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Activation of the stress protein response (SPR) inhibits iNOS-dependent release of NO from alveolar macrophages by blocking the activation of the STAT1 pathway in response to IFN-γ, a major cytokine present in the airspace of patients with acute lung injury. Inhibition of STAT1 phosphorylation after heat stress was associated with detergent insolubilization of the STAT1 protein and its proteasomal degradation which was reversed with the pretreatment of cells with glycerol, a chemical chaperone that reduces the extent of heat-induced protein denaturation. This early effect of SPR is the result of the disruption of the Hsp90 binding to the STAT1 protein. Our results also demonstrated that a late effect of SPR activation involves the regulation of iNOS function by inducible Hsp70 (Hsp70i). The STAT1 signaling pathway recovered function within 12 hours post-SPR activation and synthesis of the iNOS protein, however, NO production did not occur until 72 hours later. Inhibiting Hsp70i expression after heat stress recovered iNOS function whereas overexpressing Hsp70i in the absence of heat stress inhibited iNOS function. In summary, heat stress-induced transient inhibition of STAT1 following its dissociation from Hsp90, and the later transient inhibition of iNOS activity by inducible Hsp70, represent novel mechanisms by which the activation of the stress protein response inhibits the IFN-γ signaling pathway in alveolar macrophages. Since Hsp90 inhibitors have been shown to be safe in humans, these results also highlight a potential clinical application for this class of drugs in modulating NO signaling during the early phase of acute lung injury.
Acute lung injury (ALI) is a devastating clinical syndrome manifested by an inflammatory response that leads to respiratory failure with an overall mortality rate of 30–40 percent 1. Clinical studies have shown that the vectorial fluid transport across the alveolar epithelium critical for maintaining fluid balance in the airspace is impaired in most patients with ALI 1. Furthermore, there is a direct correlation between the impairment of the fluid removal from the airspace and mortality in patients with ALI 2. Recent experimental evidence indicates that alveolar fluid clearance is impaired by iNOS/NO-dependent mechanisms 3. Under those circumstances, large amounts of NO are released by alveolar macrophages and epithelial cells and cause modification of ENaC, CFTR and other ion channels involved in alveolar epithelial ion transport rendering these ion channels nonfunctional 4, 5. We have previously reported that activation of the stress protein response (SPR), a highly conserved cellular defense mechanism characterized by the increased expression of heat shock proteins, restores the vectorial lung fluid transport in part by reducing inducible nitric oxide synthase (iNOS) synthesis 6. However, the mechanisms by which SPR activation affects the iNOS-dependent NO release are still not completely understood.
The heat shock or stress protein response (SPR) classically defined as a highly conserved cellular defense mechanism is characterized by the increased expression of stress proteins. This allows the cell to withstand a subsequent insult that would otherwise be lethal, a phenomenon referred as “thermotolerance” or “preconditioning”. The activation of SPR is characterized by an early phase defined by the inhibition of proinflammatory cell signaling pathways within minutes after onset of SPR and a delayed phase in which the development of tolerance to inflammatory stimuli takes several hours and requires the de novo synthesis of proteins. Heat shock proteins not only function as molecular chaperones for newly synthesized proteins, but are also essential factors in the cell signaling pathways activated by proinflammatory mediators 7, 8.
The delayed phase of SPR is associated with the expression of inducible heat shock proteins including Hsp70 and Hsp27. Several studies including our own work have shown that lung injury due to ischemia-reperfusion or hemorrhagic shock is inhibited in animals that have been preconditioned with stress 6, 9, 10 In some of these studies, the inhibitory effect of SPR could be reproduced by the adenoviral gene transfer of Hsp70 into the distal airspaces of the lung, suggesting that Hsp70 may participate in the anti-inflammatory effect induced by SPR 11
During the course of ALI, interferon-gamma (IFN-γ) an activator of the STAT1 pathway is found in high levels in the pulmonary edema fluid of these patients 12. The STAT proteins are members of a family of transcription factors that transmit signals from the extracellular surface of the cell to the nucleus where they activate transcription of various genes including iNOS 13. While there is considerable information about the activation of STAT proteins and their role in transcription, much less is known about the regulation of the STAT1 signaling pathway and how the activation of SPR effects STAT1 and iNOS function. Thus, the objective of this study was to determine the mechanisms by which heat stress regulates the STAT1 pathway, the expression of iNOS and the release of NO by alveolar macrophages after exposure to IFN-γ.
An alveolar macrophage cell line, MH-S, were used in these studies.
MH-S cells were incubated at 43°C for 30 minutes and allowed to recover at 37°C for the hours indicated in the text prior to further analysis.
Cells were seeded and cultured for 24 hours prior to any treatment. After treatment with IFN-γ (10 ng/ml) for the indicated time, the cells were washed three times with phosphate buffered saline (PBS) on ice, lysed in 1x LSB, electrophoresed and transferred to nitrocellulose. STAT1 antibody (1:1,000), HRP-GAM (1:2,000).
MH-S cells were stimulated with IFN-γ for 24 hours. The extracellular medium was used for analysis of the presence of nitrite, a stable end-product of nitric oxide using the Griess reagent.
Real-time RT-PCR primers and probes are designed using Primer Express software (PE-Applied Biosystems (PE-ABI), Warrington, UK). The real-time RT-PCR probes are labelled with a fluorophore reporter dye (6-carboxy-fluorescein, FAM) at the 5′ end and a quencher dye (BHQ, Biosearch Technologies, Inc., Novato CA) at the 3′ end. Quantitative real time RT-PCR is performed as previously published 14. Mouse RT-PCR primers: iNOS TAGF: GAGCATCCCAAGTACGAGTGGT, iNOS TAGR: GGCCACGGCAGGCAG, iNOS TAGP: CCAGGAGCTCGGGTTGAAGTGGTATG. Mouse RT-PCR primers: Hsp70i TAGF: CTGTAGGAAGGATTTGTACACTTTAAACTC, Hsp70i TAGR: TTAAGGGTCAGCTCCTGAAGGT, Hsp70i TAGP: TCTGAGTCCCACACTCTCACCACCCA.
Nuclear protein isolation and EMSA was performed as described previously 7. TheSTAT1 consensu s oligonucleotide probe: 5′-ATG TGA GGG GAC TTT CCC AGG C-3′.
MH-S cells were scraped into a hypotonic buffer (10 mM HEPES, 10 mM NaCl, 5 mM MgCl2, 1 mM DTT, protease inhibitors, phosphatase inhibitors) and processed as previously described 15 .
MH-S cells were lysed as described for western blots with the addition of 40mM sodium molybdate for the immunoprecipitation of STAT1/Hsp90 complexes.
siRNA to Hsp70i was transfected into MH-S cells using X-tremeGene® according to manufactures protocol using a ratio of 10:2 of transfection reagent in microliters to micrograms of siRNA.
Adenovirus infection of macrophages was performed as previously described 16 .
Alveolar macrophages exposed to increasing times of heat stress at 43°C and recovered at 37°C for 1 hour before IFN-γ stimulation for 24h, released decreasing amounts of NO (nitrite) measured in the extracellular medium (Figure 1A). This decrease in NO production was not due to cell death as determined by alamar blue on the same cells (Figure 1B).
STAT1 activated by IFN-γ is an essential activator of iNOS transcription 17. After one-hour recovery from heat stress, the non-phosphorylated STAT1 protein was degraded (Figure 2A). Some cells were pretreated with the proteasome inhibitor MG132 for 1 hour prior to heat stress and exposure to IFN-γ. MG132 inhibited STAT1 degradation, however there was no phosphorylation of STAT1 on tyrosine 701 (Figure 2A). Thus, proteosomal degradation of the STAT1 protein was not the only mechanism of attenuation of the IFN-γ-mediated STAT1 activation by SPR. Further analysis showed that STAT1 did not translocate to the nucleus. As shown in Figure 2B, in the absence of heat stress, STAT1 protein extracted from the nucleus after IFN-γstimulation, bound to the STAT1 consensus oligonucleotide whereas after heat stress, a 1 hour recovery and IFN-γ stimulation, no binding was observed. Finally, cell fractionation experiments confirmed that heat stress caused the partition of STAT1 into the triton detergent insoluble fraction prior to its proteosomal degradation and that STAT1 insolubilization was not affected by IFN-γstimulation (Figure 2C).
We examined whether insolubilization of the STAT1 complex might be prevented by pretreating the cell monolayers with glycerol, a chemical chaperone that reduces thermal denaturation of proteins 18. The results showing the triton-soluble fraction of the cell lysates indicate that pretreatment with glycerol not only prevents the degradation of some of the STAT1 protein, but also restores phosphorylation of STAT1 in cells that have been exposed to heat stress and IFN-γ( Figure 2D).
Hsp90 is known to bind various proteins to maintain protein conformation necessary for function. We tested whether STAT1 was an Hsp90 client protein. Hsp90 co-immunoprecipitated with STAT1 under non-stimulated conditions in MH-S cells, but not after SPR activation with heat (Figure 3A). In a second series of experiments, we performed a differential centrifugation to isolate the membrane-enriched fraction of MH-S cells in the absence of IFN-γ stimulation and after increasing times of IFN-γ stimulation. Both STAT1 and Hsp90 were recruited to this membrane enriched cell fraction 10–15 min after exposure to IFN-γ. This recruitment was associated with an increase in the level of phosphorylated STAT1 on tyrosine 701 (Figure 3B). Further analysis of the Hsp90/STAT1 association showed that treatment of macrophages for 1 hour with 17-AAG, an inhibitor of Hsp90 binding, resulted in the loss of STAT1 phosphorylation on tyrosine 701 after IFN-γ stimulation (Figure 3C). This loss of activated STAT1 by 17-AAG correlated with the absence of iNOS gene transcription (Figure 3D).
We next determined the time-course of the recovery of iNOS function after heat stress. Nitric oxide production was observed only after 48 hours following heat stress and reached normal values after 72 hours recovery post heat stress (Figure 4).
We determined whether the slow recovery of the IFN-γ-mediated NO production after SPR activation was caused by a prolonged inhibition of the STAT1 pathway. Figure 5A shows that with a 12 hour recovery post-heat stress, the STAT1 protein was phosphorylated. Since the STAT1 protein was phosphorylated, the presence of nuclear STAT1 protein and its DNA binding function after a 12 hour recovery from heat stress was tested. As shown in Figure 5B, in the absence of heat stress, STAT1 protein extracted from the nucleus after IFN-γ stimulation, bound to the STAT1 consensus oligonucleotide. Likewise, after heat stress, 12 hours recovery and IFN-γ stimulation, STAT1 binding was observed. Further analysis confirmed that the IFN-γdependent activation of the STAT1 signaling pathway fully recovered after 12 hours recovery from heat stress as iNOS mRNA was detected in these cells using real-time RT-PCR (Figure 5C) as well as the iNOS protein as determined by western blot (Figure 5D). These results show that after a 12-hour recovery from heat stress, alveolar macrophages stimulated with IFN-γ are able to activate the STAT1 pathway resulting in synthesis of both iNOS mRNA and protein. In contrast, the release of NO from these cells after SPR activation and subsequent IFN-γ stimulation did not follow the same temporal recovery. Indeed, there was an inhibition of NO production 12 hours after SPR activation even though the iNOS mRNA and protein was synthesized. Thus, SPR regulates iNOS-dependent NO release by mechanisms that are independent of iNOS gene and protein expression.
Since the inducible form of Hsp70 is expressed at high levels after heat stress, we hypothesized that in the late phase of SPR activation, Hsp70i expression transiently inhibits iNOS function. First, we determined the temporal expression of Hsp70i. As shown in Figure 6A, Hsp70i expression was expressed at high levels after 1 hour recovery and these levels decreased by 25% after 24 hours recovery and by more than 50% after 48 hours recovery. There was an inverse correlation between the SPR-induced Hsp70 protein expression and NO release by alveolar macrophages (Compare Figures 4A&6A). To further explore the importance of SPR-induced Hsp70 expression on iNOS function, Hsp70 expression was inhibited using a specific siRNA. Alveolar macrophages were transfected with a siRNA control or a specific siRNA to Hsp70 after 30 minutes of heat stress. The cells were allowed to recover for 24 hours and then stimulated with IFN-γ. As seen in Figure 6B, siRNA transfection after heat stress resulted in a 65 to 70% reduction in Hsp70 expression. This reduction in Hsp70i expression resulting from siRNA inhibition was sufficient to allow NO release by alveolar macrophages stimulated with IFN-γ (Figure 6C). Control and Hsp70i siRNA transfections had no effect on NO production (data not shown). Lastly, we determined whether overexpression of the inducible Hsp70i protein would prevent the release of NO from non-heat stressed alveolar macrophages when stimulated with IFN-γ. Infection of MH-S cells with a adenovirus encoding Hsp70i, but not with a control adenovirus, significantly increased the expression of both Hsp70i mRNA and protein (Figure 6D&E) and decreased the release of NO from these cells after stimulation with IFN-γ( Figure 6F).
In this study, we show for the first time that in mouse alveolar macrophages (1) activation of the stress protein response inhibits the STAT1/iNOS signaling pathway; (2) STAT1 and Hsp90 form a complex in the unstimulated cell while IFN-γ stimulation led to recruitment of both STAT1 and Hsp90 to the plasma membrane; (3) the disruption of STAT1/Hsp90 binding inhibits IFN-γ activation of STAT1 and results in the insolubility and subsequent degradation of STAT1 via the proteosome; and lastly (4) the late inhibition of iNOS function after heat stress is due to the expression of inducible Hsp70 and not additional biochemical changes associated with heat stress 19.
Activation of the stress protein response due to thermal or non-thermal stress results in the increased synthesis of molecular chaperones or heat shock proteins 19. These chaperones bind to proteins that have unfolded due to stress-induced partial denaturation, directing them to refold. During this period, the cell enters a senescent phase and is non-responsive to external stimulation and only after a period of time do the cells return to a “normal” state in which they respond again to stimuli. In this study, we show that a non-lethal heat stress can inhibit IFN-γmediated nitric oxide production without directing the alveolar macrophages into the apoptotic pathway. Under these conditions, the STAT1 signaling pathway is inhibited and refractive to activation by IFN-γ. In the early phase of SPR activation, heat stress resulted in the degradation of the STAT1 protein. Similar to our previous study on heat stress and the NF-κB signaling pathway in which the IKKα and IKKβ become detergent insoluble after heat stress 7, the STAT1 protein also is found first in a nonionic detergent insoluble fraction and subsequently degraded by the proteasome. Interestingly, this insolubility can be reversed by the chemical chaperone glycerol.
What is the mechanism that maintains STAT1 in a phosphorylation competent conformation? Heat shock proteins not only function as molecular chaperones for newly synthesized proteins, but are also essential factors in the cell signaling pathways activated by inflammatory mediators 7, 8. Hsp90 is a highly conserved and essential stress protein that functions as a positive regulator of cell signaling pathways by modifying or maintaining the conformation of its client proteins for active signaling. Moreover, blocking the ATPase site of Hsp90 using geldanamycin or its derivative 17-AAG inhibits the function of these client proteins 20. We previously showed that the binding of Hsp90 to its client protein IKKα/β may be necessary to maintain solubility and thus function of the protein 7. In other words, solubility of NF-κB and now STAT1 directly relates to its conformation and thus its function. Furthermore, a previous study reported that STAT3 is a client protein of Hsp90 and that this interaction is required for STAT3 phosphorylation 15. We found that STAT1 and Hsp90 form a complex under non-stimulated conditions, albeit a fraction of the total STAT1 population, and that this complex is disrupted after heat stress. JAK1/2, Tyk2, STAT1 and STAT3 as well as the cytokine receptors have been found in a complex in the plasma membrane 15, 21-23, we likewise found that STAT1 and Hsp90 were present in a complex under non-stimulated conditions and recruited to the plasma membrane under IFN-γ stimulated conditions. Thus, we postulate that in alveolar macrophages, Hsp90 binding to STAT1 is necessary for STAT1 function.
Studies carried out by Shang and Tomasi recently demonstrated that JAK1, interacts with Hsp90 and disruption of this binding resulted in proteasome degradation of JAK1 24. Our studies do not preclude that Hsp90 can also bind to JAK1 and it is indeed possible that Hsp90, STAT1 and its kinase JAK1 form an active complex at the plasma membrane in response to the binding of IFN-γ to its receptor. Finally, the results of this study show that (1) heat stress disrupts the binding of Hsp90 to its client protein STAT1 and subsequently attenuates STAT1 function and that (2) treatment with an Hsp90 inhibitor in the absence of heat stress and thus Hsp70i expression are consistent with our previous studies on IKKα/β and PDK-1 in which we identified both of these proteins as novel client proteins of Hsp90 and that their function is indeed regulated by Hsp90 binding 7, 25. We therefore can add STAT1 protein to the growing list of Hsp90 client proteins 26.
While the STAT1 signaling pathway recovers function 12 hours post-activation of the stress protein response, the recovery of iNOS function takes 48 hours. The iNOS protein has been shown to be a client protein of Hsp90 27; however, the length of the recovery of iNOS function indicates that another mechanism could also be involved in the inhibition of iNOS after heat stress. Previous studies using overexpression of recombinant Hsp70i have shown that Hsp70i can inhibit iNOS function 28, 29. However, the role of the SPR-mediated Hsp70i expression in modulating iNOS function is unknown. Thus, we tested whether the SPR-mediated induction of Hsp70i was inhibiting iNOS function. Our initial experiment showed that it was a 48-hour recovery from heat stress before alveolar macrophages would release NO in response to IFN-γ stimulation. The NO release inversely correlated with the level of inducible Hsp70 protein. Further investigation using either siRNA to Hsp70i to inhibit the heat stress-induced expression of Hsp70i or recombinant adenovirus to overexpress Hsp70i in the absence of SPR activation showed that in alveolar macrophages, it is Hsp70i expression that modulates iNOS function. What is the mechanism of Hsp70i attenuation of iNOS-dependent NO release in alveolar macrophages? Some studies have shown that Hsp70i can inhibit iNOS expression 28, 30,31. This does not appear to be the mechanism of inhibition of iNOS-dependent NO release in alveolar macrophages since we can detect iNOS protein in the detergent soluble fraction. In support of our findings, one recent study identified iNOS/Hsp70i complex formation in mouse intestinal tissue lysates and suggested that Hsp70i may regulate iNOS function 29. We are currently investigating possible mechanisms of attenuation of iNOS function by Hsp70i in alveolar macrophages. In summary, our working hypothesis is that Hsp90 is a critical part of the cell signaling pathways such as STAT1/iNOS that mediate lung fluid balance abnormalities induced by ALI. SPR activation may protect the integrity of the alveolar-capillary barrier by two mechanisms: (a) first by an immediate dissociation of Hsp90 from its client proteins rendering these proteins nonfunctional, then (b) by a delayed phase involving the de novo synthesis of Hsp70 that binds to and temporarily inhibits the function of some of the Hsp90 client proteins until Hsp90 can re-complex with these proteins.
What are the clinical implications of our findings? The airspace release of iNOS-dependent NO is deleterious during the early phase of acute lung injury. Indeed, the release of NO by alveolar macrophages and lung epithelial cells inhibits ion channel function via post-translational modification, thus preventing removal of edema fluid from the airspace of the lungs 32. Several studies have reported that prior activation of the stress protein response prevents the abnormalities in the lung fluid balance associated with ALI 6, 9, 33. These studies may have an important therapeutic significance in humans. We recently reported that SPR activation occurs in patients with ALI and correlates with the preservation of alveolar fluid clearance 34. Thus, the stress response could be activated as a prophylactic therapy to protect from lung fluid balance abnormalities associated with ALI, using pharmacologic inhibitors of Hsp90 that have been shown to be safe in humans 35.
The authors thank Drs. Kimberly Mace and Nancy Boudreau for critical review of the manuscript. This work was primarily supported by UCSF Academic Senate Grant (M. Howard), NIH Grant GM 62188 (J.F. Pittet), ALA Senior Research Training Fellowship and T32 GM008440 (J. Roux).