Mitogen-activated protein kinase (MAPK) phosphatase-3 (MKP-3) displays a p-JNK-MAPK substrate preference in astrocytes in vitro

Abstract

Mitogen-activated protein kinases (MAPKs) play critical roles in the central nervous system immune responses through glial function, which are regulated with relative selectivity (or preference) by MAPK phosphatases (MKP). Phosphorylated extracellular signal-regulated protein kinase (p-ERK) is preferentially dephosphorylated by MKP-3, which display little activity over p-p38 and p-c-Jun NH2-terminal kinases (p-JNK). It has been proposed that these substrate preferences may vary depending on tissue or functional cellular processes. Since astrocytes display a prominent activity of JNK>ERK under stressed or reactive phenotype, we hypothesize that MKP-3 possess a similar or differential substrate preference in astrocytes for JNK and ERK (ERK=JNK or JNK>ERK). We generated transient expression of MKP-3 by transfecting a specific cDNA in primary rat neonatal brain cortex astrocytes. Cells were stimulated with lipopolysaccharide (LPS), and MAPKs and downstream pro-inflammatory products were measured by Western blot and ELISA analyses. MKP-3 expression in primary astrocytes reduced LPS-induced p-ERK and p-p38 by ∼50%, and p-JNK by ∼75%, and moderately reduced nitrite oxide (NO), while completely blocked Interleukin (IL)-6 and tumor necrosis factor alpha (TNFα). We confirmed MKP-3 specific activity by developing a BV-2 microglia cell line stably overexpressing MKP-3 and using a specific siRNA against MKP-3. Our data demonstrate MKP-3 has differential substrate preference in astrocytes compared to other cells types, since it preferentially dephosphorylated p-JNK over p-ERK. Our results indicate also that astrocytic immune functions can be modulated by MKP-3 induction, a strategy that could be beneficial in neurological conditions in which astrocytes play a pathophysiological role, i.e. persistent pain.

Keywords: Astrocytes; Cytokines; Glia; MKP-3; Microglia; Phosphatase.

Fig. 1.

In vitro characterization of MKP-3 and MAPKs in primary astrocytes cells transiently expressing MKP-3. MKP-3 mRNA levels determined by qRT-PCR (A), representative Western blot (B) and quantification of MKP-3 protein (C) in normal primary astrocytes (control) and primary astrocytes transiently overexpressing MKP-3 (pMKP-3). Representative Western blots of p-JNK (D), p-p38 (E), and p-ERK (F), and quantification of p-JNK (G), p-p38 (H), and p-ERK (I) protein in normal primary astrocytes (control) and primary astrocytes transiently overexpressing MKP-3 (pMKP-3), following 1 h incubation in medium (control alone) or lipopolysaccharide (LPS) stimulation (1 μg/ml). Results are expressed as mean ± s.e.m. of three experiments in triplicate. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control primary astrocytes.

Fig. 2.

Overexpression of MKP-3 reduces inflammatory effectors in primary astrocytes cells and reversal effects of MKP-3 mRNA knockdown in BV-2 microglia cells. (A) Nitrite oxide (measured as nitrite), (B) IL-6 and (C) TNFα concentration in cell culture supernatant in control (normal) and transiently expressing MKP-3 primary astrocytes cells challenged with LPS (1 μg/ml) for 12, 24 or 48 h. MKP-3 mRNA levels (D) in BV-2 cells stably expressing MKP-3 after 24 h transfection with MKP-3 siRNA compared to negative control siRNA and vehicle treated cells. Nitrite oxide (measured as nitrite) (E), IL-6 (F), and TNFα (G) cytokines release in control (normal) and stably expressing MKP-3 BV-2 cells transfected 24 h with a negative siRNA or a siRNA against MKP-3 and challenged with LPS (1 μg/ml) for 24 h. Results are expressed as mean ± s.e.m. of three experiments in duplicate. * P < 0.05, ** P < 0.01, *** P < 0.001 vs. control primary astrocytes or BV-2 cells stably expressing MKP-3.