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SR-A ligand and M-CSF dynamically regulate SR-A expression and function in primary macrophages via p38 MAPK activation.

Nikolic D, Calderon L, Du L, Post SR - BMC Immunol. (2011)

Bottom Line: These changes are associated with altered SR-A expression in macrophages; however, the intracellular signal pathways involved and the extent to which SR-A ligands regulate SR-A expression are not well defined.These results demonstrate that in resident macrophages SR-A expression and function can be dynamically regulated by changes in the macrophage microenvironment that are typical of inflammatory processes.In particular, our results indicate a previously unrecognized role for ligand binding to SR-A in up-regulating SR-A expression and activating p38 MAPK.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.

ABSTRACT

Background: Inflammation is characterized by dynamic changes in the expression of cytokines, such as M-CSF, and modifications of lipids and proteins that result in the formation of ligands for Class A Scavenger Receptors (SR-A). These changes are associated with altered SR-A expression in macrophages; however, the intracellular signal pathways involved and the extent to which SR-A ligands regulate SR-A expression are not well defined. To address these questions, SR-A expression and function were examined in resident mouse peritoneal macrophages incubated with M-CSF or the selective SR-A ligand acetylated-LDL (AcLDL).

Results: M-CSF increased SR-A expression and function, and required the specific activation of p38 MAPK, but not ERK1/2 or JNK. Increased SR-A expression and function returned to basal levels 72 hours after removing M-CSF. We next determined whether prolonged incubation of macrophages with SR-A ligand alters SR-A expression. In contrast to most receptors, which are down-regulated by chronic exposure to ligand, SR-A expression was reversibly increased by incubating macrophages with AcLDL. AcLDL activated p38 in wild-type macrophages but not in SR-A-/- macrophages, and p38 activation was specifically required for AcLDL-induced SR-A expression.

Conclusions: These results demonstrate that in resident macrophages SR-A expression and function can be dynamically regulated by changes in the macrophage microenvironment that are typical of inflammatory processes. In particular, our results indicate a previously unrecognized role for ligand binding to SR-A in up-regulating SR-A expression and activating p38 MAPK. In this way, SR-A may modulate inflammatory responses by enhancing macrophage uptake of modified protein/lipid, bacteria, and cell debris; and by regulating the production of inflammatory cytokines, growth factors, and proteolytic enzymes.

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M-CSF-stimulates SR-A expression by upregulating SR-A mRNA and protein synthesis which requires p38 MAPK activation. (A) MPM were incubated with or without M-CSF (25 ng/ml) for 10 min at 37°C. Cells were then lysed with MBST/OG buffer and phosphorylation of MAPK quantified by immunobloting. The results from a single experiment are shown and are representative of at least four separate experiments. (B) MPM were treated as indicated with specific inhibitors of p38 [SB203580 (10 μM)], JNK [SP600125 (20 μM)] or ERK1/2 [PD98059 (10 μM)] for 20 minutes at 37°C. Cells were then treated with or without M-CSF (25 ng/ml) for 24 hrs at 37°C and SR-A protein quantified by immunoblotting. Data represent the mean ± SEM from at least three independent experiments.* denotes significant difference (p < 0.05) from untreated control value (ANOVA with Dunnett's multiple comparison). (C) MPM were treated with specific inhibitors of p38 [SB203580 (10 μM)], JNK [SP600125 (20 μM)] or ERK1/2 [PD98059 (10 μM)] for 20 minutes at 37°C, and then incubated with M-CSF (25 ng/ml) for 24 hrs. SR-A-specific macrophage association was quantified by flow cytometry as described in Materials and Methods. Data represent the mean ± SEM from at least three separate experiments. * denotes significant difference (p < 0.05) from M-CSF treated control value (ANOVA with Dunnett's multiple comparison).
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Figure 2: M-CSF-stimulates SR-A expression by upregulating SR-A mRNA and protein synthesis which requires p38 MAPK activation. (A) MPM were incubated with or without M-CSF (25 ng/ml) for 10 min at 37°C. Cells were then lysed with MBST/OG buffer and phosphorylation of MAPK quantified by immunobloting. The results from a single experiment are shown and are representative of at least four separate experiments. (B) MPM were treated as indicated with specific inhibitors of p38 [SB203580 (10 μM)], JNK [SP600125 (20 μM)] or ERK1/2 [PD98059 (10 μM)] for 20 minutes at 37°C. Cells were then treated with or without M-CSF (25 ng/ml) for 24 hrs at 37°C and SR-A protein quantified by immunoblotting. Data represent the mean ± SEM from at least three independent experiments.* denotes significant difference (p < 0.05) from untreated control value (ANOVA with Dunnett's multiple comparison). (C) MPM were treated with specific inhibitors of p38 [SB203580 (10 μM)], JNK [SP600125 (20 μM)] or ERK1/2 [PD98059 (10 μM)] for 20 minutes at 37°C, and then incubated with M-CSF (25 ng/ml) for 24 hrs. SR-A-specific macrophage association was quantified by flow cytometry as described in Materials and Methods. Data represent the mean ± SEM from at least three separate experiments. * denotes significant difference (p < 0.05) from M-CSF treated control value (ANOVA with Dunnett's multiple comparison).

Mentions: Many effects of M-CSF including macrophage migration, differentiation, survival, and cytokine production are mediated, in part, via activation of MAPKs, a family of kinases that include ERK1/2, JNK, and p38 [34-36]. Activation of MAPKs, in particular JNK and p38 MAPK, regulates the activity of several transcription factors including AP-1 [23]. The binding of AP-1 to an upstream enhancer element is sufficient to direct specific macrophage SR-A expression in inflammatory cells [1,21,22]. Therefore, we examined M-CSF-dependent MAPK activation and whether MAPK activation was required for M-CSF-induced SR-A expression. For this, resident MPMs were treated with M-CSF and the activation of ERK, p38, and JNK assessed by immunoblotting with phospho-specific MAPK antibodies. As shown in Figure 2A, M-CSF induced the phosphorylation of both p38 and ERK1/2. In contrast, JNK phosphorylation was not detectable in either the presence or absence of M-CSF (data not shown). To determine if activation of MAPK was specifically required for M-CSF-induced SR-A expression and function, the ability of M-CSF to stimulate SR-A expression and AcLDL association was assessed in MPMs pretreated with specific inhibitors of p38 MAPK (SB203580), JNK (SP600125), and MEK1 (PD98059), which inhibits ERK1/2 activation. Inhibiting JNK or ERK1/2 activation had no effect on either M-CSF-induced SR-A expression (Figure 2B) or M-CSF-induced uptake of modified lipoprotein (Figure 2C). In contrast, pretreating macrophages with SB203580 inhibited both M-CSF-induced SR-A expression and modified lipoprotein uptake (Figure 2C). Together, these data define a specific requirement for activation of p38 MAPK, but not ERK1/2 and JNK, in M-CSF-induced SR-A expression and function.


SR-A ligand and M-CSF dynamically regulate SR-A expression and function in primary macrophages via p38 MAPK activation.

Nikolic D, Calderon L, Du L, Post SR - BMC Immunol. (2011)

M-CSF-stimulates SR-A expression by upregulating SR-A mRNA and protein synthesis which requires p38 MAPK activation. (A) MPM were incubated with or without M-CSF (25 ng/ml) for 10 min at 37°C. Cells were then lysed with MBST/OG buffer and phosphorylation of MAPK quantified by immunobloting. The results from a single experiment are shown and are representative of at least four separate experiments. (B) MPM were treated as indicated with specific inhibitors of p38 [SB203580 (10 μM)], JNK [SP600125 (20 μM)] or ERK1/2 [PD98059 (10 μM)] for 20 minutes at 37°C. Cells were then treated with or without M-CSF (25 ng/ml) for 24 hrs at 37°C and SR-A protein quantified by immunoblotting. Data represent the mean ± SEM from at least three independent experiments.* denotes significant difference (p < 0.05) from untreated control value (ANOVA with Dunnett's multiple comparison). (C) MPM were treated with specific inhibitors of p38 [SB203580 (10 μM)], JNK [SP600125 (20 μM)] or ERK1/2 [PD98059 (10 μM)] for 20 minutes at 37°C, and then incubated with M-CSF (25 ng/ml) for 24 hrs. SR-A-specific macrophage association was quantified by flow cytometry as described in Materials and Methods. Data represent the mean ± SEM from at least three separate experiments. * denotes significant difference (p < 0.05) from M-CSF treated control value (ANOVA with Dunnett's multiple comparison).
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Figure 2: M-CSF-stimulates SR-A expression by upregulating SR-A mRNA and protein synthesis which requires p38 MAPK activation. (A) MPM were incubated with or without M-CSF (25 ng/ml) for 10 min at 37°C. Cells were then lysed with MBST/OG buffer and phosphorylation of MAPK quantified by immunobloting. The results from a single experiment are shown and are representative of at least four separate experiments. (B) MPM were treated as indicated with specific inhibitors of p38 [SB203580 (10 μM)], JNK [SP600125 (20 μM)] or ERK1/2 [PD98059 (10 μM)] for 20 minutes at 37°C. Cells were then treated with or without M-CSF (25 ng/ml) for 24 hrs at 37°C and SR-A protein quantified by immunoblotting. Data represent the mean ± SEM from at least three independent experiments.* denotes significant difference (p < 0.05) from untreated control value (ANOVA with Dunnett's multiple comparison). (C) MPM were treated with specific inhibitors of p38 [SB203580 (10 μM)], JNK [SP600125 (20 μM)] or ERK1/2 [PD98059 (10 μM)] for 20 minutes at 37°C, and then incubated with M-CSF (25 ng/ml) for 24 hrs. SR-A-specific macrophage association was quantified by flow cytometry as described in Materials and Methods. Data represent the mean ± SEM from at least three separate experiments. * denotes significant difference (p < 0.05) from M-CSF treated control value (ANOVA with Dunnett's multiple comparison).
Mentions: Many effects of M-CSF including macrophage migration, differentiation, survival, and cytokine production are mediated, in part, via activation of MAPKs, a family of kinases that include ERK1/2, JNK, and p38 [34-36]. Activation of MAPKs, in particular JNK and p38 MAPK, regulates the activity of several transcription factors including AP-1 [23]. The binding of AP-1 to an upstream enhancer element is sufficient to direct specific macrophage SR-A expression in inflammatory cells [1,21,22]. Therefore, we examined M-CSF-dependent MAPK activation and whether MAPK activation was required for M-CSF-induced SR-A expression. For this, resident MPMs were treated with M-CSF and the activation of ERK, p38, and JNK assessed by immunoblotting with phospho-specific MAPK antibodies. As shown in Figure 2A, M-CSF induced the phosphorylation of both p38 and ERK1/2. In contrast, JNK phosphorylation was not detectable in either the presence or absence of M-CSF (data not shown). To determine if activation of MAPK was specifically required for M-CSF-induced SR-A expression and function, the ability of M-CSF to stimulate SR-A expression and AcLDL association was assessed in MPMs pretreated with specific inhibitors of p38 MAPK (SB203580), JNK (SP600125), and MEK1 (PD98059), which inhibits ERK1/2 activation. Inhibiting JNK or ERK1/2 activation had no effect on either M-CSF-induced SR-A expression (Figure 2B) or M-CSF-induced uptake of modified lipoprotein (Figure 2C). In contrast, pretreating macrophages with SB203580 inhibited both M-CSF-induced SR-A expression and modified lipoprotein uptake (Figure 2C). Together, these data define a specific requirement for activation of p38 MAPK, but not ERK1/2 and JNK, in M-CSF-induced SR-A expression and function.

Bottom Line: These changes are associated with altered SR-A expression in macrophages; however, the intracellular signal pathways involved and the extent to which SR-A ligands regulate SR-A expression are not well defined.These results demonstrate that in resident macrophages SR-A expression and function can be dynamically regulated by changes in the macrophage microenvironment that are typical of inflammatory processes.In particular, our results indicate a previously unrecognized role for ligand binding to SR-A in up-regulating SR-A expression and activating p38 MAPK.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Pathology, University of Arkansas for Medical Sciences, Little Rock, Arkansas 72205, USA.

ABSTRACT

Background: Inflammation is characterized by dynamic changes in the expression of cytokines, such as M-CSF, and modifications of lipids and proteins that result in the formation of ligands for Class A Scavenger Receptors (SR-A). These changes are associated with altered SR-A expression in macrophages; however, the intracellular signal pathways involved and the extent to which SR-A ligands regulate SR-A expression are not well defined. To address these questions, SR-A expression and function were examined in resident mouse peritoneal macrophages incubated with M-CSF or the selective SR-A ligand acetylated-LDL (AcLDL).

Results: M-CSF increased SR-A expression and function, and required the specific activation of p38 MAPK, but not ERK1/2 or JNK. Increased SR-A expression and function returned to basal levels 72 hours after removing M-CSF. We next determined whether prolonged incubation of macrophages with SR-A ligand alters SR-A expression. In contrast to most receptors, which are down-regulated by chronic exposure to ligand, SR-A expression was reversibly increased by incubating macrophages with AcLDL. AcLDL activated p38 in wild-type macrophages but not in SR-A-/- macrophages, and p38 activation was specifically required for AcLDL-induced SR-A expression.

Conclusions: These results demonstrate that in resident macrophages SR-A expression and function can be dynamically regulated by changes in the macrophage microenvironment that are typical of inflammatory processes. In particular, our results indicate a previously unrecognized role for ligand binding to SR-A in up-regulating SR-A expression and activating p38 MAPK. In this way, SR-A may modulate inflammatory responses by enhancing macrophage uptake of modified protein/lipid, bacteria, and cell debris; and by regulating the production of inflammatory cytokines, growth factors, and proteolytic enzymes.

Show MeSH
Related in: MedlinePlus