Kazu Matsumoto

Distinct Interaction of Versican/PG-M with Hyaluronan and Link Protein

The proteoglycan aggregate is the major structural component of the cartilage matrix, comprising hyaluronan (HA), link protein (LP), and a large chondroitin sulfate (CS) proteoglycan, aggrecan. Here, we found that another member of aggrecan family, versican, biochemically binds to both HA and LP. Functional analyses of recombinant looped domains (subdomains) A, B, and B′ of the N-terminal G1 domain revealed that the B-B′ segment of versican is adequate for binding to HA and LP, whereas A and B-B′ of aggrecan bound to LP and HA, respectively. BIAcore™ analyses showed that the A subdomain of versican G1 enhances HA binding but has a negligible effect on LP binding. Overlay sensorgrams demonstrated that versican G1 or its B-B′ segment forms a complex with both HA and LP. We generated a molecular model of the B-B′ segment, in which a deletion and an insertion of B′ and B are critical for stable structure and HA binding. These results provide important insights into the mechanisms of formation of the proteoglycan aggregate and HA binding of molecules containing the link module.

Conditional inactivation of Has2 reveals a crucial role for hyaluronan in skeletal growth, patterning, chondrocyte maturation and joint formation in the developing limb

The glycosaminoglycan hyaluronan (HA) is a structural component of extracellular matrices and also interacts with cell surface receptors to directly influence cell behavior. To explore functions of HA in limb skeletal development, we conditionally inactivated the gene for HA synthase 2, Has2, in limb bud mesoderm using mice that harbor a floxed allele of Has2 and mice carrying a limb mesoderm-specific Prx1-Cre transgene. The skeletal elements of Has2-deficient limbs are severely shortened, indicating that HA is essential for normal longitudinal growth of all limb skeletal elements. Proximal phalanges are duplicated in Has2 mutant limbs indicating an involvement of HA in patterning specific portions of the digits. The growth plates of Has2-deficient skeletal elements are severely abnormal and disorganized, with a decrease in the deposition of aggrecan in the matrix and a disruption in normal columnar cellular relationships. Furthermore, there is a striking reduction in the number of hypertrophic chondrocytes and in the expression domains of markers of hypertrophic differentiation in the mutant growth plates, indicating that HA is necessary for the normal progression of chondrocyte maturation. In addition, secondary ossification centers do not form in the central regions of Has2 mutant growth plates owing to a failure of hypertrophic differentiation. In addition to skeletal defects, the formation of synovial joint cavities is defective in Has2-deficient limbs. Taken together, our results demonstrate that HA has a crucial role in skeletal growth, patterning, chondrocyte maturation and synovial joint formation in the developing limb.

A mouse model of chondrocyte-specific somatic mutation reveals a role for Ext1 loss of heterozygosity in multiple hereditary exostoses

Multiple hereditary exostoses (MHE) is one of the most common skeletal dysplasias, exhibiting the formation of multiple cartilage-capped bony protrusions (osteochondroma) and characteristic bone deformities. Individuals with MHE carry heterozygous loss-of-function mutations in Ext1 or Ext2, genes which together encode an enzyme essential for heparan sulfate synthesis. Despite the identification of causative genes, the pathogenesis of MHE remains unclear, especially with regard to whether osteochondroma results from loss of heterozygosity of the Ext genes. Hampering elucidation of the pathogenic mechanism of MHE, both Ext1+/− and Ext2+/− heterozygous mutant mice, which mimic the genetic status of human MHE, are highly resistant to osteochondroma formation, especially in long bones. To address these issues, we created a mouse model in which Ext1 is stochastically inactivated in a chondrocyte-specific manner. We show that these mice develop multiple osteochondromas and characteristic bone deformities in a pattern and a frequency that are almost identical to those of human MHE, suggesting a role for Ext1 LOH in MHE. Surprisingly, however, genotyping and fate mapping analyses reveal that chondrocytes constituting osteochondromas are mixtures of mutant and wild-type cells. Moreover, osteochondromas do not possess many typical neoplastic properties. Together, our results suggest that inactivation of Ext1 in a small fraction of chondrocytes is sufficient for the development of osteochondromas and other skeletal defects associated with MHE. Because the observed osteochondromas in our mouse model do not arise from clonal growth of chondrocytes, they cannot be considered true neoplasms.

Reliabilities of and Correlations Among Five Standard Methods of Assessing the Sagittal Alignment of the Cervical Spine

Study Design. The reliabilities of and correlations among 5 standard methods of assessing cervical sagittal alignment were evaluated. Objective. To investigate the reliabilities of and correlations among 5 standard methods of assessing cervical sagittal alignment. Summary of Background Data. Although various cervical sagittal alignment assessment methods are widely used, their relative reliability and intercorrelation have not been reported. Methods. From 442 lateral cervical radiographs, 40 with lordotic, 40 with straight or sigmoid, and 40 with kyphotic alignment were selected. Two orthopedic surgeons independently evaluated the sagittal alignment in each group twice using CCL, C1–C7 Cobb, C2–C7 Cobb, sagittal tangent, and the Ishihara methods. Intraobserver and interobserver reliabilities were confirmed and the correlations among the 5 methods were measured. Results. Intraobserver and interobserver reliabilities for all 5 methods were good. In the lordotic group, the correlations among all 5 methods were consistently strong (r = 0.731 to 0.922). In the straight or sigmoid group, the correlations were weak to moderate among the CCL, C2–C7 Cobb, sagittal tangent, and Ishihara methods but tended to be weak between these 4 methods and the C1–C7 Cobb method (r = −0.245 to 0.777). In the kyphotic group, the correlations were also weak to moderate among the same 4 methods, and were statistically insignificant between them and the C1–C7 Cobb. Conclusions. The correlations among the CCL, C1–C7 Cobb, C2–C7 Cobb, sagittal tangent, and Ishihara methods are strong when lordosis is retained; otherwise, they are moderate to poor. In the kyphotic group, C1–C7 Cobb has no significant correlation with the other 4 methods.

Heparan sulfate regulates ephrin-A3/EphA receptor signaling.

Increasing evidence indicates that many signaling pathways involve not only ligands and receptors but also various types of coreceptors and matrix components as additional layers of regulation. Signaling by Eph receptors and their ephrin ligands plays a key role in a variety of biological processes, such as axon guidance and topographic map formation, synaptic plasticity, angiogenesis, and cancer. Little is known about whether the ephrin-Eph receptor signaling system is subject to such additional layers of regulation. Here, we show that ephrin-A3 binds to heparan sulfate, and that the presence of cell surface heparan sulfate is required for the full biological activity of ephrin-A3. Among the ephrins tested, including ephrin-A1, -A2, -A5, -B1, and -B2, only ephrin-A3 binds heparin or heparan sulfate. Ephrin-A3-dependent EphA receptor activation is reduced in mutant cells that are defective in heparan sulfate synthesis, in wild-type cells from which cell surface heparan sulfate has been removed, and in the hippocampus of conditional knockout mice defective in heparan sulfate synthesis. Ephrin-A3-dependent cell rounding is impaired in CHO cells lacking heparan sulfate, and cortical neurons lacking heparan sulfate exhibit impaired growth cone collapse. In contrast, cell rounding and growth cone collapse in response to ephrin-A5, which does not bind heparan sulfate, are not affected by the absence of heparan sulfate. These results show that heparan sulfate modulates ephrin/Eph signaling and suggest a physiological role for heparan sulfate proteoglycans in the regulation of ephrin-A3-dependent biological processes.

Conditional Ablation of the Heparan Sulfate-synthesizing Enzyme Ext1 Leads to Dysregulation of Bone Morphogenic Protein Signaling and Severe Skeletal Defects

Increasing evidence indicates that heparan sulfate (HS) is an integral component of many morphogen signaling pathways. However, its mechanisms of action appear to be diverse, depending on the type of morphogen and the developmental contexts. To define the function of HS in skeletal development, we conditionally ablated Ext1, which encodes an essential glycosyltransferase for HS synthesis, in limb bud mesenchyme using the Prx1-Cre transgene. These conditional Ext1 mutant mice display severe limb skeletal defects, including shortened and malformed limb bones, oligodactyly, and fusion of joints. In developing limb buds of mutant mice, chondrogenic differentiation of mesenchymal condensations is delayed and impaired, whereas the area of differentiation is diffusely expanded. Correspondingly, the distribution of both bone morphogenic protein (BMP) signaling domains and BMP2 immunoreactivity in the mutant limb mesenchyme is broadened and diffuse. In micromass cultures, chondrogenic differentiation of mutant chondrocytes is delayed, and the responsiveness to exogenous BMPs is attenuated. Moreover, the segregation of the pSmad1/5/8-expressing chondrocytes and fibronectin-expressing perichondrium-like cells surrounding chondrocyte nodules is disrupted in mutant micromass cultures. Together, our results show that HS is essential for patterning of limb skeletal elements and that BMP signaling is one of the major targets for the regulatory role of HS in this developmental context.

Identification and Characterization of Versican/PG-M Aggregates in Cartilage

Versican/PG-M is a large chondroitin sulfate proteoglycan of the extracellular matrix with a common domain structure to aggrecan and is present in cartilage at low levels. Here, we characterized cartilage versican during development and growth. Immunostaining showed that versican was mainly localized in the interterritorial zone of the articular surface at 2 weeks in mice, whereas aggrecan was in the pericellular zone of prehypertrophic and hypertrophic cells of the growth plate. Although its transcription level rapidly diminished during growth, versican remained in the articular cartilage. Biochemical analysis of normal articular cartilage and aggrecan-null cartilage from cmd (cartilage matrix deficiency)/cmd mice revealed that versican was present as a proteoglycan aggregate with both link protein and hyaluronan. Chondroitin sulfate chains of versican digested with chondroitinase ABC contained 71% nonsulfated and 28% 4-sulfated unsaturated disaccharides, whereas those of aggrecan contained 25% nonsulfated and 70% 4-sulfated. Link protein overexpression in chondrocytic N1511 cells at the early stage of differentiation, in which versican is expressed, enhanced versican deposition in the matrix and prevented subsequent aggrecan deposition. These results suggest that versican is present as an aggregate distinct from the aggrecan aggregate and may play specific roles in the articular surface.

Wrist fractures from snowboarding: a prospective study for 3 seasons from 1998 to 2001.

ObjectivesTo elucidate the epidemiology and the mechanisms of snowboarding wrist injuries, especially distal radial fractures. DesignA prospective survey of snowboarders with distal radial fractures. ParticipantsFrom November 21, 1998, to April 22, 2001, we analyzed and interviewed 5110 injured snowboarders, and a total of 740 snowboarders with distal radial fractures were studied. On the basis of the medical records and radiographs, the severity of distal radial fracture was analyzed according to the AO classification. ResultsDistal radial fractures occurred at a rate of 0.31 per 1000 snowboarder visits and were assessed in 740 snowboarders in this study. Most of the injured snowboarders were either of beginner (42.0%) or intermediate level (48.1%). The most common events leading to an injury in snowboarding were falling (59.6%) and jumping (36.1%). Comminuted and articular fractures classified as AO types A3, B, and C, which required surgical treatment, made up 63.2% of distal radial fractures in snowboarders. The most remarkable differences between the first-time or beginner group and the intermediate or expert group were that the former had a significantly higher proportions of extra-articular fractures classified as AO type A (P < 0.05), and the latter were significantly more likely to have compression or complex intra-articular fractures such as AO type C (P < 0.05). Furthermore, first-time or beginner snowboarders were more likely to be injured because of a simple fall than were the intermediates or experts (P < 0.05). Over 60% of distal radial fractures classified as AO type C in the intermediate or expert group resulted from jumping. Furthermore, the side opposite to the snowboarders preferred direction of stance was more often affected. A high incidence of injury during opposite-side edging, which is used more frequently in snowboarding, was found in novice female snowboarders. ConclusionsThis study suggested several patterns in the nature of wrist injuries sustained while snowboarding, and these facts should be taken into consideration in the diagnosis of wrist injuries in snow-boarders.

TRPV4-pathy manifesting both skeletal dysplasia and peripheral neuropathy: A report of three patients†‡

Heterozygous missense mutations of transient receptor potential vanilloid 4 channel (TRPV4) cause a spectrum of skeletal disorders, including brachyolmia, spondylometaphyseal dysplasia Kozlowski type, metatropic dysplasia, parastremmatic dysplasia, and spondyloepimetaphyseal dysplasia Maroteaux type. Similarly, heterozygous missense mutations of TRPV4 cause a spectrum of peripheral neuropathy, including hereditary motor and sensory neuropathy type IIC, congenital spinal muscular atrophy, and scapuloperoneal spinal muscular atrophy. There are no apparent differences in the amino acid positions affected or type of change predicted by the TRPV4 mutations responsible for the two disease spectrums; nevertheless, no fundamental phenotypic overlap has been shown between the two spectrums. Here, we report on three patients who had both skeletal dysplasia and peripheral neuropathy caused by heterozygous TRPV4 missense mutations. The skeletal and neurologic phenotypes of these patients covered the wide spectrum of reported TRPV4‐pathies (disease caused by TRPV4 mutations). The molecular data are complementary, proving that “neuropathic” mutations can cause skeletal dysplasia but also the “skeletopathic” mutations can lead to neuropathies. Our findings suggest that pathogenic mechanisms of TRPV4‐pathies in skeletal and nervous systems are not always mutually exclusive and provide further evidence that there is no clear genotype–phenotype correlation for either spectrum. Co‐occurrence of skeletal dysplasia and degenerative neuropathy should be kept in mind in clinical practice including diagnostic testing, surgical evaluation, and genetic counseling.

Traumatic Spinal Cord Injuries From Snowboarding

Background Little information has yet been made available on the types and mechanisms of snowboard-related spinal cord injuries or their neurologic involvement. Purpose To review the cause and types of spinal cord injuries seen in snowboarders. Study Design Case series; Level of evidence, 4. Methods The subjects were 18 patients (mean age, 24.0 years) referred to the authors’ institution for neurologic deficits associated with spinal injuries between November 1, 1995, and April 9, 2005. The clinical features of these patients were reviewed with respect to epidemiologic factors, mechanism of injury, fracture pattern, and neurologic status. Results The 18 snowboarders with spinal cord injuries constituted a very homogeneous group. First, almost all patients (94.4%) were young men. Second, most of the patients were intermediate or expert boarders. Third, the most common cause of injury was a failure of intentional jumping (83.3%). Fourth, the most commonly affected site was the thoracolumbar junction (66.7%), and the most common type of fracture was an anterior dislocation fracture (66.7%). Finally, in the thoracolumbar group, most patients (83.3%) were classed as Frankel grade A or B. Conclusion It is fundamentally important that snowboarders, especially young men, be made aware of the spinal injury risk associated with jumping.