Yasunori Miyamoto

Interactions of Wnt/β-Catenin Signaling and Sonic Hedgehog Regulate the Neurogenesis of Ventral Midbrain Dopamine Neurons

Signaling mechanisms involving Wnt/β-catenin and sonic hedgehog (Shh) are known to regulate the development of ventral midbrain (vMB) dopamine neurons. However, the interactions between these two mechanisms and how such interactions can be targeted to promote a maximal production of dopamine neurons are not fully understood. Here we show that conditional mouse mutants with region-specific activation of β-catenin signaling in vMB using the Shh–Cre mice show a marked expansion of Sox2-, Ngn2-, and Otx2-positive progenitors but perturbs their cell cycle exit and reduces the generation of dopamine neurons. Furthermore, activation of β-catenin in vMB also results in a progressive loss of Shh expression and Shh target genes. Such antagonistic effects between the activation of Wnt/β-catenin and Shh can be recapitulated in vMB progenitors and in mouse embryonic stem cell cultures. Notwithstanding these antagonistic interactions, cell-type-specific activation of β-catenin in the midline progenitors using the tyrosine hydroxylase–internal ribosomal entry site–Cre (Th-IRES-Cre) mice leads to increased dopaminergic neurogenesis. Together, these results indicate the presence of a delicate balance between Wnt/β-catenin and Shh signaling mechanisms in the progression from progenitors to dopamine neurons. Persistent activation of β-catenin in early progenitors perturbs their cell cycle progression and antagonizes Shh expression, whereas activation of β-catenin in midline progenitors promotes the generation of dopamine neurons.

Snail Regulates Cell-Matrix Adhesion by Regulation of the Expression of Integrins and Basement Membrane Proteins

Snail, a transcriptional repressor of E-cadherin expression, plays a role in the process of epithelial-mesenchymal transition. However, the molecular basis of the role of snail in epithelial-mesenchymal transition has not been fully clarified. Here we show that the expression of snail in epithelial Madin-Darby canine kidney (MDCK) and A431 cells enhances both cell detachment and attachment. Snail did not confer resistance to anoikis induced by loss of contact but instead enhanced cell attachment to extracellular matrices such as fibronectin. This attachment was inhibited by Arg-Gly-Asp (RGD) peptides. Up-regulation of the promoter activity of integrin αV was observed in snail-expressing MDCK (MDCK/snail) cells. Snail also enhanced MDCK cell migration toward osteopontin that is a ligand for integrin αVβ3. We confirmed the reduction of basement membrane proteins such as laminin (LN) α3, β3, and γ2 (laminin-5/LN-5) and of receptors for LN-5 such as integrins α3, α6, or β4 in MDCK/snail or in snail-expressing A431 (A431/snail) cells. Nevertheless, suppression of LN-α3 chain by transient transfection of small interference RNAs resulted in no enhancement of cell detachment. We also found an induction of matrix metalloproteinase-3 in MDCK/snail and A431/snail cells. However, the inhibition of matrix metalloproteinase-3 showed no significant effect on the detachment of MDCK/snail cells. These results suggest that snail enhances cell detachment by multiple mechanism and leads to cell migration and reattachment at a second site, at least in part, by changing the expression of integrins in the cells.

Multiple roles of β-catenin in controlling the neurogenic niche for midbrain dopamine neurons

Stem cell-based replacement therapy has emerged as a potential strategy to alleviate specific features of movement disorder in Parkinsons disease. However, the current strategy to produce dopamine (DA) neurons from embryonic stem cells has many limitations, including the difficulty of generating DA neurons with high yields. Further insights into the mechanisms that control the neurogenesis of DA neurons will reduce or mitigate such limitations. It is well established that the ventral midbrain (vMB) contains the neurogenic niche that produces DA neurons. However, it is unclear how the microenvironment within this niche controls DA neurogenesis. Here, we show that β-catenin controls DA neurogenesis by maintaining the integrity of the neurogenic niche and the progression from progenitors to DA neurons. Using conditional gene targeting approaches, we show that regional deletion of β-catenin in the vMB by using Shh-Cre disrupts adherent junctions of progenitors and the integrity of radial glia in the vMB, which leads to a severe reduction in DA neurogenesis and perturbs the migration and segregation of DA neurons. By contrast, Th-IRES-Cre removes β-catenin in a subset of neural progenitor cells without perturbing the cellular and structural integrity of the vMB. Interestingly, loss of β-catenin in Th-IRES-Cre;β-Ctnfl/fl mutants negatively regulates neurogenesis by interfering with the progression of committed progenitors to DA neurons. Taken together, these results provide new insights into the indispensable functions of β-catenin at multiple stages during DA neurogenesis. They also suggest that β-catenin-mediated signaling pathways can be targeted to promote and expand DA neurons in cell-based therapeutic strategies.

Mouse integrin αv promoter is regulated by transcriptional factors Ets and Sp1 in melanoma cells

A 17-bp region between the -31 and -15 bp region of the mouse integrin alphav gene is known to be one of the cis-acting elements for promoter activity. Experimental binding of nuclear proteins to the -31/-15 region reveals that the -27/-16 region mediates the binding. The -27/-16 region, GGCTCCTCCTCC, has a TCCTCC motif, one of the Sp1 binding motifs. An anti-Sp1 IgG and an Sp1-binding oligonucleotide interfered with the binding of nuclear proteins to the -27/-16 oligonucleotide, demonstrating that Sp1 binds to the -27/-16 region. In addition to the -27/-16 region, two other regions, -108/-89 and -64/-44, were found to bind to nuclear proteins within the -108/+1 alphav promoter region. An oligonucleotide containing the Ets-binding consensus sequence of CAGGAAGT interfered with their binding, indicating that both regions have a functional Ets-binding site; which is ACGGAAGT from -106 to -99 bp and ACTTCCTC from -61 to -54 bp, as deduced from the sequence. Mutations in or deletions from any one of three cis-acting elements, the two Ets-binding sites or one Sp1-binding site, remarkably decreased the promoter activity detected in the -108/+1 region. Cotransfection of both Sp1 and Ets-1 cDNAs with the -108/+1 region into B16F10 cells increased the promoter activity 2.9-fold. These results demonstrate that Sp1 and Ets cooperate to activate the -108/+1-alphav promoter region.

N-cadherin-based adherens junction regulates the maintenance, proliferation, and differentiation of neural progenitor cells during development

This review addresses our current understanding of the regulatory mechanism by which N-cadherin, a classical cadherin, affects neural progenitor cells (NPCs) during development. N-cadherin is responsible for the integrity of adherens junctions (AJs), which develop in the sub-apical region of NPCs in the neural tube and brain cortex. The apical domain, which contains the sub-apical region, is involved in the switching from symmetric proliferative division to asymmetric neurogenic division of NPCs. In addition, N-cadherin-based AJ is deeply involved in the apico-basal polarity of NPCs and the regulation of Wnt-β-catenin, hedgehog (Hh), and Notch signaling. In this review, we discuss the roles of N-cadherin in the maintenance, proliferation, and differentiation of NPCs through components of AJ, β-catenin and αE-catenin.

N-cadherin regulates the proliferation and differentiation of ventral midbrain dopaminergic progenitors.

Adherens junction (AJ) between dopaminergic (DA) progenitors maintains the structure of ventricular zone and polarity of radial glia cells in the ventral midbrain (vMB) during embryonic development. However, it is unclear how loss of N‐cadherin might influence the integrity of the AJ and the process of DA neurogenesis. Here, we used conditional gene targeting approaches to perform the region‐specific removal of N‐cadherin in the neurogenic niche of DA neurons in the vMB. Removal of N‐cadherin in the vMB using Shh‐Cre disrupts the AJs of DA progenitors and radial glia processes in the vMB. Surprisingly, loss of N‐cadherin in the vMB leads to a significant expansion of DA progenitors, including those expressing Sox2, Ngn2, and Otx2. Cell cycle analyses reveal that the cell cycle exit in the progenitor cells is decreased in the mutants from E11.5 to E12.5. In addition, the efficiency of DA progenitors in differentiating into DA neurons is decreased from E10.5 to E12.5, leading to a marked reduction in the number of DA neurons at E11.5, E12.5, and E17.5. Loss of N‐cadherin leads to the diffuse distribution of β‐catenin proteins, which are a critical component of AJ and Wnt signaling, from the AJ throughout the entire cytoplasm in neuroepithelial cells, suggesting that canonical Wnt signaling might be activated in the DA progenitors in vMB. Taken together, these results support the notion that N‐cadherin regulates the proliferation of DA progenitors and the differentiation of DA neurons through canonical Wnt‐β‐catenin signaling in the vMB.

Survival Signals of Hepatic Stellate Cells in Liver Regeneration Are Regulated by Glycosylation Changes in Rat Vitronectin, Especially Decreased Sialylation

The extracellular matrix (ECM) molecules play important roles in many biological and pathological processes. During tissue remodeling, the ECM molecules that are glycosylated are different from those of normal tissue owing to changes in the expression of many proteins that are responsible for glycan synthesis. Vitronectin (VN) is a major ECM molecule that recognizes integrin on hepatic stellate cells (HSCs). The present study attempted to elucidate how changes in VN glycans modulate the survival of HSCs, which play a critical role in liver regeneration. Plasma VN was purified from partially hepatectomized (PH) and sham-operated (SH) rats at 24 h after operation and non-operated (NO) rats. Adhesion of rat HSCs (rHSCs), together with phosphorylation of focal adhesion kinase, in PH-VN was decreased to one-half of that in NO- or SH-VN. Spreading of rHSCs on desialylated NO-VN was decreased to one-half of that of control VN, indicating the importance of sialylation of VN for activation of HSCs. Liquid chromatography/multiple-stage mass spectrometry analysis of Glu-C glycopeptides of each VN determined the site-specific glycosylation. In addition to the major biantennary complex-type N-glycans, hybrid-type N-glycans were site-specifically present at Asn167. Highly sialylated O-glycans were found to be present in the Thr110–Thr124 region. In PH-VN, the disialyl O-glycans and complex-type N-glycans were decreased while core-fucosylated N-glycans were increased. In addition, immunodetection after two-dimensional PAGE indicated the presence of hyper- and hyposialylated molecules in each VN and showed that hypersialylation was markedly attenuated in PH-VN. This study proposes that the alteration of VN glycosylation modulates the substrate adhesion to rat HSCs, which is responsible for matrix restructuring.

Cell-type dependency of two Foxa/HNF3 sites in the regulation of vitronectin promoter activity.

The mouse vitronectin promoter has two consensus sequences of the Foxa/hepatocyte nuclear factor (HNF) 3-binding site (from -34 to -25, site A, and +15 to +26 base pairs (bp), site B). Site-directed mutagenesis of site B inhibited binding of nuclear proteins from mouse neuroblastoma Neuro2a and reduced the promoter activity to 4.6% in a 101-bp fragment (from -48 to +53 bp) in Neuro2a cells. The nuclear proteins of site B were identified as the Foxa1/HNF3alpha and Foxa2/HNF3beta proteins by supershift assay. Next, we examined site A. Mutation of site A in Neuro2a cells did not affect the promoter activity, and binding of nuclear proteins was not detected. Overexpression of Foxa1 or Foxa2 protein activated the mutated site B promoter, but failed to activate the sites A and B double-mutated promoter in Neuro2a cells, indicating that site A is a potential transcription regulatory site. Recombinant Foxa1 and Foxa2 proteins and nuclear extract from mouse liver bound not only to site B, but also to site A. In human hepatoma HepG2 cells, mutation of sites A and B decreased the promoter activity to 82% and 38%, respectively, in the wild promoter, and double mutation of sites A and B decreased the wild promoter activity to 5%, indicating that sites A and B contribute to the promoter activity in HepG2 cells. These results demonstrate that the two Foxa-binding sites regulate the vitronectin promoter activity in cell type-dependent manner.

Vitronectin promotes the progress of the initial differentiation stage in cerebellar granule cells

Vitronectin (VN), which is an extracellular matrix protein, is known to be involved in the proliferation and differentiation of primary cultured cerebellar granule cell precursors (CGCPs); however, the effect of VN is not fully understood. In this study, we analyzed the effects of VN loss on the proliferation and differentiation of CGCPs in VN knockout (VNKO) mice in vivo. First, immunohistochemistry showed that VN was distributed in the region from the inner external granule layer (iEGL) through the internal granule layer (IGL) in wild-type (WT) mice. Next, we observed the formation of the cerebellar cortex using sagittal sections of VNKO mice at postnatal days (P) 5, 8 and 11. Loss of VN suppressed the ratio of NeuN, a neuronal differentiation marker, to positive cerebellar granule cells (CGCs) in the external granule layer (EGL) and the ratio of CGCs in the IGL at P8, indicating that the loss of VN suppresses the differentiation into CGCs. However, the loss of VN did not significantly affect the proliferation of CGCPs. Next, the effect of VN loss on the initial differentiation stage of CGCPs was examined. The loss of VN increased the expression levels of Transient axonal glycoprotein 1 (TAG1), a marker of neurons in the initial differentiation stage, in the cerebella of VNKO mice at P5 and 8 and increased the ratio of TAG1-positive cells in the primary culture of VNKO-derived CGCPs, indicating that the loss of VN accumulates the CGCPs in the initial differentiation stage. Taken together, these results demonstrate that VN promotes the progress of the initial differentiation stage of CGCPs.

Regulation of myelopoiesis by proinflammatory cytokines in infectious diseases

Hematopoiesis is hierarchically orchestrated by a very small population of hematopoietic stem cells (HSCs) that reside in the bone-marrow niche and are tightly regulated to maintain homeostatic blood production. HSCs are predominantly quiescent, but they enter the cell cycle in response to inflammatory signals evoked by severe systemic infection or injury. Thus, hematopoietic stem and progenitor cells (HSPCs) can be activated by pathogen recognition receptors and proinflammatory cytokines to induce emergency myelopoiesis during infection. This emergency myelopoiesis counterbalances the loss of cells and generates lineage-restricted hematopoietic progenitors, eventually replenishing mature myeloid cells to control the infection. Controlled generation of such signals effectively augments host defense, but dysregulated stimulation by these signals is harmful to HSPCs. Such hematopoietic failure often results in blood disorders including chronic inflammatory diseases and hematological malignancies. Recently, we found that interleukin (IL)-27, one of the IL-6/IL-12 family cytokines, has a unique ability to directly act on HSCs and promote their expansion and differentiation into myeloid progenitors. This process resulted in enhanced production of neutrophils by emergency myelopoiesis during the blood-stage mouse malaria infection. In this review, we summarize recent advances in the regulation of myelopoiesis by proinflammatory cytokines including type I and II interferons, IL-6, IL-27, granulocyte colony-stimulating factor, macrophage colony-stimulating factor, and IL-1 in infectious diseases.