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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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Actin rearrangement and Rho A activation upon LFA1 engagement in NK cells.(A). Rearrangement of actin microfilaments was analyzed in NK cells cultured in the indicated conditions (solvent of fluvastatin, DMSO: solvent; or fluvastatin at different doses 10-1.0-0.1 µM) for 6d in the presence of IL2 (10 ng/ml). LFA1 crosslinking was induced on ICAM1 protein coated slides for 20 min at 37°C. Then cells were fixed and permeabilized and incubated with alexafluor 488-phallotoxin to stain actin. Bar in each subpanel: 5 µm. Samples were analyzed by confocal microscopy (Olympus FluoView 500) and images were taken with PlanApo objective 60x/1.20NA and analyzed with FluoView computer program. Actin rearrangement is characterized by well-marked lines more evident at the periphery of a cell (A, upper left, white arrow). No actin rearrangement in NK cells treated with fluvastatin at 10 µM (A, upper right). Results are representative of three independent experiments with NK cells from different donors. (B). Kinetic of RhoA activation by specific C-Lisa kit. Black bars: LFA1 engagement using anti-LFA1mAb followed by GAM (LFA1-XL), white bars level of RhoA activated in basal conditions at the indicated time points from two healthy donors. (C). Activation of RhoA on NK cells cultured in the solvent of fluvastatin (solvent) or with the indicated doses of fluvastatin (10-1.0-0.1 µM) or with 10 µM fluvastatin and 1 mM mevalonate. Results in panels B and C are expressed as OD at 490 nm from two donors (B) or mean±SD of data from six different donors (C). (D,E): effect of fluvastatin on membrane rafts containing GM1. NK cells cultured 6d in IL2 with solvent (DMSO) or fluvastatin (10-1.0-0.1 µM) were incubated with anti-NKG2D mAb followed by GAM for 20 min and stained with alexafluor 488 subunit A of cholera toxin which reacts with GM1. Membrane rafts with GM1 were displayed as patches (D, white arrow). (E). Rafts with GM1 in NK cells incubated with the indicated mAbs followed by GAM. Basal: rafts in NK cells without any cross-linking. Results are shown as % of cells with GM1 rafts for each culture conditions. At least 200 cells were counted for each experiments and results are the mean±SD of six independent experiments.
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pone-0062932-g001: Actin rearrangement and Rho A activation upon LFA1 engagement in NK cells.(A). Rearrangement of actin microfilaments was analyzed in NK cells cultured in the indicated conditions (solvent of fluvastatin, DMSO: solvent; or fluvastatin at different doses 10-1.0-0.1 µM) for 6d in the presence of IL2 (10 ng/ml). LFA1 crosslinking was induced on ICAM1 protein coated slides for 20 min at 37°C. Then cells were fixed and permeabilized and incubated with alexafluor 488-phallotoxin to stain actin. Bar in each subpanel: 5 µm. Samples were analyzed by confocal microscopy (Olympus FluoView 500) and images were taken with PlanApo objective 60x/1.20NA and analyzed with FluoView computer program. Actin rearrangement is characterized by well-marked lines more evident at the periphery of a cell (A, upper left, white arrow). No actin rearrangement in NK cells treated with fluvastatin at 10 µM (A, upper right). Results are representative of three independent experiments with NK cells from different donors. (B). Kinetic of RhoA activation by specific C-Lisa kit. Black bars: LFA1 engagement using anti-LFA1mAb followed by GAM (LFA1-XL), white bars level of RhoA activated in basal conditions at the indicated time points from two healthy donors. (C). Activation of RhoA on NK cells cultured in the solvent of fluvastatin (solvent) or with the indicated doses of fluvastatin (10-1.0-0.1 µM) or with 10 µM fluvastatin and 1 mM mevalonate. Results in panels B and C are expressed as OD at 490 nm from two donors (B) or mean±SD of data from six different donors (C). (D,E): effect of fluvastatin on membrane rafts containing GM1. NK cells cultured 6d in IL2 with solvent (DMSO) or fluvastatin (10-1.0-0.1 µM) were incubated with anti-NKG2D mAb followed by GAM for 20 min and stained with alexafluor 488 subunit A of cholera toxin which reacts with GM1. Membrane rafts with GM1 were displayed as patches (D, white arrow). (E). Rafts with GM1 in NK cells incubated with the indicated mAbs followed by GAM. Basal: rafts in NK cells without any cross-linking. Results are shown as % of cells with GM1 rafts for each culture conditions. At least 200 cells were counted for each experiments and results are the mean±SD of six independent experiments.

Mentions: It has been reported that lipophilic statins affect granule polarization at the site of contact between NK and tumor cells suggesting a possible inhibition of cytoskeleton reorganization and LFA1-mediated triggering [26]. To clarify the molecular mechanism involved, we analyzed the effect of fluvastatin on actin distribution, on activation of the small GTP-binding protein RhoA needed for actin assembling [37], [38], and on ganglioside M1 raft formation in NK cells. In ex-vivo isolated or IL2-activated (6d) NK cells actin was not assembled beneath the plasma membrane but dispersed in the cytoplasm and not easily detectable (not shown). To stimulate actin assembling, NK cells cultured for 6d with IL2, with or without fluvastatin at different doses (fig. 1A), were harvested and the engagement of LFA1 was achieved using glass slides coated with purified ICAM1. A 15–30 min incubation at 37°C, led to actin redistribution, evidenced by detectable lines beneath the membrane marking the limit of each NK cell (fig. 1A, upper left). The assembling and redistribution was markedly reduced in NK cells treated with 10 µM fluvastatin (fig. 1A, upper right). This inhibiting effect was less evident at 1 µM and undetectable at 0.1 µM concentration. In parallel experiments, we found that the kinetics of RhoA activation upon cross-linking of LFA1 peaked at 5 min (fig. 1B), but strikingly decreased in NK cells cultured with fluvastatin (fig. 1C: 15%, 30% and 70% RhoA activation in 10-1-0.1 µM fluvastatin respectively vs untreated cells). The addition of mevalonate together with fluvastatin reverted the effect of the statin (fig. 1C). Then, we analyzed whether fluvastatin can affect the formation of membrane rafts containing ganglioside M1 [39], [40]. No rafts were found in ex-vivo isolated NK cells and upon culture with IL2, 20–30% of NK cells displayed rafts (fig. 1E). Engagement of the NK cell surface activating receptors NKG2D (fig. 1D,E) or CD16 or DNAM1 or LFA1 (fig. 1E) triggered the formation of GM1 rafts in almost all NK cells (90–100%). These rafts were not present in NK cells treated with 10 µM fluvastatin; at 1.0 µM fluvastatin, we found about 70% of NK cells with rafts, while no effect was detected at 0.1 µM concentration. In NK cells cultured with 1 mM mevalonate lattone the inhibiting effects of fluvastatin on RhoA activation and on GM1 rafts were abolished (not shown).


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)

Actin rearrangement and Rho A activation upon LFA1 engagement in NK cells.(A). Rearrangement of actin microfilaments was analyzed in NK cells cultured in the indicated conditions (solvent of fluvastatin, DMSO: solvent; or fluvastatin at different doses 10-1.0-0.1 µM) for 6d in the presence of IL2 (10 ng/ml). LFA1 crosslinking was induced on ICAM1 protein coated slides for 20 min at 37°C. Then cells were fixed and permeabilized and incubated with alexafluor 488-phallotoxin to stain actin. Bar in each subpanel: 5 µm. Samples were analyzed by confocal microscopy (Olympus FluoView 500) and images were taken with PlanApo objective 60x/1.20NA and analyzed with FluoView computer program. Actin rearrangement is characterized by well-marked lines more evident at the periphery of a cell (A, upper left, white arrow). No actin rearrangement in NK cells treated with fluvastatin at 10 µM (A, upper right). Results are representative of three independent experiments with NK cells from different donors. (B). Kinetic of RhoA activation by specific C-Lisa kit. Black bars: LFA1 engagement using anti-LFA1mAb followed by GAM (LFA1-XL), white bars level of RhoA activated in basal conditions at the indicated time points from two healthy donors. (C). Activation of RhoA on NK cells cultured in the solvent of fluvastatin (solvent) or with the indicated doses of fluvastatin (10-1.0-0.1 µM) or with 10 µM fluvastatin and 1 mM mevalonate. Results in panels B and C are expressed as OD at 490 nm from two donors (B) or mean±SD of data from six different donors (C). (D,E): effect of fluvastatin on membrane rafts containing GM1. NK cells cultured 6d in IL2 with solvent (DMSO) or fluvastatin (10-1.0-0.1 µM) were incubated with anti-NKG2D mAb followed by GAM for 20 min and stained with alexafluor 488 subunit A of cholera toxin which reacts with GM1. Membrane rafts with GM1 were displayed as patches (D, white arrow). (E). Rafts with GM1 in NK cells incubated with the indicated mAbs followed by GAM. Basal: rafts in NK cells without any cross-linking. Results are shown as % of cells with GM1 rafts for each culture conditions. At least 200 cells were counted for each experiments and results are the mean±SD of six independent experiments.
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pone-0062932-g001: Actin rearrangement and Rho A activation upon LFA1 engagement in NK cells.(A). Rearrangement of actin microfilaments was analyzed in NK cells cultured in the indicated conditions (solvent of fluvastatin, DMSO: solvent; or fluvastatin at different doses 10-1.0-0.1 µM) for 6d in the presence of IL2 (10 ng/ml). LFA1 crosslinking was induced on ICAM1 protein coated slides for 20 min at 37°C. Then cells were fixed and permeabilized and incubated with alexafluor 488-phallotoxin to stain actin. Bar in each subpanel: 5 µm. Samples were analyzed by confocal microscopy (Olympus FluoView 500) and images were taken with PlanApo objective 60x/1.20NA and analyzed with FluoView computer program. Actin rearrangement is characterized by well-marked lines more evident at the periphery of a cell (A, upper left, white arrow). No actin rearrangement in NK cells treated with fluvastatin at 10 µM (A, upper right). Results are representative of three independent experiments with NK cells from different donors. (B). Kinetic of RhoA activation by specific C-Lisa kit. Black bars: LFA1 engagement using anti-LFA1mAb followed by GAM (LFA1-XL), white bars level of RhoA activated in basal conditions at the indicated time points from two healthy donors. (C). Activation of RhoA on NK cells cultured in the solvent of fluvastatin (solvent) or with the indicated doses of fluvastatin (10-1.0-0.1 µM) or with 10 µM fluvastatin and 1 mM mevalonate. Results in panels B and C are expressed as OD at 490 nm from two donors (B) or mean±SD of data from six different donors (C). (D,E): effect of fluvastatin on membrane rafts containing GM1. NK cells cultured 6d in IL2 with solvent (DMSO) or fluvastatin (10-1.0-0.1 µM) were incubated with anti-NKG2D mAb followed by GAM for 20 min and stained with alexafluor 488 subunit A of cholera toxin which reacts with GM1. Membrane rafts with GM1 were displayed as patches (D, white arrow). (E). Rafts with GM1 in NK cells incubated with the indicated mAbs followed by GAM. Basal: rafts in NK cells without any cross-linking. Results are shown as % of cells with GM1 rafts for each culture conditions. At least 200 cells were counted for each experiments and results are the mean±SD of six independent experiments.
Mentions: It has been reported that lipophilic statins affect granule polarization at the site of contact between NK and tumor cells suggesting a possible inhibition of cytoskeleton reorganization and LFA1-mediated triggering [26]. To clarify the molecular mechanism involved, we analyzed the effect of fluvastatin on actin distribution, on activation of the small GTP-binding protein RhoA needed for actin assembling [37], [38], and on ganglioside M1 raft formation in NK cells. In ex-vivo isolated or IL2-activated (6d) NK cells actin was not assembled beneath the plasma membrane but dispersed in the cytoplasm and not easily detectable (not shown). To stimulate actin assembling, NK cells cultured for 6d with IL2, with or without fluvastatin at different doses (fig. 1A), were harvested and the engagement of LFA1 was achieved using glass slides coated with purified ICAM1. A 15–30 min incubation at 37°C, led to actin redistribution, evidenced by detectable lines beneath the membrane marking the limit of each NK cell (fig. 1A, upper left). The assembling and redistribution was markedly reduced in NK cells treated with 10 µM fluvastatin (fig. 1A, upper right). This inhibiting effect was less evident at 1 µM and undetectable at 0.1 µM concentration. In parallel experiments, we found that the kinetics of RhoA activation upon cross-linking of LFA1 peaked at 5 min (fig. 1B), but strikingly decreased in NK cells cultured with fluvastatin (fig. 1C: 15%, 30% and 70% RhoA activation in 10-1-0.1 µM fluvastatin respectively vs untreated cells). The addition of mevalonate together with fluvastatin reverted the effect of the statin (fig. 1C). Then, we analyzed whether fluvastatin can affect the formation of membrane rafts containing ganglioside M1 [39], [40]. No rafts were found in ex-vivo isolated NK cells and upon culture with IL2, 20–30% of NK cells displayed rafts (fig. 1E). Engagement of the NK cell surface activating receptors NKG2D (fig. 1D,E) or CD16 or DNAM1 or LFA1 (fig. 1E) triggered the formation of GM1 rafts in almost all NK cells (90–100%). These rafts were not present in NK cells treated with 10 µM fluvastatin; at 1.0 µM fluvastatin, we found about 70% of NK cells with rafts, while no effect was detected at 0.1 µM concentration. In NK cells cultured with 1 mM mevalonate lattone the inhibiting effects of fluvastatin on RhoA activation and on GM1 rafts were abolished (not shown).

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