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Overexpression of heat shock protein 72 attenuates NF-?B activation using a combination of regulatory mechanisms in microglia.

ABSTRACT: Overexpression of the inducible heat shock protein 70, Hsp72, has broadly cytoprotective effects and improves outcome following stroke. A full understanding of how Hsp72 protects cells against injury is elusive, though several distinct mechanisms are implicated. One mechanism is its anti-inflammatory effects. We study the effects of Hsp72 overexpression on activation of the transcription factor NF-?B in microglia combining experimentation and mathematical modeling, using TNF? to stimulate a microglial cell line stably overexpressing Hsp72. We find that Hsp72 overexpression reduces the amount of NF-?B DNA binding activity, activity of the upstream kinase IKK, and amount of I?B? inhibitor phosphorylated following TNF? application. Simulations evaluating several proposed mechanisms suggest that inhibition of IKK activation is an essential component of its regulatory activities. Unexpectedly we find that Hsp72 overexpression reduces the initial amount of the RelA/p65 NF-?B subunit in cells, contributing to the attenuated response. Neither mechanism in isolation, however, is sufficient to attenuate the response, providing evidence that Hsp72 relies upon multiple mechanisms to attenuate NF-?B activation. An additional observation from our study is that the induced expression of I?B? is altered significantly in Hsp72 expressing cells. While the mechanism responsible for this observation is not known, it points to yet another means by which Hsp72 may alter the NF-?B response. This study illustrates the multi-faceted nature of Hsp72 regulation of NF-?B activation in microglia and offers further clues to a novel mechanism by which Hsp72 may protect cells against injury.

SUBMITTER: Sheppard PW

PROVIDER: S-EPMC3916226 | biostudies-other | 2014 Feb

REPOSITORIES: biostudies-other

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Overexpression of heat shock protein 72 attenuates NF-κB activation using a combination of regulatory mechanisms in microglia.

Sheppard Patrick W PW Sun Xiaoyun X Khammash Mustafa M Giffard Rona G RG

PLoS computational biology 20140206 2

Overexpression of the inducible heat shock protein 70, Hsp72, has broadly cytoprotective effects and improves outcome following stroke. A full understanding of how Hsp72 protects cells against injury is elusive, though several distinct mechanisms are implicated. One mechanism is its anti-inflammatory effects. We study the effects of Hsp72 overexpression on activation of the transcription factor NF-κB in microglia combining experimentation and mathematical modeling, using TNFα to stimulate a micr ...[more]

PMID: 24516376

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Project description:Background: Intervention of neuroinflammation in central nervous system (CNS) represents a potential therapeutic strategy for a host of brain disorders. The scorpion Buthus martensii Karsch (BmK) and its venom have long been used in the Orient to treat inflammation-related diseases such as rhumatoid arthritis and chronic pain. Scorpion venom heat-resistant peptide (SVHRP), a component from BmK venom, has been shown to reduce seizure susceptibility in a rat epileptic model and protect against cerebral ischemia-reperfusion injury. As neuroinflammation has been implicated in chronic neuronal hyperexcitability, epileptogenesis and cerebral ischemia-reperfusion injury, the present study aimed to investigate whether SVHRP has anti-inflammatory property in brain. Methods: An animal model of neuroinflammation induced by lipopolysacchride (LPS) injection was employed to investigate the effect of SVHRP (125 µg/kg, intraperitoneal injection) on inflammagen-induced expression of pro-inflammatory factors and microglia activation. The effect of SVHRP (2-20 μg/ml) on neuroinflammation was further investigated in primary brain cell cultures containing microglia as well as the immortalized BV2 microglia culture stimulated with LPS. Real-time quantitative PCR were used to measure mRNA levels of inducible nitric oxide synthase (iNOS), tumor necrosis factor-α (TNF-α), interleukin (IL)-1β and IL-6 in hippocampus of animals. Protein levels of TNF-α, iNOS, P65 subunit of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs) were examined by ELISA or western blot. Microglia morphology in animal hippocampus or cell cultures and cellular distribution of p65 were shown by immunostaining. Results: Morphological study demonstrated that activation of microglia, the main component that mediates the neuroinflammatory process, was inhibited by SVHRP in both LPS mouse and cellular model. Our results also showed dramatic increases in the expression of iNOS and TNF-α in hippocampus of LPS-injected mice, which was significantly attenuated by SVHRP treatment. In vitro results showed that SVHRP attenuated LPS-elicited expression of iNOS and TNF-α in different cultures without cell toxicity, which might be attributed to suppression of NF-κB and MAPK pathways by SVHRP. Conclusion: Our study demonstrates that SVHRP is able to inhibit neuroinflammation and microglia activation, which may underlie the therapeutic effects of BmK-derived materials, suggesting that BmK venom could be a potential source for CNS drug development.

Project description:Elevated temperature induces the heat shock (HS) response, which modulates cell proliferation, apoptosis, the immune and inflammatory responses. However, specific mechanisms linking the HS response pathways to major cellular signaling systems are not fully understood. Here we used integrated computational and experimental approaches to quantitatively analyze the crosstalk mechanisms between the HS-response and a master regulator of inflammation, cell proliferation, and apoptosis the Nuclear Factor ?B (NF-?B) system. We found that populations of human osteosarcoma cells, exposed to a clinically relevant 43°C HS had an attenuated NF-?B p65 response to Tumor Necrosis Factor ? (TNF?) treatment. The degree of inhibition of the NF-?B response depended on the HS exposure time. Mathematical modeling of single cells indicated that individual crosstalk mechanisms differentially encode HS-mediated NF-?B responses while being consistent with the observed population-level responses. In particular "all-or-nothing" encoding mechanisms were involved in the HS-dependent regulation of the IKK activity and I?B? phosphorylation, while others involving transport were "analogue". In order to discriminate between these mechanisms, we used live-cell imaging of nuclear translocations of the NF-?B p65 subunit. The single cell responses exhibited "all-or-nothing" encoding. While most cells did not respond to TNF? stimulation after a 60 min HS, 27% showed responses similar to those not receiving HS. We further demonstrated experimentally and theoretically that the predicted inhibition of IKK activity was consistent with the observed HS-dependent depletion of the IKK? and IKK? subunits in whole cell lysates. However, a combination of "all-or-nothing" crosstalk mechanisms was required to completely recapitulate the single cell data. We postulate therefore that the heterogeneity of the single cell responses might be explained by the cell-intrinsic variability of HS-modulated IKK signaling. In summary, we show that high temperature modulates NF-?B responses in single cells in a complex and unintuitive manner, which needs to be considered in hyperthermia-based treatment strategies.

Dataset Information

Overexpression of heat shock protein 72 attenuates NF-?B activation using a combination of regulatory mechanisms in microglia.

Publications

Overexpression of heat shock protein 72 attenuates NF-κB activation using a combination of regulatory mechanisms in microglia.

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