Abstract
We have demonstrated that A375 melanoma cells express mRNA for both types of tumor necrosis factor (TNF) receptors and receptor proteins on their plasma membranes. Specific agonist and blocking antibodies to either 55-kDa (TNF-R1) or 75-kDa (TNF-R2) TNF receptors combined with two-dimensional gel analysis were employed to determine which receptor type is responsible for mediating the induction of individual melanoma proteins. Our results indicate that the enhanced synthesis of proteins 21/>7 (M(r)/pI), 28/5.6, and 41/5.7 is selectively induced through TNF-R1. TNF induces these proteins; antagonist antibody to TNF-R1 prevents their induction by TNF, and TNF-R1 agonist induces them in the absence of TNF. Identification of these proteins by immunoblot analysis proved that 21/>7 is manganese superoxide dismutase, protein 28/5.6 is unrelated to 27/28-kDa heat shock protein, and protein 41/5.7 is plasminogen activator inhibitor-2. Furthermore, TNF cytotoxicity for A375 cells is also mediated by TNF-R1. These studies indicate that TNF-R1 is a critical signaling receptor for TNF action on A375 cells and demonstrate the potential use of TNF-R1 antibodies to selectively block or enhance specific effects of TNF on melanoma cells.
Highlights
We have demonstrated that A375 melanoma cells ex- genes, and transcriptionfactors
Identification of these proteins by Two distinct high-affinity receptors for TNF havebeen idenimmunoblot analysis proved that 21/>7is manganese superoxide dismutase,protein 28/5.6 is unrelated to 27128kDa heat shock protein, and protein 41/5.7 is plasminogen activator inhibitor-2
(13).Both TNF-R1 and TNF-R2 have thetypical structure of a membrane-spanning protein witha single transmembrane region, and both are membersof a new superfamily of cell surface Tumor necrosis factor (TNF),’ originally defined for its abil- proteins which include the p75 chain of nerve growth factor ity to cause hemorragicnecrosis of certain tumors (11, is a receptor, the B cell antigen CD40, the OX-40 T cell activation hormone-like protein that binds tospecific receptors on a vari- marker, theSFV-T2open reading frameof Shope fibromavirus ety of different cell types and subsequently transducesa broad (6-9, 141, and the Fas antigen[15]. TNF binds with spectrum of immunological, inflammatory, and regulatory ac- high affinity to bothreceptors, the extracellular domainosf the tivities
Summary
9898-9905, 1994 Printed in U.S.A. Enhanced Synthesisof Tumor Necrosis Factor-inducible Proteins, Plasminogen Activator Inhibitor-2, Manganese Superoxide Dismutase, and Protein2W5.6, Is Selectively Triggeredby the 55-kDa TumorNecrosis Factor Receptorin Human Melanoma Cells*. The diversity of biological effects attributed to TNF tors combined with two-dimensionalgel analysis were suggests that thsepecificity of its action probably resides in the employed to determine which receptor type is respon- receptors to which it binds andor the intracellucloamr ponents sible for mediating the induction of individual mela- that become activated asa result of TNF binding to itsrecepnoma proteins. 55-kDa TNF receptor; TNF-R2, 75-kDa TNFreceptor; 2D, two-dimensional; IFN-y, interferon-y; PCR, polymerase chain reactionM; nSOD, manganese superoxide dismutase, hsp 27, 27-kDa heat shock protein; type of TNF receptor is responsible for triggering the enhanced synthesis of individual melanoma proteinbsy TNF. In view of the fact that we find TNF cytotoxicity for A375 cells is induced through TNF-Rl, and our 2D gel analysis shows no proteins to be significantly induced by TNF through TNF-R2, it is probable that TNF-Rl plays a major role in mediating TNF effects in A375 melanoma cells
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