monorden and Inflammation

Pyroptosis is a recently discovered inflammatory form of programmed cell death which is mostly triggered by infection with intracellular pathogens and critically contributes to inflammation. Mitigating pyroptosis may be a potential therapeutic target in inflammatory diseases. However, small chemicals to reduce pyroptosis is still elusive. In the present study, we screened 155 chemicals from a microbial natural product library and found Geldanamycin, an HSP90 inhibitor, profoundly rescued THP-1 cells from pyroptosis induced by LPS plus Nigericin treatment. Consistently, other HSP90 inhibitors, including Radicicol, 17-DMAG and 17-AAG, all ameliorated pyroptosis in THP-1 cells by suppressing the inflammasome/Caspase-1/GSDMD signal pathway in pyroptosis. HSP90 inhibition compromised the protein stability of NLRP3, a critical component of the inflammasome. Moreover, up-regulated HSP70 may also contribute to this effect. HSP90 inhibition may thus be a potential therapeutic strategy in the treatment of inflammatory diseases in which pyroptosis plays a role.

Topics: Benzoquinones; Caspase 1; Cell Survival; HSP72 Heat-Shock Proteins; HSP90 Heat-Shock Proteins; Humans; Inflammasomes; Inflammation; Intracellular Signaling Peptides and Proteins; Lactams, Macrocyclic; Lipopolysaccharides; Macrolides; Nigericin; NLR Family, Pyrin Domain-Containing 3 Protein; Phosphate-Binding Proteins; Proteasome Endopeptidase Complex; Protein Stability; Pyroptosis; Signal Transduction; THP-1 Cells; Up-Regulation

Severe sepsis is the leading cause of death for patients in intensive care units. Patients with severe sepsis develop multiple organ failure, including acute lung injury (ALI), resulting from a deregulated inflammatory response. Inhibitors of the ubiquitous chaperone, heat shock protein 90 (Hsp90), block the activity of certain proinflammatory mediators in vitro. We hypothesized that Hsp90 inhibitors may ameliorate the inflammation and ALI associated with severe sepsis.. To test the hypothesis that Hsp90 inhibitors prolong survival, attenuate inflammation, and reduce lung injury in a murine model of sepsis.. Male C57BL/6 mice received either one of two Hsp90 inhibitors, radicicol or 17-allylaminodemethoxygeldanamycin (17-AAG), 24, 12, 6, and 0 hours before receiving a lethal dose of endotoxin (6.75 x 10(4) endotoxin units/g body weight). Outcomes included survival and parameters of systemic inflammation (plasma neutrophil, cytokine, chemokine, and nitrite/nitrate levels), pulmonary inflammation (lung nuclear factor-kappaB and myeloperoxidase activities, inducible nitric oxide synthase expression, inducible nitric oxide synthase-Hsp90 complex formation, and leukocyte infiltration), and lung injury (pulmonary capillary leak and lung function).. Mice pretreated with vehicle and receiving endotoxin exhibited 100% 24-hour lethality, a dramatic increase in all parameters of systemic and pulmonary inflammation, increased capillary leak, and reduced lung function. Compared with them, mice receiving either radicicol or 17-AAG before endotoxin exhibited prolonged survival, reduced or abolished increases in systemic and pulmonary inflammatory parameters, attenuated capillary leak, and restored, normal lung function.. Hsp90 inhibitors may offer a new pharmacological tool in the management of severe sepsis and severe sepsis-induced ALI.

Topics: Animals; Benzoquinones; Chemokines; Cytokines; HSP90 Heat-Shock Proteins; Inflammation; Inflammation Mediators; Lactams, Macrocyclic; Lung; Macrolides; Male; Mice; Mice, Inbred C57BL; NF-kappa B; Nitrates; Nitric Oxide Synthase Type II; Nitrites; Peroxidase; Respiratory Distress Syndrome; Sepsis; Survival Rate

The molecular chaperone Hsp90 affects the function and fate of a number of signaling molecules. We have investigated the Hsp90 requirement for constitutive and inducible activity of the IkappaB kinase (IKK) complex and of NF-kappaB. Inhibition by the Hsp90 ATPase inhibitors, geldanamycin (GA) and radicicol (RC), revealed that Hsp90 controls IKKs at two levels, inducibility of enzymatic activity and biogenesis, which can be discriminated by short- and long-time GA incubation, respectively. Short-time inhibition of Hsp90 resulted in impaired IKK kinase activation by TNFalpha, IL-1beta or phorbolester PMA. Furthermore, GA inhibited constitutive activation of IKK and NF-kappaB in Hodgkin's lymphoma cells. Hsp90 function was also required for trans- and autophosphorylation of transfected IKKbeta. GA exposure for several hours resulted in a downmodulation of IKK complex alpha, beta and gamma subunits to various extent. Proteasome inhibition interfered with GA mediated IKK depletion and Hsp90 inhibition induced polyubiquitination of IKKalpha and beta during protein synthesis. In fact, GA blocked biogenesis of IKKalpha and IKKbeta but did not interfere with post-translational turnover. Together, these results define a dual requirement for Hsp90 as a regulator of NF-kappaB signaling by its general involvement in IKK activation and by its role in IKK homeostasis.

Topics: Animals; Benzoquinones; Cell Line; Cell Line, Tumor; COS Cells; Dose-Response Relationship, Drug; Enzyme Inhibitors; HeLa Cells; HSP90 Heat-Shock Proteins; Humans; I-kappa B Kinase; Inflammation; Interleukin-1; Lactams, Macrocyclic; Lactones; Lymphoma; Macrolides; Molecular Chaperones; NF-kappa B; Phosphorylation; Precipitin Tests; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Quinones; Signal Transduction; Tetradecanoylphorbol Acetate; Time Factors; Transfection; Tumor Necrosis Factor-alpha; Ubiquitin

Two isoforms of cyclooxygenase (COX) have been identified in eukaryotic cells: a constitutively expressed COX-1 and mitogen-inducible COX-2, which is selectively expressed in response to various inflammatory stimuli. Thus, COX-2 instead of COX-1 is implicated to produce prostanoids mediating inflammatory responses. Major efforts have been focused on identifying nonsteroidal anti-inflammatory drugs (NSAIDS) which can selectively inhibit the enzyme activity of COX-2. Such NSAIDS would be more desirable anti-inflammatory agents in comparison to NSAIDS which inhibit both COX-1 and COX-2. Other than glucocorticoids, pharmacological agents which can selectively suppress the expression of COX-2 without affecting that of COX-1 have not been identified. We report here that radicicol, a fungal antibiotic, is a potent protein tyrosine kinase inhibitor, and that it inhibits the expression of COX-2 without affecting COX-1 expression in lipopolysaccharide (LPS)-stimulated macrophages with the IC50 value of 27 nM. Radicicol inhibited tyrosine phosphorylation of p53/56lyn, a Src family tyrosine kinase and one of the major tyrosine-phosphorylated proteins in LPS-stimulated macrophages. Radicicol also inhibited COX-2 expression in vivo in glomeruli of rats with experimental glomerulonephritis induced by the anti-glomerular basement membrane antibodies, in which COX-2 expression is known to be enhanced. The enzyme activity of COX-1 or COX-2 was not affected by radicicol in macrophages. Radiciciol also suppressed the COX-2 expression induced by IL-1 beta in rat smooth muscle cells. Other protein tyrosine kinase inhibitors suppressed the LPS-induced COX-2 expression in macrophages but at much higher concentrations than needed for radicicol. Radicicol did not inhibit the COX-2 expression induced by phorbol 12-myristate 13-acetate in macrophages. These results suggest that the activation of tyrosine-specific protein kinases is the proximal obligatory step in the LPS-induced signal transduction pathway leading to the induction of COX-2 expression in macrophages. The magnitude of the inhibition of COX-2 protein synthesis by radicicol was much greater than that of the steady state levels of COX-2 mRNA. These results suggest that radicicol inhibits COX-2 expression mainly at post-transcriptional steps.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antibodies; Antifungal Agents; Basement Membrane; Blotting, Western; Gene Expression; Glomerulonephritis; Glyceraldehyde-3-Phosphate Dehydrogenases; Inflammation; Isoenzymes; Kidney Glomerulus; Kinetics; Lactones; Lipopolysaccharides; Macrolides; Macrophages, Alveolar; Mitogens; Molecular Structure; Prostaglandin-Endoperoxide Synthases; Protein-Tyrosine Kinases; Rats; Rats, Sprague-Dawley; RNA, Messenger; Tetradecanoylphorbol Acetate; Time Factors