Kazuya Terasawa

The gut microbiota suppresses insulin-mediated fat accumulation via the short-chain fatty acid receptor GPR43

The gut microbiota affects nutrient acquisition and energy regulation of the host, and can influence the development of obesity, insulin resistance, and diabetes. During feeding, gut microbes produce short-chain fatty acids, which are important energy sources for the host. Here we show that the short-chain fatty acid receptor GPR43 links the metabolic activity of the gut microbiota with host body energy homoeostasis. We demonstrate that GPR43-deficient mice are obese on a normal diet, whereas mice overexpressing GPR43 specifically in adipose tissue remain lean even when fed a high-fat diet. Raised under germ-free conditions or after treatment with antibiotics, both types of mice have a normal phenotype. We further show that short-chain fatty acid-mediated activation of GPR43 suppresses insulin signalling in adipocytes, which inhibits fat accumulation in adipose tissue and promotes the metabolism of unincorporated lipids and glucose in other tissues. These findings establish GPR43 as a sensor for excessive dietary energy, thereby controlling body energy utilization while maintaining metabolic homoeostasis.

Intra-Platform Repeatability and Inter-Platform Comparability of MicroRNA Microarray Technology

Over the last decade, DNA microarray technology has provided a great contribution to the life sciences. The MicroArray Quality Control (MAQC) project demonstrated the way to analyze the expression microarray. Recently, microarray technology has been utilized to analyze a comprehensive microRNA expression profiling. Currently, several platforms of microRNA microarray chips are commercially available. Thus, we compared repeatability and comparability of five different microRNA microarray platforms (Agilent, Ambion, Exiqon, Invitrogen and Toray) using 309 microRNAs probes, and the Taqman microRNA system using 142 microRNA probes. This study demonstrated that microRNA microarray has high intra-platform repeatability and comparability to quantitative RT-PCR of microRNA. Among the five platforms, Agilent and Toray array showed relatively better performances than the others. However, the current lineup of commercially available microRNA microarray systems fails to show good inter-platform concordance, probably because of lack of an adequate normalization method and severe divergence in stringency of detection call criteria between different platforms. This study provided the basic information about the performance and the problems specific to the current microRNA microarray systems.

Regulation of the activity of the transcription factor Runx2 by two homeobox proteins, Msx2 and Dlx5

Background Runx2, formerly called PEBP2αA or Cbfa1, is a transcription factor whose deletion causes a complete lack of ossification. It directly regulates the expression of osteoblast‐specific genes through the osteoblast‐specific cis‐acting element found in the promoter region of these genes.

Sustained activation of ERK1/2 by NGF induces microRNA‐221 and 222 in PC12 cells

MicroRNAs (miRNAs) are small non‐coding RNAs that regulate gene expression by inhibiting translation and/or inducing degradation of target mRNAs, and they play important roles in a wide variety of biological functions including cell differentiation, tumorigenesis, apoptosis and metabolism. However, there is a paucity of information concerning the regulatory mechanism of miRNA expression. Here we report identification of growth factor‐regulated miRNAs using the PC12 cell line, an established model of neuronal growth and differentiation. We found that expression of miR‐221 and miR‐222 expression were induced by nerve growth factor (NGF) stimulation in PC12 cells, and that this induction was dependent on sustained activation of the extracellular signal‐regulated kinase 1 and 2 (ERK1/2) pathway. Using a target prediction program, we also identified a pro‐apototic factor, the BH3‐only protein Bim, as a potential target of miR‐221/222. Overexpression of miR‐221 or miR‐222 suppressed the activity of a luciferase reporter activity fused to the 3′ UTR of Bim mRNA. Furthermore, overexpression of miR‐221/222 decreased endogenous Bim mRNA expression. These results reveal that the ERK signal regulates miR‐221/222 expression, and that these miRNAs might contribute to NGF‐dependent cell survival in PC12 cells.

MicroRNA-34a inhibits cell proliferation by repressing mitogen-activated protein kinase kinase 1 during megakaryocytic differentiation of K562 cells.

Phorbol 12-myristate 13-acetate (PMA) induces megakaryocytic differentiation of the human chronic myelocytic leukemia cell line K562. We examined the potential regulatory role of microRNAs (miRNAs) in this process. Genome-wide expression profiling identified 21 miRNAs (miRs) that were induced by the treatment of K562 cells with PMA. Among them, the expression of miR-34a, miR-221, and miR-222 was induced in the early stages and maintained throughout the late stages of differentiation. Cell signaling analysis showed that the activation of extracellular signal-regulated protein kinase (ERK) in response to PMA strongly induced miR-34a expression by transactivation via the activator protein-1 binding site in the upstream region of the miR-34a gene. Reporter gene assays identified mitogen-activated protein kinase kinase 1 (MEK1) as a direct target of miR-34a and c-fos as a direct target of miR-221/222. Although overexpression of the three miRNAs had little effect on cell differentiation, overexpression of miR-34a significantly repressed the proliferation of K562 cells with a concomitant reduction in MEK1 protein expression. Conversely, a locked nucleic acid probe against miR-34a significantly enhanced the proliferation of PMA-treated K562 cells. Taken together, the results show that PMA activates the MEK-ERK pathway and strongly induces miRNA-34a expression, which in turn inhibits cell proliferation by repressing the expression of MEK1. Thus, the results highlight an important regulatory role for miR-34a in the process of megakaryocytic differentiation, especially in the arrest of cell growth, which is a prerequisite for cells to enter differentiation.

Regulation of c‐Fos and Fra‐1 by the MEK5‐ERK5 pathway

Background: ERK5 is the newest subfamily member of the mitogen‐activated protein kinase (MAPK) family, and is activated by various extracellular signals including growth factors. MEK5 is a specific activator of ERK5. c‐Fos and Fra‐1, well‐known immediate early gene products, are members of the AP‐1 family. We previously reported that activation of the MEK5‐ERK5 pathway is able to induce expression of c‐Fos.

Regulation of Nuclear Translocation of Extracellular Signal-Regulated Kinase 5 by Active Nuclear Import and Export Mechanisms

ABSTRACT Extracellular signal-regulated kinase 5 (ERK5), a member of the mitogen-activated protein kinase family, plays an important role in growth factor signaling to the nucleus. However, molecular mechanisms regulating subcellular localization of ERK5 have remained unclear. Here, we show that nucleocytoplasmic shuttling of ERK5 is regulated by a bipartite nuclear localization signal-dependent nuclear import mechanism and a CRM1-dependent nuclear export mechanism. Our results show that the N-terminal half of ERK5 binds to the C-terminal half and that this binding is necessary for nuclear export of ERK5. They further show that the activating phosphorylation of ERK5 by MEK5 results in the dissociation of the binding between the N- and C-terminal halves and thus inhibits nuclear export of ERK5, causing its nuclear import. These results reveal the mechanism by which the activating phosphorylation of ERK5 induces its nuclear import and suggest a novel example of a phosphorylation-dependent control mechanism for nucleocytoplasmic shuttling of proteins.

Trastuzumab Produces Therapeutic Actions by Upregulating miR-26a and miR-30b in Breast Cancer Cells

Objective Trastuzumab has been used for the treatment of HER2-positive breast cancer (BC). However, a subset of BC patients exhibited resistance to trastuzumab therapy. Thus, clarifying the molecular mechanism of trastuzumab treatment will be beneficial to improve the treatment of HER2-positive BC patients. In this study, we identified trastuzumab-responsive microRNAs that are involved in the therapeutic effects of trastuzumab. Methods and Results RNA samples were obtained from HER2-positive (SKBR3 and BT474) and HER2-negetive (MCF7 and MDA-MB-231) cells with and without trastuzumab treatment for 6 days. Next, we conducted a microRNA profiling analysis using these samples to screen those microRNAs that were up- or down-regulated only in HER2-positive cells. This analysis identified miR-26a and miR-30b as trastuzumab-inducible microRNAs. Transfecting miR-26a and miR-30b induced cell growth suppression in the BC cells by 40% and 32%, respectively. A cell cycle analysis showed that these microRNAs induced G1 arrest in HER2-positive BC cells as trastuzumab did. An Annexin-V assay revealed that miR-26a but not miR-30b induced apoptosis in HER2-positive BC cells. Using the prediction algorithms for microRNA targets, we identified cyclin E2 (CCNE2) as a target gene of miR-30b. A luciferase-based reporter assay demonstrated that miR-30b post-transcriptionally reduced 27% (p = 0.005) of the gene expression by interacting with two binding sites in the 3′-UTR of CCNE2. Conclusion In BC cells, trastuzumab modulated the expression of a subset of microRNAs, including miR-26a and miR-30b. The upregulation of miR-30b by trastuzumab may play a biological role in trastuzumab-induced cell growth inhibition by targeting CCNE2.

Activation of a C-terminal Transcriptional Activation Domain of ERK5 by Autophosphorylation

ERK5 plays a crucial role in many biological processes by regulating transcription. ERK5 has a large C-terminal-half that contains a transcriptional activation domain. However, it has remained unclear how its transcriptional activation activity is regulated. Here, we show that the activated kinase activity of ERK5 is required for the C-terminal-half to enhance the AP-1 activity, and that the activated ERK5 undergoes autophosphorylation on its most C-terminal region. Changing these phosphorylatable threonine and serine residues to unphosphorylatable alanines significantly reduces the transcriptional activation activity of ERK5. Moreover, phosphomimetic mutants of the C-terminal-half of ERK5 without an N-terminal kinase domain are shown to be able to enhance the AP-1 activity in fibroblastic cells. These results reveal the role of the stimulus-induced ERK5 autophosphorylation in regulation of gene expression.

miRNAs and regulation of cell signaling

MicroRNAs (miRNAs) regulate gene expression post‐transcriptionally by binding to target mRNAs in a sequence‐specific manner. A large number of genes appear to be the target of miRNAs, and an essential role for miRNAs in the regulation of various conserved cell signaling cascades, such as mitogen‐activated protein kinase, Notch and Hedgehog, is emerging. Extensive studies have also revealed the spatial and temporal regulation of miRNA expression by various cell signaling cascades. The insights gained in such studies support the idea that miRNAs are involved in the highly complex network of cell signaling pathways. In this minireview, we present an overview of these complex networks by providing examples of recent findings.