Glypican-6 stimulates intestinal elongation by simultaneously regulating Hedgehog and non-canonical Wnt signaling.

We report here that Glypican-6 (GPC6)-null mice display at birth small intestines that are 75% shorter than those of normal littermates. Notably, we demonstrate that the role of GPC6 in intestinal elongation is mediated by both Hedgehog (Hh) and non-canonical Wnt signaling. Based on results from in vitro experiments, we had previously proposed that GPC6 stimulates Hh signaling by interacting with Hh and Patched1 (Ptc1), and facilitating/stabilizing their interaction. Here we provide strong support to this hypothesis by showing that GPC6 binds to Ptc1 in the mesenchymal layer of embryonic intestines. This study also provides experimental evidence that strongly suggests that GPC6 inhibits the activity of Wnt5a on the intestinal epithelium by binding to this growth factor, and reducing its release from the surrounding mesenchymal cells. Finally, we show that whereas the mesenchymal layer of GPC6-null intestines displays reduced cell proliferation and a thinner smooth muscle layer, epithelial cell differentiation is not altered in the mutant gut.

Glypican-6 promotes the growth of developing long bones by stimulating Hedgehog signaling.

Autosomal-recessive omodysplasia (OMOD1) is a genetic condition characterized by short stature, shortened limbs, and facial dysmorphism. OMOD1 is caused by loss-of-function mutations of glypican 6 (GPC6). In this study, we show that GPC6-null embryos display most of the abnormalities found in OMOD1 patients and that Hedgehog (Hh) signaling is significantly reduced in the long bones of these embryos. The Hh-stimulatory activity of GPC6 was also observed in cultured cells, where this GPC increased the binding of Hh to Patched 1 (Ptc1). Consistent with this, GPC6 interacts with Hh through its core protein and with Ptc1 through its glycosaminoglycan chains. Hh signaling is triggered at the primary cilium. In the absence of Hh, we observed that GPC6 is localized outside of the cilium but moves into the cilium upon the addition of Hh. We conclude that GPC6 stimulates Hh signaling by binding to Hh and Ptc1 at the cilium and increasing the interaction of the receptor and ligand.

Modulation of apical constriction by Wnt signaling is required for lung epithelial shape transition.

In lung development, the apically constricted columnar epithelium forms numerous buds during the pseudoglandular stage. Subsequently, these epithelial cells change shape into the flat or cuboidal pneumocytes that form the air sacs during the canalicular and saccular (canalicular-saccular) stages, yet the impact of cell shape on tissue morphogenesis remains unclear. Here, we show that the expression of Wnt components is decreased in the canalicular-saccular stages, and that genetically constitutive activation of Wnt signaling impairs air sac formation by inducing apical constriction in the epithelium as seen in the pseudoglandular stage. Organ culture models also demonstrate that Wnt signaling induces apical constriction through apical actomyosin cytoskeletal organization. Mathematical modeling reveals that apical constriction induces bud formation and that loss of apical constriction is required for the formation of an air sac-like structure. We identify MAP/microtubule affinity-regulating kinase 1 (Mark1) as a downstream molecule of Wnt signaling and show that it is required for apical cytoskeletal organization and bud formation. These results suggest that Wnt signaling is required for bud formation by inducing apical constriction during the pseudoglandular stage, whereas loss of Wnt signaling is necessary for air sac formation in the canalicular-saccular stages.

Basolateral secretion of Wnt5a in polarized epithelial cells is required for apical lumen formation.

Wnt5a regulates planar cell polarity in epithelial cells, but it remains to be determined whether Wnt5a and its receptors are sorted apically or basolaterally, and how Wnt5a signaling is involved in apical and basolateral polarization. We found that Wnt5a was secreted basolaterally in polarized kidney epithelial cells. The basolateral secretion of Wnt5a required Wntless (Wls), clathrin and adaptor protein 1 (AP-1). Wnt5a receptors were also localized to the basolateral membranes, but their sorting did not require Wls. Wnt5a-induced signaling was stimulated more efficiently at the basolateral side than the apical side of epithelial cells. Knockdown of Wnt5a delayed apical lumen formation of the epithelial cyst, and these phenotypes were rescued by wild-type Wnt5a, but not by a Wnt5a mutant that is secreted apically. Although apoptosis was not required for apical lumen formation in a wild-type cyst, apoptosis was necessary for eliminating luminal cells in a Wnt5a-depleted cyst. These results suggest that Wnt5a and its receptors are sorted to their correct destination by different mechanisms and that the basolateral secretion of Wnt5a is necessary for apical lumen formation in the epithelial cyst.

Identification of amino acid residues required for the substrate specificity of human and mouse chondroitin sulfate hydrolase (conventional hyaluronidase-4).

Human hyaluronidase-4 (hHYAL4), a member of the hyaluronidase family, has no hyaluronidase activity, but is a chondroitin sulfate (CS)-specific endo-ß-N-acetylgalactosaminidase. The expression of hHYAL4 is not ubiquitous but restricted to placenta, skeletal muscle, and testis, suggesting that hHYAL4 is not involved in the systemic catabolism of CS, but rather has specific functions in particular organs or tissues. To elucidate the function of hyaluronidase-4 in vivo, mouse hyaluronidase-4 (mHyal4) was characterized. mHyal4 was also demonstrated to be a CS-specific endo-ß-N-acetylgalactosaminidase. However, mHyal4 and hHYAL4 differed in the sulfate groups they recognized. Although hHYAL4 strongly preferred GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)-containing sequences typical in CS-D, where GlcUA represents d-glucuronic acid, mHyal4 depolymerized various CS isoforms to a similar extent, suggesting broad substrate specificity. To identify the amino acid residues responsible for this difference, a series of human/mouse HYAL4 chimeric proteins and HYAL4 point mutants were generated, and their preference for substrates was investigated. A combination of the amino acid residues at 261-265 and glutamine at 305 was demonstrated to be essential for the enzymatic activity as well as substrate specificity of mHyal4.

Hyaluronidases Have Strong Hydrolytic Activity toward Chondroitin 4-Sulfate Comparable to that for Hyaluronan.

Chondroitin sulfate (CS) chains are involved in the regulation of various biological processes. However, the mechanism underlying the catabolism of CS is not well understood. Hyaluronan (HA)-degrading enzymes, the hyaluronidases, are assumed to act at the initial stage of the degradation process, because HA is similar in structure to nonsulfated CS, chondroitin (Chn). Although human hyaluronidase-1 (HYAL1) and testicular hyaluronidase (SPAM1) can degrade not only HA but also CS, they are assumed to digest CS to only a limited extent. In this study, the hydrolytic activities of HYAL1 and SPAM1 toward CS-A, CS-C, Chn, and HA were compared. HYAL1 depolymerized CS-A and HA to a similar extent. SPAM1 degraded CS-A, Chn, and HA to a similar extent. CS is widely distributed from very primitive organisms to humans, whereas HA has been reported to be present only in vertebrates with the single exception of a mollusk. Therefore, a genuine substrate of hyaluronidases appears to be CS as well as HA.

Dermatan sulfate epimerase 2 is the predominant isozyme in the formation of the chondroitin sulfate/dermatan sulfate hybrid structure in postnatal developing mouse brain.

Chondroitin sulfate (CS) and dermatan sulfate (DS) are expressed in significant amounts in the brain and play important roles in the development of the central nervous system in mammals. CS and DS structures are often found in a single CS/DS hybrid chain. The l-iduronic acid (IdoA)-containing domain, which defines a DS-type domain, appears key to the biological functions of the CS/DS hybrid chain. In this study, to clarify the distribution of the DS-type structure in the brain during development, the expression patterns of DS epimerase 1 (DS-epi1) and DS-epi2, both of which convert d-glucuronic acid into IdoA, were investigated by in situ hybridization. DS-epi2 was ubiquitously expressed in the developing brain after birth, whereas the expression of DS-epi1 was faint and obscure at all developmental stages. Quantitative real-time polymerase chain reaction revealed the expression of DS-epi2 to be higher than that of DS-epi1 throughout development, suggesting that DS-epi2 but not DS-epi1 is mostly expressed in the brain and plays key roles in the epimerization of CS/DS during its biosynthesis. Moreover, an analysis of the disaccharides of CS/DS demonstrated significant amounts of IdoA-containing iD units [IdoA(2S)-GalNAc(6S)] and iB units [IdoA(2S)-GalNAc(4S)], where 2S, 4S and 6S stand for 2-O-, 4-O- and 6-O-sulfate, respectively, in every region of the brain examined. The proportion of these units in cerebellar CS/DS was greatly altered during postnatal development. These results suggest that the IdoA-containing structures in the developing brain are mainly produced by the actions of DS-epi2 and play crucial roles in postnatal development.

Identification of human hyaluronidase-4 as a novel chondroitin sulfate hydrolase that preferentially cleaves the galactosaminidic linkage in the trisulfated tetrasaccharide sequence.

Human hyaluronidases have been considered to be the enzymes acting at the initial step in the catabolism of chondroitin sulfate (CS) in vivo. However, human hyaluronidase-1 digests CS more slowly than hyaluronan (HA), and its preferred substrate is HA rather than CS. We have identified a chondroitin hydrolase in Caenorhabditis elegans, which effectively degrades chondroitin but depolymerizes HA to a much lesser extent (Kaneiwa T, Yamada S, Mizumoto S, Montaño AM, Mitani S, Sugahara K. 2008. Identification of a novel chondroitin hydrolase in Caenorhabditis elegans. J Biol Chem. 283:14971-14979), suggesting the existence of CS-specific endoglycosidases in mammalian systems. In this study, human hyaluronidase-4 was demonstrated to be a CS-specific endo-beta-N-acetylgalactosaminidase. This is the first demonstration of a CS hydrolase in higher organisms. The specificity of a purified recombinant form of the enzyme was investigated in detail through the characterization of degradation products. The best substrate of the CS hydrolase was the galactosaminidic linkage in the sequence of a trisulfated tetrasaccharide GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate)-GlcUA-GalNAc(4-O- or 6-O-sulfate), where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively. The disaccharide unit on the nonreducing side, GlcUA(2-O-sulfate)-GalNAc(6-O-sulfate) (D unit), is rich in shark fin cartilage CS-D among various CS isoforms. CS hydrolase will be a useful tool for investigating CS-specific functions in tissues and cells. In addition, it may well be applicable to the treatment of acute spinal cord injuries as in the case of, or instead of, the bacterial CS lyase which has been used for recent clinical trials.

Important role of heparan sulfate in postnatal islet growth and insulin secretion.

Heparan sulfate (HS) binds with several signaling molecules and regulates ligand-receptor interactions, playing an essential role in embryonic development. Here we showed that HS was intensively expressed in pancreatic islet beta-cells after 1 week of age in mice. The enzymatic removal of HS in isolated islets resulted in attenuated glucose-induced insulin secretion with a concomitant reduction in gene expression of several key components in the insulin secretion machinery. We further depleted islet HS by inactivating the exostosin tumor-like 3 gene specifically in beta-cells. These mice exhibited abnormal islet morphology with reduced beta-cell proliferation after 1 week of age and glucose intolerance due to defective insulin secretion. These results demonstrate that islet HS is involved in the regulation of postnatal islet maturation and required to ensure normal insulin secretion.

Differential distribution of aggrecan isoforms in perineuronal nets of the human cerebral cortex.

Aggrecan is a component of the CNS extracellular matrix (ECM) and we show here that the three primary alternative spliced transcripts of the aggrecan gene found in cartilage are also present in the adult CNS. Using a unique panel of core protein-directed antibodies against human aggrecan we further show that different aggrecan isoforms are deposited in perineuronal nets (PNNs) and neuropil ECM of Brodmann's area 6 of the human adult cerebral cortex. According to their distribution pattern, the identified cortical aggrecan isoforms were subdivided into five clusters spanning from cluster 1, comprised isoforms that appeared widespread throughout the cortex, to cluster 5, which was an aggrecan-free subset. Comparison of brain and cartilage tissues showed a different relative abundance of aggrecan isoforms, with cartilage-specific isoforms characterizing cluster 5, and PNN-associated isoforms lacking keratan sulphate chains. In the brain, isoforms of cluster 1 were disclosed in PNNs surrounding small-medium interneurons of layers II-V, small-medium pyramidal neurons of layers III and V and large interneurons of layer VI. Aggrecan PNNs enveloped both neuron bodies and neuronal processes, encompassing pre-terminal nerve fibres, synaptic boutons and terminal processes of glial cells and aggrecan was also observed in continuous 'coats' associated with satellite, neuron-associated cells of a putative glial nature. Immunolabelling for calcium-binding proteins and glutamate demonstrated that aggrecan PNNs were linked to defined subsets of cortical interneurons and pyramidal cells. We suggest that in the human cerebral cortex, discrete, layer-specific PNNs are assembled through the participation of selected aggrecan isoforms that characterize defined subsets of cortical neurons.

Identification of a novel chondroitin hydrolase in Caenorhabditis elegans.

Hyaluronidases have been postulated to be the enzyme acting at the initial step of chondroitin sulfate (CS) catabolism in vivo. Since chondroitin (Chn) but not hyaluronic acid (HA) has been detected in Caenorhabditis elegans, the nematode is a good model for elucidating the mechanism of the degradation of CS/Chn in vivo. Here we cloned the homolog of human hyaluronidase in C. elegans, T22C8.2. The Chn-degrading activity in vitro was first demonstrated when it was expressed in COS-7 cells. The enzyme cleaved preferentially Chn. CS-A and CS-C were also depolymerized but to lesser extents, and HA was hardly degraded. In order of preference, the substrates ranked Chn >> CS-A > CS-C >> HA. The products of the degradation of Chn by the enzyme were characterized by anion-exchange high performance liquid chromatography and delayed extraction matrix-assisted laser desorption ionization time-of-flight mass spectrometry. The structure of the major component in the digest was determined as GlcUAbeta1-3GalNAcbeta1-4GlcUAbeta1-3GalNAc, where GlcUA and GalNAc represent D-glucuronic acid and N-acetyl-D-galactosamine, respectively, indicating that this enzyme is a Chn hydrolase, an endo-beta-galactosaminidase specific for Chn. Investigation of the effects of pH on the activity revealed the optimum pH of Chn hydrolase to be 6.0. Since Chn in C. elegans has been demonstrated to play critical roles in cell division, Chn hydrolase possibly regulates the function of Chn in vivo. This is the first demonstration of a Chn hydrolase in an animal.