Akiko Hata

SMAD PROTEINS CONTROL DROSHA-MEDIATED MIRNA MATURATION

MicroRNAs (miRNAs) are small non-coding RNAs that participate in the spatiotemporal regulation of messenger RNA and protein synthesis. Aberrant miRNA expression leads to developmental abnormalities and diseases, such as cardiovascular disorders and cancer; however, the stimuli and processes regulating miRNA biogenesis are largely unknown. The transforming growth factor β (TGF-β) and bone morphogenetic protein (BMP) family of growth factors orchestrates fundamental biological processes in development and in the homeostasis of adult tissues, including the vasculature. Here we show that induction of a contractile phenotype in human vascular smooth muscle cells by TGF-β and BMPs is mediated by miR-21. miR-21 downregulates PDCD4 (programmed cell death 4), which in turn acts as a negative regulator of smooth muscle contractile genes. Surprisingly, TGF-β and BMP signalling promotes a rapid increase in expression of mature miR-21 through a post-transcriptional step, promoting the processing of primary transcripts of miR-21 (pri-miR-21) into precursor miR-21 (pre-miR-21) by the DROSHA (also known as RNASEN) complex. TGF-β- and BMP-specific SMAD signal transducers are recruited to pri-miR-21 in a complex with the RNA helicase p68 (also known as DDX5), a component of the DROSHA microprocessor complex. The shared cofactor SMAD4 is not required for this process. Thus, regulation of miRNA biogenesis by ligand-specific SMAD proteins is critical for control of the vascular smooth muscle cell phenotype and potentially for SMAD4-independent responses mediated by the TGF-β and BMP signalling pathways.

Smad Proteins Bind a Conserved RNA Sequence to Promote MicroRNA Maturation by Drosha

The signal transducers of the transforming growth factor beta (TGFbeta)/bone morphogenetic protein (BMP), the Smads, promote the expression of a subset of miRNAs by facilitating the cleavage reaction by Drosha. The mechanism that limits Smad-mediated processing to a selective group of miRNAs remained hitherto unexplored. In this study, we expand the number of TGFbeta/BMP-regulated miRNAs (T/B-miRs) to 20. Of interest, a majority of T/B-miRs contain a consensus sequence (R-SBE) within the stem region of the primary transcripts of T/B-miRs (pri-T/B-miRs). Here, we demonstrate that Smads directly bind the R-SBE. Mutation of the R-SBE abrogates TGFbeta/BMP-induced recruitment of Smads, Drosha, and DGCR8 to pri-T/B-miRs and impairs their processing, whereas introduction of R-SBE to unregulated pri-miRNAs is sufficient to recruit Smads and to allow regulation by TGFbeta/BMP. Thus, Smads are multifunctional proteins that modulate gene expression transcriptionally through DNA binding and posttranscriptionally through pri-miRNA binding and regulation of miRNA processing.

Induction of microRNA-221 by platelet-derived growth factor signaling is critical for modulation of vascular smooth muscle phenotype.

The platelet-derived growth factor (PDGF) signaling pathway is a critical regulator of animal development and homeostasis. Activation of the PDGF pathway leads to neointimal proliferative responses to artery injury; it promotes a switch of vascular smooth muscle cells (vSMC) to a less contractile phenotype by inhibiting the SMC-specific gene expression and increasing the rate of proliferation and migration. The molecular mechanism for these pleiotropic effects of PDGFs has not been fully described. Here, we identify the microRNA-221 (miR-221), a small noncoding RNA, as a modulator of the phenotypic change of vSMCs in response to PDGF signaling. We demonstrate that miR-221 is transcriptionally induced upon PDGF treatment in primary vSMCs, leading to down-regulation of the targets c-Kit and p27Kip1. Down-regulation of p27Kip1 by miR-221 is critical for PDGF-mediated induction of cell proliferation. Additionally, decreased c-Kit causes inhibition of SMC-specific contractile gene transcription by reducing the expression of Myocardin (Myocd), a potent SMC-specific nuclear coactivator. Our study demonstrates that PDGF signaling, by modulating the expression of miR-221, regulates two critical determinants of the vSMC phenotype; they are SMC gene expression and cell proliferation.

Molecular basis for antagonism between PDGF and the TGFβ family of signalling pathways by control of miR-24 expression

Modulation of the vascular smooth‐muscle‐cell (vSMC) phenotype from a quiescent ‘contractile’ phenotype to a proliferative ‘synthetic’ phenotype has been implicated in vascular injury repair, as well as pathogenesis of vascular proliferative diseases. Both bone morphogenetic protein (BMP) and transforming growth factor‐β (TGFβ)‐signalling pathways promote a contractile phenotype, while the platelet‐derived growth factor‐BB (PDGF‐BB)‐signalling pathway promotes a switch to the synthetic phenotype. Here we show that PDGF‐BB induces microRNA‐24 (miR‐24), which in turn leads to downregulation of Tribbles‐like protein‐3 (Trb3). Repression of Trb3 coincides with reduced expression of Smad proteins and decrease in BMP and TGFβ signalling, promoting a synthetic phenotype in vSMCs. Inhibition of miR‐24 by antisense oligonuclotides abrogates the downregulation of Trb3 as well as pro‐synthetic activity of the PDGF‐signalling pathway. Thus, this study provides a molecular basis for the antagonism between the PDGF and TGFβ pathways, and its effect on the control of the vSMC phenotype.

down-regulation of Kruppel-like factor-4 (KLF4) by microRNA-143/145 is critical for modulation of vascular smooth muscle cell phenotype by transforming growth factor-beta and bone morphogenetic protein 4.

In the postnatal vasculature, fully differentiated and quiescent vascular smooth muscle cells (VSMCs) in a “contractile” phenotype are required for the normal regulation of vascular tone. The transforming growth factor-β (TGF-β) superfamily of growth factors (TGF-βs and bone morphogenetic proteins (BMPs)) are potent inducers of contractile phenotype and mediate (i) induction of contractile genes, and (ii) inhibition of VSMC growth and migration. Transcription of contractile genes is positively regulated by a regulatory DNA element called a CArG box. The CArG box is activated by the binding of serum response factor and its coactivators, myocardin (Myocd) or Myocd-related transcription factors (MRTFs). Krüppel-like factor-4 (KLF4) is known to inhibit activation of the CArG box. However, the potential role of KLF4 in the contractile activities of TGF-β or BMP has not been explored. Here, we demonstrate that TGF-β and BMP4 rapidly down-regulate KLF4 through induction of microRNA-143 (miR-143) and miR-145, which leads to a reduction of KLF4 transcripts and decreased KLF4 protein expression. Inhibition of miR-145 prevents down-regulation of KLF4 and activation of contractile genes by TGF-β or BMP4, suggesting that modulation of KLF4 is a prerequisite for induction of contractile genes by TGF-β and BMP4. Interestingly, both TGF-β and BMP4 activate transcription of the miR-143/145 gene cluster through the CArG box, however, TGF-β mediates this effect through induction of Myocd expression, whereas BMP4 utilizes nuclear translocation of MRTF-A. Thus, this study sheds light on both the similarities and the differences of TGF-β and BMP4 signaling in the regulation of KLF4 and contractile genes.

Mechanisms of control of microRNA biogenesis

MicroRNAs (miRNAs) are a class of ∼22 nt non-coding RNAs that control diverse biological functions in animals, plants and unicellular eukaryotes by promoting degradation or inhibition of translation of target mRNAs. miRNA expression is often tissue specific and developmentally regulated. Aberrant expression of miRNAs has been linked to developmental abnormalities and human diseases, including cancer and cardiovascular disorders. The recent identification of mechanisms of miRNA biogenesis regulation uncovers that various factors or growth factor signalling pathways control every step of the miRNA biogenesis pathway. Here, we review the mechanisms that control the regulation of miRNA biogenesis discovered in human cells. Further understanding of the mechanisms that control of miRNA biogenesis may allow the development of tools to modulate the expression of specific miRNAs, which is crucial for the development of novel therapies for human disorders derived from aberrant expression of miRNAs.

Control of Phenotypic Plasticity of Smooth Muscle Cells by Bone Morphogenetic Protein Signaling through the Myocardin-related Transcription Factors

Vascular smooth muscle cells (VSMCs), unlike other muscle cells, do not terminally differentiate. In response to injury, VSMCs change phenotype, proliferate, and migrate as part of the repair process. Dysregulation of this plasticity program contributes to the pathogenesis of several vascular disorders, such as atherosclerosis, restenosis, and hypertension. The discovery of mutations in the gene encoding BMPRII, the type II subunit of the receptor for bone morphogenetic proteins (BMPs), in patients with pulmonary arterial hypertension (PAH) provided an indication that BMP signaling may affect the homeostasis of VSMCs and their phenotype modulation. Here we report that BMP signaling potently induces SMC-specific genes in pluripotent cells and prevents dedifferentiation of arterial SMCs. The BMP-induced phenotype switch requires intact RhoA/ROCK signaling but is not blocked by inhibitors of the TGFβ and PI3K/Akt pathways. Furthermore, nuclear localization and recruitment of the myocardin-related transcription factors (MRTF-A and MRTF-B) to a smooth muscle α-actin promoter is observed in response to BMP treatment. Thus, BMP signaling modulates VSMC phenotype via cross-talk with the RhoA/MRTFs pathway, and may contribute to the development of the pathological characteristics observed in patients with PAH and other obliterative vascular diseases.

Structural and functional diversities of a family of signal transducing protein kinases, protein kinase C family; two distinct classes of PKC, conventional cPKC and novel nPKC.

Recent molecular cloning and biochemical experiments on the nature of protein kinase C (PKC) have revealed the existence of two distinct classes of phorbol ester (and diacylglycerol) receptor/protein kinase, conventional PKC (cPKC) and novel PKC (nPKC). Each of these classes contains multiple related molecules expressed in tissues and cells in a type-specific manner. Although nPKC does not show the typical PKC activity ascribable to conventional PKCs and thus was neglected in earlier studies, several lines of evidence suggest that nPKCs are involved in a variety of cell responses to physiological stimuli and phorbol esters. It is possible that in some cases nPKC is the major mediator of the so-called PKC-activators, such as phorbol esters, mezerein, and bryostatins. In addition to the clear difference between cPKC and nPKC, functional diversity among conventional PKCs has also been demonstrated; PKC gamma differs in its competence to mediate the signal toward transcriptional activation through TPA-responsive cis-acting elements from cPKC alpha and nPKC epsilon. The differences between cPKC and nPKC and among the individual members of each of these two classes, and their specific pattern of distribution in tissues and cells, provide a rationale by which to explain the specificity and diversity of cellular responses to external stimuli generating DAG and to phorbol esters. The results presented here also provide a means to dissect the complex signaling pathway in cells and to analyze the molecular basis underlying the signal transduction processes mediated by this family of protein kinases.

MicroRNA in Cancer: The Involvement of Aberrant MicroRNA Biogenesis Regulatory Pathways

MicroRNAs (miRNAs) are small, noncoding RNAs that influence diverse biological outcomes through the repression of target genes during normal development and pathological responses. In particular, the alteration of miRNA expression has dramatic consequences for the progression of tumorigenesis. miRNAs undergo two processing steps that transform a long primary transcript into the mature miRNA. Although the general miRNA biogenesis pathway is well established, it is clear that not all miRNAs are created equally. Recent studies show that miRNA expression is controlled by diverse mechanisms in response to cellular stimuli. In this review, we discuss the mechanisms that govern the regulation of miRNA biogenesis with particular focus on how these mechanisms are perturbed in cancer.

ZNF423 Is Critically Required for Retinoic Acid-Induced Differentiation and Is a Marker of Neuroblastoma Outcome

Retinoids play key roles in differentiation, growth arrest, and apoptosis and are increasingly being used in the clinic for the treatment of a variety of cancers, including neuroblastoma. Here, using a large-scale RNA interference-based genetic screen, we identify ZNF423 (also known as Ebfaz, OAZ, or Zfp423) as a component critically required for retinoic acid (RA)-induced differentiation. ZNF423 associates with the RARalpha/RXRalpha nuclear receptor complex and is essential for transactivation in response to retinoids. Downregulation of ZNF423 expression by RNA interference in neuroblastoma cells results in a growth advantage and resistance to RA-induced differentiation, whereas overexpression of ZNF423 leads to growth inhibition and enhanced differentiation. Finally, we show that low ZNF423 expression is associated with poor disease outcome in neuroblastoma patients.