Interferon-gamma induces the synthesis and activation of cytosolic phospholipase A2.
Both IFN-alpha/beta and IFN-gamma have recently been demonstrated to induce a rapid but transient activation of phospholipase A2 (PLA2) in BALB/c 3T3 fibroblasts and a human neuroblastoma cell line. We report that IFN-gamma induces the synthesis and prolonged activation of cytosolic phospholipase A2 (cPLA2) in a human bronchial epithelial cell line (BEAS 2B). Treatment of the cells with IFN-gamma (300 U/ml) increased the release of [3H]arachidonic acid (AA) from prelabeled cells with a maximal effect at 12 h after stimulation. The increased [3H]AA release was inhibited by the PLA2 inhibitor p-bromophenacyl bromide (10(-5) M). Calcium ionophore A23187 (10(-5) M) further increased the [3H]AA release from the IFN-gamma-treated cells. Subcellular enzyme activity assay revealed that IFN-gamma increased PLA2 activity in both the cytosol and membrane fractions with a translocation of the cPLA2 to cell membranes in a Ca(2+)-free cell lysing buffer. Treatment with IFN-gamma also induced the release of 15-HETE, an arachidonic acid metabolite. Immunoblot showed that IFN-gamma induced the synthesis of cPLA2 protein. Nuclear run-on assay demonstrated that IFN-gamma initiated cPLA2 gene transcription within 15 min, and this effect was sustained at 4 h and returned to near control level at 12 h. The cPLA2 mRNA level was assayed by reverse transcription and PCR. IFN-gamma was found to increase the cPLA2 mRNA after 2-24 h treatment. Furthermore, the IFN-gamma induced cPLA2 mRNA increase was blocked by inhibitors of protein kinase C and calcium/calmodulin-dependent protein kinases, suggesting the involvement of these protein kinases in IFN-gamma-induced gene expression of cPLA2. This study shows that IFN-gamma induces the synthesis and prolonged activation of cPLA2.
Wu
T
T
Critical Care Medicine Department, National Institutes of Health, Bethesda, Maryland 20892.
Levine
S J
SJ
Lawrence
M G
MG
Logun
C
C
Angus
C W
CW
Shelhamer
J H
JH
D000098:Acetophenones / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism; D001483:Base Sequence; D001980:Bronchi; D000001:Calcimycin / Q000494:pharmacology; D002460:Cell Line; D002462:Cell Membrane / Q000201:enzymology; D002467:Cell Nucleus / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology; D017931:DNA Primers; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D006893:Hydroxyeicosatetraenoic Acids / Q000378:metabolism; D007371:Interferon-gamma / Q000494:pharmacology*; D007700:Kinetics; D008969:Molecular Sequence Data; D010741:Phospholipases A / Q000037:antagonists & inhibitors / Q000096:biosynthesis* / Q000302:isolation & purification / Q000378:metabolism; D054467:Phospholipases A2; D016133:Polymerase Chain Reaction; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D012333:RNA, Messenger / Q000096:biosynthesis; D014158:Transcription, Genetic
D000098:Acetophenones; D017931:DNA Primers; D006893:Hydroxyeicosatetraenoic Acids; D012333:RNA, Messenger; D016718:Arachidonic Acid; D000001:Calcimycin; C025984:15-hydroxy-5,8,11,13-eicosatetraenoic acid; D007371:Interferon-gamma; D010741:Phospholipases A; D054467:Phospholipases A2; C006821:4-bromophenacyl bromide