title: 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.
authors.affiliation: Geisel School of Medicine at Dartmouth, Department of Anesthesiology, Lebanon, NH, USA; Thayer School of Engineering at Dartmouth, Hanover, NH, USA.
authors.forename: Christian
authors.initials: C
authors.lastname: Ndong
authors.affiliation: Geisel School of Medicine at Dartmouth, Department of Anesthesiology, Lebanon, NH, USA.
authors.forename: Russell P
authors.initials: RP
authors.lastname: Landry
authors.affiliation: Geisel School of Medicine at Dartmouth, Department of Anesthesiology, Lebanon, NH, USA.
authors.forename: Madhurima
authors.initials: M
authors.lastname: Saha
authors.affiliation: Geisel School of Medicine at Dartmouth, Department of Anesthesiology, Lebanon, NH, USA; Geisel School of Medicine at Dartmouth, Department of Pharmacology/Toxicology, Hanover, NH, USA; Presbyterian College School of Pharmacy, Department of Pharmaceuticals and Administrative Science, Clinton, SC, USA. Electronic address: asandoval@presby.edu.
authors.forename: E Alfonso
authors.initials: EA
authors.lastname: Romero-Sandoval
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000378:metabolism*; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000166:cytology; D054642:Dual Specificity Phosphatase 6 / Q000235:genetics / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D055785:Gene Knockdown Techniques; D048031:JNK Mitogen-Activated Protein Kinases / Q000378:metabolism*; D008070:Lipopolysaccharides / Q000494:pharmacology; D017628:Microglia / Q000187:drug effects / Q000378:metabolism; D010766:Phosphorylation; D017207:Rats, Sprague-Dawley; D013379:Substrate Specificity
chemical_list: D008070:Lipopolysaccharides; D048049:Extracellular Signal-Regulated MAP Kinases; D048031:JNK Mitogen-Activated Protein Kinases; D054642:Dual Specificity Phosphatase 6; C517037:Dusp6 protein, rat
keywords: Astrocytes; Cytokines; Glia; MKP-3; Microglia; Phosphatase