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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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IKKα-targeting miRNAs affects noncanonical and canonical NF-κ.B gene expression(a) Real time PCR analysis of ELC mRNA in monocytes and macrophages, normalized to GAPDH, data representative of at least three independent experiments. Error bars, +/−standard deviation of the mean. (b) Anti-RelB immunoblots of lysates from macrophages (MΦ) treated with LPS for indicated time course (in hours). (c). Real time PCR analysis of ELC mRNA in monocytes, macrophages, or LPS-treated macrophages, normalized to GAPDH. Data representative of at least three independent experiments. Error bars, +/−standard deviation of the mean. (d)(e)(f), Macrophages (MΦ) were transfected with miRNA mimics or control mimic and 24 hours after transfection, cells were or were not challenged with LPS (1μg/ml) for another 24 hours. (d) Immunoblot showing IKKα protein level in corresponding protein samples for (e, f). (e) ELC and (f) A20 mRNA expression as measured by real time PCR, normalized to GAPDH mRNA. The relative fold increase in LPS treated cells compared to untreated cells is shown for (e) and (f). Error bars, +/− range based on standard deviation of the mean. Data representative of at least three independent experiments. TRADD and β-actin blots indicate loading of lanes, (b, d).
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Figure 7: IKKα-targeting miRNAs affects noncanonical and canonical NF-κ.B gene expression(a) Real time PCR analysis of ELC mRNA in monocytes and macrophages, normalized to GAPDH, data representative of at least three independent experiments. Error bars, +/−standard deviation of the mean. (b) Anti-RelB immunoblots of lysates from macrophages (MΦ) treated with LPS for indicated time course (in hours). (c). Real time PCR analysis of ELC mRNA in monocytes, macrophages, or LPS-treated macrophages, normalized to GAPDH. Data representative of at least three independent experiments. Error bars, +/−standard deviation of the mean. (d)(e)(f), Macrophages (MΦ) were transfected with miRNA mimics or control mimic and 24 hours after transfection, cells were or were not challenged with LPS (1μg/ml) for another 24 hours. (d) Immunoblot showing IKKα protein level in corresponding protein samples for (e, f). (e) ELC and (f) A20 mRNA expression as measured by real time PCR, normalized to GAPDH mRNA. The relative fold increase in LPS treated cells compared to untreated cells is shown for (e) and (f). Error bars, +/− range based on standard deviation of the mean. Data representative of at least three independent experiments. TRADD and β-actin blots indicate loading of lanes, (b, d).

Mentions: Unexpectedly, the basal levels of ELC mRNA in macrophages were decreased compared to matched monocyte samples (Fig. 7a). Since p52 was bound to nuclear DNA in macrophages, but not in monocytes (Fig. 5b), we had expected the levels of ELC to rise, given that the ELC gene is a known target of the noncanonical pathway. However, this data indicated that p52 could be acting as a repressor of transcription. Consistent with this hypothesis, transfection of GM-CSF-differentiated macrophages with miR-15a, miR-16, and miR-223 mimics, which decreased IKKα protein expression, increased basal ELC mRNA levels in macrophages when compared to control mimic (Supplementary Fig. 7).


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)

IKKα-targeting miRNAs affects noncanonical and canonical NF-κ.B gene expression(a) Real time PCR analysis of ELC mRNA in monocytes and macrophages, normalized to GAPDH, data representative of at least three independent experiments. Error bars, +/−standard deviation of the mean. (b) Anti-RelB immunoblots of lysates from macrophages (MΦ) treated with LPS for indicated time course (in hours). (c). Real time PCR analysis of ELC mRNA in monocytes, macrophages, or LPS-treated macrophages, normalized to GAPDH. Data representative of at least three independent experiments. Error bars, +/−standard deviation of the mean. (d)(e)(f), Macrophages (MΦ) were transfected with miRNA mimics or control mimic and 24 hours after transfection, cells were or were not challenged with LPS (1μg/ml) for another 24 hours. (d) Immunoblot showing IKKα protein level in corresponding protein samples for (e, f). (e) ELC and (f) A20 mRNA expression as measured by real time PCR, normalized to GAPDH mRNA. The relative fold increase in LPS treated cells compared to untreated cells is shown for (e) and (f). Error bars, +/− range based on standard deviation of the mean. Data representative of at least three independent experiments. TRADD and β-actin blots indicate loading of lanes, (b, d).
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Related In: Results  -  Collection

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Figure 7: IKKα-targeting miRNAs affects noncanonical and canonical NF-κ.B gene expression(a) Real time PCR analysis of ELC mRNA in monocytes and macrophages, normalized to GAPDH, data representative of at least three independent experiments. Error bars, +/−standard deviation of the mean. (b) Anti-RelB immunoblots of lysates from macrophages (MΦ) treated with LPS for indicated time course (in hours). (c). Real time PCR analysis of ELC mRNA in monocytes, macrophages, or LPS-treated macrophages, normalized to GAPDH. Data representative of at least three independent experiments. Error bars, +/−standard deviation of the mean. (d)(e)(f), Macrophages (MΦ) were transfected with miRNA mimics or control mimic and 24 hours after transfection, cells were or were not challenged with LPS (1μg/ml) for another 24 hours. (d) Immunoblot showing IKKα protein level in corresponding protein samples for (e, f). (e) ELC and (f) A20 mRNA expression as measured by real time PCR, normalized to GAPDH mRNA. The relative fold increase in LPS treated cells compared to untreated cells is shown for (e) and (f). Error bars, +/− range based on standard deviation of the mean. Data representative of at least three independent experiments. TRADD and β-actin blots indicate loading of lanes, (b, d).
Mentions: Unexpectedly, the basal levels of ELC mRNA in macrophages were decreased compared to matched monocyte samples (Fig. 7a). Since p52 was bound to nuclear DNA in macrophages, but not in monocytes (Fig. 5b), we had expected the levels of ELC to rise, given that the ELC gene is a known target of the noncanonical pathway. However, this data indicated that p52 could be acting as a repressor of transcription. Consistent with this hypothesis, transfection of GM-CSF-differentiated macrophages with miR-15a, miR-16, and miR-223 mimics, which decreased IKKα protein expression, increased basal ELC mRNA levels in macrophages when compared to control mimic (Supplementary Fig. 7).

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.

Show MeSH