TMEM2 is a bona fide hyaluronidase possessing intrinsic catalytic activity.

Transmembrane protein 2 (TMEM2) was originally identified as a membrane-anchored protein of unknown function. We previously demonstrated that TMEM2 can degrade hyaluronan (HA). Furthermore, we showed that induced global knockout of Tmem2 in adult mice results in rapid accumulation of incompletely degraded HA in bodily fluids and organs, supporting the identity of TMEM2 as a cell surface hyaluronidase. In spite of these advances, no direct evidence has been presented to demonstrate the intrinsic hyaluronidase activity of TMEM2. Here, we directly establish the catalytic activity of TMEM2. The ectodomain of TMEM2 (TMEM2ECD) was expressed as a His-tagged soluble protein and purified by affinity and size-exclusion chromatography. Both human and mouse TMEM2ECD robustly degrade fluorescein-labeled HA into 5 to 10 kDa fragments. TMEM2ECD exhibits this HA-degrading activity irrespective of the species of TMEM2 origin and the position of epitope tag insertion. The HA-degrading activity of TMEM2ECD is more potent than that of HYAL2, a hyaluronidase which, like TMEM2, has been implicated in cell surface HA degradation. Finally, we show that TMEM2ECD can degrade not only fluorescein-labeled HA but also native high-molecular weight HA. In addition to these core findings, our study reveals hitherto unrecognized confounding factors, such as the quality of reagents and the choice of assay systems, that could lead to erroneous conclusions regarding the catalytic activity of TMEM2. In conclusion, our results demonstrate that TMEM2 is a legitimate functional hyaluronidase. Our findings also raise cautions regarding the choice of reagents and methods for performing degradation assays for hyaluronidases.

The cell surface hyaluronidase TMEM2 plays an essential role in mouse neural crest cell development and survival.

Hyaluronan (HA) is a major extracellular matrix component whose tissue levels are dynamically regulated during embryonic development. Although the synthesis of HA has been shown to exert a substantial influence on embryonic morphogenesis, the functional importance of the catabolic aspect of HA turnover is poorly understood. Here, we demonstrate that the transmembrane hyaluronidase TMEM2 plays an essential role in neural crest development and the morphogenesis of neural crest derivatives, as evidenced by the presence of severe craniofacial abnormalities in Wnt1-Cre-mediated Tmem2 knockout (Tmem2CKO) mice. Neural crest cells (NCCs) are a migratory population of cells that gives rise to diverse cell lineages, including the craniofacial complex, the peripheral nervous system, and part of the heart. Analysis of Tmem2 expression during NCC formation and migration reveals that Tmem2 is expressed at the site of NCC delamination and in emigrating Sox9-positive NCCs. In Tmem2CKO embryos, the number of NCCs emigrating from the neural tube is greatly reduced. Furthermore, linage tracing reveals that the number of NCCs traversing the ventral migration pathway and the number of post-migratory neural crest derivatives are both significantly reduced in a Tmem2CKO background. In vitro studies using Tmem2-depleted mouse O9-1 neural crest cells demonstrate that Tmem2 expression is essential for the ability of these cells to form focal adhesions on and to migrate into HA-containing substrates. Additionally, we show that Tmem2-deficient NCCs exhibit increased apoptotic cell death in NCC-derived tissues, an observation that is corroborated by in vitro experiments using O9-1 cells. Collectively, our data demonstrate that TMEM2-mediated HA degradation plays an essential role in normal neural crest development. This study reveals the hitherto unrecognized functional importance of HA degradation in embryonic development and highlights the pivotal role of Tmem2 in the developmental process.

The cell surface hyaluronidase TMEM2 is essential for systemic hyaluronan catabolism and turnover.

As a major component of the extracellular matrix, hyaluronan (HA) plays an important role in defining the biochemical and biophysical properties of tissues. In light of the extremely rapid turnover of HA and the impact of this turnover on HA biology, elucidating the molecular mechanisms underlying HA catabolism is key to understanding the in vivo functions of this unique polysaccharide. Here, we show that TMEM2, a recently identified cell surface hyaluronidase, plays an essential role in systemic HA turnover. Employing induced global Tmem2 knockout mice (Tmem2iKO), we determined the effects of Tmem2 ablation not only on the accumulation of HA in bodily fluids and organs, but also on the process of HA degradation in vivo. Within 3 weeks of tamoxifen-induced Tmem2 ablation, Tmem2iKO mice exhibit pronounced accumulation of HA in circulating blood and various organs, reaching levels as high as 40-fold above levels observed in control mice. Experiments using lymphatic and vascular injection of fluorescent HA tracers demonstrate that ongoing HA degradation in the lymphatic system and the liver is significantly impaired in Tmem2iKO mice. We also show that Tmem2 is strongly expressed in endothelial cells in the subcapsular sinus of lymph nodes and in the liver sinusoid, two primary sites implicated in systemic HA turnover. Our results establish TMEM2 as a physiologically relevant hyaluronidase with an essential role in systemic HA catabolism in vivo, acting primarily on the surface of endothelial cells in the lymph nodes and liver.

Heparan sulfate promotes differentiation of white adipocytes to maintain insulin sensitivity and glucose homeostasis.

Heparan sulfate (HS), a highly sulfated linear polysaccharide, is involved in diverse biological functions in various tissues. Although previous studies have suggested a possible contribution of HS to the differentiation of white adipocytes, there has been no direct evidence supporting this. Here, we inhibited the synthesis of HS chains in 3T3-L1 cells using CRISPR-Cas9 technology, resulting in impaired differentiation of adipocytes with attenuated bone morphogenetic protein 4 (BMP4)-fibroblast growth factor 1 (FGF1) signaling pathways. HS reduction resulted in reduced glucose uptake and decreased insulin-dependent intracellular signaling. We then made heterozygous mutant mice for the Ext1 gene, which encodes an enzyme essential for the HS biosynthesis, specifically in the visceral white adipose tissue (Fabp4-Cre+::Ext1flox/WT mice, hereafter called Ext1Δ/WT) to confirm the importance of HS in vivo. The expression levels of transcription factors that control adipocyte differentiation, such as peroxisome proliferator-activated receptor gamma, were reduced in Ext1Δ/WT adipocytes, which contained smaller, unilocular lipid droplets, reduced levels of enzymes involved in lipid synthesis, and altered expression of BMP4-FGF1 signaling molecules. Furthermore, we examined the impact of HS reduction in visceral white adipose tissue on systemic glucose homeostasis. We observed that Ext1Δ/WT mice showed glucose intolerance because of insulin resistance. Our results demonstrate that HS plays a crucial role in the differentiation of white adipocytes through BMP4-FGF1 signaling pathways, thereby contributing to insulin sensitivity and glucose homeostasis.

The cell surface hyaluronidase TMEM2 regulates cell adhesion and migration via degradation of hyaluronan at focal adhesion sites.

The extracellular matrix (ECM) plays an important role in maintaining tissue homeostasis and poses a significant physical barrier to in vivo cell migration. Accordingly, as a means of enhancing tissue invasion, tumor cells use matrix metalloproteinases to degrade ECM proteins. However, the in vivo ECM is comprised not only of proteins but also of a variety of nonprotein components. Hyaluronan (HA), one of the most abundant nonprotein components of the interstitial ECM, forms a gel-like antiadhesive barrier that is impenetrable to particulate matter and cells. Mechanisms by which tumor cells penetrate the HA barrier have not been addressed. Here, we demonstrate that transmembrane protein 2 (TMEM2), the only known transmembrane hyaluronidase, is the predominant mediator of contact-dependent HA degradation and subsequent integrin-mediated cell-substrate adhesion. We show that a variety of tumor cells are able to eliminate substrate-bound HA in a tightly localized pattern corresponding to the distribution of focal adhesions (FAs) and stress fibers. This FA-targeted HA degradation is mediated by TMEM2, which itself is localized at site of FAs. TMEM2 depletion inhibits the ability of tumor cells to attach and migrate in an HA-rich environment. Importantly, TMEM2 directly binds at least two integrins via interaction between extracellular domains. Our findings demonstrate a critical role for TMEM2-mediated HA degradation in the adhesion and migration of cells on HA-rich ECM substrates and provide novel insight into the early phase of FA formation.

Integrity of White Matter is Compromised in Mice with Hyaluronan Deficiency.

Brain white matter is the means of efficient signal propagation in brain and its dysfunction is associated with many neurological disorders. We studied the effect of hyaluronan deficiency on the integrity of myelin in murine corpus callosum. Conditional knockout mice lacking the hyaluronan synthase 2 were compared with control mice. Ultrastructural analysis by electron microscopy revealed a higher proportion of myelin lamellae intruding into axons of knockout mice, along with significantly slimmer axons (excluding myelin sheath thickness), lower g-ratios, and frequent loosening of the myelin wrappings, even though the myelin thickness was similar across the genotypes. Analysis of extracellular diffusion of a small marker molecule tetramethylammonium (74 MW) in brain slices prepared from corpus callosum showed that the extracellular space volume increased significantly in the knockout animals. Despite this vastly enlarged volume, extracellular diffusion rates were significantly reduced, indicating that the compromised myelin wrappings expose more complex geometric structure than the healthy ones. This finding was confirmed in vivo by diffusion-weighted magnetic resonance imaging. Magnetic resonance spectroscopy suggested that water was released from within the myelin sheaths. Our results indicate that hyaluronan is essential for the correct formation of tight myelin wrappings around the axons in white matter.

TMEM2: A missing link in hyaluronan catabolism identified?

Hyaluronan (HA) is a glycosaminoglycan (GAG) composed of repeating disaccharide units of glucuronic acid and N-acetylglucosamine. HA is an extremely long, unbranched polymer, which often exceeds 106 Da and sometimes reaches 107 Da. A feature that epitomizes HA is its rapid turnover; one-third of the total body HA is turned over daily. The current model of HA catabolism postulates that high-molecular weight HA in the extracellular space is first cleaved into smaller fragments by a hyaluronidase(s) that resides at the cell surface, followed by internalization of fragments and their degradation into monosaccharides in lysosomes. Over the last decade, considerable research has shown that the HYAL family of hyaluronidases plays significant roles in HA catabolism. Nonetheless, the identity of a hyaluronidase responsible for the initial step of HA cleavage on the cell surface remains elusive, as biochemical and enzymological properties of HYAL proteins are not entirely consistent with those expected of cell surface hyaluronidases. Recent identification of transmembrane 2 (TMEM2) as a cell surface protein that possesses potent hyaluronidase activity suggests that it may be the "missing" cell surface hyaluronidase, and that novel models of HA catabolism should include this protein.

Osteoblastic heparan sulfate regulates osteoprotegerin function and bone mass.

Bone remodeling is a highly coordinated process involving bone formation and resorption, and imbalance of this process results in osteoporosis. It has long been recognized that long-term heparin therapy often causes osteoporosis, suggesting that heparan sulfate (HS), the physiological counterpart of heparin, is somehow involved in bone mass regulation. The role of endogenous HS in adult bone, however, remains unclear. To determine the role of HS in bone homeostasis, we conditionally ablated Ext1, which encodes an essential glycosyltransferase for HS biosynthesis, in osteoblasts. Resultant conditional mutant mice developed severe osteopenia. Surprisingly, this phenotype is not due to impairment in bone formation but to enhancement of bone resorption. We show that osteoprotegerin (OPG), which is known as a soluble decoy receptor for RANKL, needs to be associated with the osteoblast surface in order to efficiently inhibit RANKL/RANK signaling and that HS serves as a cell surface binding partner for OPG in this context. We also show that bone mineral density is reduced in patients with multiple hereditary exostoses, a genetic bone disorder caused by heterozygous mutations of Ext1, suggesting that the mechanism revealed in this study may be relevant to low bone mass conditions in humans.

Palovarotene Inhibits Osteochondroma Formation in a Mouse Model of Multiple Hereditary Exostoses.

Multiple hereditary exostoses (MHE), also known as multiple osteochondromas (MO), is an autosomal dominant disorder characterized by the development of multiple cartilage-capped bone tumors (osteochondromas). The large majority of patients with MHE carry loss-of-function mutations in the EXT1 or EXT2 gene, which encodes a glycosyltransferase essential for heparan sulfate (HS) biosynthesis. Increasing evidence suggests that enhanced bone morphogenetic protein (BMP) signaling resulting from loss of HS expression plays a role in osteochondroma formation in MHE. Palovarotene (PVO) is a retinoic acid receptor γ selective agonist, which is being investigated as a potential drug for fibrodysplasia ossificans progressiva (FOP), another genetic bone disorder with features that overlap with those of MHE. Here we show that PVO inhibits osteochondroma formation in the Fsp1Cre ;Ext1flox/flox model of MHE. Four-week daily treatment with PVO starting at postnatal day (P) 14 reduced the number of osteochondromas that develop in these mice by up to 91% in a dose-dependent manner. An inhibition of long bone growth observed in animals treated from P14 was almost entirely abrogated by delaying the initiation of treatment to P21. We also found that PVO attenuates BMP signaling in Fsp1Cre ;Ext1flox/flox mice and that aberrant chondrogenic fate determination of Ext1-deficient perichondrial progenitor cells in these mice is restored by PVO. Together, the present data support further preclinical and clinical investigations of PVO as a potential therapeutic agent for MHE. © 2017 American Society for Bone and Mineral Research.

Aberrant perichondrial BMP signaling mediates multiple osteochondromagenesis in mice.

Multiple hereditary exostoses (MHE) is characterized by the development of numerous benign bony tumors (osteochondromas). Although it has been well established that MHE is caused by mutations in EXT1 and EXT2, which encode glycosyltransferase essential for heparan sulfate (HS) biosynthesis, the cellular origin and molecular mechanisms of MHE remain elusive. Here, we show that in Ext1 mutant mice, osteochondromas develop from mesenchymal stem cell-like progenitor cells residing in the perichondrium, and we show that enhanced BMP signaling in these cells is the primary signaling defect that leads to osteochondromagenesis. We demonstrate that progenitor cells in the perichondrium, including those in the groove of Ranvier, highly express HS and that Ext1 ablation targeted to the perichondrium results in the development of osteochondromas. Ext1-deficient perichondrial progenitor cells show enhanced BMP signaling and increased chondrogenic differentiation both in vitro and in vivo. Consistent with the functional role for enhanced BMP signaling in osteochondromagenesis, administration of the small molecule BMP inhibitor LDN-193189 suppresses osteochondroma formation in two MHE mouse models. Together, our results demonstrate a role for enhanced perichondrial BMP signaling in osteochondromagenesis in mice, and they suggest the possibility of pharmacological treatment of MHE with BMP inhibitors.

A mammalian homolog of the zebrafish transmembrane protein 2 (TMEM2) is the long-sought-after cell-surface hyaluronidase.

Hyaluronan (HA) is an extremely large polysaccharide (glycosaminoglycan) involved in many cellular functions. HA catabolism is thought to involve the initial cleavage of extracellular high-molecular-weight (HMW) HA into intermediate-size HA by an extracellular or cell-surface hyaluronidase, internalization of intermediate-size HA, and complete degradation into monosaccharides in lysosomes. Despite considerable research, the identity of the hyaluronidase responsible for the initial HA cleavage in the extracellular space remains elusive. HYAL1 and HYAL2 have properties more consistent with lysosomal hyaluronidases, whereas CEMIP/KIAA1199, a recently identified HA-binding molecule that has HA-degrading activity, requires the participation of the clathrin-coated pit pathway of live cells for HA degradation. Here we show that transmembrane protein 2 (TMEM2), a mammalian homolog of a protein playing a role in zebrafish endocardial cushion development, is a cell-surface hyaluronidase. Live immunostaining and surface biotinylation assays confirmed that mouse TMEM2 is expressed on the cell surface in a type II transmembrane topology. TMEM2 degraded HMW-HA into ∼5-kDa fragments but did not cleave chondroitin sulfate or dermatan sulfate, indicating its specificity to HA. The hyaluronidase activity of TMEM2 was Ca2+-dependent; the enzyme's pH optimum is around 6-7, and unlike CEMIP/KIAA1199, TMEM2 does not require the participation of live cells for its hyaluronidase activity. Moreover, TMEM2-expressing cells could eliminate HA immobilized on a glass surface in a contact-dependent manner. Together, these data suggest that TMEM2 is the long-sought-after hyaluronidase that cleaves extracellular HMW-HA into intermediate-size fragments before internalization and degradation in the lysosome.

Hyaluronan deficiency due to Has3 knock-out causes altered neuronal activity and seizures via reduction in brain extracellular space.

Hyaluronan (HA), a large anionic polysaccharide (glycosaminoglycan), is a major constituent of the extracellular matrix of the adult brain. To address its function, we examined the neurophysiology of knock-out mice deficient in hyaluronan synthase (Has) genes. Here we report that these Has mutant mice are prone to epileptic seizures, and that in Has3(-/-) mice, this phenotype is likely derived from a reduction in the size of the brain extracellular space (ECS). Among the three Has knock-out models, namely Has3(-/-), Has1(-/-), and Has2(CKO), the seizures were most prevalent in Has3(-/-) mice, which also showed the greatest HA reduction in the hippocampus. Electrophysiology in Has3(-/-) brain slices demonstrated spontaneous epileptiform activity in CA1 pyramidal neurons, while histological analysis revealed an increase in cell packing in the CA1 stratum pyramidale. Imaging of the diffusion of a fluorescent marker revealed that the transit of molecules through the ECS of this layer was reduced. Quantitative analysis of ECS by the real-time iontophoretic method demonstrated that ECS volume was selectively reduced in the stratum pyramidale by ∼ 40% in Has3(-/-) mice. Finally, osmotic manipulation experiments in brain slices from Has3(-/-) and wild-type mice provided evidence for a causal link between ECS volume and epileptiform activity. Our results provide the first direct evidence for the physiological role of HA in the regulation of ECS volume, and suggest that HA-based preservation of ECS volume may offer a novel avenue for development of antiepileptogenic treatments.

Autism-like socio-communicative deficits and stereotypies in mice lacking heparan sulfate.

Heparan sulfate regulates diverse cell-surface signaling events, and its roles in the development of the nervous system recently have been increasingly uncovered by studies using genetic models carrying mutations of genes encoding enzymes for its synthesis. On the other hand, the role of heparan sulfate in the physiological function of the adult brain has been poorly characterized, despite several pieces of evidence suggesting its role in the regulation of synaptic function. To address this issue, we eliminated heparan sulfate from postnatal neurons by conditionally inactivating Ext1, the gene encoding an enzyme essential for heparan sulfate synthesis. Resultant conditional mutant mice show no detectable morphological defects in the cytoarchitecture of the brain. Remarkably, these mutant mice recapitulate almost the full range of autistic symptoms, including impairments in social interaction, expression of stereotyped, repetitive behavior, and impairments in ultrasonic vocalization, as well as some associated features. Mapping of neuronal activation by c-Fos immunohistochemistry demonstrates that neuronal activation in response to social stimulation is attenuated in the amygdala in these mice. Electrophysiology in amygdala pyramidal neurons shows an attenuation of excitatory synaptic transmission, presumably because of the reduction in the level of synaptically localized AMPA-type glutamate receptors. Our results demonstrate that heparan sulfate is critical for normal functioning of glutamatergic synapses and that its deficiency mediates socio-communicative deficits and stereotypies characteristic for autism.

Heparan sulfate regulates intraretinal axon pathfinding by retinal ganglion cells.

PURPOSE. Heparan sulfate (HS) is abundantly expressed in the developing neural retina; however, its role in the intraretinal axon guidance of retinal ganglion cells (RGCs) remains unclear. In this study, the authors examined whether HS was essential for the axon guidance of RGCs toward the optic nerve head. METHODS. The authors conditionally ablated the gene encoding the exostosin-1 (Ext1) enzyme, using the dickkopf homolog 3 (Dkk3)-Cre transgene, which disrupted HS expression in the mouse retina during directed pathfinding by RGC axons toward the optic nerve head. In situ hybridization, immunohistochemistry, DiI tracing, binding assay, and retinal explant assays were performed to evaluate the phenotypes of the mutants and the roles of HS in intraretinal axon guidance. RESULTS. Despite no gross abnormality in RGC distribution, the mutant RGC axons exhibited severe intraretinal guidance errors, including optic nerve hypoplasia, ectopic axon penetration through the full thickness of the neural retina and into the subretinal space, and disturbance of the centrifugal projection of RGC axons toward the optic nerve head. These abnormal phenotypes shared similarities with the RGC axon misguidance caused by mutations of genes encoding Netrin-1 and Slit-1/2. Explant assays revealed that the mutant RGCs exhibited disturbed Netrin-1-dependent axon outgrowth and Slit-2-dependent repulsion. CONCLUSIONS. The present study demonstrated that RGC axon projection toward the optic nerve head requires the expression of HS in the neural retina, suggesting that HS in the retina functions as an essential modulator of Netrin-1 and Slit-mediated intraretinal RGC axon guidance.

Endoplasmic reticulum protein BI-1 modulates unfolded protein response signaling and protects against stroke and traumatic brain injury.

Bax-Inhibitor-1 (BI-1) is an evolutionarily conserved cytoprotective protein that resides in membranes of the endoplasmic reticulum (ER). BI-1's cytoprotective activity is manifested in the context of ER stress, with previous studies showing that BI-1 modulates several ER-associated functions, including Unfolded Protein Response (UPR) signaling. Here we investigated the role of BI-1 in neuroprotection by generating transgenic mice in which BI-1 was constitutively expressed from a neuronal-specific promoter. Cultured primary cortical neurons from BI-1 transgenic mouse embryos exhibited greater resistance to cell death induced by agents known to cause ER stress compared to their non-transgenic counterparts. While brain morphology and vasculature of BI-1 mice appeared to be unchanged from normal non-transgenic mice, BI-1 transgenic mice showed reduced brain lesion volumes and better performance in motoric tests, compared with non-transgenic littermates, in two models of acute brain injury: stroke caused by middle cerebral artery occlusion (MCAO) and traumatic brain injury (TBI) caused by controlled cortical impact. Furthermore, brain tissue from BI-1 transgenic mice showed reduced levels of apoptotic cells and reduced induction of markers of ER stress after brain injury, including CHOP protein expression. In summary, our findings demonstrate that enforced neuronal expression of BI-1 reduces ER stress and provides protection from acute brain injury, suggesting that strategies for enhancing BI-1 expression or activity should be considered for development of new therapies for counteracting the consequences of stroke and acute brain trauma.

Roles of heparan sulfate in mammalian brain development current views based on the findings from Ext1 conditional knockout studies.

Development of the mammalian central nervous system proceeds roughly in four major steps, namely the patterning of the neural tube, generation of neurons from neural stem cells and their migration to genetically predetermined destinations, extension of axons and dendrites toward target neurons to form neural circuits, and formation of synaptic contacts. Earlier studies on spatiotemporal expression patterns and in vitro function of heparan sulfate (HS) suggested that HS is functionally involved in various aspects of neural development. Recent studies using knockout of genes involved in HS biosynthesis have provided more physiologically relevant information as to the role of HS in mammalian neural development. This chapter reviews the current understanding of the in vivo function of HS deduced from the phenotypes of conditional Ext1 knockout mice.

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.

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.

Heparan sulfate deficiency leads to Peters anomaly in mice by disturbing neural crest TGF-beta2 signaling.

During human embryogenesis, neural crest cells migrate to the anterior chamber of the eye and then differentiate into the inner layers of the cornea, the iridocorneal angle, and the anterior portion of the iris. When proper development does not occur, this causes iridocorneal angle dysgenesis and intraocular pressure (IOP) elevation, which ultimately results in developmental glaucoma. Here, we show that heparan sulfate (HS) deficiency in mouse neural crest cells causes anterior chamber dysgenesis, including corneal endothelium defects, corneal stroma hypoplasia, and iridocorneal angle dysgenesis. These dysfunctions are phenotypes of the human developmental glaucoma, Peters anomaly. In the neural crest cells of mice embryos, disruption of the gene encoding exostosin 1 (Ext1), which is an indispensable enzyme for HS synthesis, resulted in disturbed TGF-beta2 signaling. This led to reduced phosphorylation of Smad2 and downregulated expression of forkhead box C1 (Foxc1) and paired-like homeodomain transcription factor 2 (Pitx2), transcription factors that have been identified as the causative genes for developmental glaucoma. Furthermore, impaired interactions between HS and TGF-beta2 induced developmental glaucoma, which was manifested as an IOP elevation caused by iridocorneal angle dysgenesis. These findings suggest that HS is necessary for neural crest cells to form the anterior chamber via TGF-beta2 signaling. Disturbances of HS synthesis might therefore contribute to the pathology of developmental glaucoma.

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.