Sense and antisense cDNA transfection of CD36 (glycoprotein IV) in melanoma cells. Role of CD36 as a thrombospondin receptor.
Thrombospondin (TSP) is a multifunctional matrix and platelet glycoprotein that interacts with cell surfaces and may play a role in mediating cell adhesion, platelet aggregation, platelet-monocyte interactions, cell proliferation, angiogenesis, tumor metastasis, and protease generation. To clarify and confirm the function of CD36 (glycoprotein IV) as a TSP receptor, we now describe a transfected cell model using human melanoma cells genetically manipulated by sense or antisense cDNA transfection to express either high or near zero levels of CD36. Surface expression was confirmed by flow cytometry with monoclonal anti-CD36 IgG and quantified by measuring radiolabeled antibody binding. Bowes melanoma cells, which in their wild type did not express CD36 and did not bind radiolabeled TSP, when transfected with the sense construct bound TSP in a 1:1 stoichiometric ratio with CD36 expression. Conversely, C32 melanoma cells, which in their wild type expressed high levels of CD36 and bound radiolabeled TSP at a 1:1 stoichiometric ratio, did not express CD36 and did not bind TSP when transfected with an antisense construct. In addition, transfected Bowes cells and wild type C32 cells, unlike wild type Bowes cells, adhered to activated platelets in a TSP-dependent manner. These data, i.e. the gain of function with sense cDNA transfection and loss of function with antisense transfection, strongly support the TSP receptor function of CD36. The distribution of this protein in vascular cells and tissues and observations that it may participate in signal transduction events suggest that TSP-CD36 interactions may play a role in mediating some of the pathophysiological processes associated with TSP.
Silverstein
R L
RL
Department of Medicine, Cornell University Medical College, New York, New York 10021.
Baird
M
M
Lo
S K
SK
Yesner
L M
LM
D000911:Antibodies, Monoclonal; D015703:Antigens, CD / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D018955:CD36 Antigens; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D016373:DNA, Antisense / Q000235:genetics*; D006801:Humans; D008545:Melanoma; D010980:Platelet Membrane Glycoproteins / Q000378:metabolism*; D012313:RNA / Q000235:genetics / Q000302:isolation & purification; D015347:RNA Probes; D011956:Receptors, Cell Surface / Q000378:metabolism*; D016024:Receptors, Cytoadhesin / Q000378:metabolism*; D015183:Restriction Mapping; D019699:Thrombospondins; D014158:Transcription, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured
D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D018955:CD36 Antigens; D016373:DNA, Antisense; D010980:Platelet Membrane Glycoproteins; D015347:RNA Probes; D011956:Receptors, Cell Surface; D016024:Receptors, Cytoadhesin; D019699:Thrombospondins; D012313:RNA; D004247:DNA