Fig 1.
Ligand-bound SREC-I colocalized with TLR4 after LPS treatment.
A, SREC-I and TLR4 did not interact in the absence of LPS. Raw 264.7 cells were transfected with FLAG-SREC-I for 22 hours. Cells were then fixed and stained with anti TLR4 ab (green) and anti-FLAG ab (red). B, TLR4 colocalized with SREC-I in the presence of LPS. Raw 264.7 cells overexpressing FLAG-SREC-I were exposed to LPS (1 μg/ml) for 20–30 min at 4°C. Cells were then fixed and stained for TLR4 (green) and FLAG (red). Percent colocalization with or without LPS is shown in the adjacent histogram. C, D, LPS, TLR4 and SREC-I were internalized at 37°C. Raw 264.7 cells overexpressing FLAG-SREC-I were incubated with Alexa LPS (1 μg/ml) at 4°C for 20 mins and then medium was replaced with warm medium. Cells were then incubated at 37°C for 10–15 mins. Cells were fixed and stained for TLR4 (green, C, D) and FLAG-SREC-I (red in C, purple in D). Alexa LPS was shown in red (D). E, F, Endogenous TLR4 and SREC-I did not colocalize in the absence of LPS. Raw 264.7 cells were treated for 30 mins with LPS (1 μg/ml) and then fixed and stained for TLR4 (green) and SREC-I (red). E, TLR4 and SREC-I colocalize in the presence of LPS. Cells were labeled with LPS (1μg/ml) for 20–30 minutes at 4°C then fixed and stained for TLR4 (green) and SREC-I (red) (F). G, SREC-I expression level in Raw 264.7 cells and in cells overexpressing FLAG-SREC-I. H, SREC-I interacted with TLR4 physically in the presence of LPS (1 μg/ml). HEK 293 cells expressing FLAG-SREC-I and TLR4s were treated with or without Hsp90 or LPS. FLAG-SREC-I was then immunoprecipitated (IP) with FLAG ab. The IP complex was separated with SDS-PAGE and blotted for TLR4. The amount of FLAG-SREC-I immunoprecipitated was determined and β-actin was used as a loading control. All images were representative of 3 different planes from each sample. Each experiment was performed 3 times reproducibly. Scale bar, 5 μm.
Fig 2.
SREC-I supported LPS-TLR4 mediated NF-kB (phospho-p65).
A, Phospho-p65, (S536/Rel) level was increased in cells expressing SREC-I with TLR4 in the presence of LPS. HEK 293 cells expressing TLR4-MD2-CD14 and/or SREC-I, SREC-I only were treated with or without LPS (1 μg/ml) or Hsp90 for 5–7 hours. Cell lysates were then collected and SDS-PAGE was performed. Phospho-p65 levels were measured. Total level of p65 was measured in the same lysate. Total p65 level was determined. B, HEK 293 cells expressing SREC-I and TLR4 or TLR4 only were transfected with NF-kB-SEAP and incubated with LPS (1 μg/ml) for 5 hours. NF-kB activity was measured as instructed by NF-kB-SEAporter assay kit. C, Raw 264.7 cells were transfected with siRNA for SREC-I/TLR4 for 72 hours and incubated with LPS (1 μg/ml) with or without CD14 neutralizing peptide (inhibitor). Phospho-p65 level is increased with LPS incubation in cells expressing both TLR4 and SREC-I. D, Raw 264.7 cells were transfected with ctl (scr) siRNA or TLR4 siRNA/SREC-I siRNA for 72 hours. Cell lysates were isolated and later SDS-PAGE was performed. E, HEK 293 cells expressing TLR4, MD-2, CD14 or TLR4, MD-2, CD14 and SREC-I were incubated with LPS (1 μg/ml) for indicated time. Cells lysates were collected and equal amount of protein was loaded for SDS-PAGE experiment. For blocking CD14 activity, cells were treated with 10 μg/ml of anti CD14 neutralizing antibody. Error bars in graph show S.D. between three replicate experiments. P <0.0001 values were generated by ANOVA using the Bonferroni post-test.
Fig 3.
SREC-I expression led to enhanced NF-kB and MAPK activities in cells expressing TLR4 in the presence of LPS.
A, B, C, SREC-I could increase LPS-TLR4 activation of MAPK. HEK 293 cells expressing TLR4-MD2-CD14, TLR4-MD2-CD14-SREC-I were incubated with LPS (1 μg/ml) for 5 hours (CD14 neutralizing peptide added to SREC-I incubation). Cell lysates were collected and levels of phosphorylated JNK (A), p38 (B), ERK1/2 MAPK assayed (C). D, SREC-I was shown to activate LPS-TLR4 induced MAPK signaling. Raw 264.7 cells were transfected with siRNA SREC-I/TLR4 and then incubated with or without LPS in the presence of CD14 neutralizing ab (inhibitor) or not. Cell lysates were collected and subjected to SDS-PAGE and Western Blotting. E, SREC-I was involved and IRF3 activity in the absence of CD14. Raw 264.7 cells were treated as in D and then cell lysates were collected and subjected to SDS-PAGE and Western blotting with appropriate antibodies. F, Raw 264.7 cells were transfected with ctl siRNA (scr) or TLR4/SREC-I for 72 hours. Cell lysates were collected and equal protein was subjected to SDS-PAGE and Western Blotting. G, Phospho-p65 levelswere high in cells expressing TLR4-SREC-I. HEK 293 cells expressing TLR4-MD2, SREC-I-MD2, TLR4-MD2-SREC-I were incubated with mBSA (10 μg/ml), LPS (1 μg/ml), Hsp90 (10 μg/ml), Hsp90 (10 μg/ml) for 3–5 hours. Cell lysates were collected and levels of phospho-p65, p65, phospho-p38, p38, phospho-JNK and JNK measured. Densitometric analysis of gel intensity was performed using Image J software. Experiments were repeated reproducibly three times.
Fig 4.
SREC-I requiredadaptor proteins MyD88 and TRIF for downstream NF-kB activation.
A, B, C, MyD88 was involved in LPS-TLR4-SREC-I (A), LPS-SREC-I (B) or LPS-TLR4 (C) activation of NF-kB. HEK 293 cells expressing TLR4-MD2-SREC-I-MyD88, SREC-I-MD2-MyD88 and TLR4-MD2-MyD88-CD14 were treated with MyD88 blocking peptide (100 μM for 24 hours), or control peptide (100 μM for 24 hrs) as indicated. Cells were then treated with LPS (1 μg/ml) for 5 hours in the presence of blocking peptides. Densitometric analysis of bands in gels is shown. D, E, F, TRIF was involved in LPS-TLR4-SREC-I, LPS-TLR4 or LPS-SREC-I activation of NF-kB. HEK 293 cells (+MD2) expressing receptors and adaptor TRIF and CD14 as indicated were incubated with TRIF blocking peptide or control peptide (50 μM for 6 hours) and incubated with LPS for 5 hours. Phospho-p65 levels in cell lysates were then analyzed by SDS-PAGE and Western blotting. TLR4 only cells also express CD14 as shown. Densitometric analysis of band intensity is shown below. Each experiment was repeated 3 times. G, H, I, Protein lysates from HEK 293 cells stably expressing TLR4 and SREC-I, TLR4 only and SREC-I only were analyzed by SDS-PAGE and Western blotting. Relative protein expression levels are thus shown in the blot.
Fig 5.
SREC-I became localized in detergent insoluble lipid microdomainsafter LPS exposure.
A,B, FLAG-SREC-I were localized to lipid microdomains marked by the GPI anchored protein CD59 on the plasma membrane in the presence of LPS (B). HeLa cells expressing FLAG-SREC-I were incubated with LPS (1 μg/ml) and FITC labeled (red, in figure) anti CD59 ab (10 μg/ml for 30 minutes on ice at 4°C). Cells were then fixed on ice and stained for FLAG (green) using anti FLAG M2 ab. FLAG-SREC-I expressing cells were also incubated with FITC-anti-CD59 ab at 4°C for 30 mins. Cells were stained with anti-FLAG ab (secondary antibody, red). Confocal Microscopy was then used to analyze the localization of the proteins. C, SREC-I was localized to lipid microdomains after LPS exposure. Raw 264.7 cells were stimulated with LPS (1 μg/ml) or incubated without LPS and lipid microdomains were isolated using Optiprep density gradient centrifugation. 1 ml fractions (total 4) were collected from the top of the centrifuge tubes. Fractions containing detergent soluble membrane proteins were also indicated. All fractions were later separated on polyacrylamide gels, electrophoretically blotted and probed with antibodies as indicated. Flotillin was used as marker for lipid microdomain rich fractions (shown in lane 3 and 4).
Fig 6.
Intact lipid microdomains were essential for LPS-SREC-I-TLR4-induced MAPK signaling.
A, HEK 293 cells expressing TLR4-MD2-CD14, SREC-I-TLR4-MD2-CD14 were treated with or without CD14 neutralizing peptide (inhibitor). Cells were then incubated with LPS (1 μg/ml) for 3 hrs. Cell lysates were run on SDS-PAGE and then immunoblotted with phospho-specific antibodies for JNK, p38, p65 and anti-JNK ab, p38 ab and anti-p65 ab. Drugs including PP2 (10 μM Srcinhibitor), MβCD (MBD, 10 mM, cholesterol sequestering agent), TxB (2 ng/ml, Clostridium Toxin B) were added to inhibit the functions of Src kinase, lipid microdomain formation and Rho GTPase activities as indicated. Raw 264.7 cells were transfected with siRNA for TLR4/SREC-I and then treated as described above. These experiments were repeated reproducibly 2 times. B, Raw 264.7 cells expressing TLR4, SREC-I or TLR4 only was incubated with LPS (1 μg/ml) for 3 hrs. Cell lysates were collected and then subjected to SDS-PAGE and Western Blotting. C, D, Raw 264.7 cells were transfected with indicated siRNA for 72 hours. Cell lysates were collected and equal amount of protein was subjected to SDS-PAGE and Western Blotting. Expression of SREC-I and TLR4 is shown in Raw 264.7 cells.
Fig 7.
SREC-I inactivation reduced LPS-TLR4 mediated proinflammatory cytokine release in BMDM.
A, TLR4 KO and SREC-I KD in macrophages led to reduced IL-6 release compared with WT cells. Primary macrophages from WT and TLR4 KO mice were transfected with siRNA-SREC-I or control hairpins for 72 hours and then incubated with LPS for 12 hours with or without SR-A blocking antibody. IL-6 release by cells was measured in the collected medium by ELISA. B, TLR4KO and SREC-I KD in BMDMs led to reduced TNF-α compared to WT cells. Primary macrophages from WT and TLR4 KO mice were transfected with siRNA-SREC-I or a control hairpin for 72 hours and then incubated with LPS for 12 hours with or without SR-A blocking antibody. TNF-α release was measured in medium by ELISA. C, SREC-I KO and TLR4KD in BMDM led to reduced IL-6 release compared with WT cells. Primary macrophages from WT and SREC-I KO mice were transfected with or without siRNA-TLR4 for 72 hours. IL-6 release into the medium was measured according to manufacturer's instructions. F, SREC-I KO and TLR4 KD BMDMs led to reduced release of TNF-α compared to WT cells. Primary macrophages from WT and SREC-I KO mice were transfected with or without siRNA-TLR4 for 72 hours. TNF-α release by was then assayed as above. E,F, TLR4 KO and SREC-I KO BMDMs led to reduced release of IFN-β compared to WT cells. Primary macrophages from WT and TLR4 KO (E), SREC-I KO were transfected or not with SREC-I siRNA (E) and TLR4 siRNA (F) for 72 hours. IFN-β release in media was assayed as above. G, H, I, Primary BMDM cells (WT, TLR4/SREC-I KO mice, TLR4/SREC-I KO + SREC-I siRNA/TLR4 siRNA) were trypsinized and cell lysates were collected. Equal amount of protein was subjected to SDS-PAGE and Western blotting. All experiments were performedreproducibly 3 times. Error bars in graph show S.D. between three replicate experiments. ***P<0.0001 when compared to the control, and #P<0.0001 when compared to the WT. Values were generated by ANOVA using the Bonferroni post-test.
Fig 8.
Rho GTPase activity was required for LPS-SREC-I-TLR4 induced proinflammatory cytokine release.
BMDM cells from WT and TLR4 KO mice were transfected with siRNA SREC-I or control RNA. Cells were treated with or without Toxin B (2 ng/ml) and incubated withor without LPS. Experiments were performed in the presence of SR-A blocking antibodies. Cell media were collected, clarified by centrifugation and the IL-6 ELISA assay was performed. Experiments were repeated reproducibly 3 times. The height of the error bars represents the average of three independent measurements. The error bars represent one standard deviation from the mean. ***P<0.0001 when compared to the control, and #P<0.0001 when compared to the WT. Values were generated by ANOVA using the Bonferroni post-test.
Fig 9.
Schematic diagram of the proposed pathway of LPS-SREC-I-TLR4 mediated activation of proinflammatory cytokine expression.
This cartoon depicts LPS binding to SREC-I (1) followed by LPS-SREC-I complex recruitment of TLR4 into discrete lipid microdomains (thick line) (2). Localization of LPS-SREC-I-TLR4 complexes to such lipid microdomains then led to activation of downstream proinflammatory cytokine release through adaptor proteins MyD88 and TRIF. LPS exposure could thus mediate activation of the NF-kB and MAPK pathways. In addition, LPS has been shown to be recognized by CD14, the primary responder to the endotoxin, which then bound to TLR4 and triggered proinflammatory signaling through NF-kB and MAPK (3). We have additionally shown that LPS-SREC-I-TLR4 signaling led to activation of IRF3. Engagement of these signaling pathways could then lead to activation of transcription factors NF-kB, AP-1 and IRF3 that have been shown to function combinatorially in cytokine gene transcription.