Overexpression of Heat Shock Protein 72 Attenuates NF-κB Activation Using a Combination of Regulatory Mechanisms in Microglia

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 microglial cell line stably overexpressing Hsp72. We find that Hsp72 overexpression reduces the amount of NF-κB DNA binding activity, activity of the upstream kinase IKK, and amount of IκBα inhibitor phosphorylated following TNFα 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-κB 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-κB activation. An additional observation from our study is that the induced expression of IκBα 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-κB response. This study illustrates the multi-faceted nature of Hsp72 regulation of NF-κB activation in microglia and offers further clues to a novel mechanism by which Hsp72 may protect cells against injury.Author Summary: Inducing heat shock or overexpressing certain heat shock proteins (HSPs) is known to protect against brain injury, such as that resulting from stroke. Understanding the mechanisms underlying protection at the cellular and molecular level is a subject of intense research, as such knowledge may prove beneficial in designing future therapies. Regulation of the activation of the key inflammatory transcription factor Nuclear Factor κB (NF-κB) is believed to be one critical mechanism. However how its activation is altered by Hsp72 remains unresolved. Here we examine NF-κB signaling in microglia cells overexpressing Hsp72, combining experimentation and mathematical modeling. We show that Hsp72 affects signaling using at least two essential and distinct mechanisms: attenuation of upstream kinase (IKK) activity and reduction of steady state NF-κB protein levels. We provide numerical evidence suggesting that neither mechanism in isolation is sufficient to account for the observed signaling. Furthermore, our observations suggest an intriguing additional level of regulation of gene expression and protein synthesis of the IκBα inhibitor, which opens interesting new avenues of research. These results provide novel insight into the mechanisms by which Hsp72 may regulate inflammation and protect brain cells from injury.