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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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Related in: MedlinePlus

Scheme of the effects of HMG-CoA reductase inhibitors on NK cell function.Adhesion between NK and target cells triggers an activating signal in NK cells, leading to RhoA activation which favours reorganization of actin microfilaments and granule polarization to NK-target contact (A,B). At the site of contact are formed membrane microdomains (rafts) containing GM1 where activating receptor (i.e. NKG2D)-mediated [Ca2+]i increase and activation of akt leads to the degranulation of perforin (P) and granzymes (G) and consequent lysis of target cells (B). Cholesterol is essential for membrane mobility and correct aggregation of signalling molecules in membrane rafts. Fluvastatin-mediated inhibition of HMG-CoA reductase inhibiting mevalonate synthesis causes a low cholesterol content of cell membrane, a lower mobility and aggregation of activating receptors which cannot allow granule polarization. C: Apparently, both FasL and TNFα release are conserved in fluvastatin-incubated NK cells. This may depends on their low dependency of [Ca2+]i increase and akt activation and on low relevance of contact site–directed secretion. Indeed, when released FasL and TNFα may kill targets if these targets express the corresponding receptors. NK cell activation via FcγRIIIa (CD16), as it can occur with therapeutic antibodies rituximab or trastuzumab, has a quite low threshold of activation and only very high doses of fluvastatin are effective. Thus, ADCC is efficient also when the cholesterol content of cell membrane is reduced.
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pone-0062932-g007: Scheme of the effects of HMG-CoA reductase inhibitors on NK cell function.Adhesion between NK and target cells triggers an activating signal in NK cells, leading to RhoA activation which favours reorganization of actin microfilaments and granule polarization to NK-target contact (A,B). At the site of contact are formed membrane microdomains (rafts) containing GM1 where activating receptor (i.e. NKG2D)-mediated [Ca2+]i increase and activation of akt leads to the degranulation of perforin (P) and granzymes (G) and consequent lysis of target cells (B). Cholesterol is essential for membrane mobility and correct aggregation of signalling molecules in membrane rafts. Fluvastatin-mediated inhibition of HMG-CoA reductase inhibiting mevalonate synthesis causes a low cholesterol content of cell membrane, a lower mobility and aggregation of activating receptors which cannot allow granule polarization. C: Apparently, both FasL and TNFα release are conserved in fluvastatin-incubated NK cells. This may depends on their low dependency of [Ca2+]i increase and akt activation and on low relevance of contact site–directed secretion. Indeed, when released FasL and TNFα may kill targets if these targets express the corresponding receptors. NK cell activation via FcγRIIIa (CD16), as it can occur with therapeutic antibodies rituximab or trastuzumab, has a quite low threshold of activation and only very high doses of fluvastatin are effective. Thus, ADCC is efficient also when the cholesterol content of cell membrane is reduced.

Mentions: Herein, we show that the inhibition of HMG-CoA reductase with fluvastatin, leading to the block of mevalonate pathway, affects perforin/granzyme release but not FasL and TNFα secretion in human NK cells. Consequently, NK cells can still kill tumor targets through FasL/Fas interaction and/or TNFα-mediated cytotoxicity. Altogether, these findings indicate that interference with mevalonate pathway and cholesterol synthesis can affect some but not all the cellular immune mechanisms involved in the elimination of tumor cells. Fluvastatin inhibits LFA1-dependent RhoA activation, actin redistribution and formation of membrane rafts containing ganglioside M1; this may elucidate the molecular mechanism of the reported inhibiting effect on LFA1-mediated polarization of cytotoxic granules [26]. Indeed, during the recognition phase of target cells LFA1 is localized at the site of contact between NK and target cells and delivers the signal responsible for granule polarization that follows cytoskeleton reorganization; these processes are impaired in fluvastatin-cultured NK cells, so that the lethal hit to target cells is not efficiently delivered (fig. 7). It has been previously shown that only the lipophilic mevastatin, but not the hydrophylic pravastatin, can downregulate perforin-mediated cytolysis [26] and this is accompanied by down-regulation of LFA1 expression [25]; also, it has been shown that LFA1-dependent polarization of perforin containing granules is impaired in mevastatin-treated NK cells [26]. In our hands, lipophilic fluvastatin, but not hydrophilic pravastatin can efficiently reduce cholesterol membrane content in NK cells. This finding would suggest that the inefficacy of pravastatin vs mevastatin and/or fluvastatin depends on the different efficiency of these drugs in blocking HMG-CoA reductase activity. Further, we show that in NK cells fluvastatin can alter actin redistribution and activation of RhoA consequent to LFA1 engagement by its ligand ICAM1, more than the intensity of LFA1 surface expression. This indicates that blocking of mevalonate synthesis leads to the impairment of cytoskeleton assembly needed for the polarization of cytotoxic granules [26] (fig. 7). Thus, our and previous findings consistently point out the relevant role of mevalonate pathway in LFA1-dependent interaction between NK cells and tumor targets.


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)

Scheme of the effects of HMG-CoA reductase inhibitors on NK cell function.Adhesion between NK and target cells triggers an activating signal in NK cells, leading to RhoA activation which favours reorganization of actin microfilaments and granule polarization to NK-target contact (A,B). At the site of contact are formed membrane microdomains (rafts) containing GM1 where activating receptor (i.e. NKG2D)-mediated [Ca2+]i increase and activation of akt leads to the degranulation of perforin (P) and granzymes (G) and consequent lysis of target cells (B). Cholesterol is essential for membrane mobility and correct aggregation of signalling molecules in membrane rafts. Fluvastatin-mediated inhibition of HMG-CoA reductase inhibiting mevalonate synthesis causes a low cholesterol content of cell membrane, a lower mobility and aggregation of activating receptors which cannot allow granule polarization. C: Apparently, both FasL and TNFα release are conserved in fluvastatin-incubated NK cells. This may depends on their low dependency of [Ca2+]i increase and akt activation and on low relevance of contact site–directed secretion. Indeed, when released FasL and TNFα may kill targets if these targets express the corresponding receptors. NK cell activation via FcγRIIIa (CD16), as it can occur with therapeutic antibodies rituximab or trastuzumab, has a quite low threshold of activation and only very high doses of fluvastatin are effective. Thus, ADCC is efficient also when the cholesterol content of cell membrane is reduced.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3646988&req=5

pone-0062932-g007: Scheme of the effects of HMG-CoA reductase inhibitors on NK cell function.Adhesion between NK and target cells triggers an activating signal in NK cells, leading to RhoA activation which favours reorganization of actin microfilaments and granule polarization to NK-target contact (A,B). At the site of contact are formed membrane microdomains (rafts) containing GM1 where activating receptor (i.e. NKG2D)-mediated [Ca2+]i increase and activation of akt leads to the degranulation of perforin (P) and granzymes (G) and consequent lysis of target cells (B). Cholesterol is essential for membrane mobility and correct aggregation of signalling molecules in membrane rafts. Fluvastatin-mediated inhibition of HMG-CoA reductase inhibiting mevalonate synthesis causes a low cholesterol content of cell membrane, a lower mobility and aggregation of activating receptors which cannot allow granule polarization. C: Apparently, both FasL and TNFα release are conserved in fluvastatin-incubated NK cells. This may depends on their low dependency of [Ca2+]i increase and akt activation and on low relevance of contact site–directed secretion. Indeed, when released FasL and TNFα may kill targets if these targets express the corresponding receptors. NK cell activation via FcγRIIIa (CD16), as it can occur with therapeutic antibodies rituximab or trastuzumab, has a quite low threshold of activation and only very high doses of fluvastatin are effective. Thus, ADCC is efficient also when the cholesterol content of cell membrane is reduced.
Mentions: Herein, we show that the inhibition of HMG-CoA reductase with fluvastatin, leading to the block of mevalonate pathway, affects perforin/granzyme release but not FasL and TNFα secretion in human NK cells. Consequently, NK cells can still kill tumor targets through FasL/Fas interaction and/or TNFα-mediated cytotoxicity. Altogether, these findings indicate that interference with mevalonate pathway and cholesterol synthesis can affect some but not all the cellular immune mechanisms involved in the elimination of tumor cells. Fluvastatin inhibits LFA1-dependent RhoA activation, actin redistribution and formation of membrane rafts containing ganglioside M1; this may elucidate the molecular mechanism of the reported inhibiting effect on LFA1-mediated polarization of cytotoxic granules [26]. Indeed, during the recognition phase of target cells LFA1 is localized at the site of contact between NK and target cells and delivers the signal responsible for granule polarization that follows cytoskeleton reorganization; these processes are impaired in fluvastatin-cultured NK cells, so that the lethal hit to target cells is not efficiently delivered (fig. 7). It has been previously shown that only the lipophilic mevastatin, but not the hydrophylic pravastatin, can downregulate perforin-mediated cytolysis [26] and this is accompanied by down-regulation of LFA1 expression [25]; also, it has been shown that LFA1-dependent polarization of perforin containing granules is impaired in mevastatin-treated NK cells [26]. In our hands, lipophilic fluvastatin, but not hydrophilic pravastatin can efficiently reduce cholesterol membrane content in NK cells. This finding would suggest that the inefficacy of pravastatin vs mevastatin and/or fluvastatin depends on the different efficiency of these drugs in blocking HMG-CoA reductase activity. Further, we show that in NK cells fluvastatin can alter actin redistribution and activation of RhoA consequent to LFA1 engagement by its ligand ICAM1, more than the intensity of LFA1 surface expression. This indicates that blocking of mevalonate synthesis leads to the impairment of cytoskeleton assembly needed for the polarization of cytotoxic granules [26] (fig. 7). Thus, our and previous findings consistently point out the relevant role of mevalonate pathway in LFA1-dependent interaction between NK cells and tumor targets.

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