Manganese modulation of MAPK pathways: effects on upstream mitogen activated protein kinase kinases and mitogen activated kinase phosphatase-1 in microglial cells

Abstract

Multiple studies demonstrate that manganese (Mn) exposure potentiates inflammatory mediator output from activated glia; this increased output is associated with enhanced mitogen activated protein kinase (MAPK: p38, ERK and JNK) activity. We hypothesized that Mn activates MAPK by activating the kinases upstream of MAPK, i.e. MKK-3/6, MKK-1/2 and MKK-4 (responsible for activation of p38, ERK, and JNK, respectively), and/or by inhibiting a major phosphatase responsible for MAPK inactivation, MKP-1. Exposure of N9 microglia to Mn (250 µm), LPS (100 ng ml⁻¹) or Mn + LPS increased MKK-3/6 and MKK-4 activity at 1 h; the effect of Mn + LPS on MKK-4 activation was greater than the rest. At 4 h, Mn, LPS, and Mn + LPS increased MKK-3/6 and MKK-1/2 phosphorylation, whereas MKK-4 was activated only by Mn and Mn + LPS. Besides activating MKK-4 via Ser257/Thr261 phosphorylation, Mn (4 h) prevented MKK-4's phosphorylation on Ser80, which negatively regulates MKK-4 activity. Exposure to Mn or Mn + LPS (1 h) decreased both mRNA and protein expression of MKP-1, the negative MAPK regulator. In addition, we observed that at 4 h, but not at 1 h, a time point coinciding with increased MAPK activity, Mn + LPS markedly increased TNF-α, IL-6 and Cox-2 mRNA, suggesting a delayed effect. The fact that all three major groups of MKKs, MKK-1/2, MKK-3/6 and MKK-4, are activated by Mn suggests that Mn-induced activation of MAPK occurs via traditional mechanisms, which perhaps involve the MAPKs furthest upstream, MKKKs (MAP3Ks). In addition, for all MKKs, Mn-induced activation was persistent at least for 4 h, indicating a long-term effect.

Figure 1

Effects of Mn and/or LPS on MKK-3/6 activity. Shown are quantification and representative western blots of phosphorylated MKK-3/6 (pMKK-3/6; Ser 189) and total MKK-3/6 in N9 microglia following exposure to vehicle, 250 μM Mn, 100 ng/ml LPS, or 250 μM Mn + 100 ng/ml LPS for 1 (A) and 4 (B) h. Densitometric data were normalized as a ratio of phosphorylated to total MKK-3/6 protein. All data points represent means ± SEM from at least 3 independent experiments. Data were analyzed with ANOVA and means were separated using Fisher’s LSD multiple comparison post hoc test. ^a Letters denote statistically significant difference from control at p ≤ 0.05.

Figure 2

Effects of Mn and/or LPS on MKK-1/2 activity. Shown are quantification and representative western blots of phosphorylated MKK-1/2 (pMKK-1/2; Ser 218/Ser 222) and total MKK-1/2 in N9 microglia following exposure to vehicle, 250 μM Mn, 100 ng/ml LPS, or 250 μM Mn + 100 ng/ml LPS for 1 (A) and 4 (B) h. Densitometric data were normalized as a ratio of phosphorylated to total MKK-1/2 protein. All data points represent means ± SEM from at least 3 independent experiments. Data were analyzed with ANOVA and means were separated using Fisher’s LSD multiple comparison post hoc test. ^a Letters denote statistically significant difference from control at p ≤ 0.05.

Figure 3

Effects of Mn and/or LPS on MKK-4 activity modulated at Ser 257/Thr 261. Shown are quantification and representative western blots of Ser 257/Thr 261-phosphorylated MKK-4 (pMKK-4; Ser 257/Thr 261) and total MKK-4 in N9 microglia following exposure to vehicle, 250 μM Mn, 100 ng/ml LPS, or 250 μM Mn + 100 ng/ml LPS for 1 (A) and 4 (B) h. Densitometric data were normalized as a ratio of Ser 257/Thr 261-pMKK-4 to total MKK-4 protein. All data points represent means ± SEM from at least 3 independent experiments. Data were analyzed with ANOVA and means were separated using Fisher’s LSD multiple comparison post hoc test. ^{a ,b} Letters denote statistically significant difference from control, with different letters also being different from each other at p ≤ 0.05.

Figure 4

Effects of Mn and/or LPS on MKK-4 activity modulated at Ser 80. Shown are quantification and representative western blots of Ser 80-phosphorylated MKK-4 (pMKK-4; Ser 80) and total MKK-4 in N9 microglia following exposure to vehicle, 250 μM Mn, 100 ng/ml LPS, or 250 μM Mn + 100 ng/ml LPS for 1 (A) and 4 (B) h. Densitometric data were normalized as a ratio of Ser 80-pMKK-4 to total MKK-4 protein. All data points represent means ± SEM from at least 3 independent experiments. Data were analyzed with ANOVA and means were separated using Fisher’s LSD multiple comparison post hoc test. ^a Letters denote statistically significant difference from control at p ≤ 0.05.

Figure 5

Effects of Mn and/or LPS on MKP-1. Shown are representative western blots and quantification of MKP-1 protein (A) and mRNA (B) levels following exposure to vehicle, 250 μM Mn, 100 ng/ml LPS, or 250 μM Mn + 100 ng/ml LPS for 1 h. Densitometric data were normalized as a ratio of MPK-1 to α-tubulin protein. All data points represent means ± SEM from at least 3 (protein) or 4 (mRNA) independent experiments. Data were analyzed with ANOVA and means were separated using Fisher’s LSD multiple comparison post hoc test. ^{a ,b,c} Letters denote statistically significant difference from control, with different letters also being different from each other at p ≤ 0.05.

Figure 6

Fold change (up/down from vehicle control) in TNF-α (A and B), IL-6 (C and D), and Cox-2 (E and F) mRNA expression following exposure to 250 μM Mn, 100 ng/ml LPS, or 250μM Mn + 100 ng/ml LPS for 1 (left-hand graphs) and 4 (right-hand graphs) h. All data points represent means ± SEM from 4 independent experiments. Data were analyzed with ANOVA and means were separated using Fisher’s LSD multiple comparison post hoc test. ^{a ,b} Letters denote statistically significant difference from control, with different letters also being different from each other at p ≤ 0.05.

Figure 7

Simplified MAPK signaling pathway diagram depicting places where Mn has (demonstrated previously and in this study) an effect. In microglial cells, this pathway can be activated by a variety of stimuli, including inflammagens, such as LPS. Arrows beside components of the pathway denote increases or decreases caused by Mn (↑, ↓, respectively). Potential effects of Mn on MAP3K have not been studied yet and are hence marked with a question mark (?). Abbreviations: Mitogen activated protein kinase kinase kinase (MAP3K, i.e., MLK3, TAK, ASK1); mitogen activated protein kinase kinase (MAP2K, i.e. MKK-1/2, MKK-4, MKK-3/6); mitogen activated protein kinase (MAPK, i.e., extracellular regulated protein kinase [ERK], p38, c-Jun N-terminal kinase [JNK]); mitogen activated kinase phosphatase-1 (MKP-1); interleukin-6 (IL-6); tumor necrosis factor-alpha (TNF-α ); cyclooxygenase-2 (Cox-2).