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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Endotoxin-mediated proinflammatory cytokines play a significant role in the pathogenesis of acute and chronic liver diseases. Heat shock protein 90 (molecular weight, 90 kDa) (hsp90) functions as an important chaperone of lipopolysaccharide (LPS) signaling and is required for the production of proinflammatory cytokines. We hypothesized that inhibition of hsp90 would prevent LPS-induced liver injury by decreasing proinflammatory cytokines. C57BL/6 mice were injected intraperitoneally with an hsp90 inhibitor, 17-dimethylamino-ethylamino-17-demethoxygeldanamycin (17-DMAG), and LPS. Parameters of liver injury, proinflammatory cytokines, and associated mechanisms were studied by in vivo and in vitro experiments. Inhibition of hsp90 by 17-DMAG prevented LPS-induced increases in serum alanine aminotransferase activity and significantly reduced serum tumor necrosis factor alpha (TNFα) and interleukin-6 (IL-6) protein as well as messenger RNA (mRNA) in liver. Enhanced DNA-binding activity of heat shock transcription factor 1 (HSF1) and induction of target gene heat shock protein 70 (molecular weight, 70 kDa) confirmed hsp90 inhibition in liver. 17-DMAG treatment decreased cluster of differentiation 14 mRNA and LPS-induced nuclear factor kappa light-chain enhancer of activated B cells (NFκB) DNA binding without affecting Toll-like receptor 4 mRNA in liver. Mechanistic studies revealed that 17-DMAG-mediated inhibition of TNFα showed no effect on LPS-induced NFκB promoter-driven reporter activity, but significantly decreased TNFα promoter-driven reporter activity. Chromatin immunoprecipitation assays showed that 17-DMAG enhanced HSF1 binding to the TNFα promoter, but not the IL-6 promoter, suggesting HSF1 mediated direct inhibition of TNFα, but not IL-6. We show that HSF1 indirectly regulates IL-6 by the induction of another transcription factor, activating transcription factor 3. Inhibition of HSF1, using small interfering RNA, prevented 17-DMAG-mediated down-regulation of NFκB-binding activity, TNFα, and IL-6 induction, supporting a repressive role for HSF1 on proinflammatory cytokine genes during hsp90 inhibition. Conclusion: Hsp90 inhibition in vivo reduces proinflammatory cytokines and prevents LPS-induced liver injury likely through repressive action of HSF1. Our results suggest a novel application for 17-DMAG in alleviating LPS-induced liver injury. (HEPATOLOGY 2011)

The importance of macrophage activation and endotoxin-mediated proinflammatory cytokine production in liver injury is evident from numerous models of acute and chronic liver disease.1 For instance, in nonalcoholic steatohepatitis (NASH), endotoxin or lipopolysaccharide (LPS) triggers tumor necrosis factor alpha (TNFα) and other proinflammatory cytokines.2 Exposure of genetically obese mice to LPS exhibit hepatotoxicity and develop steatohepatitis.3 In alcoholic liver disease (ALD), gut-derived endotoxin (i.e., LPS) activates liver macrophages and the production of proinflammatory cytokines TNFα, interleukin-6 (IL-6), and interleukin-1 beta (IL-1β) that contribute to the pathogenesis of liver injury.4-6 Acetaminophen-mediated liver injury,7 ischemia-reperfusion injury,8 and liver cancer9 are all linked to LPS, macrophage activation, and proinflammatory cytokines. It is thus evident, from the literature, that exposure to LPS induces proinflammatory cytokines and reactive oxygen species,10 leading to the development and progression of liver injury.1, 11, 12

The significance of stress-induced heat shock proteins as molecular chaperones of the LPS-signaling pathway in macrophage activation has been reported.13-16 Heat shock protein 70 (molecular weight, 70 kDa) (Hsp70) and heat shock protein 90 (molecular weight, 90 kDa) (hsp90) bind to LPS-signaling molecules, culminating in the activation of nuclear factor kappa light-chain enhancer of activated B cells (NFκB) and expression of proinflammatory cytokines TNFα, IL-1β, and IL-6 in macrophages.17-20 Hsp90, an important molecular chaperone, is responsible for the tertiary folding of client proteins, such as IkappaB kinase,21 interleukin-1 receptor-associated kinase 1,22 and mitogen-activated protein kinase,23 and inhibition of hsp90 diminishes innate immune responses through Toll-like receptor (TLR) signaling.14 Targeting hsp90 as an attractive therapeutic strategy was evaluated in the treatment of cancers and is currently in clinical trials.24-27 Preclinical data also suggest that hsp90 inhibition is an effective treatment approach for alleviating chronic inflammatory diseases, such as uveitis18 and rheumatoid arthritis.28

The role of hsp90 in liver diseases remains elusive. Our earlier studies reported that chronic alcohol-induced macrophage activation and liver disease is associated with increased hsp90.17 Based on the requirement of hsp90 in the LPS pathway, we hypothesized that inhibition of hsp90 would prevent LPS-induced liver injury through decreased proinflammatory cytokine production. To this end, we tested the effect of hsp90 inhibition in vivo using 17-dimethylamino-ethylamino-17-demethoxygeldanamycin (17-DMAG), a water-soluble derivative of the benzoquinone ansamycin antibiotic, geldanamycin, on endotoxin-mediated liver injury and proinflammatory cytokine production in mice. To dissect the molecular mechanisms underlying the inhibition of proinflammatory cytokines by 17-DMAG, we performed in vitro studies in RAW 264.7 macrophages. Here, we show that hsp90 inhibition prevents LPS-induced liver injury by down-regulation of proinflammatory cytokine, TNFα, and IL-6, likely by heat shock transcription factor 1 (HSF1) activation in the liver.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Animals and Experimental Protocol.

All animals received proper care in agreement with animal protocols approved by the Institutional Animal Care and Use Committee at the University of Massachusetts Medical School (Worcester, MA). Six-week-old C57BL/6 female and male mice were purchased from Jackson Labs (Bar Harbor, ME). Before 17-DMAG injection, mice were injected intraperitoneally (IP) with either 0.1 mL of saline or 0.5 mg/kg body weight (BW) of LPS in 0.1 mL of saline (from Escherichia coli 0111:B4; Sigma-Aldrich, St. Louis, MO). Mice were IP administered a single dose of hsp90 inhibitor 17-DMAG (NSC 707545; National Cancer Institute, Bethesda, MD) at 2.5, 5, or 30 mg/kg BW. Mice were sacrificed at 2 or 18 hours after 17-DMAG and LPS administration. Serum was separated from whole blood and frozen at −80°C. Liver tissue was rapidly excised, and a portion was snap-frozen in liquid nitrogen and stored at −80°C. Additional portions of the livers were stored in the RNA stabilization reagent, RNAlater (Qiagen GmbH, Hilden, Germany), for RNA extraction.

Other Methods.

The following methods are described in the Supporting Materials, including serum biochemical assay and cytokines, electrophoretic mobility shift assay (EMSA), RNA extraction and real-time polymerase chain reaction (PCR), western blotting analysis, cell-culture reagents and stimulations, transfections and luciferase reporter assay, and chromatin immunoprecipitation (ChIP).

HSF1 Small Interfering RNA Transfection.

RAW macrophages were transiently transfected with 20 pM of HSF1 small interfering RNA (siRNA) (Invitrogen, Carlsbad, CA) in Opti-MEM for 6 hours (sequence listed in Supporting Table 1) using Lipofectamine 2000 (Invitrogen). RNA and nuclear protein extraction were done as reported in the Supporting Materials.

Statistical Analysis.

Statistical significance was determined using the t test or nonparametric analysis of variance, followed by the Kruskal-Wallis test. Data are presented as mean ± standard error of the mean (SEM) and were considered statistically significant at P < 0.05.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Hsp90 Inhibitor 17-DMAG Reduces Serum Alanine Aminotransferase Levels.

The significance of hsp90 in liver inflammatory responses is unknown. Here, we determined the effect of 17-DMAG, a water-soluble hsp90 inhibitor, in vivo on liver inflammatory responses and injury. Levels of serum alanine aminotransferase (ALT), a marker of liver injury, were assessed after 18 hours of 17-DMAG and LPS administration in vivo. Figure 1 shows that LPS injection in vivo at 0.5 mg/kg BW significantly induced high serum ALT levels, as compared to saline-injected controls, after 18 hours. Hsp90 inhibition by 17-DMAG, administered at 2.5, 5, and 30 mg/kg BW, exhibited significant reduction of serum ALT at all three doses (Fig. 1), independent of the dose used. These experiments suggest that hsp90 inhibition prevented LPS-induced liver injury. Because all the doses showed similar effects, subsequent experiments were performed with lower doses (2.5 and 5 mg/kg BW) of 17-DMAG in vivo.

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Figure 1. Hsp90 inhibitor 17-DMAG reduces LPS-induced serum ALT. LPS (equation image 0.5 mg/kg BW) and 17-DMAG (equation image 2.5, equation image 5, and equation image 30 mg/kg BW) were injected IP in C57BL/6 mice. All mice were sacrificed 18 hours post injection, and ALT activity was determined in serum, as described in the Supporting Materials. Values are shown as mean ± SEM (5 mice per group). *P < 0.05 versus LPS-injected mice.

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Hsp90 Inhibition Decreases Proinflammatory Cytokine Production in the Liver.

Because LPS-induced liver injury is largely mediated by proinflammatory cytokines, we determined whether 17-DMAG would have any effect on proinflammatory cytokine production in the liver. First, we analyzed messenger RNA (mRNA) levels of proinflammatory cytokines by real-time PCR in whole livers after treatment with 17-DMAG in vivo. Proinflammatory cytokine TNFα mRNA (Fig. 2A) was significantly reduced at 2.5 and 5 mg/kg of 17-DMAG treatment, compared to LPS alone, whereas IL-6 mRNA (Fig. 2B) was decreased at the higher dose (5 mg/kg) of 17-DMAG, compared to LPS alone, in the liver. Second, we measured serum cytokine levels by enzyme-linked immunosorbent assay (ELISA) and observed that TNFα (Fig. 2C) was significantly reduced at both doses of 17-DMAG, whereas IL-6 (Fig. 2D) showed significant reduction only at the 5-mg/kg 17-DMAG dose, compared to LPS alone. These results suggest that hsp90 inhibition by 17-DMAG prevented the LPS-induced proinflammatory cytokines, TNFα and IL-6, at both mRNA and protein levels in the liver.

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Figure 2. Hsp90 inhibition decreases proinflammatory cytokine production in the liver. C57BL/6 mice were injected IP with LPS (equation image 0.5 mg/kg BW) and 17-DMAG (equation image 2.5 and equation image 5 mg/kg BW). All mice were sacrificed 2 hours post injection, and total RNA was extracted from liver. mRNA levels of liver (A) TNFα and (B) IL-6 were analyzed by quantitative real-time PCR and normalized to 18S rRNA. Results are expressed as mean fold change ± SEM over mice injected with saline (5 mice per group). Another set of C57BL/6 mice were injected IP with LPS (equation image 0.5 mg/kg BW) and 17-DMAG (equation image 2.5 and equation image 5 mg/kg BW) for 18 hours, and serum TNFα (C) and IL-6 (D) were analyzed by ELISA. #P < 0.001; *P < 0.05 versus LPS-injected mice. rRNA, ribosomal RNA.

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Hsp90 Inhibition Induces HSF1 DNA-Binding Activity and Up-regulates hsp70 Expression in the Liver.

Hsp90 sequesters HSF1 in an inactive state in cytoplasm,29 and inhibition of hsp90 dissociates this complex and releases HSF1, which translocates to the nucleus.30 To confirm the inhibition of hsp90 activity in the liver, we analyzed the DNA-binding activity of HSF1 by EMSA and expression of the target gene, hsp70. Hsp90 inhibition by 17-DMAG significantly up-regulated HSF1 binding to DNA in a dose-dependent manner (Fig. 3A) in the liver. Complementary to HSF1 activation, hsp90 inhibition resulted in subsequent induction of hsp70 mRNA (Fig. 3B) and protein levels (Fig. 3C) in the liver. In accord with the reported action of 17-DMAG on hsp90 chaperone function,31 no effect was observed on protein levels of hsp90 in the liver (Fig. 3D). Our results suggest that 17-DMAG up-regulates HSF1 DNA-binding activity and induces target gene hsp70, without affecting hsp90 levels, confirming the inhibition of hsp90 function after 17-DMAG treatment in the liver.

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Figure 3. Hsp90 inhibition induces HSF1 DNA binding activity and up-regulates hsp70 expression in the liver. C57BL/6 mice were injected IP with LPS (equation image 0.5 mg/kg BW) and 17-DMAG (equation image 2.5 and equation image 5 mg/kg BW), and livers were collected at the end of 2 hours. DNA-binding activity of HSF1 was detected in nuclear extracts of liver cells by EMSA using a 32P-labeled, double-stranded HSE oligonucleotide. (A) Representative EMSA picture is shown in the upper panel, and the bar graph in the lower panel shows mean relative density ± SEM (5 mice per group). #P < 0.01; *P < 0.05, compared to LPS-injected mice. Hsp70 mRNA levels in the liver (B) were analyzed by quantitative real-time PCR and normalized to 18S rRNA. Results are expressed as mean fold change ± SEM over mice injected with saline (5 mice per group). #P < 0.001; *P < 0.0001 versus LPS-injected mice. Hsp70 (C) and hsp90 (D) protein was detected in liver whole cell lysates by western blotting. β-tubulin is shown as the internal loading control. A representative gel picture is shown with mean relative density ± SEM (3 mice per group). *P < 0.001, #P < 0.0001 compared to LPS-injected mice. ns, not significant; HSE, heat shock element; rRNA, ribosomal RNA.

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17-DMAG Affects NFκB DNA-Binding Activity and Down-regulates Cluster of Differentiation 14 mRNA in the Liver.

Hsp90 chaperones the LPS receptors, cluster of differentiation 14 (CD14) and TLR4, resulting in the activation of downstream signaling and proinflammatory cytokine production.14 We assessed CD14 and TLR4 mRNA levels, as a measure of total cellular expression, in response to hsp90 inhibition. Liver CD14 mRNA was significantly down-regulated in response to hsp90 inhibition by 17-DMAG, compared to LPS alone (Fig. 4A), whereas TLR4 mRNA was unaffected (Fig. 4A). Subsequently, to determine the effect of 17-DMAG on downstream activation, we analyzed NFκB, a pivotal transcription factor in CD14/TLR4 signaling. Our results show that 17-DMAG treatment significantly decreased LPS-induced NFκB DNA-binding activity in a dose-dependent manner (Fig. 4B). Next, we determined whether 17-DMAG-mediated down-regulation of NFκB was IκBα dependent or the result of alterations in NFκB p65 levels. We observed that 17-DMAG prevented the LPS-induced degradation of cytoplasmic IκBα (Fig. 4C) concomitant to reduced NFκB binding observed in the liver (Fig. 4B), whereas total cellular NFκB p65 (Fig. 4D) was unchanged. Furthermore, 17-DMAG did not alter nuclear phospho-p65 levels, indicating a phosphorylation-independent effect of NFκB inhibition (Fig. 4E). Together, these results suggest that hsp90 inhibition reduces CD14/TLR4 signaling and culminates in decreased NFκB DNA binding in an IκBα-dependent manner.

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Figure 4. 17-DMAG affects NFκB DNA binding activity and down-regulates CD14 in the liver. (A) Liver CD14 (left vertical axis) and TLR4 (right vertical axis) mRNA levels were analyzed by quantitative real-time PCR and normalized to 18S rRNA. Results are expressed as mean fold change ± SEM over mice injected with saline (5 mice per group). *P < 0.05 versus LPS-injected mice. ns, not significant. (B) C57BL/6 mice were injected IP with LPS (equation image 0.5 mg/kg BW) and 17-DMAG (equation image 2.5 and equation image 5 mg/kg BW), and livers were collected at the end of 2 hours. NFκB DNA-binding activity was detected in nuclear extracts of whole livers by EMSA using a 32P-labeled, double-stranded NFκB consensus oligonucleotide. A representative EMSA picture is shown in the upper panel, and the bar graph in the lower panel shows mean relative density ± SEM (5 mice per group). #P < 0.001; *P < 0.05, compared to LPS-injected mice. Cytoplasmic IκBα (C), cellular NFκB p65 (D), and nuclear NFκB phospho p65 (E) were detected in livers collected at the end of 2 hours. A representative western blotting picture is shown in the upper panel, and the bar graph in the lower panel shows mean relative density ± SEM (5 mice per group). *P < 0.05 versus LPS-injected mice. ns, not significant; rRNA, ribosomal RNA.

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HSF1 Regulates LPS-Induced TNFα Expression in Response to hsp90 Inhibition.

To further delineate whether 17-DMAG-mediated inhibition of proinflammatory cytokines was linked to reduced NFκB activity, we determined the effect of 17-DMAG on NFκB promoter-driven reporter gene activity in RAW 264.7 macrophages. RAW macrophages showed a similar effect of 17-DMAG-mediated inhibition of proinflammatory cytokines and NFκB activity as observed in the liver (data not shown) and were used as an in vitro model for subsequent mechanistic transfection experiments. LPS-induced NFκB promoter-driven luciferase reporter activity was significantly upregulated in RAW macrophages, whereas treatment with 17-DMAG had no significant effect (Fig. 5A), indicating that inhibition of proinflammatory cytokines was not solely dependent on the NFκB promoter. Next, we determined whether 17-DMAG treatment had any effect on TNFα promoter-driven reporter activity. LPS stimulation induced TNFα promoter-driven reporter activity, which was significantly decreased by 17-DMAG treatment in RAW macrophages (Fig. 5B). These results suggest that 17-DMAG did not affect NFκB promoter-driven reporter activity, but reduced TNFα promoter-driven reporter activity, suggesting that mechanisms other than NFκB binding may be involved in negatively regulating TNFα expression in response to hsp90 inhibition. Hsp70 induced during hsp90 inhibition (shown in Fig. 3C) interacts with NFκB proteins to suppress TNFα expression in heat-shocked cells.32 Here, we determined whether NFκB-p50 would bind to hsp70 in macrophages after 17-DMAG treatment. There was no significant induction in the NFκB-p50-hsp70 complex formation after LPS and/or 17-DMAG treatment, as compared to untreated samples (Supporting Fig. 1), ruling out the possibility of a hsp70-mediated mechanism of inhibition of proinflammatory cytokines after treatment with 17-DMAG.

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Figure 5. HSF1 regulates TNFα and hsp70 expression in response to hsp90 inhibition. RAW macrophages were transfected with either NFκB (A) or TNFα (B) promoter constructs separately. The next day, cells were stimulated with LPS (equation image 100 ng/ml), 17-DMAG (equation image 0.5 μM), or both (equation image) for 6 hours. Fold induction in NFκB (A) and TNFα (B) promoter activity over unstimulated cells is shown as the bar graph. *P < 0.05 versus unstimulated cells; #P < 0.001 versus LPS-stimulated cells. Data represent mean of three experiments ± SEM. ChIP assay was performed using anti-HSF1 antibody, and semiquantitative PCR was carried out using hsp70 (C), TNFα (D), and IL-6 (E) promoter-specific primers. A representative gel picture for each gene is shown. The densitometry graph represents the average of three independent experiments. *P < 0.0001 versus LPS-stimulated cells; #P < 0.001 versus unstimulated cells.

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Next, we sought to determine whether another transcription factor was involved in the modulation of 17-DMAG-mediated reduction of proinflammatory cytokine production. Earlier studies have shown that HSF1 serves as a transcriptional repressor for proinflammatory cytokine expression during heat stress by NFκB inhibition.33 To determine whether HSF1 would bind to the TNFα or IL-6 promoter, we performed ChIP of DNA-protein complexes using an anti-HSF1 antibody, followed by semiquantitative PCR, using HSF1-binding-site–specific primers in TNFα,33 IL-6 promoter,34 and hsp70 promoter.56 Positive control heat-shocked macrophages showed a significant up-regulation in the binding of HSF1 to the hsp70 promoter (7- to 8-fold) (Fig. 5C) and moderate binding to the TNFα promoter (3-fold) (Fig. 5D) without changes in LPS-treated macrophages. HSF1 binding to the TNFα promoter was up-regulated in response to hsp90 inhibition by 17-DMAG and LPS treatment (Fig. 5D). Interestingly, we observed that HSF1 binding to the IL-6 promoter was not affected after hsp90 inhibition (Fig. 5E). Thus, our results here show that HSF1 binds to the TNFα, but not IL-6, promoter and likely serves as a key transcriptional repressor down-regulating TNFα expression in response to hsp90 inhibition by 17-DMAG.

Knockdown of HSF1 Restores TNFα Transcription.

To confirm whether HSF1 down-regulates, and has a direct effect on TNFα expression during hsp90 inhibition, siRNA experiments targeting HSF1 were performed. Using specific HSF1 siRNA,35 transfection was performed in RAW macrophages, followed by treatment with LPS ± 17-DMAG. An approximate 80% knockdown of HSF1 mRNA was achieved (Fig. 6A). RAW cells were then treated with LPS in the absence or presence of 17-DMAG, and TNFα mRNA was measured by real-time PCR. Knockdown of HSF1 prevented 17-DMAG-mediated down-regulation of LPS-induced TNFα expression (Fig. 6B). Previous studies showed that HSF1 could bind to the 5′ end of the TNFα promoter36 and likely reduce NFκB DNA binding as a result of inaccessible chromatin after HSF1 binding. We thus analyzed the effect of HSF1 knockdown on LPS-induced NFκB DNA-binding activity in macrophages after hsp90 inhibition. Knockdown of HSF1 inhibited reduced LPS-induced NFκB DNA-binding activity in 17-DMAG-treated cells (Fig. 6C). These results indicate that HSF1 plays a significant role in the down-regulation of NFκB DNA binding and, ultimately, proinflammatory cytokine response after hsp90 inhibition by 17-DMAG in macrophages.

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Figure 6. HSF1 knockdown restores TNFα expression. RAW macrophages were transfected with HSF1 siRNA and were stimulated the next day with LPS, 17-DMAG, or both for 2 hours. HSF1 (A) and TNFα (B) mRNA were analyzed by real-time PCR. (A) Bar graph represents mean percent knockdown of HSF1 mRNA ± SEM of a total of three experiments. (B) Data represent mean fold change in TNFα mRNA of three experiments ± SEM. *P < 0.0001 versus nontransfected, LPS+17-DMAG-stimulated cells. NFκB DNA-binding activity (C) was detected in nuclear extracts of RAW macrophages 24 hours after HSF1 siRNA transfection and 2 hours of treatment with LPS±17-DMAG. A representative EMSA picture is shown in the upper panel, and the bar graph in the lower panel shows mean relative density ± SEM. *P < 0.01, compared to negative control siRNA-transfected, LPS+17-DMAG-stimulated cells.

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17-DMAG-Induced HSF-1 Indirectly Mediates IL-6 Suppression Through ATF3.

Recent studies show that heat-shock–induced HSF1 indirectly negatively regulates the IL-6 promoter through the induction of activating transcription factor 3 (ATF3).37 To explore the possibility of this mechanism, we analyzed ATF3 mRNA (Fig. 7A) and protein levels (Fig. 7B) after 17-DMAG treatment in the liver. We observed a significant induction of ATF3 mRNA and protein in 17-DMAG-treated livers, suggesting an ATF3-mediated IL-6 suppression. Furthermore, we determined whether inhibition of HSF1 using siRNA would affect IL-6 mRNA levels in RAW macrophages. Figure 7C shows that HSF-1 knockdown prevented the down-regulation of LPS-induced IL-6 mRNA during 17-DMAG treatment, suggesting a role for HSF1 in the regulation of IL-6, likely through ATF3.

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Figure 7. 17-DMAG-induced HSF-1 indirectly mediates IL-6 suppression through ATF. C57BL/6 mice were injected IP with LPS (equation image 0.5 mg/kg BW) and 17-DMAG (equation image 2.5 and equation image 5 mg/kg BW), and livers were collected at the end of 2 hours. mRNA levels of liver ATF3 (A) were analyzed by quantitative real-time PCR and normalized to 18S rRNA. Results are expressed as mean fold change ± SEM over mice injected with saline (5 mice per group). Nuclear ATF3 protein (B) was detected in livers collected at the end of 2 hours. A representative western blotting picture is shown in the upper panel, and the bar graph in the lower panel shows mean relative density ± SEM (5 mice per group). *P < 0.05 versus LPS-injected mice. RAW macrophages were transfected with HSF1 siRNA and were stimulated the next day with LPS, 17-DMAG, or both for 2 hours. IL-6 mRNA was analyzed by real-time PCR. (C) Data represent mean fold change in IL-6 mRNA of three experiments ± SEM. #P < 0.001 versus nontransfected, LPS+17-DMAG-stimulated cells. rRNA, ribosomal RNA.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Intracellular chaperones are necessary for the stability and function of signaling molecules down-stream to the LPS receptor.14, 15, 19 The role of hsp90, an important molecular chaperone in the LPS-signaling pathway, has been recognized.13, 19, 20 The significance of endotoxin (i.e., LPS)-mediated macrophage activation and inflammatory responses in acute and chronic liver diseases is well known.1 In this study, we targeted hsp90 to inhibit LPS signaling in the liver and reduce proinflammatory cytokine production, thereby preventing liver injury. Through experiments performed in vivo, using a water-soluble, less toxic hsp90-specific inhibitor, 17-DMAG, we show that hsp90 inhibition decreases proinflammatory cytokine production and alleviates LPS-induced liver injury. Previous limitations for in vivo use of geldanamycin and its derivatives have been their dose-limiting toxicity, leading to weight loss, hematologic, hepatic, and renal toxicity, and cell death.38, 39 Pharmacodynamic studies showed that the medium tolerated dose of 17-DMAG in vivo is 75 mg/kg with minimal toxicity.40, 41 Here, we used a single dose of 17-DMAG, ranging from 2.5 mg/kg to 30 mg/kg, with less concern for nonspecific or toxic effects. Our data here suggest hsp90 as an attractive therapeutic target in liver diseases.

Although inhibitors of hsp90 were primarily identified for their therapeutic importance in cancer, their role in inflammatory diseases,42 such as rheumatoid arthritis,28 endotoxin-mediated uveitis,18 sepsis,43 and atherosclerosis,44, 45 is emerging. Because LPS-mediated inflammatory responses are crucial to the development of liver diseases, strategies that prevent this response in the liver could have a beneficial effect. The inhibitory function of hsp90 inhibitors on liver inflammatory responses could be a dual mechanism: either loss of client proteins resulting from loss of chaperone function19, 21 or induction of anti-inflammatory transcription factor HSF1 and hsp70 expression.46 Here, we report that inhibition of hsp90 in the liver induces HSF1 and inhibits LPS-induced NFκB activation and proinflammatory cytokine production, thereby alleviating liver injury (Fig. 8). The chaperone function of hsp90 on LPS-signaling intermediates explains its effect on the expression of down-stream proinflammatory cytokines. In this context, 17-DMAG could affect the transcription of cytokine genes and their production. We observed that the proinflammatory cytokines, TNFα and IL-6, were significantly inhibited, both at the mRNA and protein level, in whole livers treated with 17-DMAG and LPS. Concomitant reduction of serum TNFα and IL-6 paralleled the liver cytokine profile. We predict that hsp90 inhibition in the liver alters LPS-signaling events proximal to proinflammatory cytokine gene transcription.

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Figure 8. Mechanism of proinflammatory cytokine inhibition and alleviation of LPS-induced liver injury by 17-DMAG. Inhibition of hsp90 by 17-DMAG induces HSF1 in the liver, which hinders the binding of NFκB to the promoter region of proinflammatory cytokine target genes, resulting in the reduction of LPS-induced proinflammatory cytokines and the prevention of liver injury.

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The transcription factor, NFκB, is a key downstream signaling intermediate of the LPS receptors, CD14 and TLR4. Earlier studies showed that 17-DMAG reduces NFκB activity in respiratory epithelial cells,47 either directly or through inhibition of upstream the LPS receptors, CD14 and TLR4.14 Hsp90 associates with the LPS receptor complex,14 and its inhibition decreases CD14 expression.48 Our results showed significant down-regulation in CD14 mRNA without changes in TLR4 mRNA in the liver. Furthermore, 17-DMAG significantly inhibited NFκB DNA binding, but did not affect NFκB-driven reporter activity. Interestingly, 17-DMAG had a profound effect on TNFα promoter-driven reporter activity, pointing to the likely involvement of other repressive transcription factors in 17-DMAG-mediated proinflammatory cytokine reduction in the liver.

Although inhibition of Hsp90 releases HSF1 from its inactive state to induce target gene expression,29, 30 HSF1 also negatively regulates the induction of proinflammatory cytokine genes.49-51 Consistent with previous findings,31 no change in hsp90 protein levels was observed after 17-DMAG treatment in the liver. On the other hand, hsp90 inhibition resulted in a significant up-regulation of HSF1 DNA binding and induction of hsp70 mRNA and protein in the liver, confirming the inhibition of hsp90 chaperone function. The repressive function of HSF1 on the transcription of proinflammatory cytokine gene TNFα in macrophages during exposure to febrile temperatures has been shown.51-53 The TNFα promoter is reported to have a binding site for HSF1.33 We postulated that activated HSF1 in the liver may serve as a repressor of TNFα gene induction during treatment with 17-DMAG. Using the ChIP assay, we showed the binding of HSF1 to the TNFα promoter in the presence of 17-DMAG treatment in macrophages. This observation correlates with elevated DNA-binding activity of HSF1 in response to hsp90 inhibition by 17-DMAG in the liver. Furthermore, whereas HSF1 bound to the hsp70 promoter, 17-DMAG treatment did not induce the binding of HSF1 to the IL-6 promoter, indicating an HSF1 indirect or independent down-regulation of IL-6 during 17-DMAG treatment. Previous studies showed that HSF1 indirectly negatively regulates the IL-6 promoter through the induction of ATF3.37 Our studies exhibited an up-regulation of LPS-induced ATF3 mRNA and protein in the liver during 17-DMAG treatment, suggesting that HSF1 negatively regulates IL-6, likely through ATF3 induction. Future studies will determine the role of ATF3 in 17-DMAG-treated macrophages and liver inflammatory responses. Finally, using HSF1 siRNA, we also confirmed the direct repressive role for HSF1 in TNFα inhibition and an indirect regulation of IL-6 in 17-DMAG-treated macrophages. Thus, HSF1 appears to play a significant role in the down-regulation of proinflammatory cytokine responses in the liver on treatment with 17-DMAG, a specific hsp90 inhibitor.

The clinical significance of our study is related to the emerging function of hsp90 as a potential therapeutic target in different diseases.18, 28, 43, 44, 54 Compelling approaches using hsp90 inhibitors in hepatocellular carcinoma41 and hepatitis C virus replication54, 55 have been reported. Our results here, for the first time, suggest a novel application for hsp90 inhibitor 17-DMAG in alleviating LPS-mediated liver injury, providing a solid basis for clinical investigations using hsp90 inhibitors in acute and chronic liver diseases. A significant role for hsp90 in chronic alcohol-mediated proinflammatory cytokine induction has been shown earlier.17 Future studies in our laboratory are under way to target hsp90 in liver diseases regulated by proinflammatory responses, such as ALD, NAFLD, and liver fibrosis.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

The authors thank Karen Kodys for labeling oligonucleotides for EMSA analysis.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
  • 1
    Nolan JP. The role of intestinal endotoxin in liver injury: a long and evolving history. HEPATOLOGY 2010; 52: 1829-1835.
  • 2
    Kudo H, Takahara T, Yata Y, Kawai K, Zhang W, Sugiyama T. Lipopolysaccharide triggered TNF-alpha-induced hepatocyte apoptosis in a murine non-alcoholic steatohepatitis model. J Hepatol 2009; 51: 168-175.
  • 3
    Yang SQ, Lin HZ, Lane MD, Clemens M, Diehl AM. Obesity increases sensitivity to endotoxin liver injury: implications for the pathogenesis of steatohepatitis. Proc Natl Acad Sci U S A 1997; 94: 2557-2562.
  • 4
    Enomoto N, Ikejima K, Bradford BU, Rivera CA, Kono H, Goto M, et al. Role of kupffer cells and gut-derived endotoxins in alcoholic liver injury. J Gastroenterol Hepatol 2000; 15( Suppl): D20-D25.
  • 5
    Uesugi T, Froh M, Arteel GE, Bradford BU, Wheeler MD, Gabele E, et al. Role of lipopolysaccharide-binding protein in early alcohol-induced liver injury in mice. J Immunol 2002; 168: 2963-2969.
  • 6
    Enomoto N, Schemmer P, Ikejima K, Takei Y, Sato N, Brenner DA, Thurman RG. Long-term alcohol exposure changes sensitivity of rat kupffer cells to lipopolysaccharide. Alcohol Clin Exp Res 2001; 25: 1360-1367.
  • 7
    Su GL, Hoesel LM, Bayliss J, Hemmila MR, Wang SC. Lipopolysaccharide binding protein inhibitory peptide protects against acetaminophen-induced hepatotoxicity. Am J Physiol Gastrointest Liver Physiol 2010; 299: G1319-G1325.
  • 8
    Colletti LM, Green M. Lung and liver injury following hepatic ischemia/reperfusion in the rat is increased by exogenous lipopolysaccharide which also increases hepatic TNF production in vivo and in vitro. Shock 2001; 16: 312-319.
  • 9
    Yu LX, Yan HX, Liu Q, Yang W, Wu HP, Dong W, et al. Endotoxin accumulation prevents carcinogen-induced apoptosis and promotes liver tumorigenesis in rodents. HEPATOLOGY 2010; 52: 1322-1333.
  • 10
    Uchikura K, Wada T, Hoshino S, Nagakawa Y, Aiko T, Bulkley GB, et al. Lipopolysaccharides induced increases in fas ligand expression by kupffer cells via mechanisms dependent on reactive oxygen species. Am J Physiol Gastrointest Liver Physiol 2004; 287: G620-G626.
  • 11
    Lu Y, Cederbaum AI. CYP2E1 potentiation of LPS and TNFalpha-induced hepatotoxicity by mechanisms involving enhanced oxidative and nitrosative stress, activation of MAP kinases, and mitochondrial dysfunction. Genes Nutr 2010; 5: 149-167.
  • 12
    Szabo G, Bala S. Alcoholic liver disease and the gut-liver axis. World J Gastroenterol 2010; 16: 1321-1329.
  • 13
    Mandrekar P. Signaling mechanisms in alcoholic liver injury: role of transcription factors, kinases, and heat shock proteins. World J Gastroenterol 2007; 13: 4979-4985.
  • 14
    Triantafilou M, Triantafilou K. Heat-shock protein 70 and heat-shock protein 90 associate with toll-like receptor 4 in response to bacterial lipopolysaccharide. Biochem Soc Trans 2004; 32: 636-639.
  • 15
    Hsu HY, Wu HL, Tan SK, Li VP, Wang WT, Hsu J, Cheng CH. Geldanamycin interferes with the 90-kDa heat shock protein, affecting lipopolysaccharide-mediated interleukin-1 expression and apoptosis within macrophages. Mol Pharmacol 2007; 71: 344-356.
  • 16
    Triantafilou K, Triantafilou M, Ladha S, Mackie A, Dedrick RL, Fernandez N, Cherry R. Fluorescence recovery after photobleaching reveals that LPS rapidly transfers from CD14 to hsp70 and hsp90 on the cell membrane. J Cell Sci 2001; 114: 2535-2545.
  • 17
    Mandrekar P, Catalano D, Jeliazkova V, Kodys K. Alcohol exposure regulates heat shock transcription factor binding and heat shock proteins 70 and 90 in monocytes and macrophages: implication for TNF-alpha regulation. J Leukoc Biol 2008; 84: 1335-1345.
  • 18
    Poulaki V, Iliaki E, Mitsiades N, Mitsiades CS, Paulus YN, Bula DV, et al. Inhibition of Hsp90 attenuates inflammation in endotoxin-induced uveitis. FASEB J 2007; 21: 2113-2123.
  • 19
    Salminen A, Paimela T, Suuronen T, Kaarniranta K. Innate immunity meets with cellular stress at the IKK complex: regulation of the IKK complex by HSP70 and HSP90. Immunol Lett 2008; 117: 9-15.
  • 20
    Joly AL, Wettstein G, Mignot G, Ghiringhelli F, Garrido C. Dual role of heat shock proteins as regulators of apoptosis and innate immunity. J Innate Immun 2010; 2: 238-247.
  • 21
    Broemer M, Krappmann D, Scheidereit C. Requirement of Hsp90 activity for IkappaB kinase (IKK) biosynthesis and for constitutive and inducible IKK and NF-kappaB activation. Oncogene 2004; 23: 5378-5386.
  • 22
    De Nardo D, Masendycz P, Ho S, Cross M, Fleetwood AJ, Reynolds EC, et al. A central role for the Hsp90.Cdc37 molecular chaperone module in interleukin-1 receptor-associated-kinase-dependent signaling by toll-like receptors. J Biol Chem 2005; 280: 9813-9822.
  • 23
    Bandyopadhyay S, Chiang CY, Srivastava J, Gersten M, White S, Bell R, et al. A human MAP kinase interactome. Nat Methods 2010; 7: 801-805.
  • 24
    Pacey S, Wilson RH, Walton M, Eatock MM, Hardcastle A, Zetterlund A, et al. A phase I study of the heat shock protein 90 inhibitor alvespimycin (17-DMAG) given intravenously to patients with advanced solid tumors. Clin Cancer Res 2011; 17: 1561-1570.
  • 25
    Richardson PG, Mitsiades CS, Laubach JP, Lonial S, Chanan-Khan AA, Anderson KC. Inhibition of heat shock protein 90 (HSP90) as a therapeutic strategy for the treatment of myeloma and other cancers. Br J Haematol 2011; 152: 367-379.
  • 26
    Sedlackova L, Spacek M, Holler E, Imryskova Z, Hromadnikova I. Heat-shock protein expression in leukemia. Tumour Biol 2011; 32: 33-44.
  • 27
    Allegra A, Sant'antonio E, Penna G, Alonci A, D'Angelo A, Russo S, et al. Novel therapeutic strategies in multiple myeloma: role of the heat shock protein inhibitors. Eur J Haematol 2011; 86: 93-110.
  • 28
    Rice JW, Veal JM, Fadden RP, Barabasz AF, Partridge JM, Barta TE, et al. Small molecule inhibitors of Hsp90 potently affect inflammatory disease pathways and exhibit activity in models of rheumatoid arthritis. Arthritis Rheum 2008; 58: 3765-3775.
  • 29
    Conde R, Belak ZR, Nair M, O'Carroll RF, Ovsenek N. Modulation of Hsf1 activity by novobiocin and geldanamycin. Biochem Cell Biol 2009; 87: 845-851.
  • 30
    Kim HR, Kang HS, Kim HD. Geldanamycin induces heat shock protein expression through activation of HSF1 in K562 erythroleukemic cells. IUBMB Life 1999; 48: 429-433.
  • 31
    Roe SM, Prodromou C, O'Brien R, Ladbury JE, Piper PW, Pearl LH. Structural basis for inhibition of the Hsp90 molecular chaperone by the antitumor antibiotics radicicol and geldanamycin. J Med Chem 1999; 42: 260-266.
  • 32
    Guzhova IV, Darieva ZA, Melo AR, Margulis BA. Major stress protein Hsp70 interacts with NF-kB regulatory complex in human T-lymphoma cells. Cell Stress Chaperones 1997; 2: 132-139.
  • 33
    Singh IS, He JR, Hester L, Fenton MJ, Hasday JD. Bacterial endotoxin modifies heat shock factor-1 activity in RAW 264.7 cells: implications for TNF-alpha regulation during exposure to febrile range temperatures. J Endotoxin Res 2004; 10: 175-184.
  • 34
    Inouye S, Fujimoto M, Nakamura T, Takaki E, Hayashida N, Hai T, Nakai A. Heat shock transcription factor 1 opens chromatin structure of interleukin-6 promoter to facilitate binding of an activator or a repressor. J Biol Chem 2007; 282: 33210-33217.
  • 35
    Jacobs AT, Marnett LJ. HSF1-mediated BAG3 expression attenuates apoptosis in 4-hydroxynonenal-treated colon cancer cells via stabilization of anti-apoptotic bcl-2 proteins. J Biol Chem 2009; 284: 9176-9183.
  • 36
    Singh IS, Viscardi RM, Kalvakolanu I, Calderwood S, Hasday JD. Inhibition of tumor necrosis factor-alpha transcription in macrophages exposed to febrile range temperature. A possible role for heat shock factor-1 as a negative transcriptional regulator. J Biol Chem 2000; 275: 9841-9848.
  • 37
    Takii R, Inouye S, Fujimoto M, Nakamura T, Shinkawa T, Prakasam R, et al. Heat shock transcription factor 1 inhibits expression of IL-6 through activating transcription factor 3. J Immunol 2010; 184: 1041-1048.
  • 38
    Solit DB, Zheng FF, Drobnjak M, Munster PN, Higgins B, Verbel D, et al. 17-allylamino-17-demethoxygeldanamycin induces the degradation of androgen receptor and HER-2/neu and inhibits the growth of prostate cancer xenografts. Clin Cancer Res 2002; 8: 986-993.
  • 39
    Hollingshead M, Alley M, Burger AM, Borgel S, Pacula-Cox C, Fiebig HH, Sausville EA. In vivo antitumor efficacy of 17-DMAG (17-dimethylaminoethylamino-17-demethoxygeldanamycin hydrochloride), a water-soluble geldanamycin derivative. Cancer Chemother Pharmacol 2005; 56: 115-125.
  • 40
    Egorin MJ, Lagattuta TF, Hamburger DR, Covey JM, White KD, Musser SM, Eiseman JL. Pharmacokinetics, tissue distribution, and metabolism of 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (NSC 707545) in CD2F1 mice and fischer 344 rats. Cancer Chemother Pharmacol 2002; 49: 7-19.
  • 41
    Breinig M, Caldas-Lopes E, Goeppert B, Malz M, Rieker R, Bergmann F, et al. Targeting heat shock protein 90 with non-quinone inhibitors: a novel chemotherapeutic approach in human hepatocellular carcinoma. HEPATOLOGY 2009; 50: 102-112.
  • 42
    Yun TJ, Harning EK, Giza K, Rabah D, Li P, Arndt JW, et al. EC144, a synthetic inhibitor of heat shock protein 90, blocks innate and adaptive immune responses in models of inflammation and autoimmunity. J Immunol 2011; 186: 563-575.
  • 43
    Chatterjee A, Dimitropoulou C, Drakopanayiotakis F, Antonova G, Snead C, Cannon J, et al. Heat shock protein 90 inhibitors prolong survival, attenuate inflammation, and reduce lung injury in murine sepsis. Am J Respir Crit Care Med 2007; 176: 667-675.
  • 44
    Madrigal-Matute J, Lopez-Franco O, Blanco-Colio LM, Munoz-Garcia B, Ramos-Mozo P, Ortega L, et al. Heat shock protein 90 inhibitors attenuate inflammatory responses in atherosclerosis. Cardiovasc Res 2010; 86: 330-337.
  • 45
    Brouet A, Sonveaux P, Dessy C, Moniotte S, Balligand JL, Feron O. Hsp90 and caveolin are key targets for the proangiogenic nitric oxide-mediated effects of statins. Circ Res 2001; 89: 866-873.
  • 46
    Bagatell R, Paine-Murrieta GD, Taylor CW, Pulcini EJ, Akinaga S, Benjamin IJ, Whitesell L. Induction of a heat shock factor 1-dependent stress response alters the cytotoxic activity of hsp90-binding agents. Clin Cancer Res 2000; 6: 3312-3318.
  • 47
    Malhotra V, Shanley TP, Pittet JF, Welch WJ, Wong HR. Geldanamycin inhibits NF-kappaB activation and interleukin-8 gene expression in cultured human respiratory epithelium. Am J Respir Cell Mol Biol 2001; 25: 92-97.
  • 48
    Vega VL, De Maio A. Geldanamycin treatment ameliorates the response to LPS in murine macrophages by decreasing CD14 surface expression. Mol Biol Cell 2003; 14: 764-773.
  • 49
    Singh IS, Gupta A, Nagarsekar A, Cooper Z, Manka C, Hester L, et al. Heat shock co-activates interleukin-8 transcription. Am J Respir Cell Mol Biol 2008; 39: 235-242.
  • 50
    Xie Y, Chen C, Stevenson MA, Auron PE, Calderwood SK. Heat shock factor 1 represses transcription of the IL-1beta gene through physical interaction with the nuclear factor of interleukin 6. J Biol Chem 2002; 277: 11802-11810.
  • 51
    Cooper ZA, Singh IS, Hasday JD. Febrile range temperature represses TNF-alpha gene expression in LPS-stimulated macrophages by selectively blocking recruitment of Sp1 to the TNF-alpha promoter. Cell Stress Chaperones 2010; 15: 665-673.
  • 52
    Xiao X, Zuo X, Davis AA, McMillan DR, Curry BB, Richardson JA, Benjamin IJ. HSF1 is required for extra-embryonic development, postnatal growth, and protection during inflammatory responses in mice. EMBO J 1999; 18: 5943-5952.
  • 53
    Cooper ZA, Ghosh A, Gupta A, Maity T, Benjamin IJ, Vogel SN, et al. Febrile-range temperature modifies cytokine gene expression in LPS-stimulated macrophages by differentially modifying NF-{kappa}B recruitment to cytokine gene promoters. Am J Physiol Cell Physiol 2010; 298: C171-C181.
  • 54
    Nakagawa S, Umehara T, Matsuda C, Kuge S, Sudoh M, Kohara M. Hsp90 inhibitors suppress HCV replication in replicon cells and humanized liver mice. Biochem Biophys Res Commun 2007; 353: 882-888.
  • 55
    Ujino S, Yamaguchi S, Shimotohno K, Takaku H. Heat-shock protein 90 is essential for stabilization of the hepatitis C virus nonstructural protein NS3. J Biol Chem 2009; 284: 6841-6846.
  • 56
    Singh IS, He JR, Calderwood S, Hasday JD. A high affinity HSF-1 binding site in the 5′-untranslated region of the murine tumor necrosis factor-alpha gene is a transcriptional repressor. J Biol Chem 2002; 277: 4981-4988.

Supporting Information

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

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