DiGeorge Syndrome Critical Region 8 (DGCR8) Protein-mediated microRNA Biogenesis Is Essential for Vascular Smooth Muscle Cell Development in Mice

Department of Physiology, Campbell Clinic, University of Tennessee Health Science Center, Memphis, Tennessee 38163, USA.
Journal of Biological Chemistry (Impact Factor: 4.57). 04/2012; 287(23):19018-28. DOI: 10.1074/jbc.M112.351791
Source: PubMed


DiGeorge Critical Region 8 (DGCR8) is a double-stranded RNA-binding protein that interacts with Drosha and facilitates microRNA (miRNA) maturation. However, the role of DGCR8 in vascular smooth muscle cells (VSMCs) is not well understood. To investigate whether DGCR8 contributes to miRNA maturation in VSMCs, we generated DGCR8 conditional knockout (cKO) mice by crossing VSMC-specific Cre mice (SM22-Cre) with DGCR8(loxp/loxp) mice. We found that loss of DGCR8 in VSMCs resulted in extensive liver hemorrhage and embryonic mortality between embryonic days (E) 12.5 and E13.5. DGCR8 cKO embryos displayed dilated blood vessels and disarrayed vascular architecture. Blood vessels were absent in the yolk sac of DGCR8 KOs after E12.5. Disruption of DGCR8 in VSMCs reduced VSMC proliferation and promoted apoptosis in vitro and in vivo. In DGCR8 cKO embryos and knockout VSMCs, differentiation marker genes, including αSMA, SM22, and CNN1, were significantly down-regulated, and the survival pathways of ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated. Knockout of DGCR8 in VSMCs has led to down-regulation of the miR-17/92 and miR-143/145 clusters. We further demonstrated that the miR-17/92 cluster promotes VSMC proliferation and enhances VSMC marker gene expression, which may contribute to the defects of DGCR8 cKO mutants. Our results indicate that the DGCR8 gene is required for vascular development through the regulation of VSMC proliferation, apoptosis, and differentiation.

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    • "Thus, targeted disruption of the Dgcr8 locus in mice leads to embryonic lethality (Wang et al., 2007; Stark et al., 2008) and defects in proliferation and differentiation in embryonic stem cells (Wang et al., 2007, 2008; Stark et al., 2008). Similarly, specific ablation of DGCR8 in diverse adult mouse tissues results in severe proliferation and differentiation defects in the affected organs (Yi et al., 2009; Fenelon et al., 2011; Chen et al., 2012). Our data suggest that the arrest triggered by DGCR8 depletion is relayed mainly by the direct action of specific miRNAs on the expression of senescence regulators, although some contribution of alternative pathways remains possible. "
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    ABSTRACT: The regulation of gene expression by microRNAs (miRNAs) is critical for normal development and physiology. Conversely, miRNA function is frequently impaired in cancer, and other pathologies, either by aberrant expression of individual miRNAs or dysregulation of miRNA synthesis. Here, we have investigated the impact of global disruption of miRNA biogenesis in primary fibroblasts of human or murine origin, through the knockdown of DGCR8, an essential mediator of the synthesis of canonical miRNAs. We find that the inactivation of DGCR8 in these cells results in a dramatic antiproliferative response, with the acquisition of a senescent phenotype. Senescence triggered by DGCR8 loss is accompanied by the upregulation of the cell-cycle inhibitor p21CIP1. We further show that a subset of senescence-associated miRNAs with the potential to target p21CIP1 is downregulated during DGCR8-mediated senescence. Interestingly, the antiproliferative response to miRNA biogenesis disruption is retained in human tumor cells, irrespective of p53 status. In summary, our results show that defective synthesis of canonical microRNAs results in cell-cycle arrest and cellular senescence in primary fibroblasts mediated by specific miRNAs, and thus identify global miRNA disruption as a novel senescence trigger. This article is protected by copyright. All rights reserved.
    Aging cell 06/2013; 12(5). DOI:10.1111/acel.12117 · 6.34 Impact Factor
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    • "Recent studies indicated that some splicing-independent mirtron-like miRNA (simtrons) maturation does not require the canonical miRNA biogenesis pathway, which skips the processing of DGCR8, exportin-5, Dicer, and Ago2, but is Drosha-dependent [6], [7], [8]. In our previous studies, we showed that disruption of DGCR8 in VSMCs results in embryonic death 2–3 days earlier than that of Dicer, although both DGCR8 and Dicer cKO embryos share some phenotypic similarity [9], [10]. Our studies demonstrated that DGCR8 plays a more important role than does Dicer in maintaining the VSMC functions by participating in the miRNA process in the upstream biogenesis pathway. "
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    ABSTRACT: miRNA biogenesis enzyme Drosha cleaves double-stranded primary miRNA by interacting with double-stranded RNA binding protein DGCR8 and processes primary miRNA into precursor miRNA to participate in the miRNA biogenesis pathway. The role of Drosha in vascular smooth muscle cells (VSMCs) has not been well addressed. We generated Drosha conditional knockout (cKO) mice by crossing VSMC-specific Cre mice, SM22-Cre, with Drosha loxp/loxp mice. Disruption of Drosha in VSMCs resulted in embryonic lethality at E14.5 with severe liver hemorrhage in mutant embryos. No obvious developmental delay was observed in Drosha cKO embryos. The vascular structure was absent in the yolk sac of Drosha homozygotes at E14.5. Loss of Drosha reduced VSMC proliferation in vitro and in vivo. The VSMC differentiation marker genes, including aSMA, SM22, and CNN1, and endothelial cell marker CD31 were significantly downregulated in Drosha cKO mice compared to controls. ERK1/2 mitogen-activated protein kinase and the phosphatidylinositol 3-kinase/AKT were attenuated in VSMCs in vitro and in vivo. Disruption of Drosha in VSMCs of mice leads to the dysregulation of miRNA expression. Using bioinformatics approach, the interactions between dysregulated miRNAs and their target genes were analyzed. Our data demonstrated that Drosha is required for VSMC survival by targeting multiple signaling pathways.
    PLoS ONE 04/2013; 8(4):e60888. DOI:10.1371/journal.pone.0060888 · 3.23 Impact Factor
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    • "Increased expression of mir-21 is linked to glioblastoma (Wang et al., 2012). Deregulated DGCR8 expression, which is associated with DiGeorge syndrome (Chen et al., 2012), is involved in miRNA processing and in learning disability. Tourette's syndrome is associated with sequence variations in mir-189, which targets SLIT (axonal growth-controlling protein SLIT) in a mechanism that includes alterations in the miR-189 binding site in SLIT and Trk-like family member1 (SLITRK-1) mRNA (Abelson et al., 2005), a protein that is essential for neuronal growth, guidance, and neurite branching. "
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    ABSTRACT: RNA editing is an alteration in the primary nucleotide sequences resulting from a chemical change in the base. RNA editing is observed in eukaryotic mRNA, transfer RNA, ribosomal RNA, and non-coding RNAs (ncRNA). The most common RNA editing in the mammalian central nervous system is a base modification, where the adenosine residue is base-modified to inosine (A to I). Studies from ADAR (adenosine deaminase that act on RNA) mutants in Caenorhabditis elegans, Drosophila, and mice clearly show that the RNA editing process is an absolute requirement for nervous system homeostasis and normal physiology of the animal. Understanding the mechanisms of editing and findings of edited substrates has provided a better knowledge of the phenotype due to defective and hyperactive RNA editing. A to I RNA editing is catalyzed by a family of enzymes knows as ADARs. ADARs modify duplex RNAs and editing of duplex RNAs formed by ncRNAs can impact RNA functions, leading to an altered regulatory gene network. Such altered functions by A to I editing is observed in mRNAs, microRNAs (miRNA) but other editing of small and long ncRNAs (lncRNAs) has yet to be identified. Thus, ncRNA and RNA editing may provide key links between neural development, nervous system function, and neurological diseases. This review includes a summary of seminal findings regarding the impact of ncRNAs on biological and pathological processes, which may be further modified by RNA editing. NcRNAs are non-translated RNAs classified by size and function. Known ncRNAs like miRNAs, smallRNAs (smRNAs), PIWI-interacting RNAs (piRNAs), and lncRNAs play important roles in splicing, DNA methylation, imprinting, and RNA interference. Of note, miRNAs are involved in development and function of the nervous system that is heavily dependent on both RNA editing and the intricate spatiotemporal expression of ncRNAs. This review focuses on the impact of dysregulated A to I editing and ncRNAs in neurodegeneration.
    Frontiers in Genetics 01/2012; 3:326. DOI:10.3389/fgene.2012.00326
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