Dyssegmental dysplasia Rolland-Desbuquois type is caused by pathogenic variants in HSPG2 - a founder haplotype shared in five patients.

Dyssegmental dysplasia (DD) is a severe skeletal dysplasia comprised of two subtypes: lethal Silverman-Handmaker type (DDSH) and nonlethal Rolland-Desbuquois type (DDRD). DDSH is caused by biallelic pathogenic variants in HSPG2 encoding perlecan, whereas the genetic cause of DDRD remains undetermined. Schwartz-Jampel syndrome (SJS) is also caused by biallelic pathogenic variants in HSPG2 and is an allelic disorder of DDSH. In SJS and DDSH, 44 and 8 pathogenic variants have been reported in HSPG2, respectively. Here, we report that five patients with DDRD carried four pathogenic variants in HSPG2: c.9970 G > A (p.G3324R), c.559 C > T (p.R187X), c7006 + 1 G > A, and c.11562 + 2 T > G. Two patients were homozygous for p.G3324R, and three patients were heterozygous for p.G3324R. Haplotype analysis revealed a founder haplotype spanning 85,973 bp shared in the five patients. SJS, DDRD, and DDSH are allelic disorders with pathogenic variants in HSPG2.

Beyond 2D: A scalable and highly sensitive method for a comprehensive 3D analysis of kidney biopsy tissue.

The spatial organization of various cell populations is critical for the major physiological and pathological processes in the kidneys. Most evaluation of these processes typically comes from a conventional 2D tissue cross-section, visualizing a limited amount of cell organization. Therefore, the 2D analysis of kidney biopsy introduces selection bias. The 2D analysis potentially omits key pathological findings outside a 1- to 10-µm thin-sectioned area and lacks information on tissue organization, especially in a particular irregular structure such as crescentic glomeruli. In this study, we introduce an easy-to-use and scalable method for obtaining high-quality images of molecules of interest in a large tissue volume, enabling a comprehensive evaluation of the 3D organization and cellular composition of kidney tissue, especially the glomerular structure. We show that CUBIC and ScaleS clearing protocols could allow a 3D analysis of the kidney tissues in human and animal models of kidney disease. We also demonstrate that the paraffin-embedded human biopsy specimens previously examined via 2D evaluation could be applicable to 3D analysis, showing a potential utilization of this method in kidney biopsy tissue collected in the past. In summary, the 3D analysis of kidney biopsy provides a more comprehensive analysis and a minimized selection bias than 2D tissue analysis. Additionally, this method enables a quantitative evaluation of particular kidney structures and their surrounding tissues, with the potential utilization from basic science investigation to applied diagnostics in nephrology.

Impact of the heparan sulfate proteoglycan perlecan on human disease and health.

Perlecan, a basement membrane-type heparan sulfate proteoglycan, is an important molecule in the functional diversity of organisms because of the diversity of its glycan chains and the multifunctionality of its core proteins. Human diseases associated with perlecan have been identified using gene-deficient mice. Two human diseases related to perlecan have been reported. One is Silverman-Handmaker type dyssegmental dysplasia, resulting from the complete loss of function of the HSPG2 gene that encodes perlecan core protein, which is mapped to chromosome 1p36. The other is Schwartz-Jampel syndrome resulting from the partial loss of function of the HSPG2 gene. Subsequent in vivo and in vitro studies have revealed the organ-specific functions of perlecan, suggesting its involvement in the pathogenesis of various human diseases. In this review, we discuss the role of perlecan in human diseases and summarize our knowledge about perlecan as a future therapeutic target to treat related diseases and for healthy longevity.

α-1,6-Fucosyltransferase Is Essential for Myogenesis in Zebrafish.

Glycosylation is an important mechanism regulating various biological processes, including intercellular signaling and adhesion. α-1,6-fucosyltransferase (Fut8) belongs to a family of enzymes that determine the terminal structure of glycans. Fut8 is widely conserved from Caenorhabditis elegans to humans, and its mutants have been reported in humans, mice, and zebrafish. Although mutants show various symptoms, such as spinal deformity and growth retardation, its effects on skeletal muscles are unknown. We aimed to elucidate the function of Fut8 in skeletal muscle using zebrafish and C2C12 cells for evaluation. We observed that most fut8a morphants died at 2 days post-fertilization (dpf) or in earlier developmental stages even at low concentrations of morpholino oligonucleotides (MOs). Mutant juveniles also had small body sizes, and abnormal myocepta and sarcomere structures, suggesting that Fut8a plays important roles in myogenesis. Moreover, treatment of C2C12 cells with 2-fluorofucose (2FF), a fucosylation inhibitor, during cell differentiation dramatically reduced the expression of myogenic genes, such as Myomaker and other myogenic fusion genes, and inhibited myotube formation. These results indicate that Fut8 is an important factor in myogenesis, and myofusion in particular.

Perlecan Facilitates Neuronal Nitric Oxide Synthase Delocalization in Denervation-Induced Muscle Atrophy.

Perlecan is an extracellular matrix molecule anchored to the sarcolemma by a dystrophin-glycoprotein complex. Perlecan-deficient mice are tolerant to muscle atrophy, suggesting that perlecan negatively regulates mechanical stress-dependent skeletal muscle mass. Delocalization of neuronal nitric oxide synthase (nNOS) from the sarcolemma to the cytosol triggers protein degradation, thereby initiating skeletal muscle atrophy. We hypothesized that perlecan regulates nNOS delocalization and activates protein degradation during this process. To determine the role of perlecan in nNOS-mediated mechanotransduction, we used sciatic nerve transection as a denervation model of gastrocnemius muscles. Gastrocnemius muscle atrophy was significantly lower in perinatal lethality-rescued perlecan-knockout (Hspg2-/--Tg) mice than controls (WT-Tg) on days 4 and 14 following surgery. Immunofluorescence microscopy showed that cell membrane nNOS expression was reduced by denervation in WT-Tg mice, with marginal effects in Hspg2-/--Tg mice. Moreover, levels of atrophy-related proteins-i.e., FoxO1a, FoxO3a, atrogin-1, and Lys48-polyubiquitinated proteins-increased in the denervated muscles of WT-Tg mice but not in Hspg2-/--Tg mice. These findings suggest that during denervation, perlecan promotes nNOS delocalization from the membrane and stimulates protein degradation and muscle atrophy by activating FoxO signaling and the ubiquitin-proteasome system.

A nationwide observational study of locomotive syndrome in Japan using the ResearchKit: The Locomonitor study.

BACKGROUND: We developed the Locomonitor application (app), the world's first iOS app to study locomotive syndrome, using the ResearchKit and examined the prevalence and risk factors for locomotive syndrome in Japanese general individuals 20-69 years old in a nationwide cross-sectional observational study. METHODS: The participants were recruited from February to August 2016. The outcome measures for the locomotive function were evaluated by locomotive syndrome risk tests (LSRTs) using the Locomonitor app. The chi-squared test, a linear-by-linear association trend analysis, and Spearman's correlation test were performed as statistical analyses. RESULTS: A total of 2177 subjects from all prefectures in Japan were included (average 42.2 years old). The Locomo25 and Stand-Up test scores in female participants and the Two-Step test scores in male participants showed age-dependent deterioration. In the overall population, the incidence of Locomo stage 1 and 2, as evaluated by the Locomo25, Stand-Up test or Two-Step test, was 30.2% and 29.2%, respectively. In subjects without locomotive syndrome (40.5%), LSRT scores showed age-dependent deterioration in both sexes. Locomotive syndrome in participants with a body mass index (BMI) of ≥25 kg/m2 was more frequent than in those with a BMI of <25 kg/m2 (age- and gender-adjusted odds ratio [OR] 1.344 [95% confidence interval {CI} 1.03-1.75, p = 0.027]). Locomotive syndrome in participants with an exercise habit was less frequent than in those without an exercise habit (age- and gender-adjusted OR 0.499 [95% CI 0.33-0.755, p < 0.0001]). CONCLUSIONS: The Locomonitor app, a newly developed remote platform, revealed that approximately 20%-30% of Japanese individuals 20-69 years old in the general population met the definition of locomotive syndrome. Locomotive syndrome in participants with obesity was more frequent than those without obesity, while locomotive syndrome in participants with an exercise habit was less frequent than those without an exercise habit.

Fibulin-7 is overexpressed in glioblastomas and modulates glioblastoma neovascularization through interaction with angiopoietin-1.

Glioblastoma (GBM) is pathologically characterized by highly malignant neoplastic cells, focal necrosis and aberrant blood vessels composed of disorganized endothelial cells and pericytes. The recent cancer microarray database revealed upregulation of fibulin-7 (Fbln7), a member of the fibulin family, but provided no information on the tissue localization or biological function. In the present study, we demonstrated that Fbln7 is markedly overexpressed by the GBM tissue among astrocytic tumors, and immunolocalized mainly to endothelial cells and pericytes of the glomeruloid and hypertrophied microvessels. The production of Fbln7 by endothelial cells and pericytes was confirmed in cultured human umbilical vein endothelial cells (HUVEC) and human brain vascular pericytes (HBVP) and vascular endothelial growth factor (VEGF) stimulated the Fbln7 expression in HUVEC. Fbln7 bound to angiopoietin-1, but not angiopoietin-2 or Tie2 receptor, through interaction between the N-terminal portions of Fbln7 and angiopoietin-1, and it blocked phosphorylation of Tie2 receptor in HUVEC. In a coculture assay using HUVEC and HBVP, multilayered and irregular-shaped tube-like structures of HUVEC were induced by treatment with a high concentration of VEGF. This was accompanied by Fbln7 overproduction by HUVEC and angiopoietin-1 expression by HBVP. The production of aberrant VEGF-induced tube-like structures was attenuated by treatment with antibody or synthetic peptides specific to the Fbln7 N-terminal domain or knockdown of Fbln7. These data demonstrate that Fbln7 is overexpressed by endothelial cells and pericytes of the abnormal microvessels in GBM, and suggest that Fbln7 may contribute to the aberrant vessel formation by modulation of the angiopoietin-1/angiopoietin-2-Tie2 axis.

Extracellular Protein Fibulin-7 and Its C-Terminal Fragment Have In Vivo Antiangiogenic Activity.

Angiogenesis is crucial for tissue development and homeostasis; however, excessive angiogenesis can lead to diseases, including arthritis and cancer metastasis. Some antiangiogenic drugs are available, but side effects remain problematic. Thus, alternative angiogenesis inhibition strategies are needed. Fibulin-7 (Fbln7) is a newly discovered member of the fibulin protein family, a group of cell-secreted glycoproteins, that functions as a cell adhesion molecule and interacts with other extracellular matrix (ECM) proteins as well as cell receptors. We previously showed that a recombinant C-terminal Fbln7 fragment (Fbln7-C) inhibits tube formation by human umbilical vein endothelial cells (HUVECs) in vitro. In the present study, we examined the in vivo antiangiogenic activity of recombinant full-length Fbln7 (Fbln7-FL) and Fbln7-C proteins using a rat corneal angiogenesis model. We found that both Fbln7-FL and Fbln7-C inhibited neovascularization. Fbln7-C bound to vascular endothelial growth factor receptor 2 (VEGFR2), inhibiting VEGFR2 and ERK phosphorylation and resulting in reduced HUVEC motility. HUVEC attachment to Fbln7-C occurred through an interaction with integrin α5ß1 and regulated changes in cellular morphology. These results suggest that Fbln7-C action may target neovascularization by altering cell/ECM associations. Therefore, Fbln7-C could have potential as a therapeutic agent for diseases associated with angiogenesis.

Altered expression of laminin alpha1 in aganglionic colon of endothelin receptor-B null mouse model of Hirschsprung's disease.

PURPOSE: Laminin, an extracellular matrix molecule, is essential for normal development of the nervous system. The alpha1 subunit of laminin-1 (LAMA1) has been reported to promote neurites and outgrowth and is expressed only during embryogenesis. Previously, we developed a Sox10 transgenic version of the Endothelin receptor-B (Ednrb) mouse to visualize Enteric neural crest-derived cell (ENCC)s with a green fluorescent protein, Venus. We designed this study to investigate the expression of LAMA1 using Sox10-VENUS mice gut. METHODS: We harvested the gut on days 13.5 (E13.5) and 15.5 (E15.5) of gestation. Sox10-VENUS+/Ednrb -/- mice (n = 8) were compared with Sox10-VENUS+/Ednrb +/+ mice (n = 8) as controls. Gene expression of LAMA1 was analysed by real-time RT-PCR. Fluorescent immunohistochemistry was performed to assess protein distribution. RESULTS: The relative mRNA expression levels of LAMA1 were significantly increased in HD in the proximal and distal colon on E15.5 compared to controls (p < 0.05), whereas there were no significant differences on E13.5. LAMA1 was expressed in the serosa, submucosa and basal lamina in the gut, and was markedly increased in the proximal and distal colon of HD on E15.5. CONCLUSIONS: Altered LAMA1 expression in the aganglionic region may contribute to impaired ENCC migration, resulting in HD. These data could help in understanding the pathophysiologic interactions between LAMA1 and ENCC migration.

Perlecan is required for the chondrogenic differentiation of synovial mesenchymal cells through regulation of Sox9 gene expression.

We previously reported that perlecan, a heparan-sulfate proteoglycan (Hspg2), expressed in the synovium at the cartilage-synovial junction, is required for osteophyte formation in knee osteoarthritis. To examine the mechanism underlying this process, we examined the role of perlecan in the proliferation and differentiation of synovial mesenchymal cells (SMCs), using a recently established mouse synovial cell culture method. Primary SMCs isolated from Hspg2-/- -Tg (Hspg2-/- ;Col2a1-Hspg2Tg/- ) mice, in which the perlecan-knockout was rescued from perinatal lethality, lack perlecan. The chondrogenic-, osteogenic-, and adipogenic-potentials were examined in the Hspg2-/- -Tg SMCs compared to the control SMCs prepared from wild-type Hspg2+/+ -Tg (Hspg2+/+ ;Col2a1-Hspg2Tg/- ) littermates. In a culture condition permitting proliferation, both control and Hspg2-/- -Tg SMCs showed similar rates of proliferation and expression of cell surface markers. However, in micromass cultures, the cartilage matrix production and Sox9 and Col2a1 mRNA levels were significantly reduced in Hspg2-/- -Tg SMCs, compared with control SMCs. The reduced level of Sox9 mRNA was restored by the supplementation with exogenous perlecan protein. There was no difference in osteogenic differentiation between the control and Hspg2-/- -Tg SMCs, as measured by the levels of Runx2 and Col1a1 mRNA. The adipogenic induction and PPARγ mRNA levels were significantly reduced in Hspg2-/- -Tg SMCs compared to control SMCs. The reduction of PPARγ mRNA levels in Hspg2-/- -Tg SMCs was restored by supplementation of perlecan. Perlecan is required for the chondrogenic and adipogenic differentiation from SMCs via its regulation of the Sox9 and PPARγ gene expression, but not for osteogenic differentiation via Runx2. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:837-846, 2017.

Perlecan inhibits autophagy to maintain muscle homeostasis in mouse soleus muscle.

The autophagy-lysosome system is essential for muscle protein synthesis and degradation equilibrium, and its dysfunction has been linked to various muscle disorders. It has been reported that a diverse collection of extracellular matrix constituents, including decorin, collagen VI, laminin α2, endorepellin, and endostatin, can modulate autophagic signaling pathways. However, the association between autophagy and perlecan in muscle homeostasis remains unclear. The mechanical unloading of perlecan-deficient soleus muscles resulted in significantly decreased wet weights and cross-section fiber area compared with those of control mice. We found that perlecan deficiency in slow-twitch soleus muscles enhanced autophagic activity. This was accompanied by a decrease in autophagic substrates, such as p62, and an increase in LC3II levels. Furthermore, perlecan deficiency caused a reduction in the phosphorylation levels of p70S6k and Akt and increased the phosphorylation of AMPKα. Our findings suggested that perlecan inhibits the autophagic process through the activation of the mTORC1 pathway. This autophagic response may be a novel target for enhancing the efficacy of skeletal muscle atrophy treatment.

A missense mutation in domain III in HSPG2 in Schwartz-Jampel syndrome compromises secretion of perlecan into the extracellular space.

Schwartz-Jampel syndrome (SJS) type 1 is characterized by short stature, myotonia, and chondrodysplasia, and is caused by partial loss-of-function mutations in HSPG2 encoding perlecan. Six missense mutations have been reported in SJS to date and only one has been characterized using a recombinant protein. We report an 11-year-old Japanese boy with SJS, who shows "rigid" walking with less flexion of knees/ankles and protruded mouth. His intelligence is normal. We identified by whole genome resequencing a heterozygous missense p.Leu1088Pro in domain III-2 and a heterozygous nonsense p.Gln3061Ter in domain IV of perlecan. Expression studies revealed that p.Leu1088Pro markedly reduces the cellular expression of domain III-2 and almost nullifies its secretion into the culture medium. As five of the seven missense mutations in SJS affect domain III of perlecan, domain III is likely to be essential for secretion of perlecan into the extracellular space.

A C-terminal fragment of fibulin-7 interacts with endothelial cells and inhibits their tube formation in culture.

We have previously demonstrated that fibulin-7 (Fbln7) is expressed in teeth by pre-odontoblast and odontoblast cells, localized in the basement membrane and dentin matrices, and is an adhesion molecule for dental mesenchyme cells and odontoblasts. Fbln7 is also expressed in blood vessels by endothelial cells. In this report, we show that a recombinant C-terminal Fbln7 fragment (Fbln7-C) bound to Human Umbilical Vein Endothelial Cells (HUVECs) but did not promote cell spreading and actin stress fiber formation. Fbln7-C binding to HUVECs induced integrin clustering at cell adhesion sites with other focal adhesion molecules, and sustained activation of FAK, p130Cas, and Rac1. In addition, RhoA activation was inhibited, thereby preventing HUVEC spreading. As endothelial cell spreading is an important step for angiogenesis, we examined the effect of Fbln7-C on angiogenesis using in vitro assays for endothelial cell tube formation and vessel sprouting from aortic rings. We found that Fbln7-C inhibited the HUVEC tube formation and the vessel sprouting in aortic ring assays. Our findings suggest potential anti-angiogenic activity of the Fbln7 C-terminal region.

Teneurin-4 promotes cellular protrusion formation and neurite outgrowth through focal adhesion kinase signaling.

Teneurin-4 (Ten-4), a transmembrane protein, is highly expressed in the central nervous system; however, its cellular and molecular function in neuronal differentiation remains unknown. In this study, we aimed to elucidate the function of Ten-4 in neurite outgrowth. Ten-4 expression was induced during neurite outgrowth of the neuroblastoma cell line Neuro-2a. Ten-4 protein was localized at the neurite growth cones. Knockdown of Ten-4 expression in Neuro-2a cells decreased the formation of the filopodia-like protrusions and the length of individual neurites. Conversely, overexpression of Ten-4 promoted filopodia-like protrusion formation. In addition, knockdown and overexpression of Ten-4 reduced and elevated the activation of focal adhesion kinase (FAK) and Rho-family small GTPases, Cdc42 and Rac1, key molecules for the membranous protrusion formation downstream of FAK, respectively. Inhibition of the activation of FAK and neural Wiskott-Aldrich syndrome protein (N-WASP), which is a downstream regulator of FAK and Cdc42, blocked protrusion formation by Ten-4 overexpression. Further, Ten-4 colocalized with phosphorylated FAK in the filopodia-like protrusion regions. Together, our findings show that Ten-4 is a novel positive regulator of cellular protrusion formation and neurite outgrowth through the FAK signaling pathway.

Laminin-1 promotes enteric nervous system development in mouse embryo.

BACKGROUND/AIM: Neuronal development is regulated by extracellular environmental factors including nerve growth factor (NGF) and laminin. We have previously demonstrated that laminin-1 promotes neurite outgrowth of dorsal root ganglion cells by modulating NGF and integrin signaling. However, information about their effects on the enteric nervous system (ENS) is limited. Recently, we succeeded in visualizing enteric neural crest-derived cell (ENCC) migration using SOX10-Venus transgenic mice, in which ENCC are labeled with a green fluorescent protein, Venus. In this study, we examine the effects of NGF and laminin-1 in ENCC migration using SOX10-Venus mice gut. METHODS: Pregnant SOX10-Venus mice were killed on day 12.5 of gestation. The colorectum was dissected from embryos (n = 10) and placed in culture medium including NGF with or without laminin-1 for 12 h. Extension rates of ENCC migration, colorectum and ENCC migration per colorectum were calculated. RESULTS: Venus positive-ENCC extension rate was significantly higher in the laminin group (n = 5) compared to control (n = 5), 22.84 and 13.96 %, respectively (p < 0.05). The extension rate of the colorectum was not significantly different between the two groups. CONCLUSIONS: Our results suggest that laminin promotes ENCC migration in mice. This technique allowed us to visualize the effects of extracellular molecules on ENCC migration and it potentially provides us with an insight into the pathophysiology of developmental disorders of the ENS, such as Hirschsprung's disease.

Isolation and characterization of multipotential mesenchymal cells from the mouse synovium.

The human synovium contains mesenchymal stem cells (MSCs), which are multipotential non-hematopoietic progenitor cells that can differentiate into a variety of mesenchymal lineages and they may therefore be a candidate cell source for tissue repair. However, the molecular mechanisms by which this can occur are still largely unknown. Mouse primary cell culture enables us to investigate the molecular mechanisms underlying various phenomena because it allows for relatively easy gene manipulation, which is indispensable for the molecular analysis. However, mouse synovial mesenchymal cells (SMCs) have not been established, although rabbit, cow, and rat SMCs are available, in addition to human MSCs. The aim of this study was to establish methods to harvest the synovium and to isolate and culture primary SMCs from mice. As the mouse SMCs were not able to be harvested and isolated using the same protocol for human, rat and rabbit SMCs, the protocol for humans was modified for SMCs from the Balb/c mouse knee joint. The mouse SMCs obtained showed superior proliferative potential, growth kinetics and colony formation compared to cells derived from muscle and bone marrow. They expressed PDGFRá and Sca-1 detected by flow cytometry, and showed an osteogenic, adipogenic and chondrogenic potential similar or superior to the cells derived from muscle and bone marrow by demonstrating in vitro osteogenesis, adipogenesis and chondrogenesis. In conclusion, we established a primary mouse synovial cell culture method. The cells derived from the mouse synovium demonstrated both the ability to proliferate and multipotentiality similar or superior to the cells derived from muscle and bone marrow.

Teneurin-4 is a novel regulator of oligodendrocyte differentiation and myelination of small-diameter axons in the CNS.

Myelination is essential for proper functioning of the CNS. In this study, we have identified a mouse mutation, designated furue, which causes tremors and hypomyelination in the CNS, particularly in the spinal cord, but not in the sciatic nerve of the PNS. In the spinal cord of the furue mice, myelination of small-diameter axons was dramatically reduced, and differentiation of oligodendrocytes, the myelin-forming cells in the CNS, was inhibited. We subsequently found that the furue mutation was associated with a transgene insertion into the teneurin-4 (Ten-4, Ten-m4/Odz4) gene, encoding a transmembrane protein of unknown function. Ten-4 was strongly expressed in the spinal cord of wild-type mice and was induced during normal oligodendrocyte differentiation. In contrast, in the furue mice, the expression of Ten-4 was absent. Differentiation and cellular process formation of oligodendrocytes were inhibited in primary cell culture from the furue mice. Cell differentiation and process formation were also inhibited in the oligodendrocyte progenitor cell line CG-4 after suppression of Ten-4 expression by shRNA. Furthermore, Ten-4 positively regulated focal adhesion kinase, an essential signaling molecule for oligodendrocyte process formation and myelination of small-diameter axons. These findings suggest that Ten-4 is a novel regulator of oligodendrocyte differentiation and that it plays a critical role in the myelination of small-diameter axons in the CNS.

Perlecan modulates VEGF signaling and is essential for vascularization in endochondral bone formation.

Perlecan (Hspg2) is a heparan sulfate proteoglycan expressed in basement membranes and cartilage. Perlecan deficiency (Hspg2(-/-)) in mice and humans causes lethal chondrodysplasia, which indicates that perlecan is essential for cartilage development. However, the function of perlecan in endochondral ossification is not clear. Here, we report the critical role of perlecan in VEGF signaling and angiogenesis in growth plate formation. The Hspg2(-/-) growth plate was significantly wider but shorter due to severely impaired endochondral bone formation. Hypertrophic chondrocytes were differentiated in Hspg2(-/-) growth plates; however, removal of the hypertrophic matrix and calcified cartilage was inhibited. Although the expression of MMP-13, CTGF, and VEGFA was significantly upregulated in Hspg2(-/-) growth plates, vascular invasion into the hypertrophic zone was impaired, which resulted in an almost complete lack of bone marrow and trabecular bone. We demonstrated that cartilage perlecan promoted activation of VEGF/VEGFR by binding to the VEGFR of endothelial cells. Expression of the perlecan transgene specific to the cartilage of Hspg2(-/-) mice rescued their perinatal lethality and growth plate abnormalities, and vascularization into the growth plate was restored, indicating that perlecan in the growth plate, not in endothelial cells, is critical in this process. These results suggest that perlecan in cartilage is required for activating VEGFR signaling of endothelial cells for vascular invasion and for osteoblast migration into the growth plate. Thus, perlecan in cartilage plays a critical role in endochondral bone formation by promoting angiogenesis essential for cartilage matrix remodeling and subsequent endochondral bone formation.

Laminin α1 is essential for mouse cerebellar development.

Laminin α1 (Lama1), which is a subunit of laminin-1 (laminin-111), a heterotrimeric ECM protein, is essential for embryonic development and promotes neurite outgrowth in culture. Because the deletion of Lama1 causes lethality at early embryonic stages in mice, the in vivo role of Lama1 in neural development and functions has not yet been possible to determine. In this study, we generated conditional Lama1 knockout (Lama1(CKO)) mice in the epiblast lineage using Sox2-Cre mice. These Lama1(CKO) mice survived, but displayed behavioral disorders and impaired formation of the cerebellum. Deficiency of Lama1 in the pial basement membrane of the meninges resulted in defects in the conformation of the meninges. During cerebellar development, Lama1 deficiency also caused a decrease in the proliferation and migration of granule cell precursors, disorganization of Bergmann glial fibers and endfeet, and a transient reduction in the activity of Akt. A marked reduction in numbers of dendritic processes in Purkinje cells was observed in Lama1(CKO) mice. Together, these results indicate that Lama1 is required for cerebellar development and functions.

Laminin-121--recombinant expression and interactions with integrins.

Laminin-121, previously referred as to laminin-3, was expressed recombinantly in human embryonic kidney (HEK) 293 cells by triple transfection of full-length cDNAs encoding mouse laminin α1, ß2 and γ1 chains. The recombinant laminin-121 was purified using Heparin-Sepharose followed by molecular sieve chromatography and shown to be correctly folded by electron microscopy and circular dichroism (CD). The CD spectra of recombinant laminin-121 were very similar to those of laminin-111 isolated from Engelbreth-Holm-Swarm tumor (EHS-laminin) but its T(m) value was smaller than EHS-laminin and recombinant lamnin-111 suggesting that the replacement of the ß chain reduced the stability of the coiled-coil structure of laminin-121. Its binding to integrins was compared with EHS-laminin, laminin-3A32 purified from murine epidermal cell line and recombinantly expressed laminins-111, -211 and -221. Laminin-121 showed the highest affinity to α6ß1 and α7ß1 integrins and furthermore, laminin-121 most effectively supported neurite outgrowth. Together, this suggests that the ß2 laminins have higher affinity for integrins than the ß1 laminins.