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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

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.
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pone.0122529.g002: 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.

Mentions: As NF-kB is the most potent proinflammatory transcription factor, we examined the potential role of SREC-I in its activation by LPS. TLR4 activation has been shown to trigger activation of NF-kB. We next examined therefore the potential activity of SREC-I in LPS triggered NF-kB signaling in HEK 293 cells. As mentioned above, in contrast to Raw 264.7, HEK 293 cells were shown to be deficient in most SR family members (including SREC-I), thus SREC-I-specific effects could be examined in isolation in transfectants. This HEK 293 cell line stably expressed CD14, TLR4 and MD2; CD14 independent signaling was determined using CD14 blocking peptides (CD14-inh, 10 μg/ml), while the role of TLR4 was probed using RNA interference. The CD14 blocking peptide ab was first tested in THP1 cells by its ability to neutralize LPS-induced TNF-α secretion. The ab neutralized >60% of LPS induced cytokine release in this cell line. NF-kB activation was assayed by determining phosphorylation of its trans-activating subunit p65/Rel on serine 536 [19]. We then compared LPS-induced NF-kB signaling in cells expressing TLR4 and the LPS recognizing and signaling adaptor proteins CD14 and MD2 (Fig 2A, lane 1) without (lane 1) or with SREC-I (lane 2). LPS activated NF-kB in the absence of CD14 but with SREC-I expression (lane 2). Minimal signaling was observed in the absence of TLR4 with or without SREC-I (lanes 3, 4). This experiment showed therefore a role for SREC-I as recognizing receptor for LPS that could interact with TLR4. We also asked if exposure to an alternative SREC-I ligand, Hsp90 could trigger p65 phosphorylation (Fig 2A, lanes 5–7). However, minimal increases in phospho-536-p65/Rel levels were induced by Hsp90 even in cells expressing SREC-I plus TLR4 (Fig 2A).


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)

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.
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pone.0122529.g002: 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.
Mentions: As NF-kB is the most potent proinflammatory transcription factor, we examined the potential role of SREC-I in its activation by LPS. TLR4 activation has been shown to trigger activation of NF-kB. We next examined therefore the potential activity of SREC-I in LPS triggered NF-kB signaling in HEK 293 cells. As mentioned above, in contrast to Raw 264.7, HEK 293 cells were shown to be deficient in most SR family members (including SREC-I), thus SREC-I-specific effects could be examined in isolation in transfectants. This HEK 293 cell line stably expressed CD14, TLR4 and MD2; CD14 independent signaling was determined using CD14 blocking peptides (CD14-inh, 10 μg/ml), while the role of TLR4 was probed using RNA interference. The CD14 blocking peptide ab was first tested in THP1 cells by its ability to neutralize LPS-induced TNF-α secretion. The ab neutralized >60% of LPS induced cytokine release in this cell line. NF-kB activation was assayed by determining phosphorylation of its trans-activating subunit p65/Rel on serine 536 [19]. We then compared LPS-induced NF-kB signaling in cells expressing TLR4 and the LPS recognizing and signaling adaptor proteins CD14 and MD2 (Fig 2A, lane 1) without (lane 1) or with SREC-I (lane 2). LPS activated NF-kB in the absence of CD14 but with SREC-I expression (lane 2). Minimal signaling was observed in the absence of TLR4 with or without SREC-I (lanes 3, 4). This experiment showed therefore a role for SREC-I as recognizing receptor for LPS that could interact with TLR4. We also asked if exposure to an alternative SREC-I ligand, Hsp90 could trigger p65 phosphorylation (Fig 2A, lanes 5–7). However, minimal increases in phospho-536-p65/Rel levels were induced by Hsp90 even in cells expressing SREC-I plus TLR4 (Fig 2A).

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