Manabu Nakayama

The Zinc Transporter SLC39A13/ZIP13 Is Required for Connective Tissue Development; Its Involvement in BMP/TGF-β Signaling Pathways

Background Zinc (Zn) is an essential trace element and it is abundant in connective tissues, however biological roles of Zn and its transporters in those tissues and cells remain unknown. Methodology/Principal Findings Here we report that mice deficient in Zn transporter Slc39a13/Zip13 show changes in bone, teeth and connective tissue reminiscent of the clinical spectrum of human Ehlers-Danlos syndrome (EDS). The Slc39a13 knockout (Slc39a13-KO) mice show defects in the maturation of osteoblasts, chondrocytes, odontoblasts, and fibroblasts. In the corresponding tissues and cells, impairment in bone morphogenic protein (BMP) and TGF-β signaling were observed. Homozygosity for a SLC39A13 loss of function mutation was detected in sibs affected by a unique variant of EDS that recapitulates the phenotype observed in Slc39a13-KO mice. Conclusions/Significance Hence, our results reveal a crucial role of SLC39A13/ZIP13 in connective tissue development at least in part due to its involvement in the BMP/TGF-β signaling pathways. The Slc39a13-KO mouse represents a novel animal model linking zinc metabolism, BMP/TGF-β signaling and connective tissue dysfunction.

Novel regulation of MHC class II function in B cells

The presence of post‐translational regulation of MHC class II (MHC II) under physiological conditions has been demonstrated recently in dendritic cells (DCs) that potently function as antigen‐presenting cells (APCs). Here, we report that MARCH‐I, an E3 ubiquitin ligase, plays a pivotal role in the post‐translational regulation of MHC II in B cells. MARCH‐I expression was particularly high in B cells, and the forced expression of MARCH‐I induced the ubiquitination of MHC II. In B cells from MARCH‐I‐deficient mice (MARCH‐I KO), the half‐life of surface MHC II was prolonged and the ubiquitinated form of MHC II completely disappeared. In addition, MARCH‐I‐deficient B cells highly expressed exogenous antigen‐loaded MHC II on their surface and showed high ability to present exogenous antigens. These results suggest that the function of MHC II in B cells is regulated through ubiquitination by MARCH‐I.

CBX8, a polycomb group protein, is essential for MLL-AF9-induced leukemogenesis.

Chromosomal translocations involving the mixed lineage leukemia (MLL) gene lead to the development of acute leukemias. Constitutive HOX gene activation by MLL fusion proteins is required for MLL-mediated leukemogenesis; however, the underlying mechanisms remain elusive. Here, we show that chromobox homolog 8 (CBX8), a Polycomb Group protein that interacts with MLL-AF9 and TIP60, is required for MLL-AF9-induced transcriptional activation and leukemogenesis. Conversely, both CBX8 ablation and specific disruption of the CBX8 interaction by point mutations in MLL-AF9 abrogate HOX gene upregulation and abolish MLL-AF9 leukemic transformation. Surprisingly, Cbx8-deficient mice are viable and display no apparent hematopoietic defects. Together, our findings demonstrate that CBX8 plays an essential role in MLL-AF9 transcriptional regulation and leukemogenesis.

Extracellular Calcium Controls Background Current and Neuronal Excitability via an UNC79-UNC80-NALCN Cation Channel Complex

In contrast to its extensively studied intracellular roles, the molecular mechanisms by which extracellular Ca(2+) regulates the basal excitability of neurons are unclear. One mechanism is believed to be through Ca(2+)s interaction with the negative charges on the cell membrane (the charge screening effect). Here we show that, in cultured hippocampal neurons, lowering [Ca(2+)](e) activates a NALCN channel-dependent Na(+)-leak current (I(L-Na)). The coupling between [Ca(2+)](e) and NALCN requires a Ca(2+)-sensing G protein-coupled receptor, an activation of G-proteins, an UNC80 protein that bridges NALCN to a large novel protein UNC79 in the same complex, and the last amino acid of NALCNs intracellular tail. In neurons from nalcn and unc79 knockout mice, I(L-Na) is insensitive to changes in [Ca(2+)](e), and reducing [Ca(2+)](e) fails to elicit the excitatory effects seen in the wild-type. Therefore, extracellular Ca(2+) influences neuronal excitability through the UNC79-UNC80-NALCN complex in a G protein-dependent fashion.

Distinctive four promoters collectively direct expression of brain-derived neurotrophic factor gene.

In order to get a deeper insight into comprehensive understanding of gene regulation of brain-derived neurotrophic factor (BDNF), we characterized the transcriptional apparatus of this gene on the basis of the genomic structure. The results in this study revealed that there are at least four distinctive promoters in the BDNF gene; two of them are neuron-specific and the rest are active in some non-neuronal tissues as well as neuronal ones. Although the analyses of the promoter usage pattern clarified many characteristic features in controlling these promoter activities, the most notable finding was that administration of kainic acid resulted in great activation of two out of the four promoters in hippocampal neurons in a regionally different manner and thus indicated the presence of two distinct signal transduction pathways for kainate-induced activation of BDNF gene expression in neurons. The analysis of BDNF gene expression in terms of the promoter usage pattern would provide a new and important insight into understanding a molecular control mechanism of this gene expression.

The epigenetic regulator Uhrf1 facilitates the proliferation and maturation of colonic regulatory T cells

Intestinal regulatory T cells (Treg cells) are necessary for the suppression of excessive immune responses to commensal bacteria. However, the molecular machinery that controls the homeostasis of intestinal Treg cells has remained largely unknown. Here we report that colonization of germ-free mice with gut microbiota upregulated expression of the DNA-methylation adaptor Uhrf1 in Treg cells. Mice with T cell–specific deficiency in Uhrf1 (Uhrf1fl/flCd4-Cre mice) showed defective proliferation and functional maturation of colonic Treg cells. Uhrf1 deficiency resulted in derepression of the gene (Cdkn1a) that encodes the cyclin-dependent kinase inhibitor p21 due to hypomethylation of its promoter region, which resulted in cell-cycle arrest of Treg cells. As a consequence, Uhrf1fl/flCd4-Cre mice spontaneously developed severe colitis. Thus, Uhrf1-dependent epigenetic silencing of Cdkn1a was required for the maintenance of gut immunological homeostasis. This mechanism enforces symbiotic host-microbe interactions without an inflammatory response.

Type II brain 4.1 (4.1B/KIAA0987), a member of the protein 4.1 family, is localized to neuronal paranodes.

Histochemical analyses of type II brain 4.1/4.1B/KIAA0987, a member of the protein 4.1 family, were carried out in rat brain. In situ hybridization (ISH) showed that type II brain 4.1 mRNA is expressed in a variety of neuronal cells. In particular, type II brain 4.1 mRNA was actively transcribed in the cells of the mesencephalon and the brainstem, which have large myelinated nerve fibers. Expression of type II brain 4.1 mRNA was not observed at least in glial cells distributed in nerve fiber tracts. In immunohistochemical studies using anti-type II brain 4.1-specific antibody, the major immunosignals appeared as brilliant pairs of dots along nerve fibers. Such immunosignals were detected throughout the brain, but were highly concentrated in nerve fiber tracts. These data suggested that type II brain 4.1 is predominantly localized to neuronal paranodes. Detailed analysis concentrating on the nodal region indicated that type II brain 4.1 is present at the paranodal membrane but not in the axoplasm. Weaker type II brain 4.1-specific immunosignals were observed along the internodal membrane of myelinated axons and in the cytoplasm of some neuronal cells. Finally, comparative immunohistochemical studies using antibodies against the other three protein 4.1 family members, type I brain 4.1/4.1N/KIAA0338, erythroid type 4.1 (4.1R) and 4.1G, demonstrated that each of these proteins is distributed in a unique pattern in the cerebellum. Our results are the first to show that type II brain 4.1 is the only member of the protein 4.1 family localized to neuronal paranodes.

Characterization of a new β-spectrin gene which is predominantly expressed in brain

We recently identified a gene which shows high similarity to the beta-spectrin gene but with a different chromosomal location from either of the two known beta-spectrin genes [T. Nagase, K.-I. Ishikawa, D. Nakajima, M. Ohira, N. Seki, N. Miyajima, A. Tanaka, H. Kotani, N. Nomura, O. Ohara, Prediction of the coding sequences of unidentified human genes: VII. The complete sequences of 100 new cDNA clones from brain which can code for large proteins in vitro, DNA Res. 4 (1997) 141-150]. In order to further characterize this new spectrin gene and its product, we isolated the rat counterpart of this gene and analyzed it in terms of its protein coding sequence, the tissue distribution of its mRNA and the product, and the regional distribution of the mRNA and the product in the brain. The results indicated that this gene was most abundantly transcribed in the brain and neurons were the predominant cell-type to express this gene. In particular, Purkinje cells were the richest in this gene product, and this new form of beta-spectrin was found more prominently in the dendrites than in the cell bodies. Since the expression pattern and the subcellular localization of this gene product were quiet distinct from those of the two beta-spectrin isoforms already characterized, this beta-spectrin gene would play an important role in neuronal membrane skeleton although it has been overlooked to date.

Regulated selection of germinal-center cells into the memory B cell compartment

Despite the importance of memory B cells in protection from reinfection, how such memory cells are selected and generated during germinal-center (GC) reactions remains unclear. We found here that light-zone (LZ) GC B cells with B cell antigen receptors (BCRs) of lower affinity were prone to enter the memory B cell pool. Mechanistically, cells in this memory-prone fraction had higher expression of the transcriptional repressor Bach2 than that of their counterparts with BCRs of higher affinity. Haploinsufficiency of Bach2 resulted in reduced generation of memory B cells, independently of suppression of the gene encoding the transcription factor Blimp-1. Bach2 expression in GC cells was inversely correlated with the strength of help provided by T cells. Thus, we propose an instructive model in which weak help from T cells maintains relatively high expression of Bach2, which predisposes GC cells to enter the memory pool.

Up-regulation of cystatin C by microglia in the rat facial nucleus following axotomy

Cystatin C, a cysteine proteinase inhibitor, is expressed in the central nervous system (CNS) as well as many other organs of mammals. However, little is known concerning whether its expression is regulated under pathological conditions of the CNS and what types of cells are responsible for this regulation. We performed differential hybridization screening of cDNA libraries derived from the rat facial nucleus and found a cDNA of rat cystatin C to be up-regulated following facial nerve axotomy. In situ hybridization using an RNA probe for rat cystatin C revealed that cystatin C mRNA in the facial nucleus was markedly increased in amount by day 7 after axotomy and was then decreased to the normal level by day 50. The intense signal for cystatin C mRNA in the damaged facial nucleus was localized in the glial cells which had the morphological characteristics of microglia. Light and electron microscopic immunohistochemistry using a rabbit antibody specific for cystatin C confirmed that microglia in the damaged facial nucleus were strongly positive for cystatin C. The immunoreactivity was also found in the extracellular space, consistent with the fact that cells producing cystatin C generally secrete this protein. These results demonstrate that cystatin C is markedly up-regulated by microglia in response to axotomy and is probably secreted by these cells into the extracellular space, suggesting that this proteinase inhibitor has (a) significant function(s) in the processes of neuronal degeneration, regeneration, and/or repair subsequent to axotomy.