Regulation of complement 5a receptor expression in U937 cells by phorbol ester.
Receptors for the anaphylactic portion of complement, C5a, are not initially expressed in the monoblastic U937 cell line, but appear as the cell is induced to differentiate by the synergistic actions of 1,25(OH)2D and cyclic adenosine monophosphate (cAMP). Phorbol myristate acetate (PMA), which activates the protein kinase C pathway (PKC), does not cause C5a receptor (C5aR) expression when used as a single agent. The induction of C5aR by the synergistic actions of 1,25(OH)2D and cAMP, however, can be augmented as much as 180% by the addition of PMA. C5aR arising in cells exposed to 1,25(OH)2D and 8,4-chlorophenylthio-cAMP have an affinity constant of about 0.4 nM as assessed by cold competition analysis. We show here that when phorbol augmentation of receptor number occurs, the affinity constant is increased by 3.6-fold. In an effort to ascertain whether the change in C5aR Kd involved a PKC-dependent event we examined whether 5-60 min exposure of C5aR-positive cells to PMA would change C5aR Kd. Acutely, PMA caused a downregulation of receptor binding with decreases in apparent receptor number out of proportion to changes in Kd. One hundred nanomolar PMA, which effects nearly complete translocation of PKC to the membrane, consistently caused a 70-90% decrease in C5a surface binding. This downregulation was proportional to PMA dose and exposure time. Micromolar concentrations of the microtubule depolymerizing agents colchicine and vinblastine caused a less drastic downregulation, about 50% of the maximal phorbol effect. Our data suggest that activation of the PKC system might acutely limit the macrophage's ability to respond to C5a; chronically, phorbols upregulate receptor expression, most likely through positive effects on C5aR gene expression.
Rubin
J
J
Veterans Administration Medical Center, Atlanta, Decatur, GA 30033.
Titus
L
L
Nanes
M S
MS
D002117:Calcitriol / Q000494:pharmacology; D003078:Colchicine / Q000494:pharmacology; D015936:Complement C5a / Q000378:metabolism; D000242:Cyclic AMP / Q000494:pharmacology; D004357:Drug Synergism; D004789:Enzyme Activation / Q000502:physiology; D015973:Gene Expression Regulation, Leukemic / Q000502:physiology*; D006801:Humans; D016403:Lymphoma, Large B-Cell, Diffuse / Q000235:genetics* / Q000473:pathology*; D011485:Protein Binding / Q000187:drug effects; D011493:Protein Kinase C / Q000502:physiology; D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement / Q000235:genetics* / Q000378:metabolism / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*; D014407:Tumor Cells, Cultured / Q000378:metabolism / Q000473:pathology / Q000648:ultrastructure; D015854:Up-Regulation / Q000187:drug effects; D014747:Vinblastine / Q000494:pharmacology
D044087:Receptor, Anaphylatoxin C5a; D011951:Receptors, Complement; D014747:Vinblastine; D015936:Complement C5a; D000242:Cyclic AMP; D011493:Protein Kinase C; D002117:Calcitriol; D013755:Tetradecanoylphorbol Acetate; D003078:Colchicine