Geldanamycin treatment ameliorates the response to LPS in murine macrophages by decreasing CD14 surface expression.
Geldanamycin (GA) is an antibiotic produced by Actinomyces, which specifically inhibits the function of the heat shock protein 90 family. Treatment of a murine macrophage cell line (J774) with GA resulted in a reduced response to Escherichia coli lipopolysaccharide (LPS) as visualized by a decrease of NF-kappaB translocation into the nucleus and secretion of tumor necrosis factor alpha (TNF-alpha). To elucidate the mechanism of this effect, the expression of CD14, the formal LPS receptor, was analyzed. Cells treated with GA showed a reduced level of surface CD14 detected by immunostaining, whereas the expression of other surface receptors, such as FC-gamma receptor and tumor necrosis factor receptors (TNF-R1 and TNF-R2), was unaffected. The reduced surface level of CD14 was not due to a reduction in its expression because CD14 steady state mRNA levels or the total cellular pool of CD14 was not altered by GA treatment. Surface CD14 was more rapidly internalized after GA treatment (2-3 h) than after incubation with cycloheximide. Immunostaining of permeabilized cells after GA treatment revealed a higher intracellular content of CD14 colocalizing with calnexin, an endoplasmic reticulum (ER) protein. These results suggest that the decrease in CD14 surface expression after GA treatment is due to rapid internalization without new replacement. These effects may be due to the inhibition of Hsp90 and Grp94 by GA in macrophages.
Vega
Virginia L
VL
Division of Pediatric Surgery and Department of Physiology, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
De Maio
Antonio
A
D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D016227:Benzoquinones; D015152:Blotting, Northern; D015153:Blotting, Western; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D003513:Cycloheximide / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D018840:HSP70 Heat-Shock Proteins / Q000378:metabolism; D018841:HSP90 Heat-Shock Proteins / Q000378:metabolism; D047029:Lactams, Macrocyclic; D018950:Lipopolysaccharide Receptors / Q000096:biosynthesis*; D008070:Lipopolysaccharides / Q000378:metabolism*; D008264:Macrophages / Q000378:metabolism*; D008565:Membrane Proteins / Q000378:metabolism; D051379:Mice; D016328:NF-kappa B / Q000378:metabolism; D011500:Protein Synthesis Inhibitors / Q000494:pharmacology; D021381:Protein Transport; D011809:Quinones / Q000494:pharmacology*; D012333:RNA, Messenger / Q000378:metabolism; D013997:Time Factors
D000903:Antibiotics, Antineoplastic; D016227:Benzoquinones; D018840:HSP70 Heat-Shock Proteins; D018841:HSP90 Heat-Shock Proteins; D047029:Lactams, Macrocyclic; D018950:Lipopolysaccharide Receptors; D008070:Lipopolysaccharides; D008565:Membrane Proteins; D016328:NF-kappa B; D011500:Protein Synthesis Inhibitors; D011809:Quinones; D012333:RNA, Messenger; C018551:glucose-regulated proteins; D003513:Cycloheximide; C001277:geldanamycin