Shuichi Hiraoka

FGF9 monomer-dimer equilibrium regulates extracellular matrix affinity and tissue diffusion

The spontaneous dominant mouse mutant, Elbow knee synostosis (Eks), shows elbow and knee joint synosotsis, and premature fusion of cranial sutures. Here we identify a missense mutation in the Fgf9 gene that is responsible for the Eks mutation. Through investigation of the pathogenic mechanisms of joint and suture synostosis in Eks mice, we identify a key molecular mechanism that regulates FGF9 signaling in developing tissues. We show that the Eks mutation prevents homodimerization of the FGF9 protein and that monomeric FGF9 binds to heparin with a lower affinity than dimeric FGF9. These biochemical defects result in increased diffusion of the altered FGF9 protein (FGF9Eks) through developing tissues, leading to ectopic FGF9 signaling and repression of joint and suture development. We propose a mechanism in which the range of FGF9 signaling in developing tissues is limited by its ability to homodimerize and its affinity for extracellular matrix heparan sulfate proteoglycans.

Nucleotide-sugar transporter SLC35D1 is critical to chondroitin sulfate synthesis in cartilage and skeletal development in mouse and human

Proteoglycans are a family of extracellular macromolecules comprised of glycosaminoglycan chains of a repeated disaccharide linked to a central core protein. Proteoglycans have critical roles in chondrogenesis and skeletal development. The glycosaminoglycan chains found in cartilage proteoglycans are primarily composed of chondroitin sulfate. The integrity of chondroitin sulfate chains is important to cartilage proteoglycan function; however, chondroitin sulfate metabolism in mammals remains poorly understood. The solute carrier-35 D1 (SLC35D1) gene (SLC35D1) encodes an endoplasmic reticulum nucleotide-sugar transporter (NST) that might transport substrates needed for chondroitin sulfate biosynthesis. Here we created Slc35d1-deficient mice that develop a lethal form of skeletal dysplasia with severe shortening of limbs and facial structures. Epiphyseal cartilage in homozygous mutant mice showed a decreased proliferating zone with round chondrocytes, scarce matrices and reduced proteoglycan aggregates. These mice had short, sparse chondroitin sulfate chains caused by a defect in chondroitin sulfate biosynthesis. We also identified that loss-of-function mutations in human SLC35D1 cause Schneckenbecken dysplasia, a severe skeletal dysplasia. Our findings highlight the crucial role of NSTs in proteoglycan function and cartilage metabolism, thus revealing a new paradigm for skeletal disease and glycobiology.

Prediction of the Coding Sequences of Mouse Homologues of FLJ Genes: The Complete Nucleotide Sequences of 110 Mouse FLJ- Homologous cDNAs Identified by Screening of Terminal Sequences of cDNA Clones Randomly Sampled from Size-Fractionated Libraries

We have been conducting a human cDNA project to predict protein-coding sequences in long cDNAs (> 4 kb) since 1994. The number of these newly identified human genes exceeds 2000 and these genes are known as KIAA genes. As an extension of this project, we herein report characterization of cDNAs derived from mouse KIAA-homologous genes. A primary aim of this study was to prepare a set of mouse. KIAA-homologous cDNAs that could be used to analyze the physiological roles of KIAA genes in mice. In addition, comparison of the structures of mouse and human KIAA cDNAs might enable us to evaluate the integrity of KIAA cDNAs more convincingly. In this study, we selected mouse KIAA-homologous cDNA clones to be sequenced by screening a library of terminal sequences of mouse cDNAs in size-fractionated libraries. We present the entire sequences of 100 cDNA clones thus selected and predict their protein-coding sequences. The average size of the 100 cDNA sequences reached 5.1 kb and that of mouse KIAA-homologous proteins predicted from these cDNAs was 989 amino acid residues.

Identification of loss-of-function mutations of SLC35D1 in patients with Schneckenbecken dysplasia, but not with other severe spondylodysplastic dysplasias group diseases

Background: Schneckenbecken dysplasia (SBD) is an autosomal recessive lethal skeletal dysplasia that is classified into the severe spondylodysplastic dysplasias (SSDD) group in the international nosology for skeletal dysplasias. The radiological hallmark of SBD is the snail-like configuration of the hypoplastic iliac bone. SLC35D1 (solute carrier-35D1) is a nucleotide-sugar transporter involved in proteoglycan synthesis. Recently, based on human and mouse genetic studies, we showed that loss-of-function mutations of the SLC35D1 gene (SLC35D1) cause SBD. Object: To explore further the range of SLC35D1 mutations in SBD and elucidate whether SLC35D1 mutations cause other skeletal dysplasias that belong to the SSDD group. Methods and results: We searched for SLC35D1 mutations in five families with SBD and 15 patients with other SSDD group diseases, including achodrogenesis type 1A, spondylometaphyseal dysplasia Sedaghatian type and fibrochondrogenesis. We identified four novel mutations, c.319C>T (p.R107X), IVS4+3A>G, a 4959-bp deletion causing the removal of exon 7 (p.R178fsX15), and c.193A>C (p. T65P), in three SBD families. Exon trapping assay showed IVS4+3A>G caused skipping of exon 4 and a frameshift (p.L109fsX18). Yeast complementation assay showed the T65P mutant protein lost the transporter activity of nucleotide sugars. Therefore, all these mutations result in loss of function. No SLC35D1 mutations were identified in all patients with other SSDD group diseases. Conclusion: Our findings suggest that SLC35D1 loss-of-function mutations result consistently in SBD and are exclusive to SBD.

CCN3 Protein Participates in Bone Regeneration as an Inhibitory Factor

Background: We previously demonstrated that CCN3 inhibits BMP-2-induced osteoblast differentiation by in vitro experiments. Results: CCN3 is up-regulated in bone regeneration and acts as a negative regulator for bone regeneration. Conclusion: CCN3 is a negative regulator for bone regeneration. Significance: This study provides the first evidence that CCN3 inhibits bone regeneration and contributes to develop new strategies for bone regeneration therapy. CCN3, a member of the CCN protein family, inhibits osteoblast differentiation in vitro. However, the role of CCN3 in bone regeneration has not been well elucidated. In this study, we investigated the role of CCN3 in bone regeneration. We identified the Ccn3 gene by microarray analysis as a highly expressed gene at the early phase of bone regeneration in a mouse bone regeneration model. We confirmed the up-regulation of Ccn3 at the early phase of bone regeneration by RT-PCR, Western blot, and immunofluorescence analyses. Ccn3 transgenic mice, in which Ccn3 expression was driven by 2.3-kb Col1a1 promoter, showed osteopenia compared with wild-type mice, but Ccn3 knock-out mice showed no skeletal changes compared with wild-type mice. We analyzed the bone regeneration process in Ccn3 transgenic mice and Ccn3 knock-out mice by microcomputed tomography and histological analyses. Bone regeneration in Ccn3 knock-out mice was accelerated compared with that in wild-type mice. The mRNA expression levels of osteoblast-related genes (Runx2, Sp7, Col1a1, Alpl, and Bglap) in Ccn3 knock-out mice were up-regulated earlier than those in wild-type mice, as demonstrated by RT-PCR. Bone regeneration in Ccn3 transgenic mice showed no significant changes compared with that in wild-type mice. Phosphorylation of Smad1/5 was highly up-regulated at bone regeneration sites in Ccn3 KO mice compared with wild-type mice. These results indicate that CCN3 is up-regulated in the early phase of bone regeneration and acts as a negative regulator for bone regeneration. This study may contribute to the development of new strategies for bone regeneration therapy.

Presenilin-1 controls the growth and differentiation of endothelial progenitor cells through its β-catenin-binding region

Presenilin‐1 (PS1) is a gene responsible for the development of early‐onset familial Alzheimers disease. Targeted disruption of the PS1 gene in mice suggested that PS1 might be involved in angiogenesis. We have used an in vitro embryonic stem (ES) cell culture system to prepare endothelial progenitor cells (EPC) lacking PS1 and investigated the roles of PS1 in endothelial cell lineage. With this system, Flk‐1+ E‐cadherin− EPC were generated from PS1‐deficient ES cells, and the EPC lacking PS1 as well as wild‐type EPC grew to form VE‐cadherin+ endothelial colonies supported by a layer of OP9 stromal cells. Although the endothelial colonies from PS1‐deficient EPC showed morphology similar to those from wild‐type EPC, the PS1‐deficient EPC formed a large number of the colonies compared to wild‐type EPC. The enhanced colony‐forming ability of PS1‐deficient EPC was attenuated by the inductions of wild‐type human PS1. To differentiate multiple activities of PS1 for colony‐forming ability, we used two types of human PS1 mutants: one (hPS1D257A) with the aspartate to alanine mutation at residue 257 that impairs the proteolytic activity of PS1, and the other (hPS1Δcat) deleting amino acids 340–371 of the cytosolic loop sequence essential for β‐catenin binding. hPS1D257A showed activity to regulate the colony‐forming ability of PS1‐deficient EPC, while hPS1Δcat failed to exhibit this activity. These results suggest that PS1 regulates the growth and differentiation of endothelial progenitor cells through its β‐catenin‐binding region and that the defect of PS1 function in endothelial cell lineage could contribute to the induction of vascular pathology.

Transcription factors Mesp2 and Paraxis have critical roles in axial musculoskeletal formation

Mesp2 and Paraxis are basic helix–loop–helix (bHLH) ‐type transcription factors coexpressed in the presomitic mesoderm (PSM) and are required for normal somite formation. Here, we show that Mesp2/Paraxis double‐null mice exhibit a distinct phenotype unexpected from either Mesp2 or Paraxis single‐null mice. In the posterior region of the body, most of the skeletal components of both the vertebral body and neural arches are severely reduced and only a rudimental lamina and ribs remain, indicating a strong genetic interaction in the sclerotomal cell lineage. However, yeast two‐hybrid analyses revealed no direct interaction between Mesp2 and Paraxis. The Mesp2/Paraxis double‐null embryo has caudalized somites, revealed by expanded Uncx4.1 expression pattern observed in the Mesp2‐null embryo, but the expression level of Uncx4.1 was significantly decreased in mature somites, indicative of hypoplasia of lateral sclerotome derivatives. By focusing on vertebral column formation, we found that expressions of Pax1, Nkx3.1, and Bapx1 are regulated by Paraxis and that Pax9 expression was severely affected in the Mesp2/Paraxis double‐null embryo. Furthermore, the expression of Pax3, a crucial factor for hypaxial muscle differentiation, is regulated by both Mesp2 and Paraxis in the anteriormost PSM and nascent somite region. The present data strongly suggest that patterning events by bHLH‐type transcription factors have deep impacts on regional chondrogenic and myogenic differentiation of somitic cells, mainly by means of control of Pax genes. Developmental Dynamics 236:1484–1494, 2007.

T1783 Kif26a-Deficient Mice Develop Megacolon Associated with Dysfunction of Enteric Neuronal Cells in the Colon

BACKGROUND AND AIMS: Kinesin superfamily proteins (KIFs) are motor proteins that transport organelles and macromolecules along microtubules. Recent reports have revealed that a total of 45 KIFs have been identified in the mouse and human genomes. Dysfunctions of KIFs underlie some human diseases. These findings have prompted us to elucidate the role of KIF26A in the gastrointestinal systems, physiological functions of which remained totally unknown. METHODS: RT-PCR and in situ hybridization (ISH) were performed for detecting expression levels of mRNA. Kif26a gene knockout mice were generated using C57BL/6 mice by deletion of consensus motif for kinesin in KIF26A molecule (exons 711). Hematoxylin-eosin (HE) staining and Massons trichrome staining were performed for exploring histological findings. Small intestinal transit was assessed by 13C breath test and charcoal method. The Magnus method was performed for examining contraction ability of the excised colonic tissues. RESULTS: RT-PCR using total RNA extracted from tissues of the wild-type mice revealed that kif26a mRNA is highly expressed in the brain, skeletal muscle, and colon. In contrast, very low expressions were detected in the liver, stomach, jejunum, and ileum. ISH for elucidating distribution of kif26a in the wild-type colonic tissues revealed that kif26amRNA was exclusively expressed in neuronal cells both in the Meissners plexus and the Auerbachs plexus. Kif26a-/mice were viable but half of them died before 8 weeks after birth. Intriguingly, kif26a-/mice developed megacolon by age 4-8 wk, whereas the morphology of the stomach and the small intestines were unchanged. HE staining and Massons trichrome staining showed neither infiltration of inflammatory cells nor overaccumulation of connective tissues in the colon of kif26a-/mice. 13C breath test and charcoal method showed no differences in the small intestinal transit between the wild-type and the homozygous mutant mice. Finally, the Magnus method revealed that contraction ability of the colonic tissues of kif26a-/mice by administration of acetylcholine was markedly disrupted as compared to those of the wild-type mice. CONCLUSIONS: To our knowledge, this is the first study demonstrating Kif26a-deficient mice develop megacolon. Deficiency of KIF26A is likely to bring about dysfunction of the enteric neuronal cells in the colonic tissues, followed by weakened contractions of the colon. We propose KIF26A play a critical role in maintenance of proper contractions of the colon. The mutant mice can be used to elucidate a novel pathogenesis for human colonic motility disorders.