Avian neural crest cell migration is diversely regulated by the two major hyaluronan-binding proteoglycans PG-M/versican and aggrecan

It has been proposed that hyaluronan-binding proteoglycans play an important role as guiding cues during neural crest (NC) cell migration, but their precise function has not been elucidated. In this study, we examine the distribution, structure and putative role of the two major hyaluronan-binding proteoglycans, PG-M/versicans and aggrecan, during the course of avian NC development. PG-M/versicans V0 and V1 are shown to be the prevalent isoforms at initial and advanced phases of NC cell movement, whereas the V2 and V3 transcripts are first detected following gangliogenesis. During NC cell dispersion, mRNAs for PG-M/versicans V0/V1 are transcribed by tissues lining the NC migratory pathways, as well as by tissues delimiting nonpermissive areas. Immunohistochemistry confirm the deposition of the macromolecules in these regions and highlight regional differences in the density of these proteoglycans. PG-M/versicans assembled within the sclerotome rearrange from an initially uniform distribution to a preferentially caudal localization, both at the mRNA and protein level. This reorganization is a direct consequence of the metameric NC cell migration through the rostral portion of the somites. As suggested by previous in situ hybridizations, aggrecan shows a virtually opposite distribution to PG-M/versicans being confined to the perinotochordal ECM and extending dorsolaterally in a segmentally organized manner eventually to the entire spinal cord at axial levels interspacing the ganglia. PG-M/versicans purified from the NC migratory routes are highly polydispersed, have an apparent M(r) of 1,200-2,000 kDa, are primarily substituted with chondroitin-6-sulfates and, upon chondroitinase ABC digestion, are found to be composed of core proteins with apparent M(r)of 360–530, 000. TEM/rotary shadowing analysis of the isolated PG-M/versicans confirmed that they exhibit the characteristic bi-globular shape, have core proteins with sizes predicted for the V0/V1 isoforms and carry relatively few extended glycosaminoglycan chains. Orthotopical implantation of PG-M/versicans immobilized onto transplantable micromembranes tend to ‘attract’ moving cells toward them, whereas similar implantations of a notochordal type-aggrecan retain both single and cohorts of moving NC cells in close proximity of the implant and thereby perturb their spatiotemporal migratory pattern. NC cells fail to migrate through three-dimensional collagen type I-aggrecan substrata in vitro, but locomote in a haptotactic manner through collagen type I-PG-M/versican V0 substrata via engagement of HNK-1 antigen-bearing cell surface components. The present data suggest that PG-M/versicans and notochordal aggrecan exert divergent guiding functions during NC cell dispersion, which are mediated by both their core proteins and glycosaminoglycan side chains and may involve ‘haptotactic-like’ motility phenomena. Whereas aggrecan defines strictly impenetrable embryonic areas, PG-M/versicans are central components of the NC migratory pathways favoring the directed movement of the cells

Syndecans Reside in Sphingomyelin-Enriched Low-Density Fractions of the Plasma Membrane Isolated from a Parathyroid Cell Line

BACKGROUND: Heparan sulfate proteoglycans (HSPGs) are one of the basic constituents of plasma membranes. Specific molecular interactions between HSPGs and a number of extracellular ligands have been reported. Mechanisms involved in controlling the localization and abundance of HSPG on specific domains on the cell surface, such as membrane rafts, could play important regulatory roles in signal transduction. METHODOLOGY/PRINCIPAL FINDINGS: Using metabolic radiolabeling and sucrose-density gradient ultracentrifugation techniques, we identified [(35)S]sulfate-labeled macromolecules associated with detergent-resistant membranes (DRMs) isolated from a rat parathyroid cell line. DRM fractions showed high specific radioactivity ([(35)S]sulfate/mg protein), implying the specific recruitment of HSPGs to the membrane rafts. Identity of DRM-associated [(35)S]sulfate-labeled molecules as HSPGs was confirmed by Western blotting with antibodies that recognize heparan sulfate (HS)-derived epitope. Analyses of core proteins by SDS-PAGE revealed bands with an apparent MW of syndecan-4 (30-33 kDa) and syndecan-1 (70 kDa) suggesting the presence of rafts with various HSPG species. DRM fractions enriched with HSPGs were characterized by high sphingomyelin content and found to only partially overlap with the fractions enriched in ganglioside GM1. HSPGs could be also detected in DRMs even after prior treatment of cells with heparitinase. CONCLUSIONS/SIGNIFICANCE: Both syndecan-1 and syndecan-4 have been found to specifically associate with membrane rafts and their association seemed independent of intact HS chains. Membrane rafts in which HSPGs reside were also enriched with sphingomyelin, suggesting their possible involvement in FGF signaling. Further studies, involving proteomic characterization of membrane domains containing HSPGs might improve our knowledge on the nature of HSPG-ligand interactions and their role in different signaling platforms

Possible Repositioning of an Oral Anti-Osteoporotic Drug, Ipriflavone, for Treatment of Inflammatory Arthritis via Inhibitory Activity of KIAA1199, a Novel Potent Hyaluronidase

KIAA1199 has a strong hyaluronidase activity in inflammatory arthritis. This study aimed to identify a drug that could reduce KIAA1199 activity and clarify its effects on inflammatory arthritis. Rat chondrosarcoma (RCS) cells were strongly stained with Alcian blue (AB). Its stainability was reduced in RCS cells, which were over-expressed with the KIAA1199 gene (RCS-KIAA). We screened the drugs that restore the AB stainability in RCS-KIAA. The effects of the drug were evaluated by particle exclusion assay, HA ELISA, RT-PCR, and Western blotting. We further evaluated the HA accumulation and the MMP1 and three expressions in fibroblast-like synoviocytes (FLS). In vivo, the effects of the drug on symptoms and serum concentration of HA in a collagen-induced arthritis mouse were evaluated. Ipriflavone was identified to restore AB stainability at 23%. Extracellular matrix formation was significantly increased in a dose-dependent manner (p = 0.006). Ipriflavone increased the HA accumulation and suppressed the MMP1 and MMP3 expression on TNF-α stimulated FLS. In vivo, Ipriflavone significantly improved the symptoms and reduced the serum concentrations of HA. Conclusions: We identified Ipriflavone, which has inhibitory effects on KIAA1199 activity. Ipriflavone may be a therapeutic candidate based on its reduction of KIAA1199 activity in inflammatory arthritis

Sucrose-density gradient analysis of DRMs obtained from a [³⁵S]sulfate labeled rat parathyroid cell line.

<p>Rat parathyroid cells (2×10⁷) were metabolically labeled with [³⁵S]sulfate, and subjected to a preparation of DRMs as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032351#s4" target="_blank"><i>Materials and Methods</i></a>. Fractions (200 µl each) were collected and counted for radioactivity after removal of free [³⁵S]sulfate. A. Graph shows total radioactivity (○) and protein concentration (•) detected in each fraction. <i>Inset</i>, the same experimental data plotted in an expanded scale, emphasizing the presence of ³⁵S-labeled material in low-density fractions. B. Graphic representation of specific radioactivity calculated for each fraction. The specific activity was expressed as total radioactivity per amount of the total protein material. DRM fractions were characterized by high specific activity suggesting enrichment with [³⁵S]sulfate labeled molecules.</p

Characterization of DRM fractions isolated from a parathyroid cell line.

<p>Confluent PTr cells were subjected to DRMs preparation as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032351#s4" target="_blank"><i>Materials and Methods</i></a>. Concentrated fractions were analyzed for presence of HSPGs by WB. They were also examined for the presence of ganglioside GM1 by the binding of cholera toxin B (CTB) subunit and sphingomyelin (SM) by HPTLC analysis. A. Staining with 3G10 antibodies confirmed the presence of HSPGs in low-density fractions. Equal volumes (13 µl) of each fraction were used for analysis. Fractions 13 and 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 16, 62 and 56 times, respectively, prior to the analysis. B. DRM markers, Lyn and Giα showed a broad distribution, but partially overlapped with low-density fractions containing HSPGs. Equal volumes (33 µl) of each fraction were used for analysis. Fractions 13 through 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 18, 72 and 64 times, respectively, prior the analysis due to high protein content. Successful preparation was confirmed by immunostaining for TfR, which was found mainly in the bottom fractions. C. An equal aliquot (2 µl) of each fraction was dot-blotted onto PVDF membrane and stained with HRP-conjugated CTB subunit. Numbers correspond to the fraction number; L, original lysate before sucrose-density gradient ultracentrifugation. D. Lipids extracted from sucrose density gradient fractions (fractions 2–11) were developed on the HPTLC plate and stained with 3% cupric acetate and 8% phosphoric acid. Chol, cholesterol; PE, phosphoethanolamine; PC, phosphocholine; SM, sphingomyelin. Non-specific staining due to the presence of traces of detergent in samples is marked with (*). E. Graphic representation of distribution of HSPGs, ganglioside GM1 and SM in DRM fractions. Low-density fractions containing HSPGs showed enrichment with SM. Density of bands detected in WB and HPTLC was measured with ImageJ and expressed in arbitrary units. Graph shows distribution of ganglioside GM1 (○); HSPGs (▴) and SM (▪).</p

Western blotting analysis of DRM fractions isolated from a rat parathyroid cell line.

<p>DRMs were prepared from confluent PTr cells as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0032351#s4" target="_blank"><i>Materials and Methods</i></a>. Collected fractions, were concentrated, treated with heparitinase I and subjected to SDS-PAGE and WB analysis. A. Staining with anti-ΔHS (3G10) antibodies confirmed the presence of HSPGs in low-density fractions. Equal volumes (13 µl) of each fraction were analyzed. Fractions 13 and 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 16, 62 and 56 times, respectively, prior to the analysis. Bands marked with (*) represent non-specific staining due to the presence of BSA at high concentration. B. Staining with antibodies against DRM markers, Lyn and Giα defined the low-density fractions as DRMs. Equal volumes (33 µl) of each fraction were used for analysis. Fractions 13 through 14, bottom fraction (pooled fractions 15 and 16, B) and lysate (L) were diluted 18, 72 and 64 times, respectively, prior the analysis due to high protein content. Staining for transferrin receptor (TfR) was used as a control for the successful preparation. C. Graphic representation of the distribution of TfR, Lyn, Giα and HSPGs in fractions obtained from sucrose-density gradient ultracentrifugation. Density of bands detected in WB analysis (A and C) was measured and expressed as arbitrary units. TfR (○); Lyn (▪); Giα (◊) and HSPG (▴).</p