Akira Kudo

Osteopetrosis and thalamic hypomyelinosis with synaptic degeneration in DAP12-deficient mice

Deletions in the DAP12 gene in humans result in Nasu-Hakola disease, characterized by a combination of bone fractures and psychotic symptoms similar to schizophrenia, rapidly progressing to presenile dementia. However, it is not known why these disorders develop upon deficiency in DAP12, an immunoreceptor signal activator protein initially identified in the immune system. Here we show that DAP12-deficient (DAP12(-/-)) mice develop an increased bone mass (osteopetrosis) and a reduction of myelin (hypomyelinosis) accentuated in the thalamus. In vitro osteoclast induction from DAP12(-/-) bone marrow cells yielded immature cells with attenuated bone resorption activity. Moreover, immature oligodendrocytes were arrested in the vicinity of the thalamus, suggesting that the primary defects in DAP12(-/-) mice are the developmental arrest of osteoclasts and oligodendrocytes. In addition, the mutant mice also showed synaptic degeneration, impaired prepulse inhibition, which is commonly observed in several neuropsychiatric diseases in humans including schizophrenia, and aberrant electrophysiological profiles in the thalami. These results provide a molecular basis for a unique combination of skeletal and psychotic characteristics of Nasu-Hakola disease as well as for schizophrenia and presenile dementia.

The divergent expression of periostin mRNA in the periodontal ligament during experimental tooth movement

A novel 90-kDa protein named periostin, which is preferentially expressed in the periosteum and the periodontal ligament (PDL), may play a role in bone metabolism and remodeling. However, the precise role of periostin in the PDL remains unclear. Therefore, we examined the expression of periostin mRNA during experimental tooth movement. Experimental tooth movement was achieved in 7-week-old male Sprague-Dawley rats. In control specimens without tooth movement, the expression of periostin mRNA was uniformly observed in the PDL surrounding the mesial and distal roots of the upper molars and was weak in the PDL of the root furcation area. The periostin mRNA-expressing cells were mainly fibroblastic cells in the PDL and osteoblastic cells on the alveolar bone surfaces. The divergent expression of periostin mRNA in the PDL began to be observed at 3xa0h and continued up to 96xa0h after tooth movement. The maximum changes, which showed stronger staining in the pressure sites than in the tension sites, were observed at 24xa0h. The expression of periostin mRNA in the PDL 168xa0h after tooth movement exhibited a similar distribution to that of the control specimens. These results suggest that periostin is one of the local contributing factors in bone and periodontal tissue remodeling following mechanical stress during experimental tooth movement.

EBF-regulating Pax5 transcription is enhanced by STAT5 in the early stage of B cells.

Pax5 is an essential transcription factor for B cell development, and it is reported that Pax5 expression was reduced in the IL‐7 receptor (IL‐7R) knockout mouse. To investigate whether signals from the IL‐7R regulate Pax5 transcription, we searched the consensus sequence of signal transducers and activators of transcription (STAT) in the Pax5 promoter region, since STAT is one of the components of cytokine signal transduction. A STAT‐binding motif, termed SBM, was identified at 1,118u2004bp upstream of the transcriptional start site, and SBM completely overlapped with the binding sitefor early B cell factor (EBF). STAT5 was phosphorylated in the presence of IL‐7 in the IL‐7‐dependent preB cell line, PreBR1, and phosphorylated‐STAT5 as well as EBF was found to bind to the SBM. Moreover, we also revealed STAT5 binding to SBM in PreBR1 cells by chromatin immunoprecipitation assay. Transient co‐transfection of reporter genes together with expression vectors of a constitutive active form of STAT5 and EBF into NIH3T3 cells demonstrated that STAT5 enhanced EBF‐regulating transcription. Our results suggest that STAT5 phosphorylated by IL‐7 can directly up‐regulate Pax5 transcription in early B cells.

TRAF5 functions in both RANKL- and TNFα-induced osteoclastogenesis

Although TRAF6 is essential for both RANKL‐ and TNFα‐induced osteoclastogenesis, it has remained unclear whether other members of the TRAF family are involved in osteoclastogenesis. We examined TRAF5 function in both RANKL‐ and TNFα‐induced osteoclastogenesis by using osteoclast progenitor cells from TRAF5‐deficient mice. The results demonstrated that RANKL or TNFα did not effectively induce osteoclast differentiation from osteoclast progenitor cells derived from these mice into mature multinucleated osteoclasts, although c‐jun N‐terminal kinase (JNK) and NF‐κB activation was apparently observed in osteoclast progenitor cells. In the parathyroid hormone (PTH)‐induced hypercalcemia model, calcium concentration peaked at day 3 after administration. However, in TRAF5‐deficient mice, this peak was delayed and found at day 5, showing less effective osteoclast differentiation. Thus, we have provided the first evidence showing that TRAF5 is involved in osteoclastogenesis.

Analysis of Wnt8 for neural posteriorizing factor by identifying Frizzled 8c and Frizzled 9 as functional receptors for Wnt8

The dorsal ectoderm of vertebrate gastrula is first specified into anterior fate by an activation signal and posteriorized by a graded transforming signal, leading to the formation of forebrain, midbrain, hindbrain and spinal cord along the anteroposterior (A-P) axis. Transplanted non-axial mesoderm rather than axial mesoderm has an ability to transform prospective anterior neural tissue into more posterior fates in zebrafish. Wnt8 is a secreted factor that is expressed in non-axial mesoderm. To investigate whether Wnt8 is the neural posteriorizing factor that acts upon neuroectoderm, we first assigned Frizzled 8c and Frizzled 9 to be functional receptors for Wnt8. We then, transplanted non-axial mesoderm into the embryos in which Wnt8 signaling is cell-autonomously blocked by the dominant-negative form of Wnt8 receptors. Non-axial mesodermal transplants in embryos in which Wnt8 signaling is cell-autonomously blocked induced the posterior neural markers as efficiently as in wild-type embryos, suggesting that Wnt8 signaling is not required in neuroectoderm for posteriorization by non-axial mesoderm. Furthermore, Wnt8 signaling, detected by nuclear localization of beta-catenin, was not activated in the posterior neuroectoderm but confined in marginal non-axial mesoderm. Finally, ubiquitous over-expression of Wnt8 does not expand neural ectoderm of posterior character in the absence of mesoderm or Nodal-dependent co-factors. We thus conclude that other factors from non-axial mesoderm may be required for patterning neuroectoderm along the A-P axis.

TRANCE together with IL-7 induces pre-B cells to proliferate

TRANCE (TNF‐related activation‐induced cytokine)‐deficient mice completely lack osteoclasts, and develop severe osteopetrosis. These mice also show a defect in their pre‐Bu2004cell differentiation. In the present study, the expression of TRANCE was examined in pre‐Bu2004cell lines using flow cytometry and reverse transcription‐PCR. Three pre‐Bu2004cell lines, 18‐81, B3P816‐1, and 38B9, expressed TRANCE on their surface, and two pre‐Bu2004cell lines, 70Z/3 and NFS5, at the late pre‐Bu2004cell stage, expressed it at low levels, although their mRNA expression was normal. Another pre‐Bu2004cell line, 38‐C‐13, at the intermediate stage between pre‐B and immature Bu2004cells, did not express TRANCE. The IL‐7‐dependent pre‐Bu2004cell line PreBR, which expresses the pre‐Bu2004cell receptor on the cellsurface, also expressed TRANCE. When differentiation of PreBR cells was induced in vitro by removing IL‐7 from cultures, TRANCE expression dropped; it was restored by the addition of IL‐7, suggesting that TRANCE functions in cooperation with IL‐7. To examine the function of TRANCE, we introduced the TRANCE gene into PreBR cells and established two transfectants that constitutively expressed TRANCE, even in the absence of IL‐7. In these transfectants, after removal of IL‐7, the number of cells that succeeded in κu2004chain rearrangement was decreased to one third; and CD40 expression decreased to less than one tenth. Moreover, the percentage of cells in the S/G2/M phase was increased by 50% over the mock transfectant. These findings indicate that, before κu2004chain rearrangement occurs, TRANCE together with IL‐7 induces pre‐Bu2004cells to proliferate and makes this rearrangement more efficient.

Expression of zisp, a DHHC zinc finger gene, in somites and lens during zebrafish embryogenesis.

The zebrafish zisp gene encodes a putative transmembrane protein with a DHHC zinc finger motif. At the segmentation period zisp is expressed in the adaxial cells and the somites in a striping pattern. The zisp transcripts are localized to the posterior parts within the individual somites. In fused somites mutants, zisp is expressed throughout the somitic mesoderm. These expression patterns are similar to those of myoD. In addition to the somitic expression, the zisp expression was observed in lens cells at the late segmentation period and the early pharyngula period.