The functional effects of ceramide on the monocyte CD36 scavenger receptor and nadph oxidase
The present study investigated whether short chain ceramides reduce NADPH oxidase in U937 monocytes by disrupting the membrane component of NADHP oxidase. Results showed that C2 ceramide alters the distribution of raft marker, flotillin and the raft environment. NADPH oxidase membrane component gp91 phox and cytosolic component p47 phox were identified in rafts. C2 ceramide reduces both gp91 and p47 phox in rafts, which leads to the decrease of peroxide production by NADPH oxidase. Ceramide is also an important second messenger involved in many different signalling pathways associated with atherogenesis from the activation of sphinogomyelinase (SMase). This thesis shows that ceramides significantly reduce CD36 surface expression on U937 monocytes, macrophages and human primary monocytes. This effect is seen using both synthetic short chain ceramide and SMase catalysed long chain ceramide treatment. To investigate whether the effect of ceramide on CD36 is functional, OxLDL uptake was measured in ceramide treated cells. Ceramide reduces the uptake of OxLDL by both U937 monocytes and PMA-differentiated macrophages. The mechanism of ceramide reduction of CD36 expression was studied by measuring the surface antigen using flow cytometry and fluorescence microscopy, whole cellular CD36 expression and shedding of CD36 by Western blotting of cell lysates and cell culture supernatants and mRNA level of CD36 using RT-PCR. Ceramide reduces shedding of CD36, activates mRNA expression of CD36 and induces intracellular CD36 accumulation probably through retaining the receptor inside cells. In summary, ceramides modulate several of the processes involved in LDL oxidation and uptake by CD36 receptors on monocytes/macrophages in a way which may protect against atherosclerosis.