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

Significance Dicer is a ribonuclease III enzyme in biosynthesis of miRNAs, regulators of gene expression involved in macrophage differentiation. We found a specific truncation of Dicer in monocytic cells resulting from apparently constitutive cleavage by a serine protease. Inhibition of this proteolytic truncation, which occurred during macrophage differentiation in presence of TLR ligands or prostaglandin E2, up-regulates full-length Dicer and promotes miR biosynthesis. Regulation of transcription of pri-miRNA is one mode to regulate biosynthesis of mature miRNA. Inhibition of constitutive proteolysis of Dicer, as described here, provides a second layer of regulation, at the level of miRNA processing. Our data provide insights to Dicer and miRNAs in macrophage polarization/differentiation, a key process in the innate immune response. 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 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 TNFα. 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.