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Direct modification of the proinflammatory cytokine macrophage migration inhibitory factor by dietary isothiocyanates.

ABSTRACT: Isothiocyanates are a class of phytochemicals with widely reported anti-cancer and anti-inflammatory activity. However, knowledge of their activity at a molecular level is limited. The objective of this study was to identify biological targets of phenethyl isothiocyanate (PEITC) using an affinity purification approach. An analogue of PEITC was synthesized to enable conjugation to a solid-phase resin. The pleiotropic cytokine macrophage migration inhibitory factor (MIF) was the major protein captured from cell lysates. Site-directed mutagenesis and mass spectrometry showed that PEITC covalently modified the N-terminal proline residue of MIF. This resulted in complete loss of catalytic tautomerase activity and disruption of protein conformation, as determined by impaired recognition by a monoclonal antibody directed to the region that receptors and interacting proteins bind to MIF. The conformational change was supported by in silico modeling. Monoclonal antibody binding to plasma MIF was disrupted in humans consuming watercress, a major dietary source of PEITC. The isothiocyanates have significant potential for development as MIF inhibitors, and this activity may contribute to the biological properties of these phytochemicals.

SUBMITTER: Brown KK

PROVIDER: S-EPMC2781657 | biostudies-literature | 2009 Nov

REPOSITORIES: biostudies-literature

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Direct modification of the proinflammatory cytokine macrophage migration inhibitory factor by dietary isothiocyanates.

Brown Kristin K KK Blaikie Frances H FH Smith Robin A J RA Tyndall Joel D A JD Lue Hongqi H Bernhagen Jürgen J Winterbourn Christine C CC Hampton Mark B MB

The Journal of biological chemistry 20090923 47

Isothiocyanates are a class of phytochemicals with widely reported anti-cancer and anti-inflammatory activity. However, knowledge of their activity at a molecular level is limited. The objective of this study was to identify biological targets of phenethyl isothiocyanate (PEITC) using an affinity purification approach. An analogue of PEITC was synthesized to enable conjugation to a solid-phase resin. The pleiotropic cytokine macrophage migration inhibitory factor (MIF) was the major protein capt ...[more]

PMID: 19776019

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Project description:BackgroundThe danger-associated molecular patterns (DAMPs) are critical contributors to the progressive neuropathology and thereafter affect the functional outcomes following spinal cord injury (SCI). Up to now, the regulatory mechanisms on their inducible production from the living cells remain elusive, aside from their passive release from the necrotic cells. Thrombin is immediately activated by the damaged or stressed central nervous system (CNS), which potently mediates inflammatory astrocytic responses through proteolytic cleavage of protease-activated receptors (PARs). Therefore, SCI-activated thrombin is conceived to induce the production of DAMPs from astrocytes at lesion site.MethodsRat SCI model was established by the cord contusion at T8-T10. The expression of thrombin and macrophage migration inhibitory factor (MIF) was determined by ELISA and Western blot. The PAR1, PAR3, and PAR4 receptors of thrombin were examined by PCR and immunohistochemistry. Primary astrocytes were isolated and purified from the spinal cord, followed by stimulation with different concentrations of thrombin either for transcriptome sequencing or for analysis of thrombin-mediated expression of MIF and related signal pathways in the presence or absence of various inhibitors. The post-injury locomotor functions were assessed using the Basso, Beattie, and Bresnahan (BBB) locomotor scale.ResultsMIF protein levels were significantly elevated in parallel with those of thrombin induced by SCI. Immunostaining demonstrated that PAR1 receptor, together with MIF, was abundantly expressed in astrocytes. By transcriptome sequencing and bioinformatical analysis of thrombin-stimulated primary astrocytes, MIF was identified to be dynamically regulated by the serine protease. Investigation of the underlying mechanism using various inhibitors revealed that thrombin-activated PAR1 was responsible for the MIF production of astrocytes through modulation of JNK/NFκB pathway. Administration of PAR1 inhibitor at lesion sites following SCI significantly reduced the protein levels of MIF and ameliorated functional deficits of rat locomotion.ConclusionSCI-activated thrombin is a robust inducer of MIF production from astrocytes. Exploring the roles of thrombin in promoting the production of DAMPs from astrocytes at lesion site will provide an alternative strategy for the clinical therapy of CNS inflammation.

Project description:Autistic spectrum disorders are childhood neurodevelopmental disorders characterized by social and communicative impairment and repetitive and stereotypical behavior. Macrophage migration inhibitory factor (MIF) is an upstream regulator of innate immunity that promotes monocyte/macrophage-activation responses by increasing the expression of Toll-like receptors and inhibiting activation-induced apoptosis. On the basis of results of previous genetic linkage studies and reported altered innate immune response in autism spectrum disorder, we hypothesized that MIF could represent a candidate gene for autism spectrum disorder or its diagnostic components.Genetic association between autism spectrum disorder and MIF was investigated in 2 independent sets of families of probands with autism spectrum disorder, from the United States (527 participants from 152 families) and Holland (532 participants from 183 families). Probands and their siblings, when available, were evaluated with clinical instruments used for autism spectrum disorder diagnoses. Genotyping was performed for 2 polymorphisms in the promoter region of the MIF gene in both samples sequentially. In addition, MIF plasma analyses were conducted in a subset of Dutch patients from whom plasma was available.There were genetic associations between known functional polymorphisms in the promoter for MIF and autism spectrum disorder-related behaviors. Also, probands with autism spectrum disorder exhibited higher circulating MIF levels than did their unaffected siblings, and plasma MIF concentrations correlated with the severity of multiple autism spectrum disorder symptoms.These results identify MIF as a possible susceptibility gene for autism spectrum disorder. Additional research is warranted on the precise relationship between MIF and the behavioral components of autism spectrum disorder, the mechanism by which MIF contributes to autism spectrum disorder pathogenesis, and the clinical use of MIF genotyping.

Dataset Information

Direct modification of the proinflammatory cytokine macrophage migration inhibitory factor by dietary isothiocyanates.

Publications

Direct modification of the proinflammatory cytokine macrophage migration inhibitory factor by dietary isothiocyanates.

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