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
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
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 ? and ? 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
107
Green, J.A.
da242068-358d-43be-8351-991a0825f994
Rand, L.
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Moores, R.
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Dholakia, S.
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Pezas, T.
8845063d-4562-4603-9ecc-0b5b339d755a
Elkington, P.T.
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Friedland, J.S.
e64a7af8-b969-4426-82e6-5ebe819799c9
August 2013
Green, J.A.
da242068-358d-43be-8351-991a0825f994
Rand, L.
561f2c9a-d763-4d16-a887-c1eae5d5dbf9
Moores, R.
cc4fe290-6419-4947-bdb0-1ae431b3ba63
Dholakia, S.
c85c39d3-e7a9-46de-88db-e5a9144d18c4
Pezas, T.
8845063d-4562-4603-9ecc-0b5b339d755a
Elkington, P.T.
60828c7c-3d32-47c9-9fcc-6c4c54c35a15
Friedland, J.S.
e64a7af8-b969-4426-82e6-5ebe819799c9
Green, J.A., Rand, L., Moores, R., Dholakia, S., Pezas, T., Elkington, P.T. and Friedland, J.S.
(2013)
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.
Journal of Neuroinflammation, 10, .
(doi:10.1186/1742-2094-10-107).
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 ? and ? 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
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Published date: August 2013
Organisations:
Clinical & Experimental Sciences
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Local EPrints ID: 359642
URI: http://eprints.soton.ac.uk/id/eprint/359642
ISSN: 1742-2094
PURE UUID: d284cdd0-ebb1-4145-9f64-49eb672fc8f1
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Date deposited: 08 Nov 2013 09:38
Last modified: 15 Mar 2024 03:43
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Author:
J.A. Green
Author:
L. Rand
Author:
R. Moores
Author:
S. Dholakia
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T. Pezas
Author:
J.S. Friedland
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