HSP-70 mitigates LPS/SKI-induced cell damage by increasing sphingosine kinase 1 (SK1).
Heat shock proteins (HSPs) are potent protectors of cellular integrity against environmental stresses, including toxic microbial products. To investigate the mechanism of HSP-70 cell protection against bacterial lipopolysaccharide (LPS), we established a stable HSP-70 gene-transfected RAW 264.7 murine macrophage model of LPS-induced cell death. Bacterial LPS increases the activity of sphingosine kinase 1 (SK1), which catalyzes formation of sphingosine-1-phosphate (S1P). S1P functions as a critical signal for initiation and maintenance of diverse aspects of immune cell activation and function. When mouse macrophages were incubated with Escherichia coli LPS (1 microg/ml) and sphingosine kinase inhibitor (SKI, 5 microM), 90% of cells died. Neither LPS nor SKI alone at these doses damaged the cells. The LPS/SKI-induced cell death was partially reversed by overexpression of HSP-70 in gene-transfected macrophages. The specificity of HSP-70 in this reversal was demonstrated by transfection of HSP-70-specific siRNA. Down-regulation of HSP-70 expression after transfection of siRNA specific for HSP-70 was associated with increased LPS/SKI-induced cell damage. Overexpression of human or murine HSP-70 (HSPA1A and Hspa1a, respectively) increased both cellular SK1 mRNA and protein levels. Cellular heat shock also increased SK1 protein. These studies confirm the importance of SK1 as a protective moiety in LPS-induced cell injury and demonstrate that HSP-70-mediated protection from cells treated with LPS/SKI is accompanied by upregulating expression of SK1. HSP-70-mediated increases in SK1 and consequent increased levels of S1P may also play a role in protection of cells from other processes that lead to programmed cell death.
Ding
Xuan Z
XZ
Division of Bacterial & Rickettsial Diseases, Walter Reed Army Institute of Research, Silver Spring, MD 20910, USA. tom.ding@amedd.army.mil
Feng
Xiao R
XR
Borschel
Richard H
RH
Nikolich
Mikeljon P
MP
Feng
Jie
J
Li
Yan S
YS
Hoover
David L
DL
D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D005786:Gene Expression Regulation / Q000187:drug effects; D055785:Gene Knockdown Techniques; D018840:HSP70 Heat-Shock Proteins / Q000172:deficiency / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008264:Macrophages / Q000166:cytology* / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D051379:Mice; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D018805:Sepsis / Q000473:pathology; D014162:Transfection; D015854:Up-Regulation / Q000187:drug effects; D000099157:Sphingosine Kinase
D018840:HSP70 Heat-Shock Proteins; D008070:Lipopolysaccharides; D017853:Phosphotransferases (Alcohol Group Acceptor); D012333:RNA, Messenger; D034741:RNA, Small Interfering; D000099157:Sphingosine Kinase