The INHIBITION OF LPS-INDUCED PRODUCTION OF INFLAMMATORY CYTOKINES BY HSP70 INVOLVES INACTIVATION OF THE NF-κB PATHWAY BUT NOT THE MAPK PATHWAYS

Abstract

The objective of this study was to evaluate the negative regulatory role of heat shock protein 70 (HSP70) on endotoxin-induced activation of inflammatory cytokine signaling pathways in a macrophage cell line. Our studies show that elevation of HSP70 either by activation of the heat shock response (HSR) or through forced expression of the hsp70.1 gene downregulates cytokine expression. Our experiments showed that activation of the HSR and HSP70 overexpression could inhibit LPS-mediated expression of the proinflammatory cytokines TNF-alpha and IL-1 at the mRNA and protein levels. We also investigated the effects of HSP70 elevation on signaling pathways downstream of LPS and its receptors, including the NF-kappaB and mitogen-activated protein kinase (MAPK) pathways. The effects of HSP70 on cytokine expression were correlated with its effects on activation of NF-kappaB, a known activator of the tnfalpha and Il-1 genes. Overexpression of HSP70 inhibited the nuclear translocation of p65, the transcriptionally active component of the NF-kappaB complex, and prevented the degradation of IkappaBalpha, the regulator of NF-kappaB activity. However, HSP70 elevation did not markedly inhibit signaling through the MAPK arm of the LPS-induced pathway, suggesting that the effects of HSP70 are mediated primarily through the NF-kappaB cascade. Our experiments therefore suggested that elevated levels of HSP70 inhibit LPS-induced production of inflammatory cytokines by a mechanisms involving inactivation of NF-kappaB but cast doubt on significant role for the MAPK pathway in these effects.

The regulation of TNFα production after heat and endotoxin stimulation is dependent on Annexin-A1 and HSP70

“…Indeed, ANXA1 has been shown to negatively regulate IL-6 expression through the modulation of MKP1 (Yang et al 2006), and HSP70 levels were also higher in WT cells expressing ANXA1 in our study, indicating that ANXA1 is required for HSP70 induction after heat. The role of HSP70 in reducing TNFα levels has previously been shown (Shi et al 2006;van der Bruggen et al 1999) and supports our postulation that in response to heat and LPS, ANXA1 increases HSP70 expression (mRNA and protein), which can increase MKP1 levels, which in turn inhibit ERK, p38, and more likely JNK activation, leading to low TNFα levels (Fig. 7).…”

“…In the current study, we show that potent pro-inflammatory cytokines TNFα and IL-6 were downregulated upon heat and LPS treatment as compared to LPS treatment. This is similar to other studies on TNFα (Shi et al 2006), IL-12 (Li et al 2001), andIL-6 (Ensor et al 1994), but opposite to other reported cytokines such as MIP2 (Garekar et al 2006). This regulation of cytokine production by heat can be related to another protective phenomenon known as endotoxin tolerance (Biswas and Lopez-Collazo 2009).…”

“…Previous studies have shown that heat pretreatment can inhibit cytokine production (Shi et al 2006). To confirm this finding, BMMOs obtained from BALB/c mice were incubated at 37 or 42°C for 1 h, after which cells were stimulated with 100 ng/ml LPS and the levels of TNFα, IL-6, and IL-12 were measured.…”

“…The main role of the heat stress response is to protect the cell during stressful conditions by promoting its cell survival. As a means of promoting cell survival, the heat stress response and HSPs are also known to play a role in inflammatory signaling by regulating the production of inflammatory cytokines (Asea et al 2002;Shi et al 2006).…”

ING1 (inhibitor of growth family, member 1)

“…This induction of HSP70 required the amino terminus of ING1b but not the PHD, PBR and NLS region (Feng et al, 2006). HSP70 is known to inhibit the NF-kB pathways (Ran et al, 2004;Shi et al, 2006). More recently, it has been shown that the C-terminal PHD of ING1 interacts with histone H3 trimethylated at Lys4 (H3K4me3).…”