastatin reduced ganglioside M1 rafts formation triggered through the engagement of NK cell activating receptors as FcγRIIIA (CD16), NKG2D and DNAM1. Cytolysis of tumor targets was inhibited up to 90% when NK cells were cultured with fluvastatin by affecting i) receptor-mediated increase of the intracellular free calcium concentration, ii) activation of akt1/PKB and iii) perforin and granzyme release. Fluvastatin displayed a stronger inhibiting effect on NKG2D, DNAM1, 2B4, NKp30, NKp44 and NKp46 than on CD16-mediated NK cell triggering. This was in line with the impairment of surface expression of all these receptors but not of CD16. Remarkably, fluvastatin did not affect the expression of the inhibiting receptors CD94, KIR2D and LAIR1. FasL release elicited by either NK-tumor cell interaction or CD16 or NKG2D engagement, as well as FasL-mediated killing, were not sensitive to fluvastatin. Moreover, TNFα secretion triggered in NK cells upon incubation with tumor target cells or engagement of NKG2D receptor was not impaired in fluvastatin-treated NK cells. Likewise, antibody dependent cellular cytotoxicity (ADCC) triggered through FcγRIIIA engagement with the humanized monoclonal antibody rituximab or trastuzumab was only marginally affected in fluvastatin-treated NK cells. Altogether these findings suggest that interference with mevalonate synthesis impairs activation and assembly of cytoskeleton, degranulation and cytotoxic effect of perforins and granzyme but not FasL- and TNFα-mediated cytotoxicity.

Show MeSH
Related in: MedlinePlus