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Lovastatin blocks Kv1.3 channel in human T cells: a new mechanism to explain its immunomodulatory properties.

Zhao N, Dong Q, Qian C, Li S, Wu QF, Ding D, Li J, Wang BB, Guo KF, Xie JJ, Cheng X, Liao YH, Du YM - Sci Rep (2015)

Bottom Line: However, 30 μM Lovastatin had no apparent effect on KCa current in human T cells.At last, Mevalonate application only partially reversed the inhibition of Lovastatin on IL-2 secretion, and the siRNA against Kv1.3 also partially reduced this inhibitory effect of Lovastatin.In conclusion, Lovastatin can exert immunodulatory properties through the new mechanism of blocking Kv1.3 channel.

View Article: PubMed Central - PubMed

Affiliation: Research Center of Ion Channelopathy, Institute of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.

ABSTRACT
Lovastatin is a member of Statins, which are beneficial in a lot of immunologic cardiovascular diseases and T cell-mediated autoimmune diseases. Kv1.3 channel plays important roles in the activation and proliferation of T cells, and have become attractive target for immune-related disorders. The present study was designed to examine the block effect of Lovastatin on Kv1.3 channel in human T cells, and to clarify its new immunomodulatory mechanism. We found that Lovastatin inhibited Kv1.3 currents in a concentration- and voltage-dependent manner, and the IC50 for peak, end of the pulse was 39.81 ± 5.11, 6.92 ± 0.95 μM, respectively. Lovastatin also accelerated the decay rate of current inactivation and negatively shifted the steady-state inactivation curves concentration-dependently, without affecting the activation curve. However, 30 μM Lovastatin had no apparent effect on KCa current in human T cells. Furthermore, Lovastatin inhibited Ca(2+) influx, T cell proliferation as well as IL-2 production. The activities of NFAT1 and NF-κB p65/50 were down-regulated by Lovastatin, too. At last, Mevalonate application only partially reversed the inhibition of Lovastatin on IL-2 secretion, and the siRNA against Kv1.3 also partially reduced this inhibitory effect of Lovastatin. In conclusion, Lovastatin can exert immunodulatory properties through the new mechanism of blocking Kv1.3 channel.

No MeSH data available.


Related in: MedlinePlus

Effect of Lovastatin on T cell proliferation and IL-2 production.(A) PBTCs were seeded and pre-incubated with 0, 3, 10, 30, or 100 μM Lovastatin. After 30 min, anti-CD3/CD28 antibodies were added to induce PBTCs proliferation. After 3 days, the relative cell number was determined by CCK-8 kit. The summarized data from at least 5 duplicates was expressed as mean ± SEM. **P < 0.01 vs. control, #P < 0.05, ##P < 0.01 vs. CD3/CD28 stimulated group without Lovastatin. (B) Jurkat cells were pre-treated with 0, 3, 10, 30, or 100 μM Lovastatin for 30 min, then stimulated with PHA + PMA for 24 h. The supernatants were collected for IL-2 measurement. **P < 0.01 vs. control, #P < 0.05, ##P < 0.01 vs. PHA + PMA stimulated group. (C) Jurkat cells were treated using 100 μM Lovastatin with or without 1 mM Mevalonate application. Then, the inhibition% of Lovastatin on IL-2 secretion was calculated and showed. (D) Con Jurkat cells and NC- or Kv1.3-siRNA-transfected cells were pre-treated with 0 or 100 μM Lovastatin for 30 min, then stimulated with PHA + PMA for 24 h. The supernatants were collected for the measurement of IL-2. **P < 0.01 vs. NC-siRNA group and N.S. represented no statistical significance. (E) Kv1.3-siRNA-transfected Jurkat cells were pre-treated with 0, 10, 30, or 100 μM Lovastatin for 30 min, then stimulated with PHA + PMA for 24h. The supernatants were collected for the measurement of IL-2. (#P < 0.05, ##P < 0.01 vs. PHA + PMA stimulated group).
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f8: Effect of Lovastatin on T cell proliferation and IL-2 production.(A) PBTCs were seeded and pre-incubated with 0, 3, 10, 30, or 100 μM Lovastatin. After 30 min, anti-CD3/CD28 antibodies were added to induce PBTCs proliferation. After 3 days, the relative cell number was determined by CCK-8 kit. The summarized data from at least 5 duplicates was expressed as mean ± SEM. **P < 0.01 vs. control, #P < 0.05, ##P < 0.01 vs. CD3/CD28 stimulated group without Lovastatin. (B) Jurkat cells were pre-treated with 0, 3, 10, 30, or 100 μM Lovastatin for 30 min, then stimulated with PHA + PMA for 24 h. The supernatants were collected for IL-2 measurement. **P < 0.01 vs. control, #P < 0.05, ##P < 0.01 vs. PHA + PMA stimulated group. (C) Jurkat cells were treated using 100 μM Lovastatin with or without 1 mM Mevalonate application. Then, the inhibition% of Lovastatin on IL-2 secretion was calculated and showed. (D) Con Jurkat cells and NC- or Kv1.3-siRNA-transfected cells were pre-treated with 0 or 100 μM Lovastatin for 30 min, then stimulated with PHA + PMA for 24 h. The supernatants were collected for the measurement of IL-2. **P < 0.01 vs. NC-siRNA group and N.S. represented no statistical significance. (E) Kv1.3-siRNA-transfected Jurkat cells were pre-treated with 0, 10, 30, or 100 μM Lovastatin for 30 min, then stimulated with PHA + PMA for 24h. The supernatants were collected for the measurement of IL-2. (#P < 0.05, ##P < 0.01 vs. PHA + PMA stimulated group).

Mentions: To investigate the functional immunosuppression effect performed by Lovastatin through inhibiting the Kv1.3 channel, we measured T cell proliferation and IL-2 production. CCK-8 kit was used to access the proliferation of anti CD3/CD28-stimulated human PBTCs, and IL-2 secretion from PHA + PMA activated Jurkat cell was measured by ELISA. In Fig. 8A,B, 3, 10, and 30 μM Lovastatin inhibited the T cell proliferation and IL-2 production concentration-dependently. Since Statins can exert immunomodulatory effect by blocking the isoprenoid pathway13, we applied 1 mM Mevalonate (the intermediate of isoprenoid pathway) along with 30 μM Lovastatin. We found that the blocking effect of Lovastatin on IL-2 secretion was partially neutralized, from 68.57% to 39.11%, which suggested that Lovastatin probably play immunomodulatory effect only partly through isoprenoid pathway. Furthermore, we knocked down Kv1.3 expression using specific Kv1.3-siRNA in Jurkat cells. The Kv1.3-siRNA transfection resulted in reduced inhibitory effect of 30 μM Lovastatin on IL-2 production (34.27 ± 4.74% vs. NC-siRNA 65.94 ± 0.80%, P < 0.01). Simultaneously, NC-siRNA transfection did not alter the block effect of Lovastatin on IL-2 secretion (65.94 ± 0.80% vs. Jurkat cell 68.57 ± 2.20%, P > 0.05). As shown in Fig. 8E, Lovastatin also concentration-dependently reduced IL-2 secretion in Kv1.3 knockdown Jurkat cells. In conclusion, Lovastatin exerted immunomodulatory effect partly through Kv1.3 channel.


Lovastatin blocks Kv1.3 channel in human T cells: a new mechanism to explain its immunomodulatory properties.

Zhao N, Dong Q, Qian C, Li S, Wu QF, Ding D, Li J, Wang BB, Guo KF, Xie JJ, Cheng X, Liao YH, Du YM - Sci Rep (2015)

Effect of Lovastatin on T cell proliferation and IL-2 production.(A) PBTCs were seeded and pre-incubated with 0, 3, 10, 30, or 100 μM Lovastatin. After 30 min, anti-CD3/CD28 antibodies were added to induce PBTCs proliferation. After 3 days, the relative cell number was determined by CCK-8 kit. The summarized data from at least 5 duplicates was expressed as mean ± SEM. **P < 0.01 vs. control, #P < 0.05, ##P < 0.01 vs. CD3/CD28 stimulated group without Lovastatin. (B) Jurkat cells were pre-treated with 0, 3, 10, 30, or 100 μM Lovastatin for 30 min, then stimulated with PHA + PMA for 24 h. The supernatants were collected for IL-2 measurement. **P < 0.01 vs. control, #P < 0.05, ##P < 0.01 vs. PHA + PMA stimulated group. (C) Jurkat cells were treated using 100 μM Lovastatin with or without 1 mM Mevalonate application. Then, the inhibition% of Lovastatin on IL-2 secretion was calculated and showed. (D) Con Jurkat cells and NC- or Kv1.3-siRNA-transfected cells were pre-treated with 0 or 100 μM Lovastatin for 30 min, then stimulated with PHA + PMA for 24 h. The supernatants were collected for the measurement of IL-2. **P < 0.01 vs. NC-siRNA group and N.S. represented no statistical significance. (E) Kv1.3-siRNA-transfected Jurkat cells were pre-treated with 0, 10, 30, or 100 μM Lovastatin for 30 min, then stimulated with PHA + PMA for 24h. The supernatants were collected for the measurement of IL-2. (#P < 0.05, ##P < 0.01 vs. PHA + PMA stimulated group).
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f8: Effect of Lovastatin on T cell proliferation and IL-2 production.(A) PBTCs were seeded and pre-incubated with 0, 3, 10, 30, or 100 μM Lovastatin. After 30 min, anti-CD3/CD28 antibodies were added to induce PBTCs proliferation. After 3 days, the relative cell number was determined by CCK-8 kit. The summarized data from at least 5 duplicates was expressed as mean ± SEM. **P < 0.01 vs. control, #P < 0.05, ##P < 0.01 vs. CD3/CD28 stimulated group without Lovastatin. (B) Jurkat cells were pre-treated with 0, 3, 10, 30, or 100 μM Lovastatin for 30 min, then stimulated with PHA + PMA for 24 h. The supernatants were collected for IL-2 measurement. **P < 0.01 vs. control, #P < 0.05, ##P < 0.01 vs. PHA + PMA stimulated group. (C) Jurkat cells were treated using 100 μM Lovastatin with or without 1 mM Mevalonate application. Then, the inhibition% of Lovastatin on IL-2 secretion was calculated and showed. (D) Con Jurkat cells and NC- or Kv1.3-siRNA-transfected cells were pre-treated with 0 or 100 μM Lovastatin for 30 min, then stimulated with PHA + PMA for 24 h. The supernatants were collected for the measurement of IL-2. **P < 0.01 vs. NC-siRNA group and N.S. represented no statistical significance. (E) Kv1.3-siRNA-transfected Jurkat cells were pre-treated with 0, 10, 30, or 100 μM Lovastatin for 30 min, then stimulated with PHA + PMA for 24h. The supernatants were collected for the measurement of IL-2. (#P < 0.05, ##P < 0.01 vs. PHA + PMA stimulated group).
Mentions: To investigate the functional immunosuppression effect performed by Lovastatin through inhibiting the Kv1.3 channel, we measured T cell proliferation and IL-2 production. CCK-8 kit was used to access the proliferation of anti CD3/CD28-stimulated human PBTCs, and IL-2 secretion from PHA + PMA activated Jurkat cell was measured by ELISA. In Fig. 8A,B, 3, 10, and 30 μM Lovastatin inhibited the T cell proliferation and IL-2 production concentration-dependently. Since Statins can exert immunomodulatory effect by blocking the isoprenoid pathway13, we applied 1 mM Mevalonate (the intermediate of isoprenoid pathway) along with 30 μM Lovastatin. We found that the blocking effect of Lovastatin on IL-2 secretion was partially neutralized, from 68.57% to 39.11%, which suggested that Lovastatin probably play immunomodulatory effect only partly through isoprenoid pathway. Furthermore, we knocked down Kv1.3 expression using specific Kv1.3-siRNA in Jurkat cells. The Kv1.3-siRNA transfection resulted in reduced inhibitory effect of 30 μM Lovastatin on IL-2 production (34.27 ± 4.74% vs. NC-siRNA 65.94 ± 0.80%, P < 0.01). Simultaneously, NC-siRNA transfection did not alter the block effect of Lovastatin on IL-2 secretion (65.94 ± 0.80% vs. Jurkat cell 68.57 ± 2.20%, P > 0.05). As shown in Fig. 8E, Lovastatin also concentration-dependently reduced IL-2 secretion in Kv1.3 knockdown Jurkat cells. In conclusion, Lovastatin exerted immunomodulatory effect partly through Kv1.3 channel.

Bottom Line: However, 30 μM Lovastatin had no apparent effect on KCa current in human T cells.At last, Mevalonate application only partially reversed the inhibition of Lovastatin on IL-2 secretion, and the siRNA against Kv1.3 also partially reduced this inhibitory effect of Lovastatin.In conclusion, Lovastatin can exert immunodulatory properties through the new mechanism of blocking Kv1.3 channel.

View Article: PubMed Central - PubMed

Affiliation: Research Center of Ion Channelopathy, Institute of Cardiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan 430022, China.

ABSTRACT
Lovastatin is a member of Statins, which are beneficial in a lot of immunologic cardiovascular diseases and T cell-mediated autoimmune diseases. Kv1.3 channel plays important roles in the activation and proliferation of T cells, and have become attractive target for immune-related disorders. The present study was designed to examine the block effect of Lovastatin on Kv1.3 channel in human T cells, and to clarify its new immunomodulatory mechanism. We found that Lovastatin inhibited Kv1.3 currents in a concentration- and voltage-dependent manner, and the IC50 for peak, end of the pulse was 39.81 ± 5.11, 6.92 ± 0.95 μM, respectively. Lovastatin also accelerated the decay rate of current inactivation and negatively shifted the steady-state inactivation curves concentration-dependently, without affecting the activation curve. However, 30 μM Lovastatin had no apparent effect on KCa current in human T cells. Furthermore, Lovastatin inhibited Ca(2+) influx, T cell proliferation as well as IL-2 production. The activities of NFAT1 and NF-κB p65/50 were down-regulated by Lovastatin, too. At last, Mevalonate application only partially reversed the inhibition of Lovastatin on IL-2 secretion, and the siRNA against Kv1.3 also partially reduced this inhibitory effect of Lovastatin. In conclusion, Lovastatin can exert immunodulatory properties through the new mechanism of blocking Kv1.3 channel.

No MeSH data available.


Related in: MedlinePlus