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Simvastatin inhibits T-cell activation by selectively impairing the function of Ras superfamily GTPases

Raffaella Ghittoni^*,, Laura Patrussi^*, Katja Pirozzi^*, Michela Pellegrini^*, Pietro E. Lazzerini, P. Leopoldo Capecchi, Franco Laghi Pasini and Cosima T. Baldari^*,1

^* Department of Evolutionary Biology, University of Siena;
Department of Clinical Medicine and Immunological Sciences, Policlinico Le Scotte, University of Siena, Siena, Italy

¹Correspondence: Department of Evolutionary Biology, Via Aldo Moro 2, Siena 53100, Italy. E-mail: baldari{at}unisi.it

SPECIFIC AIMS

Statins are widely used hypocholesterolemic drugs that inhibit 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, a rate-limiting enzyme of the mevalonate pathway, whose biosynthetic end product is cholesterol. In addition to lowering circulating cholesterol, statins perturb the composition of cell membranes, resulting in disruption of lipid rafts, which function as signaling platforms in immunoreceptor signaling. In addition, by inhibiting protein prenylation, a process also dependent on mevalonate, statins block membrane targeting and hence activity of small GTPases, which control multiple pathways triggered by these receptors. T cell activation is crucially dependent on Ras, Rho, and Rab GTPases. Furthermore, T cell antigen receptor (TCR) signaling is orchestrated at lipid rafts, thus identifying T cells as potential cellular targets of statins. Here we have investigated the effect of simvastatin treatment on the early, lipid raft-dependent signals triggered by the TCR, as well as on the specific pathways controlled by Ras superfamily GTPases.

PRINCIPAL FINDINGS

1. Simvastatin impairs Ras and Rac association with lipid rafts without significantly affecting intracellular cholesterol levels and lipid raft integrity
Because of their capacity to inhibit an intermediate step in cholesterol biosynthesis that also controls protein prenylation, statins can potentially affect two key components in TCR signaling: lipid rafts and Ras/Rho family GTPases. The effect of simvastatin on intracellular cholesterol levels and lipid raft integrity were investigated using Jurkat T cells as a model. No differences in cholesterol were detectable either in cell lysates or in purified lipid rafts when simvastatin treatment was carried out for 24 h or 48 h.

Accordingly, no significant perturbation of lipid rafts was observed, as evaluated from the levels of the raft-associated tyrosine kinases, Lck and Fyn. On the other hand, the levels of Ras and, to a lesser extent, of Rac, detectable in lipid rafts were significantly reduced after simvstatin treatment. This effect was dependent on HMG-CoA reductase inhibition by simvastatin, as it was reversed by addition of mevalonate. Hence, the effects of statins on membrane localization of Ras and Rac can be detected in conditions where intracellular cholesterol levels are unaffected, indicating that protein prenylation is exquisitely sensitive to HMG-CoA inhibition in T cells. This different threshold provides a tool to study the effects of simvastatin on Ras- and Rac-dependent events in the absence of more generalized effects dependent on lipid raft integrity.

2. Simvastatin inhibits TCR-dependent activation of the Ras/MAP kinase pathway
To address the effect on TCR signaling of the decreased lipid raft association of Ras induced by simvastatin, Ras activity was measured. Simvastatin treatment resulted in inhibition of TCR-dependent Ras activation, which was reversed by mevalonate, indicating that it was dependent on inhibition of HMG-CoA reductase (Fig. 1 ).

View larger version (23K): [in this window] [in a new window]   Figure 1. Simvastatin impairs TCR-dependent Ras/MAP kinase activation. A) Immunoblot analysis with anti-Ras mAb of in vitro binding assays of postnuclear supernatants from Jurkat cells, using agarose-conjugated GST-Raf1 (top). Equal amounts of postnuclear supernatants from the same samples were separated on the same gel (bottom). Cells were treated for 24 h with 20 µM simvastatin, in the absence or the presence of 50-200 µM mevalonate, or carrier, and either left unstimulated or activated for 5 min with anti-CD3 mAb. B–D) Immunoblot analysis of postnuclear supernatants from Jurkat cells treated and activated as above.

Activation of Ras results in the initiation of a serine-threonine cascade involving sequentially Raf, MEK, and Erk. In agreement with the block of Ras activation, activation of Raf, MEK, and Erk was dramatically inhibited by simvastatin; this effect was reversed by mevalonate (Fig. 1) . Similar results were obtained on human peripheral blood lymphocytes (PBL). Flow cytometric analysis showed that surface expression of the early activation marker CD69, which is induced by TCR engagement and is dependent on Ras, was significantly reduced after simvastatin treatment in Jurkat T cells. Inhibition of TCR-dependent expression of CD69, as well as of CD25, the high affinity IL-2 receptor, was also observed in simvastatin treated PBL. These effects were reversed by mevalonate. In agreement with this finding, peripheral T-lymphocyte proliferation induced either by CD3 aggregation, or by CD3/CD28 costimulation, was dramatically suppressed.

3. Simvastatin selectively affects Ras activity by inhibiting its prenylation in conditions that do not impair tyrosine kinase-dependent TCR signaling
Activation of Ras is the endpoint of a protein tyrosine phosphorylation cascade initiated by TCR engagement. The effect of simvastatin on the activation of tyrosine kinase-dependent events upstream of Ras was investigated. Neither phosphorylation of CD3, ZAP-70, or the LAT adaptor, which plays a primary role in Ras activation, or TCR-dependent calcium mobilization were impaired in simvastatin-treated cells, ruling out a defect in TCR signaling upstream of Ras. On the other hand, the impairment in TCR-dependent Ras/MAP kinase signaling, as well as its reversal by mevalonate, strongly suggests that simvastatin directly affects Ras function as the result of HMG-CoA reductase inhibition. Mevalonate is indeed the precursor of farnelylpyrophosphate, which is posttranslationally added to Ras, resulting in its localization to the inner leaflet of the plasma membrane. To address this issue, the most downstream event in Ras/MAP kinase signaling, Erk activation, was tested in cells treated with simvastatin in the presence of farnesylpyrophosphate (FPP). The inhibition of TCR-dependent Erk activation by simvastatin was fully reversed by FPP, but not by squalene, supporting the notion that simvastatin inhibits Ras farnesylation, resulting in impaired membrane localization and activity. Indeed, immunoblot analysis of lipid rafts purified from cells treated with simvastatin in the presence of FPP showed full recovery of Ras localization in membrane rafts.

4. Simvastatin inhibits TCR-dependent activation of the stress kinase pathway
In addition to Ras, TCR signaling involves Rho family GTPases. Among these, Rac is critically required for reorganization of cortical actin at the immunological synapse. The effect of simvastatin on TCR-dependent Rac activation was tested. Rac activity was significantly inhibited in simvastatin treated cells. The inhibition was reversed by mevalonate, indicating that simvastatin directly affects Rac function as the result of HMG-CoA reductase inhibition. In agreement with this result, TCR-dependent activation of the stress kinase p38, which is the endpoint of a serine/threonine kinase cascade triggered by Rac/Pak1 complexes, was significantly reduced in cells treated with simvastatin. Complete reversal of the inhibitory effect of simvastatin on p38 activation was achieved by mevalonate as well as geralygeranylpyrophosphate (GGPP), suggesting that the inhibitory effect of simvastatin on p38 activation results from defective geranylgeranylation of Rac and subsequent impairment in its subcellular localization. Immunoblot analysis with anti-Rac antibodies of lipid rafts purified from cells treated with simvastatin in the presence of GGPP showed indeed that GGPP reverted the impairment in membrane localization of Rac. Tyrosine kinase-dependent TCR signaling upstream of Rac, including tyrosine phosphorylation of the Rho family exchange factor Vav, was conversely not affected by simvastatin treatment.

5. Simvastatin inhibits ligand-dependent TCR internalization
TCR internalization is a key element in productive TCR signaling. Receptor endocytosis is regulated by Rab GTPases. Similarly to other small GTP binding proteins, Rab proteins are posttranslationally modified by prenylation, and as such are potential targets of statins. The effect of simvastatin on TCR internalization was addressed. Simvastatin treatment resulted in a significant reduction in receptor endocytosis. This inhibition was fully reversed by mevalonate. Hence simvastatin blocks ligand-dependent receptor endocytosis, an effect likely to result from defective membrane targeting of Rab proteins.

CONCLUSIONS AND SIGNIFICANCE

The immunosuppressive activities of statins are well documented and have potential important clinical relevance; however, the molecular basis of immunosuppression by statins is as yet not fully established. Our data show that simvastatin suppresses T cell activation and proliferation induced by TCR ligation by preventing correct positioning of Ras and Rac at the plasma membrane. This results in a dramatic impairment in the pathways regulated by these small GTPases, including the MAP kinase and stress-activated kinase pathways. Furthermore, TCR internalization, which is controlled by Rab GTPases, is inhibited by simvastatin. The results identify Ras and Ras-like proteins as strategic molecular targets in T cell immunosuppression by statins (Fig. 2 ). The expanding body of data describing the effects of statins on cellular processes, while indicating new potential therapeutic applications, underscores differences not only in the potency, but also in the panel of activities, molecular targets, and mechanism of action of statins. A more complete understanding of the intracellular activities of different statins is essential to fully exploit their therapeutic potential in pathologies as diverse as autoimmune diseases, transplant rejection, and cancer.

View larger version (46K): [in this window] [in a new window]   Figure 2. Schematic of the effects of simvastatin on TCR signaling relevant to this study.

FOOTNOTES

To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.04-2702fje

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