Abstract
We have demonstrated that lipopolysaccharide (LPS)-mediated reactive oxygen species (ROS) and signal transduction are involved in the regulation of interleukin-1 (IL-1) β gene expression within macrophages. Because the 90-kDa heat shock protein (Hsp90) plays an important role in the LPS mediation of macrophage activation, using Hsp90 inhibitor geldanamycin A (GA), we analyzed the mechanism of Hsp90 upon LPS-transduced signaling in the regulation of IL-1 expression and determined the function of Hsp90 regarding the viability of human primary macrophages and murine macrophages cell line. In essence, GA decreased LPS-induced Hsp90/pp60Src heterocomplex formation. In addition, Hsp90 is important for IL-1 protein translation, plays a minor role in IL-1 mRNA transcription, and is involved in nuclear factor-κB activation and the phosphorylation and activation of p38, c-Jun NH2-terminal kinase, and extracellular signal-regulated kinase; however, Hsp90 plays a more important role in LPS-stimulated p38 activation. In analyzing the function of Hsp90 regarding the cytotoxicity/viability of macrophages, we found that the combination of LPS and GA increases apoptosis, as evidenced by the increased caspase-3 activity and the proportion of nuclear/chromatin condensation. In contrast, N-acetyl-cysteine dramatically blocked GA/LPS-induced ROS production, simultaneously decreasing caspase-3 activity and the presence of apoptotic nuclei. We concluded that Hsp90 plays an indispensable role in the process of LPS-induced IL-1 secretion. Furthermore, we established the mechanism of GA interference with Hsp90 function for LPS-stimulated macrophages, resulting in increased ROS production and caspase-3 activation, and consequently leading to synergistic enhancement of macrophage apoptosis.
Footnotes
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This work was supported by National Science Council (NSC), Taiwan (NSC 94-2120-M-010-002 and NSC 93-2314-B-010-003 to H.-Y.H.), National Health Research Institutes, Taiwan (NHRI-EX94-9211SI to H.-Y.H.), NSC 95-2752-B-006-003-PAE (to H.-L.W.), and a grant from the Ministry of Education, Aim for the Top University Plan (95A-C-D01-PPG-10 to H.-Y.H.).
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H.-Y.H. and H.-L.W. contributed equally to this work.
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ABBREVIATIONS: LPS, lipopolysaccharide; Hsp90, 90-kDa heat shock protein; TLR, toll-like receptor; IRAK-1, interleukin-1 receptor-associated kinase-1; GA, geldanamycin A; ROS, reactive oxygen species; IL-1, interleukin-1β; pro-IL-1, prointerleukin-1β; NAC, N-acetyl-cysteine; NF-κB, nuclear factor-κB; MAPK, mitogen-activated protein kinase; ERK, extracellular signal-regulated kinase; JNK, c-Jun NH2-terminal protein kinase; p38, p38 mitogen-activated protein kinase; ATF, activating transcription factor; TNF, tumor necrosis factor; FBS, fetal bovine serum; PBMC, peripheral blood mononuclear cell; PBS, phosphate-buffered saline; CTR, control; PMSF, phenylmethylsulfonyl fluoride; HA, herbimycin A; DAPI, 4′,6-diamidino-2-phenylindole; RT-PCR, reverse transcription-polymerase chain reaction; bp, base pair; HRP, horseradish peroxidase; GAPDH, glyceraldehyde phosphate dehydrogenase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium; PAGE, polyacrylamide gel electrophoresis; DCFH, 2′,7′-dichlorofluorescein diacetate; Src, pp60Src; Ac-DEVD-AMC, N-acetyl-Asp-Glu-Val-Asp-amino-4-methylcoumarin; CM-DCFH, carboxyl-2′,7′-dichlorofluorescein diacetate.
- Received March 7, 2006.
- Accepted July 6, 2006.
- The American Society for Pharmacology and Experimental Therapeutics
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