Microglial low-density lipoprotein receptor-related protein 1 modulates c-Jun N-terminal kinase activation - PubMed (original) (raw)

Ana Pocivavsek et al. J Neuroimmunol. 2009.

Abstract

Apolipoprotein E (apoE)-induced activation of low-density lipoprotein receptor (LDL) family members reduces inflammatory responses by suppressing c-Jun N-terminal kinase (JNK) activation. We aimed to identify which specific receptor family member mediates the effect of apoE on inflammation in primary cultures of microglia. Low-density lipoprotein receptor-related protein 1 (LRP1)-deficient (LRP1-/-) microglia were derived from mice using tissue-specific loxP/Cre recombination. Using a peptide formed from the receptor-binding region of apoE (EP), we found that LRP1 mediates the effects of apoE on microglial inflammation. Microglial LRP1 was also essential for EP to suppress JNK activation induced by lipopolysaccharide.

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Figures

Figure 1

Figure 1. LRP1 knockout in primary microglia

A) Cell lysates from wild-type (wt) and LRP1 −/− (ko) microglia were analyzed by Western blotting for LRP1 expression (denoted by the asterisks). The left panel shows a representative blot probed with 5A6, recognizing the 85 kDa band for LRP1. The right panel shows a blot probed with goat α-LRP1, recognizing full length LRP1 (n = 4). B) Primary microglia from wild-type (WT) and LRP1 −/− cultures were immunostained with monoclonal Iba1 (detected with Alexa488, green) and goat anti-LRP antibody (detected with Alexa568, red). C) Cells positive for Iba1 and LRP1 expression were counted. Quantification shows that 95 % of wild-type microglia and 25 % of LRP1 −/− microglia were LRP1 positive (mean ± SEM; *** P < 0.001 compared to wild-type; n = 5 for wild-type and n = 7 for LRP1 −/−).

Figure 2

Figure 2. Stimulation of microglia with LPS increased NO through JNK activation

A) In wild-type and LRP1 −/− microglia, LPS promoted nitrite accumulation in the conditioned media at 24 h (mean ± SEM; ***P < 0.001; n = 6 for each of 3 independent experiments). B) Cell lysates from wild-type and LRP1 −/− microglia were analyzed by Western blotting with antibodies to iNOS and β-actin (as a control). A representative blot shows that LPS increased iNOS levels in both cell types (n=6 for each of 3 independent experiments). C) Wild-type and LRP1 −/− microglia were treated with LPS alone (open bars) or LPS and SP600125 (colored bars). In a dose-dependent manner, SP600125 treatment reduced nitrite production in both wild-type and LRP1 −/− microglia to levels of unstimulated microglia (defined as the line at 100 %) (mean ± SEM; **P < 0.01 compared to control cultures; # P < 0.05, ## P < 0.01 compared to corresponding cultures; n=3 for each of 2 independent experiments).

Figure 3

Figure 3. ApoE peptide attenuated LPS-induced NO production in LRP1 expressing microglia

A) Wild-type and LRP1 −/− microglia were treated with 100 ng/mL LPS and nitrite was measured in the conditioned media at 24 h. LPS induced nitrite accumulation in both cell types and EP showed attenuation of LPS-induced nitrite production only in wild-type cultures (mean ± SEM; *** P < 0.001 compared to corresponding control cultures; ## P < 0.01, ### P < 0.001 compared with indicated cultures; n = 6 for each of 4 independent experiments). B) Cell lysates from wild-type and LRP1 −/− cultures were analyzed by Western blotting for iNOS and b-actin. Representative blots show that EP and LPS treatment reduced iNOS expression compared to LPS stimulation alone in wild-type stimulated cultures but not LRP1 −/− cultures. C) Western blot data were quantified as percent of control (mean ± SEM; * P < 0.05, ** P < 0.01 compared with LPS stimulated cultures; # < 0.05 compared with indicated cultures; n = 6 for each of 4 independent experiments). D) Wild-type (left panel) and LRP1 −/− (right panel) cultures were treated LPS and nitrite was measured in the conditioned media at 24 h. LPS induced nitrite accumulation in wild-type and LRP1 −/− cultures. EP and LPS treatment attenuated LPS-induced nitrite accumulation significantly in wild-type cultures and treatment with 500 nM VLDLR blocking antibody, EP and LPS did not prevent the effects of EP. Similarly in LRP1 −/− microglia, nitrite accumulation in cultures treated with VLDLR antibody cocktail did not significantly differ from cultures treated with EP and LPS (mean ± SEM; ns means not significant; n = 4).

Figure 4

Figure 4. ApoE peptide signaling effects are LRP1 mediated

Wild-type and LRP1 −/− primary microglia were treated with PBS (c), 1 μM EP, 100 ng/mL LPS, and 1 μM EP with LPS for 1 h. Cell lysates were analyzed by Western blotting with antibodies to phospho-JNK and total JNK. A) Wild-type cells treated with EP showed decreased phospho-JNK compared with cells treated with PBS (c). Treatment of wild-type cells with LPS increased phospho-JNK. Cells treated with EP and LPS showed reduced phospho-JNK compared to cells treated with LPS alone. LRP1 −/− cells treated with LPS showed increased phospho-JNK compared with cells treated with PBS (c). In LRP1 −/− cultures, EP and LPS treatment did not reduce phospho-JNK compared to cells treated with LPS alone. B) Western blot data were quantified as percent of control phospho-JNK (mean ± SEM; *** P < 0.001 compared with control cultures; ## P < 0.01 compared with indicated cultures; n = 3 for each of 2 independent experiments).

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