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MicroRNAs modulate the noncanonical transcription factor NF-kappaB pathway by regulating expression of the kinase IKKalpha during macrophage differentiation.

Li T, Morgan MJ, Choksi S, Zhang Y, Kim YS, Liu ZG - Nat. Immunol. (2010)

Bottom Line: Here we show that during human monocyte-macrophage differentiation, expression of the microRNAs miR-223, miR-15a and miR-16 decreased considerably, which led to higher expression of the serine-threonine kinase IKKalpha in macrophages.However, proinflammatory stimuli in macrophages resulted in greater induction of noncanonical NF-kappaB target genes.Thus, a decrease in certain microRNAs probably prevents macrophage hyperactivation yet primes the macrophage for certain responses to proinflammatory stimuli.

View Article: PubMed Central - PubMed

Affiliation: Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

ABSTRACT
MicroRNAs are key regulators of many biological processes, including cell differentiation. Here we show that during human monocyte-macrophage differentiation, expression of the microRNAs miR-223, miR-15a and miR-16 decreased considerably, which led to higher expression of the serine-threonine kinase IKKalpha in macrophages. In macrophages, higher IKKalpha expression in conjunction with stabilization of the kinase NIK induced larger amounts of p52. Because of low expression of the transcription factor RelB in untreated macrophages, high p52 expression repressed basal transcription of both canonical and noncanonical NF-kappaB target genes. However, proinflammatory stimuli in macrophages resulted in greater induction of noncanonical NF-kappaB target genes. Thus, a decrease in certain microRNAs probably prevents macrophage hyperactivation yet primes the macrophage for certain responses to proinflammatory stimuli.

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p100 is constitutively processed to p52 in macrophages(a) Immunoblot analysis of IKKα and p100-p52 in monocytes (Mo) vs. macrophages (MΦ). (b) Electrophoretic Mobility Shift Assay (EMSA) from nuclear lysates of monocytes (Mo) and macrophages (MΦ) showing binding to κB or AP2 oligos. p52 antibody added where indicated. (c) Western blots from monocytes (Mo) and macrophages (MΦ) lysates analyzed by immunoblot with anti-NIK and anti-TRAF2 antibodies. (d) Macrophages (MΦ) were transfected with miRNA mimic control oligo and pooled miRNAs mimics, (miR-15a, miR16 and miR-223). Forty-eight hours after transfection, cell lyates were analyzed by immunoblot with indicated antibodies. TRADD and β-actin blots indicate loading of lanes, (a, c, d).
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Figure 5: p100 is constitutively processed to p52 in macrophages(a) Immunoblot analysis of IKKα and p100-p52 in monocytes (Mo) vs. macrophages (MΦ). (b) Electrophoretic Mobility Shift Assay (EMSA) from nuclear lysates of monocytes (Mo) and macrophages (MΦ) showing binding to κB or AP2 oligos. p52 antibody added where indicated. (c) Western blots from monocytes (Mo) and macrophages (MΦ) lysates analyzed by immunoblot with anti-NIK and anti-TRAF2 antibodies. (d) Macrophages (MΦ) were transfected with miRNA mimic control oligo and pooled miRNAs mimics, (miR-15a, miR16 and miR-223). Forty-eight hours after transfection, cell lyates were analyzed by immunoblot with indicated antibodies. TRADD and β-actin blots indicate loading of lanes, (a, c, d).

Mentions: Since IKKα is essential in noncanonical NF-κB signaling, we examined downstream noncanonical components. Though minimal in monocytes, p100 processing increased substantially in macrophages, leading to a large amount of p52 (Fig. 5a). An electrophoretic mobility shift assay showed that while κB sites were bound by various NF-κB complexes in both monocytes and macrophages in the absence of treatment, p52 was bound only to κB sites in macrophages, and not monocytes, as shown by supershifting (Fig. 5b). Therefore, increased IKKα protein expression correlates with p52 DNA binding. To further examine the activation of the noncanonical NF-κB pathway, we examined NIK stability, a key requirement in this process. Immunoblotting revealed that substantial NIK protein was present in macrophages, but not in monocytes (Fig. 5c). Additionally, TRAF2, which negatively regulates NIK protein levels, was substantially decreased in the macrophages, when compared to the monocytes (Fig. 5c). The combination of these data suggests that the noncanonical NF-κB pathway is activated upon macrophage differentiation. HeLa cells stably express p100 and NIK, resulting in constitutive p52 production. We therefore used these cells to examine what effect IKKα-targeting miRNAs have on the events downstream of stabilized NIK. Transfection of pooled miRNA mimics in HeLa cells reduced not only IKKα protein, but also reduced the amounts of p52 without meaningfully affecting p100, demonstrating that decreases of these miRNAs and an increase in IKKα protein expression could indeed substantially contribute to a rise in p52 levels downstream of NIK stabilization (Supplementary Fig. 5). In macrophages, pooled mimics reduced not only IKKα protein expression relative to the control, but also reduced the amount of NIK protein (Fig. 5d). However, the target site prediction algorithm did not predict any target sites in the NIK mRNA and when we further examined TRAF2 protein expression in these blots, we found higher amounts of TRAF2 protein in the presence of these mimics when compared to the control (Fig. 5d), suggesting that an additional target mRNA of these mimics contributes to activation of the noncanonical pathway upstream of TRAF2 degradation. Similar results in mimic pool-transfected cells were obtained in HeLa cells (Supplementary Fig. 6).


MicroRNAs modulate the noncanonical transcription factor NF-kappaB pathway by regulating expression of the kinase IKKalpha during macrophage differentiation.

Li T, Morgan MJ, Choksi S, Zhang Y, Kim YS, Liu ZG - Nat. Immunol. (2010)

p100 is constitutively processed to p52 in macrophages(a) Immunoblot analysis of IKKα and p100-p52 in monocytes (Mo) vs. macrophages (MΦ). (b) Electrophoretic Mobility Shift Assay (EMSA) from nuclear lysates of monocytes (Mo) and macrophages (MΦ) showing binding to κB or AP2 oligos. p52 antibody added where indicated. (c) Western blots from monocytes (Mo) and macrophages (MΦ) lysates analyzed by immunoblot with anti-NIK and anti-TRAF2 antibodies. (d) Macrophages (MΦ) were transfected with miRNA mimic control oligo and pooled miRNAs mimics, (miR-15a, miR16 and miR-223). Forty-eight hours after transfection, cell lyates were analyzed by immunoblot with indicated antibodies. TRADD and β-actin blots indicate loading of lanes, (a, c, d).
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Figure 5: p100 is constitutively processed to p52 in macrophages(a) Immunoblot analysis of IKKα and p100-p52 in monocytes (Mo) vs. macrophages (MΦ). (b) Electrophoretic Mobility Shift Assay (EMSA) from nuclear lysates of monocytes (Mo) and macrophages (MΦ) showing binding to κB or AP2 oligos. p52 antibody added where indicated. (c) Western blots from monocytes (Mo) and macrophages (MΦ) lysates analyzed by immunoblot with anti-NIK and anti-TRAF2 antibodies. (d) Macrophages (MΦ) were transfected with miRNA mimic control oligo and pooled miRNAs mimics, (miR-15a, miR16 and miR-223). Forty-eight hours after transfection, cell lyates were analyzed by immunoblot with indicated antibodies. TRADD and β-actin blots indicate loading of lanes, (a, c, d).
Mentions: Since IKKα is essential in noncanonical NF-κB signaling, we examined downstream noncanonical components. Though minimal in monocytes, p100 processing increased substantially in macrophages, leading to a large amount of p52 (Fig. 5a). An electrophoretic mobility shift assay showed that while κB sites were bound by various NF-κB complexes in both monocytes and macrophages in the absence of treatment, p52 was bound only to κB sites in macrophages, and not monocytes, as shown by supershifting (Fig. 5b). Therefore, increased IKKα protein expression correlates with p52 DNA binding. To further examine the activation of the noncanonical NF-κB pathway, we examined NIK stability, a key requirement in this process. Immunoblotting revealed that substantial NIK protein was present in macrophages, but not in monocytes (Fig. 5c). Additionally, TRAF2, which negatively regulates NIK protein levels, was substantially decreased in the macrophages, when compared to the monocytes (Fig. 5c). The combination of these data suggests that the noncanonical NF-κB pathway is activated upon macrophage differentiation. HeLa cells stably express p100 and NIK, resulting in constitutive p52 production. We therefore used these cells to examine what effect IKKα-targeting miRNAs have on the events downstream of stabilized NIK. Transfection of pooled miRNA mimics in HeLa cells reduced not only IKKα protein, but also reduced the amounts of p52 without meaningfully affecting p100, demonstrating that decreases of these miRNAs and an increase in IKKα protein expression could indeed substantially contribute to a rise in p52 levels downstream of NIK stabilization (Supplementary Fig. 5). In macrophages, pooled mimics reduced not only IKKα protein expression relative to the control, but also reduced the amount of NIK protein (Fig. 5d). However, the target site prediction algorithm did not predict any target sites in the NIK mRNA and when we further examined TRAF2 protein expression in these blots, we found higher amounts of TRAF2 protein in the presence of these mimics when compared to the control (Fig. 5d), suggesting that an additional target mRNA of these mimics contributes to activation of the noncanonical pathway upstream of TRAF2 degradation. Similar results in mimic pool-transfected cells were obtained in HeLa cells (Supplementary Fig. 6).

Bottom Line: Here we show that during human monocyte-macrophage differentiation, expression of the microRNAs miR-223, miR-15a and miR-16 decreased considerably, which led to higher expression of the serine-threonine kinase IKKalpha in macrophages.However, proinflammatory stimuli in macrophages resulted in greater induction of noncanonical NF-kappaB target genes.Thus, a decrease in certain microRNAs probably prevents macrophage hyperactivation yet primes the macrophage for certain responses to proinflammatory stimuli.

View Article: PubMed Central - PubMed

Affiliation: Cell and Cancer Biology Branch, Center for Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD, USA.

ABSTRACT
MicroRNAs are key regulators of many biological processes, including cell differentiation. Here we show that during human monocyte-macrophage differentiation, expression of the microRNAs miR-223, miR-15a and miR-16 decreased considerably, which led to higher expression of the serine-threonine kinase IKKalpha in macrophages. In macrophages, higher IKKalpha expression in conjunction with stabilization of the kinase NIK induced larger amounts of p52. Because of low expression of the transcription factor RelB in untreated macrophages, high p52 expression repressed basal transcription of both canonical and noncanonical NF-kappaB target genes. However, proinflammatory stimuli in macrophages resulted in greater induction of noncanonical NF-kappaB target genes. Thus, a decrease in certain microRNAs probably prevents macrophage hyperactivation yet primes the macrophage for certain responses to proinflammatory stimuli.

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