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Dicer up-regulation by inhibition of specific proteolysis in differentiating monocytic cells.

ABSTRACT: Dicer is a ribonuclease III enzyme in biosynthesis of micro-RNAs (miRNAs). Here we describe a regulation of Dicer expression in monocytic cells, based on proteolysis. In undifferentiated Mono Mac 6 (MM6) cells, full-length Dicer was undetectable; only an ?50-kDa fragment appeared in Western blots. However, when MM6 cells were treated with zymosan or LPS during differentiation with TGF-? and 1,25diOHvitD3, full-length Dicer became abundant together with varying amounts of ?170- and ?50-kDa Dicer fragments. Mass spectrometry identified the Dicer fragments and showed cleavage about 450 residues upstream from the C terminus. Also, PGE₂ (prostaglandin E2) added to differentiating MM6 cells up-regulated full-length Dicer, through EP2/EP4 and cAMP. The TLR stimuli strongly induced miR-146a-5p, while PGE₂ increased miR-99a-5p and miR-125a-5p, both implicated in down-regulation of TNF?. The Ser protease inhibitor AEBSF (4-[2-aminoethyl] benzene sulfonyl fluoride) up-regulated full-length Dicer, both in MM6 cells and in primary human blood monocytes, indicating a specific proteolytic degradation. However, AEBSF alone did not lead to a general increase in miR expression, indicating that additional mechanisms are required to increase miRNA biosynthesis. Finally, differentiation of monocytes to macrophages with M-CSF or GM-CSF strongly up-regulated full-length Dicer. Our results suggest that differentiation regimens, both in the MM6 cell line and of peripheral blood monocytes, inhibit an apparently constitutive Dicer proteolysis, allowing for increased formation of miRNAs.

SUBMITTER: Basavarajappa D

PROVIDER: S-EPMC7165444 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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Dicer up-regulation by inhibition of specific proteolysis in differentiating monocytic cells.

Basavarajappa Devaraj D Uebbing Stella S Kreiss Marius M Lukic Ana A Suess Beatrix B Steinhilber Dieter D Samuelsson Bengt B Rådmark Olof O

Proceedings of the National Academy of Sciences of the United States of America 20200327 15

Dicer is a ribonuclease III enzyme in biosynthesis of micro-RNAs (miRNAs). Here we describe a regulation of Dicer expression in monocytic cells, based on proteolysis. In undifferentiated Mono Mac 6 (MM6) cells, full-length Dicer was undetectable; only an ∼50-kDa fragment appeared in Western blots. However, when MM6 cells were treated with zymosan or LPS during differentiation with TGF-β and 1,25diOHvitD3, full-length Dicer became abundant together with varying amounts of ∼170- and ∼50-kDa Dicer ...[more]

PMID: 32220961

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Project description:Monocytic cells, including macrophages and dendritic cells, exist in different activation states that are critical to the regulation of antimicrobial immunity. Many pandemic viruses are monocytotropic, including porcine reproductive and respiratory syndrome virus (PRRSV), which directly infects subsets of monocytic cells and interferes with antiviral responses. To study antiviral responses in PRRSV-infected monocytic cells, we characterized inflammatory cytokine responses and genome-wide profiled signature genes to investigate response pathways in uninfected and PRRSV-infected monocytic cells at different activation states. Our findings showed suppressed interferon (IFN) production in macrophages in non-antiviral states and an arrest of lipid metabolic pathways in macrophages at antiviral states. Importantly, porcine monocytic cells at different activation states were susceptible to PRRSV and responded differently to viral infection. Based on Gene Ontology (GO) analysis, two approaches were used to potentiate antiviral activity: (i) pharmaceutical modulation of cellular lipid metabolism and (ii) in situ PRRSV replication-competent expression of interferon alpha (IFN-?). Both approaches significantly suppressed exogenous viral infection in monocytic cells. In particular, the engineered IFN-expressing PRRSV strain eliminated exogenous virus infection and sustained cell viability at 4 days postinfection in macrophages. These findings suggest an intricate interaction of viral infection with the activation status of porcine monocytic cells. An understanding and integration of antiviral infection with activation status of monocytic cells may provide a means of potentiating antiviral immunity.Activation statuses of monocytic cells, including monocytes, macrophages (M?s), and dendritic cells (DCs), are critically important for antiviral immunity. Unfortunately, the activation status of porcine monocytic cells or how cell activation status functionally interacts with antiviral immunity remains largely unknown. This is a significant omission because many economically important porcine viruses are monocytotropic, including our focus, PRRSV, which alone causes nearly $800 million economic loss annually in the U.S. swine industries. PRRSV is ideal for deciphering how monocytic cell activation statuses interact with antiviral immunity, because it directly infects subsets of monocytic cells and subverts overall immune responses. In this study, we systematically investigate the activation status of porcine monocytic cells to determine the intricate interaction of viral infection with activation statuses and functionally regulate antiviral immunity within the framework of the activation paradigm. Our findings may provide a means of potentiating antiviral immunity and leading to novel vaccines for PRRS prevention.

Dataset Information

Dicer up-regulation by inhibition of specific proteolysis in differentiating monocytic cells.

Publications

Dicer up-regulation by inhibition of specific proteolysis in differentiating monocytic cells.

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