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. 2009 May 28:5:25.
doi: 10.1186/1744-8069-5-25.

Cannabinoid receptor type 2 activation induces a microglial anti-inflammatory phenotype and reduces migration via MKP induction and ERK dephosphorylation

Edgar Alfonso Romero-Sandoval  1 , Ryan Horvath, Russell P Landry, Joyce A DeLeo
Affiliations

Cannabinoid receptor type 2 activation induces a microglial anti-inflammatory phenotype and reduces migration via MKP induction and ERK dephosphorylation

Edgar Alfonso Romero-Sandoval et al. Mol Pain. .

Abstract

Background: Cannabinoid receptor type 2 (CBR2) inhibits microglial reactivity through a molecular mechanism yet to be elucidated. We hypothesized that CBR2 activation induces an anti-inflammatory phenotype in microglia by inhibiting extracellular signal-regulated kinase (ERK) pathway, via mitogen-activated protein kinase-phosphatase (MKP) induction. MKPs regulate mitogen activated protein kinases, but their role in the modulation of microglial phenotype is not fully understood.

Results: JWH015 (a CBR2 agonist) increased MKP-1 and MKP-3 expression, which in turn reduced p-ERK1/2 in LPS-stimulated primary microglia. These effects resulted in a significant reduction of tumor necrosis factor-alpha (TNF) expression and microglial migration. We confirmed the causative link of these findings by using MKP inhibitors. We found that the selective inhibition of MKP-1 by Ro-31-8220 and PSI2106, did not affect p-ERK expression in LPS+JWH015-treated microglia. However, the inhibition of both MKP-1 and MKP-3 by triptolide induced an increase in p-ERK expression and in microglial migration using LPS+JWH015-treated microglia.

Conclusion: Our results uncover a cellular microglial pathway triggered by CBR2 activation. These data suggest that the reduction of pro-inflammatory factors and microglial migration via MKP-3 induction is part of the mechanism of action of CBR2 agonists. These findings may have clinical implications for further drug development.

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Figures

Figure 1
Figure 1
Effects of microglial CBR2 activation on the MKP-1/3 pathway. Representative western blots and quantification of MKP-1 (A) and MKP-3 expression (B) at different incubation time points (15–60 min, 0 = medium control group) in LPS-stimulated microglia in the presence or absence of JWH015 (1 μM). JWH015 produced a MKP-1 and MKP-3 increase (15 min). +p < 0.05 vs. medium control group; *p < 0.05 vs. LPS alone group.
Figure 2
Figure 2
Effects of microglial CBR2 activation on the ERK/TNF pathway. Representative western blots (A) and quantification of p-ERK1 (B), p-ERK2 (C) and TNF (D, 34 kD; E, 17 kD) expression at different incubation time points (15–60 min, 0 = medium control group) in LPS-stimulated microglia in the presence or absence of JWH015 (1 μM). JWH015 produced a ERK dephosphorylation (30 min) and TNF reduction (60 min). +p < 0.05 vs. medium control group; *p < 0.05 vs. LPS alone group.
Figure 3
Figure 3
Blockade of JWH015's effects on MKP-1 and MKP-3. (A) Representative western blots of MKP-1 and beta-actin in primary microglia treated with LPS in the absence or presence of JWH015 (15 min incubation, JWH). Cells were pre-incubated with or without 10 μM PSI2106 (PS), Ro-31-8220 (Ro) or triptolide (Trip) for 30 min. (B) Triptolide (10 μM) completely blocked the MKP-1 expression induced by JWH015 (1 μM). (C) PSI2106 or Ro-31-8220 partially inhibited the MKP-1 expression induced by JWH015. (D) Representative western blots of MKP-3 and beta-actin in primary microglia treated with LPS in the absence of presence of JWH015 (15 min incubation). Cells were pre-incubated with or without PSI2106, Ro-31-8220 or triptolide (10 μM) for 30 min. (E) Triptolide (10 μM) completely blocked the MKP-3 expression induced by JWH015 (1 μM). (F) PSI2106 or Ro-31-8220 did not modify the MKP-3 expression induced by JWH015. *p < 0.05 vs. LPS + medium (MED) or LPS + JWH015 + PSI2106, Ro-31-8220 or triptolide group (indicated by the connecting lines).
Figure 4
Figure 4
Effects of MKP-1 and/or MKP-3 inhibition on p-ERK1/2. (A) Representative western blots of p-ERK1/2 and beta-actin in primary microglia treated with LPS and JWH015 (15 min incubation, JWH). Cells were pre-incubated with or without 10 μM PSI2106 (PS), Ro-31-8220 (Ro) or triptolide (Trip) for 30 min. (B) Triptolide increased the expression of p-ERK1/2 in parallel with the inhibition of both MKP-1 and MKP-3. C. PSI2106 or Ro-31-8220 did not modify p-ERK1/2 expression. *p < 0.05 vs. LPS + JWH015.
Figure 5
Figure 5
Blockade of CBR2 antagonizes the MKP-3 induction by JWH015. Quantification and representative western blot of MKP-3 expression in LPS-stimulated microglia in the presence or absence of JWH015 (1 μM) with or without the CBR2 antagonist, AM630, or the CBR1 antagonist, AM281. JWH015 induced a significant increase in MKP-3 compared to the LPS alone groups at the 15 min incubation time point. However, AM630 significantly blocked JWH015's effects on MKP-3 expression. JWH015's effects on MKP-3 were not modified by AM281. *p < 0.05 vs. LPS alone and LPS + JWH015 1 μM groups.
Figure 6
Figure 6
Quantification of JWH015-reduced LPS-stimulated microglial migration. (A) JWH015 reduced the number of LPS-stimulated microglia that migrated towards ADP in a dose related fashion. *p < 0.05 vs. medium control group (0) group. (B) Blockade of the effects of JWH015 (1 μM,) on microglial migration by the selective CBR2 antagonist, AM630, but not by the selective CBR1 antagonist, AM281. Data are presented as the number of cells that migrated towards ADP. +p < 0.05 vs. medium group (MED), *p < 0.05 vs. JWH015 1 μM group (JWH) group. (C) Blockade of microglial migration by the selective MEK inhibitor UO126 in LPS-stimulated microglia. UO126 (1 μM), which selectively blocked MEK and thereby ERK phosphorylation, significantly reduced the number of LPS-stimulated microglia that migrated towards ADP. *p < 0.05 vs.medium group (MED) group.
Figure 7
Figure 7
Blockade of the JWH015 effects in microglial migration by MKP-1 and MKP-3 inhibition. (A) JWH015 1 μM (JWH 1) significantly reduced the number of LPS-stimulated microglia that migrated towards ADP and triptolide 10 μM (Trip 10) blocked this effect. Triptolide 10 μM did not modify the number of LPS-stimulated microglia that migrated towards ADP. *p < 0.05 vs. JWH015 group. (B) Representative photomicrographs of the migration chamber membranes with attached microglial cells stained with crystal violet. Cells (black) migrated through the pores (white circles) and towards ADP placed into the lower well.
Figure 8
Figure 8
Proposed hypotheses. (A) Scheme of the proposed hypothesis on the mechanisms by which p-ERK induces a pro-inflammatory phenotype in microglia and contributes to neuropathic pain. Peripheral nerve injury increases microglial p-ERK in spinal cord dorsal horn, which in turn leads to the expression of TNF and an increase in microglial motility. These pro-inflammatory microglia migrate towards the spinal injured neuron area attracted by chemoattractants, such as ATP/ADP. The enhancement of pro-inflammatory microglia sensitizes spinal nociceptive neurons by increasing the concentration of pro-algesic factors, such as TNF. (B) Scheme of the proposed hypothesis on the mechanisms by which CBR2 activation-induced MKP-3 promotes an anti-inflammatory phenotype in microglia and alleviates neuropathic pain. Microglial CBR2 are increased in microglia following peripheral nerve injury. Microglial CBR2 activation induces an anti-inflammatory phenotype in microglia by increasing MKP-3 expression, which selectively inhibits p-ERK in spinal cord dorsal horn. Subsequently, ERK dephosphorylation results in a reduction of TNF expression and microglial motility. The reduction in the migration of pro-inflammatory microglia reduces the source of pro-algesic factors, such as TNF preventing neuronal sensitization and alleviating peripheral nerve injury-induced allodynia.
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