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Abstract Background Recent fate-mapping studies establish that microglia, the resident mononuclear phagocytes of the CNS, are distinct in origin from the bone marrow-derived myeloid lineage. Interferon regulatory factor 8 (IRF8, also known as interferon consensus sequence binding protein) plays essential roles in development and function of the bone marrow-derived myeloid lineage. However, little is known about its roles in microglia. Methods The CNS tissues of IRF8-deficient mice were immunohistochemically analyzed. Pure microglia isolated from wild-type and IRF8-deficient mice were studied in vitro by proliferation, immunocytochemical and phagocytosis assays. Microglial response in vivo was compared between wild-type and IRF8-deficient mice in the cuprizon-induced demyelination model. Results Our analysis of IRF8-deficient mice revealed that, in contrast to compromised development of IRF8-deficient bone marrow myeloid lineage cells, development and colonization of microglia are not obviously affected by loss of IRF8. However, IRF8-deficient microglia demonstrate several defective phenotypes. In vivo, IRF8-deficient microglia have fewer elaborated processes with reduced expression of IBA1/AIF1 compared with wild-type microglia, suggesting a defective phenotype. IRF8-deficient microglia are significantly less proliferative in mixed glial cultures than wild-type microglia. Unlike IRF8-deficient bone marrow myeloid progenitors, exogenous macrophage colony stimulating factor (colony stimulating factor 1) (M-CSF (CSF1)) restores their proliferation in mixed glial cultures. In addition, IRF8-deficient microglia exhibit an exaggerated growth response to exogenous granulocyte-macrophage colony stimulating factor (colony stimulating factor 2) (GM-CSF (CSF2)) in the presence of other glial cells. IRF8-deficient microglia also demonstrate altered cytokine expressions in response to interferon-gamma and lipopolysaccharide in vitro. Moreover, the maximum phagocytic capacity of IRF8-deficient microglia is reduced, although their engulfment of zymosan particles is not overtly impaired. Defective scavenging activity of IRF8-deficient microglia was further confirmed in vivo in the cuprizone-induced demyelination model in mice. Conclusions This study is the first to demonstrate the essential contribution of IRF8-mediated transcription to a broad range of microglial phenotype. Microglia are distinct from the bone marrow myeloid lineage with respect to their dependence on IRF8-mediated transcription.

Activation of REarranged during Transfection (RET) proto-oncogene is responsible for various human cancers such as papillary and medullary thyroid carcinomas and non-small cell lung carcinomas. RET activation in these tumors is caused by point mutations or gene rearrangements, resulting in constitutive activation of RET tyrosine kinase. Physiologically, RET is activated by glial cell line–derived neurotrophic factor (GDNF) ligands that bind to coreceptor GDNF family receptor alphas (GFRαs), leading to RET dimerization. GDNF-GFRα1-RET signaling plays crucial roles in the development of the enteric nervous system, kidney and lower urinary tract as well as in spermatogenesis. Intracellular tyrosine phosphorylation in RET and recruitment of adaptor proteins to phosphotyrosines are essential for various biological functions. Significance of intracellular RET signaling pathways activated by GDNF is discussed and summarized in this review. [ABSTRACT FROM AUTHOR]

TheRETproto-oncogene is responsible for the development of several human inherited and non-inherited diseases. Germline point mutations were identified in multiple endocrine neoplasia types 2A and 2B, and familial medullary thyroid carcinoma. More than 10 rearranged forms ofRET, referred to as RET/PTC 1–9, ELKS/RET and RFP/RET, have been cloned from sporadic and radiation-associated papillary thyroid carcinomas. These mutations induced oncogenic activation of RET tyrosine kinase by different mechanisms. To date, various kinds of therapeutic approaches have been developed for the treatment of RET-associated cancers, including tyrosine kinase inhibitors, gene therapy with dominant negative RET mutants, and RNA interference to abrogate oncogenic mutant RET expression. RET and some signaling molecules that function downstream of RET could be potential targets for the development of selective cancer therapeutics. (Cancer Sci2005; 96: 143–148) [ABSTRACT FROM AUTHOR]

The Ret receptor tyrosine kinase plays a crucial role in the development of the enteric nervous system and the kidney. Tyrosine 1062 in Ret represents a binding site for the phosphotyrosine-binding domains of several adaptor and effector proteins that are important for the activation of intracellular signaling pathways, such as the RAS/ERK, phosphatidylinositol 3-kinase/AKT, and Jun-associated N-terminal kinase pathways. To investigate the importance of tyrosine 1062 for organogenesis in vivo, knock-in mice in which tyrosine 1062 in Ret was replaced with phenylalanine were generated. Although homozygous knock-in mice were born normally, they died by day 27 after birth and showed growth retardation. The development of the enteric nervous system was severely impaired in homozygous mutant mice, about 40% of which lacked enteric neurons in the whole intestinal tract, as observed in Ret-deficient mice. The rest of the mutant mice developed enteric neurons in the intestine to various extentS, although the size and number of ganglion cells were significantly reduced. Unlike Ret-deficient mice, a small kidney developed in all knock-in mice, accompanying a slight histological change. The reduction of kidney size was due to a decrease of ureteric bud branching during embryogenesis. Thus, these findings demonstrated that the signal via tyrosine 1062 plays an important role in histogenesis of the enteric nervous system and nephrogenesis. [ABSTRACT FROM AUTHOR]

Germline mutations in the RET proto-oncogene are responsible for the development of human hereditary diseases, including multiple endocrine neoplasia (MEN) type 2A and 2B, familial medullary thyroid carcinoma (FMTC), and Hirschsprung's disease (HSCR). It has been reported that some families developed both MEN 2A/FMTC and HSCR, in which a mutation in a cysteine residue at codon 609, 618, or 620 in the RET gene was present. Here we report a novel RET mutation detected in a Japanese family with medullary thyroid carcinoma and HSCR. A germline mutation in cysteine 611 of the RET gene was identified in this family, which introduced an amino-acid change from cysteine to serine. By biological and biochemical analyses of mutant RET proteins, we previously predicted the potentiality that amino-acid substitution for cysteine 611 as well as cysteines 609, 618, and 620 would promote the development of MEN 2A/FMTC and HSCR. This clinical case substantiates our suggestion for the mechanism of the development of both the diseases. [ABSTRACT FROM AUTHOR]

Mi-2β is the main component of the nucleosome remodeling and deacetylase complex and plays an important role in epigenetic transcriptional repression. Here we show that the amino-terminal and carboxyl-terminal regions of Mi-2β have distinct transcriptional activities and bind to BRG1, a component of the SWI/SNF complex, and the RET finger protein (RFP), respectively. Analysis by luciferase reporter assay revealed that the amino-terminal region of Mi-2β has a strong transactivating ability, whereas its carboxyl-terminal region has transcriptional repressive activity. Co-localization and association of Mi-2, RFP, and histone deacetylase 1 suggested that these proteins cooperate in transcriptional repression. Furthermore, the functional importance of the association of Mi-2β and RFP was confirmed by using Rfp[sup -/-] fibroblasts. On the other hand, we demonstrated that Mi-2 and BRG1 were associated with each other and that the bromodomain region of BRG1 strongly suppressed transactivation by the amino-terminal region of Mi-2β. The findings that Mi-2β interacts with both transactivating and repressing proteins and directly associates with another chromatin remodeling protein, BRG1, provide new insight into the formation of multiprotein supercomplex involved in transcriptional regulation. [ABSTRACT FROM AUTHOR]

Abstract: The glial cell line-derived neurotrophic factor (GDNF)/RET tyrosine kinase signaling pathway plays crucial roles in the development of the enteric nervous system (ENS) and the kidney. Tyrosine 1062 (Y1062) in RET is an autophosphorylation residue that is responsible for the activation of the PI3K/AKT and RAS/MAPK signaling pathways. Mice lacking signaling via Ret Y1062 show renal hypoplasia and hypoganglionosis of the ENS although the phenotype is milder than the Gdnf- or Ret-deficient mice. Sprouty2 (Spry2) was found to be an antagonist for fibroblast growth factor receptor (FGFR) and acts as an inhibitory regulator of ERK activation. Spry2-deficient mice exhibit hearing loss and enteric nerve hyperplasia. In the present study, we generated Spry2-deficient and Ret Y1062F knock-in (tyrosine 1062 is replaced with phenylalanine) double mutant mice to see if abnormalities of the ENS and kidney, caused by loss of signaling via Ret Y1062, are rescued by a deficiency of Spry2. Double mutant mice showed significant recovery of ureteric bud branching and ENS development in the stomach. These results indicate that Spry2 regulates downstream signaling mediated by GDNF/RET signaling complex in vivo. [ABSTRACT FROM AUTHOR]

Summary: The serine/threonine kinase Akt (also called protein kinase B) is well known as an important regulator of cell survival and growth and has also been shown to be required for cell migration in different organisms. However, the mechanism by which Akt functions to promote cell migration is not understood. Here, we identify an Akt substrate, designated Girdin/APE (Akt-phosphorylation enhancer), which is an actin binding protein. Girdin expresses ubiquitously and plays a crucial role in the formation of stress fibers and lamellipodia. Akt phosphorylates serine at position 1416 in Girdin, and phosphorylated Girdin accumulates at the leading edge of migrating cells. Cells expressing mutant Girdin, in which serine 1416 was replaced with alanine, formed abnormal elongated shapes and exhibited limited migration and lamellipodia formation. These findings suggest that Girdin is essential for the integrity of the actin cytoskeleton and cell migration and provide a direct link between Akt and cell motility. [Copyright &y& Elsevier]

Abstract: Among three different isoforms of non-muscle myosin heavy chains (NMMHCs), only NMMHCA is associated with inherited human disease, called MYH9 disorders, characterized by macrothrombocytopenia and characteristic granulocyte inclusions. Here targeted gene disruption was performed to understand fundamental as well as pathological role of the gene for NMMHCA, MYH9. Heterozygous intercrosses yielded no homozygous animals among 552 births, suggesting that MYH9 expression is required for embryonic development. In contrast, MYH9+/− mice were viable and fertile without gross anatomical, hematological, and nephrological abnormalities. Immunofluorescence analysis also showed the normal cytoplasmic distribution of NMMHCA. We further measured the auditory brainstem response and found two of six MYH9+/− mice had hearing losses, whereas the remaining four were comparable to wild-type mice. Such observation may parallel the diverse expression of Alport’s manifestations of human individuals with MYH9 disorders and suggest the limited requirement of the gene for maintenance and function of specific organs. [Copyright &y& Elsevier]

Collapsin response mediator protein-2 (CRMP-2) is a mammalian homologue of UNC-33 of Caenorhabditis elegans. Mutations of CRMP-2 result in abnormal axon termination. Recently, it was demonstrated that CRMP-2 binds to tubulin heterodimers to promote microtubule assembly that is critical for axonal differentiation and growth during development. Here we show that glial cell line-derived neurotrophic factor (GDNF) enhances CRMP-2 expression in TGW human neuroblastoma cells via activation of RET receptor tyrosine kinase. GDNF-mediated CRMP-2 expression was regulated mainly by the extracellular regulated kinase (ERK) pathway, but was independent of activation of phosphatidylinositol 3-kinase and Src family kinases. Analysis of the promoter region of the CRMP-2 gene revealed that the region 214–48 bp upstream of the transcriptional start site is important for CRMP-2 expression. The SP1, E2F, and GATA1/2 binding sites appeared to play some roles in regulation of CRMP-2 expression. As expected, the CRMP-2 protein accumulated in extended neurites of TGW cells treated with GDNF. However, neuritogenesis of TGW cells was mostly dependent on Src family kinase activity and not ERK activity, indicating that the increased expression of CRMP-2 alone was not sufficient for neuritogenesis. [Copyright &y& Elsevier]

In the developing kidney, activation of the rearrangement during transfection by glial cell line-derived neurotrophic factor (GDNF) is required for normal branching of the ureteric bud epithelium. By differential display analysis we identified a novel GI)NF-inducible gene (named GZF1) with a BTB/POZ (broad complex, tramtrack, and bric-a-brac)/(poxvirus and zinc finger) domain and 10 tandemly repeated zinc finger motifs. The up-regulation of the GZF1 gene showed two peaks at 1 h and 24-48 h after GDNF stimulation by Northern blotting. The late induction was also found at protein levels by Western blotting with anti-GZF1 antibody. As observed for other proteins with the BTB/POZ domain, the GZF1 protein had strong transcriptional repressive activity. Intriguingly, its expression was detected at high levels in branching ureteric buds and collecting ducts of mouse metanephric kidney in which RET was also expressed. Antisense phosphorothioated oligodeoxynucleotides of the GZF1 gene markedly impaired the ureteric bud branching in the metanephric organ culture, suggesting that the induction of GZF1 expression via the GDNF/RET signaling system is required for renal branching morphogenesis. [ABSTRACT FROM AUTHOR]

We recently demonstrated that RFP, which belongs to the large B-box RING finger protein family, interacts with Enhancer of Polycomb 1 (EPC1) and functions as a transcriptional repressor in human cultured cells. In this study, we examined the expression of RFP and EPC1 in mouse tissues by immunoblotting as well as their interaction by a pull-down assay. Both RFP and EPC1 proteins are expressed in several mouse tissues including testis, spleen, thymus, adrenal gland, cerebrum, and cerebellum. In addition, they were coprecipitated from the lysate of mouse testis. Pull-down assays using glutathione S-transferase (GST)-fused EPC1 proteins revealed that RFP is associated with the EPcA, EPcB, and carboxy-terminal (CT) regions of EPC1. Although RFP is highly expressed as 58- and 68-kDa proteins in mouse testis, the EPC1 CT region more strongly interacted with the 68-kDa form than the EPcA or EPcB region. Interaction of the 58-kDa form of RFP with each region was weak compared with that of the 68-kDa form with the EPC1 CT region. Because the 68-kDa form of RFP was almost completely digested with O-glycosidase but not with N-glycosidase, this suggested that O-glycosylation of RFP plays a role in its interaction with the EPC1 CT region that may be responsible for transcriptional repression. In addition, the luciferase reporter gene assay showed that expression of the EPcA region strongly impairs the transcriptional repressive activity of RFP. [Copyright &y& Elsevier]