The serine/threonine phosphatase inhibitor, calyculin A, inhibits and dissociates macrophage responses to lipopolysaccharide.
LPS-stimulated macrophages (M phi) produce inflammatory mediators that are largely responsible for the pathophysiology associated with septic shock. M phi respond to LPS with rapid protein phosphorylation and dephosphorylation on serine, threonine, and tyrosine residues. If these events are critical for the cellular response to LPS, the kinases and/or phosphatases involved may be vulnerable targets for pharmacologic intervention. Recent studies demonstrated that tyrosine kinase inhibitors block LPS-induced tyrosine phosphorylation of MAP kinases as well as TNF-alpha and IL-1 beta production. To investigate a role for serine/threonine phosphatases, we evaluated the effect of calyculin A, a potent serine/threonine phosphatase inhibitor, on LPS stimulation of murine M phi. Pretreatment of M phi with calyculin A inhibited LPS-induced expression of six immediate-early genes: TNF-alpha, IL-1 beta, IFN-beta, IP-10, IRF-1, and TNFR-2. Calyculin A added 1.5 h after LPS treatment greatly reduced accumulation of IP-10, IRF-1, and TNFR-2 mRNA, but not TNF-alpha, IL-1 beta, and IFN-beta mRNA. Calyculin A, in the absence or presence of LPS, resulted in sustained tyrosine phosphorylation of the MAP kinases. These findings suggest that an "early" serine/threonine phosphatase activity is essential for LPS stimulation of M phi and that the activation of MAP kinases is not sufficient for the induction of these immediate-early genes. The requirement for a "late" phosphatase activity for expression of a subset of LPS-inducible genes dissociates at least two regulatory pathways in LPS signal transduction.
Barber
S A
SA
Department of Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, USA.
Perera
P Y
PY
McNally
R
R
Vogel
S N
SN
D000818:Animals; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D016207:Cytokines / Q000096:biosynthesis / Q000235:genetics; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects*; D017781:Genes, Immediate-Early / Q000187:drug effects; D008070:Lipopolysaccharides / Q000494:pharmacology*; D017737:Macrophages, Peritoneal / Q000187:drug effects*; D008387:Marine Toxins; D051379:Mice; D008809:Mice, Inbred C3H; D010080:Oxazoles / Q000494:pharmacology*; D010749:Phosphoprotein Phosphatases / Q000037:antagonists & inhibitors* / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D015398:Signal Transduction / Q000187:drug effects*
D016207:Cytokines; D008070:Lipopolysaccharides; D008387:Marine Toxins; D010080:Oxazoles; C059041:calyculin A; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases