Miller - REPORTS 3). Thus, deletion of JNK2 in macrophages...

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3). Thus, deletion of JNK2 in macrophages was sufficient to decrease atherogenesis. Two receptors appear to be essential in foam cell formation and receptor-mediated binding and uptake of modified lipoproteins: CD36 and scavenger receptor A (SR-A) ( 20 ). Immunofluorescence analyses revealed that expression of CD36 was unchanged in acLDL-stimulated peritoneal ApoE j / j JNK2 j / j macrophages (Fig. 4A and fig. S7A). However, analyses with antibodies to SR-A showed increased abundance of this receptor (Fig. 4B and fig. S7B) ( P G 0.01). Protein immunoblotting confirmed increased abundance of SR-A in protein extracts prepared from macrophages stimulated with acLDL. Amounts of SR-A were not altered in response to acLDL in double knockout or control animals (fig. S7C). ApoE j / j JNK2 j / j macrophages formed filopodia-like projec- tions, which were not observed in controls (Fig. 4C). This cellular phenotype is associat- ed with increased adhesion and has been described in macrophages overexpressing SR-A ( 21 ). To examine whether increased abundance of SR-A in cultured macrophages also occurred in vivo, we used immunohisto- chemistry to detect SR-A on plaques from ApoE j / j JNK2 j / j mice and ApoE j / j con- trol mice. Increased amounts of SR-A were de- tected in macrophages in plaques of ApoE j / j JNK2 j / j mice compared to those of control mice (Fig. 4D). Alternative splicing results in three types of SR-A transcripts in humans. Occurrence of the Type III SR-A blocks modified LDL uptake ( 22 ). Therefore, we analyzed the expression of all three splicing variants in macrophages by semiquantitative RT-PCR using specific prim- ers. We could not detect Type III mRNA in macrophages of either genotype. Type I and Type II mRNA was not increased in the ab- sence of JNK2 (fig. S7D). Expression of CD36 or peroxisome proliferator–actived receptor (PPAR g )( 23 ), was also not affected. Activa- tion of the well-known JNK target c-jun in aortas from ApoE j / j and ApoE j / j JNK2 j / j mice fed either a normal or high-cholesterol diet was not affected, suggesting that c-jun- dependent gene expression was not impaired (fig. S7E). Phosphorylation of SR-A on spe- cific serines is essential for SR-A–dependent processing of modified LDL and for surface expression of SR-A ( 24–26 ). We immunopre- cipitated SR-A from total protein extracts of JNK2 j / j macrophages and corresponding wild- type cells. Western blotting of immunopre- cipitated SR-A revealed an increased amount of SR-A in JNK2 j / j cells compared to wild- type cells (Fig. 4, E and F). Blotting with phosphoserine-specific antibody indicated that the amount of serine-phosphorylated SR-A was lower in JNK2 j / j extracts even though more SR-A protein was present (Fig. 4E). We confirmed decreased phosphorylation of SR-A after labeling of JNK2 j / j macrophages with E 32 P ^ orthophosphoric acid (Fig. 4F).
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Miller - REPORTS 3). Thus, deletion of JNK2 in macrophages...

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