JNK1 Mediates Lipopolysaccharide-Induced CD14 and SR-AI Expression and Macrophage Foam Cell Formation

Abstract

Foam cell formation is the key process in the development of atherosclerosis. The uptake of oxidized low-density lipoprotein (oxLDL) converts macrophages into foam cells. We recently reported that lipopolysaccharide (LPS)-induced foam cell formation is regulated by CD14 and scavenger receptor AI (SR-AI). In this study, we employed pharmaceutical and gene knockdown approaches to determine the upstream molecular mediators, which control LPS-induced foam cell formation. Our results demonstrated that the specific c-Jun N-terminal kinase (JNK) pathway inhibitor, SP600125, but neither the specific inhibitor of extracellular signaling-regulated kinase (ERK) kinase MEK1/2, U0126, nor the specific inhibitor of p38 MAPK, SB203580, significantly blocks LPS-induced oxLDL uptake, suggesting that the JNK pathway is the upstream mediator of LPS-induced oxLDL uptake/foam cell formation. To address whether JNK pathway mediates LPS-induced oxLDL uptake is due to JNK pathway-regulated CD14 and SR-AI expression, we assessed whether the pharmaceutical inhibitor of JNK influences LPS-induced expression of CD14 and SR-AI. Our results indicate that JNK pathway mediates LPS-induced CD14 and SR-AI expression. To conclusively address the isoform role of JNK family, we depleted JNK isoforms using the JNK isoform-specific siRNA. Our data showed that the depletion of JNK1, but not JNK2 blocked LPS-induced CD14/SR-AI expression and foam cell formation. Taken together, our results reveal for the first time that JNK1 is the key mediator of LPS-induced CD14 and SR-AI expression in macrophages, leading to LPS-induced oxLDL uptake/foam cell formation. We conclude that the novel JNK1/CD14/SR-AI pathway controls macrophage oxLDL uptake/foam cell formation.

Keywords: CD14; lipopolysaccharide; macrophage; signal transduction; vascular biology.

Figure 1

LPS induction of MAPK activation in BMDMs. (A) Western blotting data showed time courses of LPS induction of phosphorylation of ERK, p38, and JNK in BMDMs. Cultured BMDMs were starved for 24 h prior to LPS (100 ng/ml) stimulation for various times. Cell lysates were analyzed by Western blotting, using specific antibodies against p-ERK, p-p38, and p-JNK. GAPDH served as the loading control. The representative Western blot results shown were from three independent experiments. (B) results of the Western blot analysis were quantified as the densitometry value analyzed by UN-SCAN-IT gel 6.1 software. ^*p < 0.05; ^**p < 0.01 vs. control.

Figure 2

The involvement of JNK pathway in LPS induction of foam cell formation. (A) LPS effects on the uptake of oxLDL and LDL were examined by the Oil Red O staining with Nikon Eclipse E600 microscopy. BMDMs were stimulated with LPS for 18 h prior to the treatment of oxLDL or LDL for 3.5 h. (B) fluorescence microscopy results showed that pretreatment with the specific JNK inhibitor SP600125, but not the specific MEK inhibitor U0126 or p38 inhibitor SB203580, significantly blocked LPS-induced Di-oxLDL uptake. Twenty-four-hour serum free starved BMDMs were pretreated with various MAPK inhibitors (SP600125, 1 μM; SB203580, 5 μM; U0126, 1 μM) for 45 min prior to LPS treatment, followed by the addition of 30 μg/ml DiI-oxLDL for 3.5 h. The results were examined by fluorescence microscopy (Nikon Eclipse E600 microscopy); the Dil-oxLDL signals were shown in red. (C) pretreatment with specific JNK inhibitor SP600125, but not the specific MEK inhibitor U0126 or p38 inhibitor SB203580, dose-dependently blocked LPS-induced Dil-oxLDL uptake. The uptake of Dil-oxLDL fluorescence value was measured in Synergy HT plate reader at 530 nm (excitation) and 590 nm (emission). The Y axis represents the increased oxLDL uptake levels by LPS compared to the basal oxLDL uptake (uptake of oxLDL alone was considered as 100%). Quantified results were from three independent experiments. ^#p < 0.05; ^##p < 0.01 vs. LPS alone group.

Figure 3

The JNK pathway contributes to CD14 and SR-AI expression. (A) Western blotting data showed that LPS dose-dependently induces CD14 and SR-AI expression in BMDMs. (B) Pretreatment with the specific JNK inhibitor SP600125, but not the specific MEK inhibitor U0126 or p38 inhibitor SB203580, dose-dependently blocked LPS-induced CD14 and SR-AI expression. Twenty-four-hour serum starved BMDMs were pretreated with various inhibitors for 45 min, and then 100 ng/ml LPS was added for 4 h. CD14 and SR-AI protein levels were determined by Western blotting. Data shown were from three independent experiments. (C) results of the Western blot analysis were quantified as the densitometry value analyzed by UN-SCAN-IT gel 6.1 software. ^#p < 0.05; ^##p < 0.01 vs. LPS alone group.

Figure 4

JNK 1 is the key mediator of LPS-induced CD14 and SR-AI expression. (A) Western blotting data showed that knockdown of JNK1 expression, but not JNK2, blocked LPS-induced CD14 and SR-AI expression in BMDMs. GAPDH served as the loading control. The representative Western blot results shown were from three independent experiments. (B) results of the Western blot analysis were quantified as the densitometry value analyzed by UN-SCAN-IT gel 6.1 software. ^##p < 0.01 vs. Non-silencing group.

Figure 5

The JNK1 activity is required for LPS-induced oxLDL uptake in macrophages/foam cell formation. (A) knockdown efficiency of JNK1 and JNK2 with specific siRNAs in BMDMs was assessed in a Western blot analysis. (B) Visualized fluorescence microscopy data showed that knockdown of expression of JNK1 with specific siRNA blocked LPS-induced Dil-oxLDL uptake in BMDMs. The results were examined by fluorescence microscopy (Nikon Eclipse E600 microscopy); the Dil-oxLDL signals were shown in red. (C) quantitative fluorescence data indicated that knockdown of JNK1 expression using the specific JNK1 siRNA blocked LPS-induced Dil-oxLDL uptake in BMDMs. The quantitative analyses of oxLDL uptake using a fluorescence microplate reader were described in Figure 2. The Y axis represents the increased oxLDL uptake levels by LPS compared to the basal oxLDL uptake (uptake of oxLDL alone was considered as 100%). Quantified results were from three independent experiments. ^##p < 0.01 vs. Non-silencing group.