Regulation of monocyte CD36 and thrombospondin-1 expression by soluble mediators.

CD36 is an 88-kD integral membrane protein expressed on platelets, monocytes, macrophages, certain microvascular endothelia, and retinal pigment epithelium. It functions as an adhesive receptor for thrombospondin-1 (TSP-1), collagen, and malaria-infected erythrocytes and as a scavenger receptor for oxidized LDL and photoreceptor outer segments. The CD36-TSP-1 interaction plays a role in cell adhesion and the phagocytosis of apoptotic cells by macrophages. Because of the potential importance of the CD36-TSP-1 interaction in mediating atherogenic and inflammatory processes, we studied their expression in human peripheral blood monocytes exposed to soluble mediators known to regulate inflammation and atherogenesis. RNase protection assays showed 6- to 12-fold increases in CD36 mRNA in response to interleukin-4, monocyte colony-stimulating factor, and phorbol myristate acetate, while lipopolysaccharide and dexamethasone strongly downregulated CD36 mRNA. The downregulation of CD36 mRNA was associated with the disappearance of surface expression of CD36 antigen and loss of TSP-1 surface-binding capacity. Upregulation of CD36 mRNA was associated with a modest increase in surface antigen expression and a larger expansion of an intracellular pool of CD36. As with CD36, monocytes treated with monocyte colony-stimulating factor showed a rapid increase in TSP-1 mRNA expression. Moreover, while dexamethasone treatment decreased CD36 expression, it resulted in a rapid increase in TSP-1 mRNA, and while PMA increased CD36 mRNA, it rapidly decreased TSP-1 expression. Interferon gamma, which had no effect on CD36 mRNA, rapidly increased steady-state TSP-1 mRNA. Thus, expression of both CD36 and its ligand TSP-1 is regulated by soluble mediators, although certain mediators induce concordant changes and others discordant changes.

Regulated expression of CD36 during monocyte-to-macrophage differentiation: potential role of CD36 in foam cell formation.

CD36 is an 88-kD integral membrane glycoprotein expressed on monocytes, platelets, and certain microvascular endothelium serving distinct cellular functions both as an adhesive receptor for thrombospondin, collagen, and Plasmodium falciparum-infected erythrocytes, and as a scavenger receptor for oxidized low-density lipoprotein and apoptotic neutrophils. In this study, we examined the expression of CD36 during in vitro differentiation of peripheral blood monocytes into culture-derived macrophages. Steady-state mRNA levels of CD36 showed a transient eightfold increase during monocyte-to-macrophage differentiation, peaking at the early macrophage stage (days 3 or 4 in culture), following a gradual decrease back to baseline levels by the mature macrophage stage (days 7 or 8 in culture). Immunoblotting with monoclonal antibodies to CD36 supported this transient, yet significant (8- to 10-fold) increase in total protein levels of CD36. The increased CD36 protein was observed at the plasma membrane, whereas an intracellular pool of CD36 was also detected from day 2 to day 6 in culture through indirect immunofluorescence. A concomitant twofold increase in the cells' ability to bind 125I-thrombospondin at the early macrophage stage (day 4) verified the functional competency of the plasma membrane localized CD36, and supported the presence of an intracellular pool of CD36. The in vitro differentiated macrophages as well as alveolar macrophages remained responsive to macrophage colony-stimulating factor (M-CSF), a known transcriptional regulator of monocyte CD36. The M-CSF-induced macrophages resulted in enhanced foam cell formation, which was inhibitable with monoclonal antibodies to CD36. Thus, the transient expression of CD36 during monocyte-to-macrophage differentiation, and the ability of M-CSF to maintain macrophage CD36 at elevated levels, may serve as a critical process in dictating the functional activity of CD36 during inflammatory responses and atherogenesis.

CD36 induction on human monocytes upon adhesion to tumor necrosis factor-activated endothelial cells.

Cell adhesion between circulating monocytes and the endothelium is a critical component of vascular thromboregulation and atherogenesis. The biochemical and genetic consequences of adhesion are poorly understood. We have found that monocyte surface expression of CD36, an integral membrane receptor for thrombospondin, collagen, and oxidized low density lipoprotein, increased dramatically upon adhesion to tumor necrosis factor-activated human umbilical vein endothelial cells (HUVEC). Expression was assessed by indirect immunofluorescence microscopy and immunoblotting using monoclonal antibodies to CD36. Steady-state CD36 mRNA levels, detected by RNase protection assay, also showed a similar pattern of up-regulation. To verify the adhesion dependence of the observed phenomenon, monocytes were co-cultured with tumor necrosis factor-activated HUVEC in a transwell apparatus that physically separated monocytes from the endothelial cells. Under these conditions, no increase in CD36 expression was detected, demonstrating that the enhanced monocyte CD36 expression observed is not due to soluble factors released by HUVEC. To characterize the specific adhesion molecules involved in the process, co-culture assays were performed on murine L cells transfected with either human E-selectin or intercellular adhesion molecule-1 cDNAs. A dramatic increase in CD36 mRNA was seen upon monocyte adhesion to E-selectin-transfected L cells compared with adhesion to intercellular adhesion molecule-1 or control transfectants. Furthermore, monoclonal antibodies to E-selectin inhibited the adhesion-dependent up-regulation of CD36 mRNA induced by transfected L cells or cytokine-activated endothelial cells. These findings demonstrate adhesion-dependent gene regulation of monocyte CD36 and suggest the possible involvement of E-selectin in initiating this process.

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.

Regulation of Tcl transposable elements in Caenorhabditis elegans.

C. elegans strains contain variable numbers of a 1.6-kb transposable genetic element. Activity of this element, which is denoted Tcl, shows regulation at at least two levels. At one level, excision of Tcl elements occurs in somatic cells at a frequency several orders of magnitude higher than in germ cells. Evidence is presented suggesting that this results from regulation at the level of trans-acting functions that are required for excision or that repress excision. At the second level, germ line transposition of Tcl occurs at greater frequency in some strains than in others. The hypothesis is proposed that this is because Tcl is one component of a two-element system, the second element of which differs between strains. Evidence for a second putative transposable element family in C. elegans is presented. This family has properties that suggest a relationship to Tcl. This possibility is currently under investigation.

High-frequency excision of transposable element Tc 1 in the nematode Caenorhabditis elegans is limited to somatic cells.

Tc 1 transposable elements in the nematode Caenorhabditis elegans undergo excision at high frequency. We show here that this excision occurs primarily or entirely in the somatic tissues of the organism. Absence of germ-line excision is demonstrated by showing that Tc 1 elements are genetically stable; elements at particular genomic sites, as well as the overall number of elements in the genome, were stably maintained during a year of continuous, nonselective propagation. Somatic excision is demonstrated by showing that empty Tc 1 sites arise during a single generation of growth of a synchronous population and are not inherited by the next generation. These results suggest that excision of Tc 1 elements is under the control of tissue-specific factors.

Evidence for a transposon in Caenorhabditis elegans.

The C. elegans genome contains a 1.7 kb repeated DNA sequence (Tc1) that is present in different numbers in various strains. In strain Bristol and 10 other strains analyzed, there are 20 +/- 5 copies of Tc1, and these are located at a nearly constant set of sites in the DNA. In Bergerac, however, there are 200 +/- 50 interspersed copies of Tc1 that have arisen by insertion of Tc1 elements into new genomic sites. The interspersed copies of Tc1 have a conserved, nonpermuted structure. The structure of genomic Tc1 elements was analyzed by the cloning of a single Tc1 element from Bergerac and the comparison of its structure with homologous genomic sequences in Bristol and Bergerac. Tc1 elements at three sites analyzed in Bergerac undergo apparently precise excision from their points of insertion at high frequency.