Akihiko Muto

Reconstituted human granulocyte-macrophage colony-stimulating factor receptor transduces growth-promoting signals in mouse NIH 3T3 cells: comparison with signalling in BA/F3 pro-B cells.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) plays a critical role in growth and differentiation of myeloid cells. We previously reconstituted high-affinity human GM-CSF receptor (hGM-CSFR) in a pro-B cell line, BA/F3, by cotransfecting alpha- and beta-chain cDNA clones and showed that the reconstituted receptor could transduce growth-promoting signals. The high-affinity hGM-CSFR was also reconstituted in mouse NIH 3T3 cells, but its ability to transduce signals in fibroblasts remained undetermined. In the present study, we further characterized signal transduction by the reconstituted hGM-CSFR in both NIH 3T3 cells and BA/F3 cells. We found that the reconstituted hGM-CSFR transduces signals in NIH 3T3 fibroblasts and BA/F3 cells in response to hGM-CSF to activate transcription of the c-fos, c-jun, and c-myc proto-oncogenes. hGM-CSF also induces protein tyrosine phosphorylation and DNA synthesis in both cell types. These results indicated that hGM-CSFR is functional in fibroblasts, that signal transduction via hGM-CSFR in fibroblasts involves tyrosine kinase(s), and that association of hGM-CSFR with a factor(s) specific to hematopoietic cell lineage is not essential to transduce growth-promoting signals.

The group E Sox genes Sox8 and Sox9 are regulated by Notch signaling and are required for Müller glial cell development in mouse retina

Although Müller glial cells play pivotal roles in the vertebrate retina, the regulation of their development is poorly understood. While Notch-Hes5 signaling has been shown to be involved in this developmental process, the presence of Müller glial cells in Hes5-deficient mice suggests the involvement of other molecules. We found that two group E Sox genes, Sox8 and Sox9, are expressed in proliferating progenitors and then exclusively in Müller glial cells in mouse retina. Knocking-down Sox8 and Sox9 by shRNA significantly reduced the population of Müller glial cells and relatively increased the proportion of rod photoreceptors, suggesting that the Sox genes play roles in the specification of Müller glial cells. Using an activated form of Notch and the gamma-secretase inhibitor DAPT, we also found that Notch signaling regulates the transcription of Sox8 and Sox9. This is the first evidence that group E Sox genes play important roles in the developing vertebrate retina.

Melk-Like Kinase Plays a Role in Hematopoiesis in the Zebra Fish

ABSTRACT A serine/threonine kinase, Melk, was initially cloned in mouse oocytes as a maternal gene, but whose function was unknown. In adult mice, Melk was strongly expressed in the thymus and bone marrow, suggesting a role for Melk in hematopoiesis. We cloned a Melk-like gene from zebra fish (zMelk). zMelk-like gene was expressed in the brain and lateral mesoderm at 12 hours postfertilization (hpf) and in several tissues of adult fish, including the kidney and spleen, both of which are known to be hematopoietic tissues in zebra fish. Abrogation of zMelk-like gene function by zMelk-like gene-specific Morpholino (MO) resulted in abnormal swelling around the tectum region. In addition, the start of blood circulation was severely delayed but, in contrast, the vessel formation seemed normal. Expression of scl, gata-1, and lmo-2 was down regulated at 12 to 14 hpf in the zMelk-like gene MO-injected embryos, and the coexpression of gata-1 rescued the anemic phenotype induced by zMelk-like gene MO. Expression of the zMelk-like gene in embryos enhanced gata-1 promoter-dependent enhanced green fluorescent protein expression, suggesting that the zMelk-like gene affects gata-1 expression at the transcriptional level. Taken together, our data suggest that the zMelk-like gene may play a role in primitive hematopoiesis by affecting the expression of genes critical for hematopoiesis.

Mouse Rab11-FIP4 regulates proliferation and differentiation of retinal progenitors in a Rab11-independent manner.

We identified Rab11‐family interacting protein 4 (Rab11‐FIP4) as a gene strongly expressed in the developing mouse retina. The major transcript encoding a full‐length protein, mRab11‐FIP4A, was expressed predominantly in neural tissues; whereas an alternative transcript encoding an N‐terminally truncated form of the protein, mRab11‐FIP4B, was expressed ubiquitously as a minor form. Gain‐of‐function of mRab11‐FIP4A in retina promoted cell cycle exit and increased subpopulations of retinal cells localized in the inner nuclear layer, such as bipolar cells and Müller glia. Reversal of the phenotype was observed in the loss‐of‐function experiment. Furthermore, Shh signaling was suggested to be involved in these functions. Analysis using truncation mutants revealed the essential role of the N‐terminal region containing a conserved EF‐hand motif for the retinal phenotypes induced by the expression of mRab11‐FIP4A, whereas binding to Rab11 was dispensable, suggesting the involvement of a novel Rab11‐independent mechanism for mRab11‐FIP4A action in the regulation of retinal development. Developmental Dynamics 236:214–225, 2007.

Function of the Common β Subunit of the GM-CSF/IL-3/IL-5 Receptors

Interleukin 3 (IL-3) and granulocyte-macrophage colony stimulating factor (GM-CSF) stimulate various lineage-committed cells as well as early multipotential progenitors while interleukin 5 (IL-5) stimulates eosinophils and basophils1. These three cytokines induce similar intracellular signals and exhibit similar functions in their common target cells. Although primary amino acid sequences of IL-3, IL-5 and GM-CSF show no obvious homology, they consist of four α-helices and their gross tertiary structures are similar. Interestingly, binding of a human cytokine to its high affinity receptor is inhibited by another cytokine on their common target cells, e.g. high affinity IL-3 binding to its receptor is inhibited by GM-CSF and vice versa2.

Roles of Jak Kinases in Human GM-CSF Receptor

The IL-3 and GM-CSF (hGMR) receptors consist of two subunits, α and β, both of which are members of the cytokine receptor superfamily. Chemical cross-linking and immunoprecipitation revealed that the α and β subunits associate following stimulation with GM-CSF, but the β subunit forms a homodimer even in the absence of the ligand. We analyzed the mechanism of c-fos mRNA activation by GM-CSF using several hGMRβ subunit mutants. In addition to boxl region, a membrane distal region (a.a. 544-589) of hGMRβ is required for c-fos activation. Only one tyrosine residue (Tyr577) exists within the region 544–589, and substitution of Tyr577 to phenylalanine in GMRβ 589 resulted in the loss of c-fos activation. In contrast, the same substitution in a wild type receptor did not affect GM-CSF-induced activities such as c-fos mRNA induction and proliferation but abolished Shc phosphorylation. These results suggest that the activation of She is not essential for c-fos activation and several tyrosine residues coordinate to activate c-fos activation.