Keisuke Hitachi

Activin signaling as an emerging target for therapeutic interventions

After the initial discovery of activins as important regulators of reproduction, novel and diverse roles have been unraveled for them. Activins are expressed in various tissues and have a broad range of activities including the regulation of gonadal function, hormonal homeostasis, growth and differentiation of musculoskeletal tissues, regulation of growth and metastasis of cancer cells, proliferation and differentiation of embryonic stem cells, and even higher brain functions. Activins signal through a combination of type I and II transmembrane serine/threonine kinase receptors. Activin receptors are shared by multiple transforming growth factor-β (TGF-β) ligands such as myostatin, growth and differentiation factor-11 and nodal. Thus, although the activity of each ligand is distinct, they are also redundant, both physiologically and pathologically in vivo. Activin receptors activated by ligands phosphorylate the receptor-regulated Smads for TGF-β, Smad2 and 3. The Smad proteins then undergo multimerization with the co-mediator Smad4, and translocate into the nucleus to regulate the transcription of target genes in cooperation with nuclear cofactors. Signaling through receptors and Smads is controlled by multiple mechanisms including phosphorylation and other posttranslational modifications such as sumoylation, which affect potein localization, stability and transcriptional activity. Non-Smad signaling also plays an important role in activin signaling. Extracellularly, follistatin and related proteins bind to activins and related TGF-β ligands, and control the signaling and availability of ligands.The functions of activins through activin receptors are pleiotrophic, cell type-specific and contextual, and they are involved in the etiology and pathogenesis of a variety of diseases. Accordingly, activin signaling may be a target for therapeutic interventions. In this review, we summarize the current knowledge on activin signaling and discuss the potential roles of this pathway as a molecular target of therapy for metabolic diseases, musculoskeletal disorders, cancers and neural damages.

Molecular links among the causative genes for ocular malformation: Otx2 and Sox2 coregulate Rax expression

The neural-related genes Sox2, Pax6, Otx2, and Rax have been associated with severe ocular malformations such as anophthalmia and microphthalmia, but it remains unclear as to how these genes are linked functionally. We analyzed the upstream signaling of Xenopus Rax (also known as Rx1) and identified the Otx2 and Sox2 proteins as direct upstream regulators of Rax. We revealed that endogenous Otx2 and Sox2 proteins bound to the conserved noncoding sequence (CNS1) located ≈2 kb upstream of the Rax promoter. This sequence is conserved among vertebrates and is required for potent transcriptional activity. Reporter assays showed that Otx2 and Sox2 synergistically activated transcription via CNS1. Furthermore, the Otx2 and Sox2 proteins physically interacted with each other, and this interaction was affected by the Sox2-missense mutations identified in these ocular disorders. These results demonstrate that the direct interaction and interdependence between the Otx2 and Sox2 proteins coordinate Rax expression in eye development, providing molecular linkages among the genes responsible for ocular malformation.

Myostatin signaling regulates Akt activity via the regulation of miR-486 expression.

Myostatin, also known as growth and differentiation factor-8, is a pivotal negative regulator of skeletal muscle mass and reduces muscle protein synthesis by inhibiting the insulin-like growth factor-1 (IGF-1)/Akt/mammalian target of rapamycin (mTOR) pathway. However, the precise mechanism by which myostatin inhibits the IGF-1/Akt/mTOR pathway remains unclear. In this study, we investigated the global microRNA expression profile in myostatin knockout mice and identified miR-486, a positive regulator of the IGF-1/Akt pathway, as a novel target of myostatin signaling. In myostatin knockout mice, the expression level of miR-486 in skeletal muscle was significantly increased. In addition, we observed increased expression of the primary transcript of miR-486 (pri-miR-486) and Ankyrin 1.5 (Ank1.5), the host gene of miR-486, in myostatin knockout mice. In C2C12 cells, myostatin negatively regulated the expression of Ank1.5. Moreover, canonical myostatin signaling repressed the skeletal muscle-specific promoter activity of miR-486/Ank1.5. This repression was partially mediated by the E-box elements in the proximal region of the promoter. We also show that overexpression of miR-486 induced myotube hypertrophy in vitro and that miR-486 was essential to maintain skeletal muscle size both in vitro and in vivo. In addition, inhibition of miR-486 led to a decrease in Akt activity in C2C12 myotubes. Our findings indicate that miR-486 is one of the intermediary molecules connecting myostatin signaling and the IGF-1/Akt/mTOR pathway in the regulation of skeletal muscle size.

Role of microRNAs in skeletal muscle hypertrophy

Skeletal muscle comprises approximately 40% of body weight, and is important for locomotion, as well as for metabolic homeostasis. Adult skeletal muscle mass is maintained by a fine balance between muscle protein synthesis and degradation. In response to cytokines, nutrients, and mechanical stimuli, skeletal muscle mass is increased (hypertrophy), whereas skeletal muscle mass is decreased (atrophy) in a variety of conditions, including cancer cachexia, starvation, immobilization, aging, and neuromuscular disorders. Recent studies have determined two important signaling pathways involved in skeletal muscle mass. The insulin-like growth factor-1 (IGF-1)/Akt pathway increases skeletal muscle mass via stimulation of protein synthesis and inhibition of protein degradation. By contrast, myostatin signaling negatively regulates skeletal muscle mass by reducing protein synthesis. In addition, the discovery of microRNAs as novel regulators of gene expression has provided new insights into a multitude of biological processes, especially in skeletal muscle physiology. We summarize here the current knowledge of microRNAs in the regulation of skeletal muscle hypertrophy, focusing on the IGF-1/Akt pathway and myostatin signaling.

Ripply2 is essential for precise somite formation during mouse early development

The regions of expression of Ripply1 and Ripply2, presumptive transcriptional corepressors, overlap at the presomitic mesoderm during somitogenesis in mouse and zebrafish. Ripply1 is required for somite segmentation in zebrafish, but the developmental role of Ripply2 remains unclear in both species. Here, we generated Ripply2 knock‐out mice to investigate the role of Ripply2. Defects in segmentation of the axial skeleton were observed in the homozygous mutant mice. Moreover, somite segmentation and expression of Notch2 and Uncx4.1 were disrupted. These findings indicate that Ripply2 is involved in somite segmentation and establishment of rostrocaudal polarity.

In vitro organogenesis from undifferentiated cells in Xenopus

Amphibians have been used for over a century as experimental animals. In the field of developmental biology in particular, much knowledge has been accumulated from studies on amphibians, mainly because they are easy to observe and handle. Xenopus laevis is one of the most intensely investigated amphibians in developmental biology at the molecular level. Thus, Xenopus is highly suitable for studies on the mechanisms of organ differentiation from not only a single fertilized egg, as in normal development, but also from undifferentiated cells, as in the case of in vitro organogenesis. Based on the established in vitro organogenesis methods, we have identified many genes that are indispensable for normal development in various organs. These experimental systems are useful for investigations of embryonic development and for advancing regenerative medicine. Developmental Dynamics 238:1309–1320, 2009.

The Xenopus Bowline/Ripply family proteins negatively regulate the transcriptional activity of T-box transcription factors

Bowline, which is a member of the Xenopus Bowline/Ripply family of proteins, represses the transcription of somitogenesis-related genes before somite segmentation, which makes Bowline indispensable for somitogenesis. Although there are three bowline/Ripply family genes in each vertebrate species, it is not known whether the Bowline/Ripply family proteins share a common role in development. To elucidate their developmental roles, we examined the expression patterns and functions of the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline, and a novel member of this protein family, xRipply3. We found that the expression patterns of bowline and ledgerline overlapped in the presomitic mesoderm (PSM), whereas ledgerline was additionally expressed in the newly formed somites. In addition, we isolated xRipply3, which is expressed in the pharyngeal region. Co-immunoprecipitation assays revealed that Ledgerline and xRipply3 interacted with T-box proteins and the transcriptional co-repressor Groucho/TLE. In luciferase assays, xRipply3 weakly suppressed the transcriptional activity of Tbx1, while Ledgerline strongly suppressed that of Tbx6. In line with the repressive role of Ledgerline, knockdown of Ledgerline resulted in enlargement of expression regions of the somitogenesis-related-genes mespb and Tbx6. Inhibition of histone deacetylase activity increased the expression of mespb, as seen in the Bowline and Ledgerline knockdown experiments. These results suggest that the Groucho-HDAC complex is required for the repressive activity of Bowline/Ripply family proteins during Xenopus somitogenesis. We conclude that although the Xenopus Bowline/Ripply family proteins Bowline, Ledgerline and xRipply3 are expressed differentially, they all act as negative regulators of T-box proteins.

Physical interaction between Tbx6 and mespb is indispensable for the activation of bowline expression during Xenopus somitogenesis.

During vertebrate somitogenesis, various transcriptional factors function coordinately to determine the position of the somite boundary. Previously, we reported on the signaling crosstalk that occurs between two major transcription factors involved in somitogenesis, Tbx6 and mespb/mesp2. These factors synergistically activated the expression of a downstream gene, bowline/Ripply2, which is essential for precise formation of the somite boundary. However, the molecular mechanism underlying this synergistic effect remains unclear. In this report, we found that the Tbx6 and mespb proteins interacted physically with each other. Pulldown assays with various deletion mutants of these proteins identified the essential domains for this physical interaction. Finally, we found that interference with the physical interaction by a dominant-negative form of mespb, mespbDeltaDBD, abrogated the expression of the bowline gene during Xenopus somitogenesis. These results indicate that the appropriate expression of bowline/Ripply2 is regulated by a direct interaction between the Tbx6 and mespb proteins during Xenopus somitogenesis.

Molecular analyses of Xenopus laevis Mesp-related genes.

During vertebrate somitogenesis, somites bud off from the anterior end of the presomitic mesoderm (PSM). Mesodermal posterior (Mesp)-related genes play essential roles in somitogenesis, particularly in the definition of the somite boundary position. Among vertebrates, two types of Mesp-related genes have been identified: Mesp1 and Mesp2 in the mouse; Meso-1 and Meso-2 in the chicken; Xl-mespa and Xl-mespb (also known as Thylacine1) in the African clawed frog (Xenopus laevis); and mesp-a and mesp-b in the zebrafish. However, the functional differences between two Mesp-related genes remain unknown. In the present study, we carried out comparative analyses of the Xl-mespa and Xl-mespb genes. The amino acid sequences of the Xl-mespa and Xl-mespb proteins showed a high level of similarity. The expression of Xl-mespa started broadly in the ventrolateral mesoderm and gradually shifted to a striped pattern of expression. In contrast, Xl-mespb showed a striped pattern of expression from the start. These expression profiles completely overlapped at the PSM during somitogenesis. To investigate the functional differences between Xl-mespa and Xl-mespb in terms of target gene regulation, we carried out a luciferase assay using the murine Lunatic fringe (L-fng) promoter. Transcription of the L-fng promoter was activated more strongly by Xl-mespb than by Xl-mespa. This same pattern was observed for the murine Mesp-related proteins. These results suggest that the functional differences between the two types of Mesp-related genes are evolutionally conserved in vertebrates.

TSC-box is essential for the nuclear localization and antiproliferative effect of XTSC-22

Transforming growth factor‐β1‐stimulated clone 22 (TSC‐22) encodes a leucine zipper‐containing protein that is highly conserved among various species. Mammalian TSC‐22 is a potential tumor suppressor gene. It translocates into nuclei and suppresses cell division upon antiproliferative stimuli. In human colon carcinoma cells, TSC‐22 inhibits cell growth by upregulating expression of the p21 gene, a cyclin‐dependent kinase (Cdk) inhibitor. We previously showed that the Xenopus laevis homologue of the TSC‐22 gene (XTSC‐22) is required for cell movement during gastrulation through cell cycle regulation. In this report, we investigated the molecular mechanism of the antiproliferative effect of XTSC‐22. Reverse transcriptase‐polymerase chain reaction (RT‐PCR) analysis suggested that XTSC‐22 did not affect the expression levels of the p21 family of Cdk inhibitors or other cell cycle regulators. Analysis of deletion mutants of XTSC‐22 revealed that nuclear localization of the N‐terminal TSC‐box is necessary for cell cycle inhibition by XTSC‐22. Further experiments suggested that p27Xic1, a key Cdk inhibitor in Xenopus, interacts with XTSC‐22. Because p27Xic1 is a cell cycle inhibitor with a nuclear localization signal, it is possible that XTSC‐22 suppresses cell division by translocating into the nucleus with p27Xic1, where it may potentiate the intranuclear action of p27Xic1.