Heat shock protein 70 negatively regulates the heat-shock-induced suppression of the IkappaB/NF-kappaB cascade by facilitating IkappaB kinase renaturation and blocking its further denaturation

Heat shock (HS) treatment has been previously shown to suppress the IkappaB/nuclear factor-kappaB (NF-kappaB) cascade by denaturing, and thus inactivating IkappaB kinase (IKK). HS is characterized by the induction of a group of heat shock proteins (HSPs). However, their role in the HS-induced suppression of the IkappaB/NF-kappaB cascade is unclear. Adenovirus-mediated HSP70 overexpression was found not to suppress the TNF-alpha-induced activation of the IkappaB/NF-kappaB pathway, thus suggesting that HSP70 is unlikely to suppress this pathway. When TNF-alpha-induced activation of the IkappaB/NF-kappaB pathway was regained 24 h after HS, HSP70 was found to be highly up-regulated. Moreover, blocking HSP70 induction delayed TNF-alpha-induced IkappaBalpha degradation and the resolubilization of IKK. In addition, HSP70 associated physically with IKK, suggesting that HSP70 is involved in the recovery process via molecular chaperone effect. Adenovirus-mediated HSP70 overexpression prior to HS blocked the IkappaBalpha stabilizing effect of HS by suppressing IKK insolubilization. Moreover, the up-regulation of endogenous HSP70 by preheating, suppressed this subsequent HS-induced IKK insolubilization, and this effect was abrogated by blocking HSP70 induction. These findings indicate that HSP70 accumulates during HS and negatively regulates the HS-induced suppression of the IkappaB/NF-kappaB cascade by facilitating the renaturation of IKK and blocking its further denaturation.     

Arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1 beta-stimulated Caco-2 cells independent of the heat shock response

Recent studies suggest that sodium arsenite downregulates NF-kappaB activity by inhibiting phosphorylation and subsequent degradation of IkappaBalpha. Many effects of sodium arsenite are secondary to induction of heat shock proteins. The role of the heat shock response in arsenite-induced inhibition of NF-kappaB, however, is not known. We examined the involvement of the heat shock response in arsenite-induced inhibition of NF-kappaB activity in IL-1beta-stimulated Caco-2 cells, a human colorectal adenocarcinoma cell line with enterocytic properties. Treatment of the cells with IL-1beta resulted in increased IkappaB kinase activity, reduced levels of IkappaBalpha and increased NF-kappaB DNA binding activity. Sodium arsenite blocked all of these responses to IL-1beta without inducing changes in heat shock factor activity or heat shock protein levels. Results from additional experiments showed that the protective effect of sodium arsenite on IkappaBalpha was not influenced by the oxygen radical scavenger catalase or by inhibitors of the MAP-kinase signaling pathway. The present results suggest that sodium arsenite stabilizes IkappaBalpha and prevents NF-kappaB activation in IL-1beta-stimulated Caco-2 cells independent of the heat shock response. In addition, stabilization of IkappaBalpha by sodium arsenite does not require oxygen radical formation or activation of the MAP kinase signaling pathway.     

Stress-induced inhibition of the NF-kappaB signaling pathway results from the insolubilization of the IkappaB kinase complex following its dissociation from heat shock protein 90

Activation of the stress response attenuates proinflammatory responses by suppressing cytokine-stimulated activation of the NF-kappaB signaling pathway. In this study, we show that the activation of the cellular stress response, either by heat shock treatment or after exposure to sodium arsenite, leads to a transient inhibition of IkappaBalpha phosphorylation. Inhibition of IkappaBalpha phosphorylation after stress was associated with the detergent insolubilization of the upstream kinases, IkappaB kinase alpha (IKKalpha) and IkappaB kinase beta, components involved in IkappaBalpha phosphorylation. Pretreatment of cells with glycerol, a chemical chaperone that reduces the extent of stress-induced protein denaturation, reduced the stress-dependent detergent insolubility of the IKK complex and restored the cytokine-stimulated phosphorylation of IkappaB. The stress-dependent insolubility of the IKK complex appeared reversible; as the cells recovered from the heat shock treatment, the IKK complex reappeared within the soluble fraction of cells and was again capable of mediating the phosphorylation of IkappaBalpha in response to added cytokines. Treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90) function, also resulted in IKK detergent insolubility and proteasome-mediated degradation of the IKK complex. Furthermore, while IKKalpha coprecipitated with Hsp90 in control cells, coprecipitation of the two proteins was greatly reduced in those cells early after stress or following exposure to geldanamycin. Stress-induced transient insolubilization of the IkappaB kinase complex following its dissociation from Hsp90 represents a novel mechanism by which the activation of the stress response inhibits the NF-kappaB signaling pathway in response to proinflammatory stimuli.     

Distinct roles of IkappaB proteins in regulating constitutive NF-kappaB activity

The inhibitor of NF-kappaB (IkappaB) family of proteins is believed to regulate NF-kappaB activity by cytoplasmic sequestration. We show that in cells depleted of IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins, a small fraction of p65 binds DNA and leads to constitutive activation of NF-kappaB target genes, even without stimulation, whereas most of the p65 remains cytoplasmic. These results indicate that although IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins could be dispensable for cytoplasmic retention of NF-kappaB, they are essential for preventing NF-kappaB-dependent gene expression in the basal state. We also show that in the absence of IkappaBalpha, IkappaBbeta and IkappaBepsilon proteins, cytoplasmic retention of NF-kappaB by other cellular proteins renders the pathway unresponsive to activation.     

Phosphatase inhibition leads to activation of IkappaB kinase in murine macrophages

We have been interested in elucidating the role of intracellular phosphatase activity in the regulation of immune cell activation. To this end, we treated RAW 264.7 murine macrophages with the phosphatase inhibitor, calyculin-A. Treatment with calyculin-A led to activation of IkappaB kinase, degradation of IkappaBalpha, and induced nuclear translocation and DNA binding of NF-kappaB. Each of these effects occurred in both a time- and dose-dependent manner. In addition, each of these effects was negatively modulated by prior induction of the heat-shock response. Despite clear activation of the IkappaB kinase/IkappaBalpha/NF-kappaB pathway, however, phosphatase inhibition did not lead to increased expression of NF-kappaB-dependent genes. Thus, intracellular phosphatase activity is a central regulator of the NF-kappaB signal transduction pathway and is negatively modulated by heat shock. Inhibition of intracellular phosphatase activity with calyculin-A is not sufficient to induce NF-kappaB-dependent gene expression, demonstrating the complexity of NF-kappaB regulation in immune cells.     

Postrepression Activation of NF-κB Requires the Amino-Terminal Nuclear Export Signal Specific to IκBα

One of the most prominent NF-kappaB target genes in mammalian cells is the gene encoding one of its inhibitor proteins, IkappaBalpha. The increased synthesis of IkappaBalpha leads to postinduction repression of nuclear NF-kappaB activity. However, it is unknown why IkappaBalpha, among multiple IkappaB family members, is involved in this process and what significance this feedback regulation has beyond terminating NF-kappaB activity. Herein, we report an important IkappaBalpha-specific function dictated by its amino-terminal nuclear export sequence (N-NES). The IkappaBalpha N-NES is necessary for the postinduction export of nuclear NF-kappaB, which is a critical event in reestablishing a permissive condition for NF-kappaB to be rapidly reactivated. We show that although IkappaBalpha and another IkappaB member, IkappaBbeta, can enter the nucleus and repress NF-kappaB DNA-binding activity during the postinduction phase, only IkappaBalpha allows the efficient export of nuclear NF-kappaB. Moreover, swapping the N-terminal region of IkappaBbeta for the corresponding IkappaBalpha sequence is sufficient for the IkappaB chimera protein to export NF-kappaB similarly to IkappaBalpha during the postinduction state. Our findings provide a mechanistic explanation of why IkappaBalpha but not other IkappaB members is crucial for postrepression activation of NF-kappaB. We propose that this IkappaBalpha-specific function is important for certain physiological and pathological conditions where NF-kappaB needs to be rapidly reactivated.     

HTLV-1 Tax-associated hTid-1, a human DnaJ protein, is a repressor of Ikappa B kinase beta subunit

hTid-1, a human DnaJ protein, is a novel cellular target for HTLV-1 Tax. Here, we show that hTid-1 represses NF-kappaB activity induced by Tax as well as other activators such as tumor necrosis factor alpha (TNFalpha) and Bcl10. hTid-1 specifically suppresses serine phosphorylation of IkappaBalpha by activated IkappaB kinase beta (IKKbeta), but the activities of other serine kinases including p38, ERK2, and JNK1 are not affected. The suppressive activity of hTid-1 on IKKbeta requires a functional J domain that mediates association with heat shock proteins and results in prolonging the half-life of the NF-kappaB inhibitors IkappaBalpha and IkappaBbeta. Collectively, our data suggest that hTid-1, in association with heat shock proteins, exerts a negative regulatory effect on the NF-kappaB activity induced by various extracellular and intracellular activators including HTLV-1 Tax.     

Characterization of the nuclear import and export functions of Ikappa B(epsilon)

Control over the nuclear localization of nuclear factor kappaB/Rel proteins is accomplished in large part through association with members of the inhibitor of kappaB (IkappaB) protein family. For example, the well studied IkappaBalpha protein actively shuttles between the nucleus and the cytoplasm and both inhibits nuclear import and mediates nuclear export of NF-kappaB/Rel proteins. In contrast, the IkappaBbeta protein can inhibit nuclear import of NF-kappaB/Rel proteins but does not remove NF-kappaB/Rel proteins from the nucleus. To further understand how the IkappaB proteins control the nuclear-cytoplasmic distribution of NF-kappaB/Rel proteins, we have characterized the nuclear import and nuclear export functions of IkappaBepsilon. Our results indicate that the IkappaBepsilon protein, like the IkappaBalpha protein, actively shuttles between the nucleus and the cytoplasm. Similar to IkappaBalpha, nuclear import of IkappaBepsilon is mediated by its ankyrin repeat domain and is not blocked by the dominant-negative RanQ69L protein. However, the nuclear import function of the IkappaBepsilon ankyrin repeat domain is markedly less efficient than that of IkappaBalpha, with the result that nuclear shuttling of IkappaBepsilon between the nucleus and the cytoplasm is significantly slower than IkappaBalpha. Nuclear export of IkappaBepsilon is mediated by a short leucine-rich nuclear export sequence (NES)-like sequence ((343)VLLPFDDLKI(352)), located between amino acids 343 and 352. This NES-like sequence is required for RanGTP-dependent binding of IkappaBepsilon to CRM1. Nuclear accumulation of IkappaB(epsilon) is increased by either leptomycin B treatment or alanine substitutions within the IkappaBepsilon-derived NES. A functional NES is required for both efficient cytoplasmic retention and post-induction control of c-Rel by IkappaBepsilon, consistent with the notion that IkappaBepsilon-mediated nuclear export contributes to control over the nucleocytoplasmic distribution of NF-kappaB/Rel proteins.