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Scavenger receptor SREC-I mediated entry of TLR4 into lipid microdomains and triggered inflammatory cytokine release in RAW 264.7 cells upon LPS activation.

Murshid A, Gong J, Prince T, Borges TJ, Calderwood SK - PLoS ONE (2015)

Bottom Line: Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation.We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors.Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Center for Life Sciences, 3 Blackfan Circle, Boston, Massachusetts, United States of America.

ABSTRACT
Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation. As our previous studies indicated that SREC-I required intact Toll-like receptor 4 (TLR4) expression for its functions in tumor immunity, we examined potential interactions between these two receptors. We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors. The receptors then became internalized together in intracellular endosomes. SREC-I promoted TLR4-induced signal transduction through the NF-kB and MAP kinase pathways, leading to enhanced inflammatory cytokine release. Activation of inflammatory signaling through SREC-I/TLR4 complexes appeared to involve recruitment of the receptors into detergent-insoluble, cholesterol-rich lipid microdomains that contained the small GTPase Cdc42 and the non-receptor tyrosine kinase c-src. Under conditions of SREC-I activation by LPS, TLR4 activity required Cdc42 as well as cholesterol and actin polymerization for signaling through NF-kB and MAP kinase pathways in RAW 264.7 cells. SREC-I appeared to respond differently to another ligand, the molecular chaperone Hsp90 that, while triggering SREC-I-TLR4 binding caused only faint activation of the NF-kB pathway. Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages.

No MeSH data available.


Related in: MedlinePlus

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.
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pone.0122529.g006: 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.

Mentions: Next we examined the potential role of SREC-I localization to CD59-marked/lipid microdomainin TLR4 signaling through the JNK, p38 MAPK and NF-kB pathways using the HEK 293 system described above (Fig 6). We incubated cells with a number of agents known to disrupt lipid microdomains and formation of the GEEC compartment. These included cholesterol sequestering agent methyl β cyclodextrin (MβCD) and other inhibitors such as PP2 (Src kinase inhibitor), cytochalasin D (actin depolymerizing agent) and Clostridium. Difficile Toxin B (Rho GTPase inhibitor). These inhibitors each blocked internalization of antigen-bound SREC-I and subsequent antigen cross presentation in our prior studies [2]. In cells transfected with CD14 and TLR4, LPS induced efficient signaling through each pathway and these events were not markedly reduced by any of the inhibitors (Fig 6, 11th-14th lanes). However LPS-induced activation of JNK and p38as well as NF-kB in cells with combined SREC-I/TLR4 expression (Fig 6, lanes 3–6) was blocked by many of the inhibitors (Fig 6). MβCD and Toxin B were particularly effective in this regard and blocked signaling through each pathway. Minimal signaling through the JNK, p38 or NF-kB pathways was observed in cells expressing SREC-I alone (Fig 6, lanes 7–10). This finding was predictable from the earlier experiments indicating a need for association with TLR4 in order for SREC-I to activate these pathways. This experiment suggested that TLR4 became co-localized with SREC-I after LPS exposure in new microdomains within the membrane and required enriched cholesterol, actin cytoskeleton function and small Rho GTPase activity for signaling pathways (Fig 6). When the experiments were repeated in Raw 264.7 cells, we again saw that inhibitors of lipid microdomain formation blocked NF-kB signaling in cells coordinately expressing TLR4 and SREC-I (Fig 6). As before, the inhibitors failed to disrupt NF-kB signaling in cells expressing TLR4 without SREC-I (Fig 6). These experiments, carried out in both HEK 293 and Raw 264.7 cells, indicated TLR4 signaling from either a lipid microdomain fraction or an internal endosomal compartment after SREC-I/TLR endocytosis via the GEEC pathway (Fig 6). We again confirmed the activation of NF-kB through phosphorylated p65 level in the presence of LPS in Raw 264.7 cells expressing TLR4 and SREC-I in the absence of inhibitors suggesting the role of lipid microdomain in activating LPS-SREC-I-TLR4 signaling (Fig 6B).


Scavenger receptor SREC-I mediated entry of TLR4 into lipid microdomains and triggered inflammatory cytokine release in RAW 264.7 cells upon LPS activation.

Murshid A, Gong J, Prince T, Borges TJ, Calderwood SK - PLoS ONE (2015)

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.
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pone.0122529.g006: 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.
Mentions: Next we examined the potential role of SREC-I localization to CD59-marked/lipid microdomainin TLR4 signaling through the JNK, p38 MAPK and NF-kB pathways using the HEK 293 system described above (Fig 6). We incubated cells with a number of agents known to disrupt lipid microdomains and formation of the GEEC compartment. These included cholesterol sequestering agent methyl β cyclodextrin (MβCD) and other inhibitors such as PP2 (Src kinase inhibitor), cytochalasin D (actin depolymerizing agent) and Clostridium. Difficile Toxin B (Rho GTPase inhibitor). These inhibitors each blocked internalization of antigen-bound SREC-I and subsequent antigen cross presentation in our prior studies [2]. In cells transfected with CD14 and TLR4, LPS induced efficient signaling through each pathway and these events were not markedly reduced by any of the inhibitors (Fig 6, 11th-14th lanes). However LPS-induced activation of JNK and p38as well as NF-kB in cells with combined SREC-I/TLR4 expression (Fig 6, lanes 3–6) was blocked by many of the inhibitors (Fig 6). MβCD and Toxin B were particularly effective in this regard and blocked signaling through each pathway. Minimal signaling through the JNK, p38 or NF-kB pathways was observed in cells expressing SREC-I alone (Fig 6, lanes 7–10). This finding was predictable from the earlier experiments indicating a need for association with TLR4 in order for SREC-I to activate these pathways. This experiment suggested that TLR4 became co-localized with SREC-I after LPS exposure in new microdomains within the membrane and required enriched cholesterol, actin cytoskeleton function and small Rho GTPase activity for signaling pathways (Fig 6). When the experiments were repeated in Raw 264.7 cells, we again saw that inhibitors of lipid microdomain formation blocked NF-kB signaling in cells coordinately expressing TLR4 and SREC-I (Fig 6). As before, the inhibitors failed to disrupt NF-kB signaling in cells expressing TLR4 without SREC-I (Fig 6). These experiments, carried out in both HEK 293 and Raw 264.7 cells, indicated TLR4 signaling from either a lipid microdomain fraction or an internal endosomal compartment after SREC-I/TLR endocytosis via the GEEC pathway (Fig 6). We again confirmed the activation of NF-kB through phosphorylated p65 level in the presence of LPS in Raw 264.7 cells expressing TLR4 and SREC-I in the absence of inhibitors suggesting the role of lipid microdomain in activating LPS-SREC-I-TLR4 signaling (Fig 6B).

Bottom Line: Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation.We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors.Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages.

View Article: PubMed Central - PubMed

Affiliation: Molecular and Cellular Radiation Oncology, Beth Israel Deaconess Medical Center, Harvard Medical School, Center for Life Sciences, 3 Blackfan Circle, Boston, Massachusetts, United States of America.

ABSTRACT
Scavenger receptor associated with endothelial cells I (SREC-I) was shown to be expressed in immune cells and to play a role in the endocytosis of peptides and antigen presentation. As our previous studies indicated that SREC-I required intact Toll-like receptor 4 (TLR4) expression for its functions in tumor immunity, we examined potential interactions between these two receptors. We have shown here that SREC-I became associated with TLR4 on binding bacterial lipopolysaccharides (LPS) in RAW 264.7 and HEK 293 cells overexpressing these two receptors. The receptors then became internalized together in intracellular endosomes. SREC-I promoted TLR4-induced signal transduction through the NF-kB and MAP kinase pathways, leading to enhanced inflammatory cytokine release. Activation of inflammatory signaling through SREC-I/TLR4 complexes appeared to involve recruitment of the receptors into detergent-insoluble, cholesterol-rich lipid microdomains that contained the small GTPase Cdc42 and the non-receptor tyrosine kinase c-src. Under conditions of SREC-I activation by LPS, TLR4 activity required Cdc42 as well as cholesterol and actin polymerization for signaling through NF-kB and MAP kinase pathways in RAW 264.7 cells. SREC-I appeared to respond differently to another ligand, the molecular chaperone Hsp90 that, while triggering SREC-I-TLR4 binding caused only faint activation of the NF-kB pathway. Our experiments therefore indicated that SREC-I could bind LPS and might be involved in innate inflammatory immune responses to extracellular danger signals in RAW 264.7 cells or bone marrow-derived macrophages.

No MeSH data available.


Related in: MedlinePlus