title: In an in vitro model of human tuberculosis, monocyte-microglial networks regulate matrix metalloproteinase-1 and -3 gene expression and secretion via a p38 mitogen activated protein kinase-dependent pathway.
abstract: BACKGROUND
  Tuberculosis (TB) of the central nervous system (CNS) is characterized by extensive tissue inflammation, driven by molecules that cleave extracellular matrix such as matrix metalloproteinase (MMP)-1 and MMP-3. However, relatively little is known about the regulation of these MMPs in the CNS.
  
  
  METHODS
  Using a cellular model of CNS TB, we stimulated a human microglial cell line (CHME3) with conditioned medium from Mycobacterium tuberculosis-infected primary human monocytes (CoMTb). MMP-1 and MMP-3 secretion was detected using ELISAs confirmed with casein zymography or western blotting. Key results of a phospho-array profile that detects a wide range of kinase activity were confirmed with phospho-Western blotting. Chemical inhibition (SB203580) of microglial cells allowed investigation of expression and secretion of MMP-1 and MMP-3. Finally we used promoter reporter assays employing full length and MMP-3 promoter deletion constructs. Student's t-test was used for comparison of continuous variables and multiple intervention experiments were compared by one-way ANOVA with Tukey's correction for multiple pairwise comparisons.
  
  
  RESULTS
  CoMTb up-regulated microglial MMP-1 and MMP-3 secretion in a dose- and time-dependent manner. The phospho-array profiling showed that the major increase in kinase activity due to CoMTb stimulation was in p38 mitogen activated protein kinase (MAPK), principally the α and γ subunits. p38 phosphorylation was detected at 15 minutes, with a second peak of activity at 120 minutes. High basal extracellular signal-regulated kinase activity was further increased by CoMTb. Secretion and expression of MMP-1 and MMP-3 were both p38 dependent. CoMTb stimulation of full length and MMP-3 promoter deletion constructs demonstrated up-regulation of activity in the wild type but a suppression site between -2183 and -1612 bp.
  
  
  CONCLUSIONS
  Monocyte-microglial network-dependent MMP-1 and MMP-3 gene expression and secretion are dependent upon p38 MAPK in tuberculosis. p38 is therefore a potential target for adjuvant therapy in CNS TB.
authors: Section of Infectious Diseases and Immunity and the Imperial College Wellcome Trust Centre for Clinical Tropical Medicine, Hammersmith Campus, Imperial College London, London W12 0NN, UK.
authors: Justin A
authors: JA
authors: Green
authors: Lucinda
authors: L
authors: Rand
authors: Rachel
authors: R
authors: Moores
authors: Shruti
authors: S
authors: Dholakia
authors: Theodore
authors: T
authors: Pezas
authors: Paul T
authors: PT
authors: Elkington
authors: Jon S
authors: JS
authors: Friedland
mesh_terms: D015153:Blotting, Western; D002364:Caseins / Q000378:metabolism; D002478:Cells, Cultured; D017077:Culture Media, Conditioned; D018076:DNA, Complementary / Q000096:biosynthesis / Q000302:isolation & purification; D004797:Enzyme-Linked Immunosorbent Assay; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics / Q000502:physiology*; D006801:Humans; D020781:Matrix Metalloproteinase 1 / Q000096:biosynthesis* / Q000235:genetics*; D019278:Matrix Metalloproteinase 3 / Q000096:biosynthesis* / Q000235:genetics*; D017628:Microglia / Q000502:physiology*; D009000:Monocytes / Q000502:physiology*; D009169:Mycobacterium tuberculosis; D011494:Protein Kinases / Q000502:physiology*; D012313:RNA / Q000096:biosynthesis / Q000302:isolation & purification; D060888:Real-Time Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000096:biosynthesis / Q000235:genetics; D019716:Tissue Inhibitor of Metalloproteinase-2 / Q000096:biosynthesis / Q000235:genetics; D019714:Tissue Inhibitor of Metalloproteinases / Q000378:metabolism; D014376:Tuberculosis / Q000201:enzymology* / Q000235:genetics*; D048051:p38 Mitogen-Activated Protein Kinases / Q000502:physiology*
chemical_list: D002364:Caseins; D017077:Culture Media, Conditioned; D018076:DNA, Complementary; D019715:Tissue Inhibitor of Metalloproteinase-1; D019714:Tissue Inhibitor of Metalloproteinases; D019716:Tissue Inhibitor of Metalloproteinase-2; D012313:RNA; D011494:Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D019278:Matrix Metalloproteinase 3; D020781:Matrix Metalloproteinase 1
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