Abstract
p38 MAPK has been strongly implicated in the development of atherosclerosis, but its role in cholesterol ester accumulation in macrophages and formation of foam cells, an early step in the development of atherosclerosis, has not been investigated. We addressed this issue and made some brand new observations. First, elevated intracellular cholesterol level induced by the exposure to LDL-activated p38 MAPK and activation of p38 MAPK with anisomycin increased the ratio of cholesterol esters over free cholesterol, whereas inhibition of p38 MAPK with SB203580 or siRNA reduced the LDL loading-induced intracellular accumulation of free cholesterol and cholesterol esters in macrophages. Second, exposure to LDL cholesterol inhibited autophagy in macrophages, and inhibition of autophagy with 3-methyladenine increased intracellular accumulation of cholesterol (free cholesterol and cholesterol esters), whereas activation of autophagy with rapamycin decreased intracellular accumulation of free cholesterol and cholesterol esters induced by the exposure to LDL cholesterol. Third, LDL cholesterol loading-induced inhibition of autophagy was prevented by blockade of p38 MAPK with SB203580 or siRNA. Neutral cholesterol ester hydrolase was co-localized with autophagosomes. Finally, LDL cholesterol loading and p38 activation suppressed expression of the key autophagy gene, ulk1, in macrophages. Together, our results provide brand new insight about cholesterol ester accumulation in macrophages and foam cell formation.
Highlights
Introduction of Small InterferingRNA—THP-1 cells were transiently transfected with 100 nM Small InterferingRNA (siRNA) against p38 MAPK␣ (Santa Cruz Biotechnology, Inc.) with Lipofectamine 2000 transfection reagents (Invitrogen) according to instructions from the manufacturer
Cholesterol Loading with Low density lipoproteins (LDLs) Activates p38 MAPK in Macrophages—To examine the effect of the exposure to a high level of cholesterol on p38 MAPK activity, primary human CD14ϩ monocytes were incubated with LDL aggregates for a different time (1, 3, 6, 12, or 24 h) as described previously [42] or for 24 h with a different amount of LDL aggregates (10 –50 g/ml), followed by evaluations of total and phosphorylated p38 MAPK as we described previously [43,44,45,46,47]
Activation of p38 MAPK Is Associated with Increased Accumulation of Cholesterol Esters in Macrophages Exposed to LDL—To determine the role of p38 MAPK activation in cholesterol accumulation, activity of p38 MAPK was either stimulated or inhibited in THP-1 macrophages that were treated with LDL as noted, followed by visualization of cholesterol accumulation in macrophages with Oil Red O staining
Summary
The direct role for p38 MAPK in foam cell formation has not been investigated. Results: Inhibition and activation of p38 MAPK alter levels of autophagy activity and cholesterol ester accumulation in macrophages. P38 MAPK has been strongly implicated in the development of atherosclerosis, but its role in cholesterol ester accumulation in macrophages and formation of foam cells, an early step in the development of atherosclerosis, has not been investigated. We addressed this issue and made some brand new observations. It is known that p38 MAPK can inhibit autophagy [40] It is currently unknown whether or not p38 MAPK inhibition of autophagy is involved in cholesterol ester accumulation within macrophages and foam cell formation, an early event in the development of atherosclerosis. We investigated the potential roles of p38 MAPK and autophagy in cholesterol ester accumulation in macrophages and defined the relationship between p38 MAPK and autophagy in the process
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