Structural analysis of glycosaminoglycans derived from axonally transported proteoglycans in regenerating goldfish optic nerve

Structural characteristics of glycosaminoglycans (GAGs) derived from axonally transported proteoglycans (PGs) were compared in 21 day regenerating and intact goldfish optic tracts. Twenty one days following unilateral optic nerve crushes, fish received intraocular injections of³⁵SO₄. Eight hours post injection, tracts were removed and the³⁵SO₄-labeled GAGs, chondroitin sulfate (CS) and heparan sulfate (HS), isolated. The HS from regenerating optic tracts had a DEAE elution profile indicative of decreased charge density, while heparitinase treatment of HS followed by Sephadex G50 analysis of the resulting fragments showed a change in the elution pattern, suggesting reduced overall sulfation. HPLC analysis of HS disaccharides revealed a difference in the sulfation pattern of regenerating tract HS, characterized by the reduced presence of tri-sulfated disaccharides. Other structural features, such as the sizes of CS and HS, and the sulfation of CS, showed no changes during regeneration. These results indicate that changes in the structure of axonally transported HS accompany regeneration of goldfish optic axons.     

Localization of chondroitin sulfate proteoglycan versican in adult brain with special reference to large projection neurons

Versican is a chondroitin sulfate proteoglycan belonging to the lectican family. Versican has two glycosaminoglycan attachment regions, named the GAGα and GAGβ domains, which are both regulated by alternative splicing and yield four protein isoforms. We have investigated the expression and localization of versican in the developing and adult brain by using anti-versican GAGα and GAGβ antibodies. Western analysis revealed that GAGα-reactive isoform was dominant in the adult brain. Immunohistochemical study demonstrated that GAGα immunoreactivity was detectable from neonatal periods to adulthood, whereas GAGβ immunoreactivity completely disappeared within 3 weeks of birth. In the adult brain, GAGα immunoreactivity was seen in the white matter regions and was also localized in the gray matter including somata and dendrites of cortical and hippocampal pyramidal neurons and cerebellar Purkinje cells. In contrast, GAGα immunoreactivity was not localized on parvalbumin-positive interneurons and cerebellar stellate cells. Furthermore, GAGα immunoreactivity was not co-localized with perineuronal net markers such as Wisteria floribunda agglutinin lectin and phosphacan. Thus, versican was localized on large projection neurons rather than small interneurons. To confirm the binding mechanism of versican to neurons, hyaluronan and chondroitin sulfates were enzymatically removed from brain sections before the immunolabeling of versican. These treatments had no effect on the labeling pattern of versican, suggesting that other versican-interactive molecules are involved in the binding of versican to neurons. This study was supported by a Grant-in-Aid for Scientific Research on Priority Areas “Advanced Brain Science Project” from the Ministry of Education, Culture, Sports, Science, and Technology, Japan.     

Association of the SNP rs2623047 in the HSPG modification enzyme SULF1 with an Australian Caucasian Breast Cancer Cohort

Breast cancer is the second most common cancer worldwide and the most common cancer reported in women. This malignant tumour is characterised by a number of specific features including uncontrolled cell proliferation. It ranks fifth in the world as a cause of cancer death overall in developed countries and is the second most frequent cause of cancer death in women. Early diagnosis increases 5-year survival rates up to 95%. Heparan sulfate proteoglycans (HSPGs) are complex proteins composed of a core protein to which a number of highly sulfated side chains attach, ubiquitous to the cell surface and within the extracellular matrix. HSPG side chains are synthesised by a highly co-ordinated process resulting in distinct sulfation patterns, which determine specific interactions with cell-signalling partners including growth factors, their receptors, ligands and morphogens. The enzymes responsible for chain initiation, elongation and sulfation are critical for creating HS chain variability conferring biological functionality. This study investigated a single nucleotide polymorphism in SULF1, the enzyme responsible for the 6-O desulfation of heparan sulfate side chains. We investigated this SNP in an Australian Caucasian case–control breast cancer population and found a significant association between SULF1 and breast cancer at both the allelic and genotypic levels (allele, p = 0.016; genotype, p = 0.032). Our results suggest that the rs2623047 SNP in SULF1 may impact breast cancer susceptibility. Specifically, the T allele of rs2623047 in SULF1 is associated with an increased risk of developing breast cancer in our cohort. The identification of markers including SULF1 may improve detection of this disease at its earliest stages improving patient treatment and prognosis.     

HSULF-1 inhibits ERK and AKT signaling and decreases cell viability in vitro in human lung epithelial cells

Background

Heparan sulfate proteoglycans (HSPGs) modulate the binding and activation of signaling pathways of specific growth factors, such as fibroblast growth factor-2 (FGF-2). Human endosulfatase 1 (HSULF-1) is an enzyme that selectively removes 6-O sulfate groups from HS side chains and alter their level and pattern of sulfation and thus biological activity. It is known that HSULF-1 is expressed at low levels in some cancer cell lines and its enhanced expression can inhibit cancer cell growth or induce apoptosis, but the mechanism(s) involved has not been identified.

Methods

HSULF-1 mRNA expression was assessed in five normal cells (primary human lung alveolar type 2 (hAT2) cells, adult lung fibroblasts (16Lu), fetal lung fibroblasts (HFL), human bronchial epithelial cells (HBE), and primary human lung fibroblasts (HLF)) and five lung cancer cell lines (A549, H292, H1975, H661, and H1703) using quantitative real time polymerase chain reaction (qRT-PCR). H292 and hAT2 cells over-expressing HSULF-1 were analyzed for cell viability, apoptosis, and ERK/Akt signaling, by MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay, TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay, and Western Blot, respectively. Apoptosis pathway activation was confirmed by PCR array in hAT2, H292, and A549 cells.

Results

HSULF-1 was expressed at a significantly lower level in epithelial cancer cell lines compared to normal cells. Infection with recombinant adenovirus for HSULF-1 over-expression resulted in decreased cell viability in H292 cells, but not in normal hAT2 cells. HSULF-1 over-expression induced apoptosis in H292 cells, but not in hAT2 cells. In addition, apoptosis pathways were activated in both H292 and A549 cells, but not in hAT2 cells. HSULF-1 over-expression reduced ERK and Akt signaling activation in H292 cells, which further demonstrated its inhibitory effects on signaling related to proliferation.

Conclusions

These results indicate that HSULF-1 is expressed at lower levels in H292 lung cancer cells than in normal human alveolar cells and that its over-expression reduced cell viability in H292 cells by inducing apoptotic pathways, at least in part by inhibiting ERK/Akt signaling. We hypothesize that HSULF-1 plays important roles in cancer cells and functions to modify cell signaling, inhibit cancer proliferation, and promote cancer cell death.     

Heparan sulfate proteoglycans exert positive and negative effects in Shh activity

Hedgehog (Hh) proteins are morphogens involved in short- and long-range effects during early embryonic development. Genetic analysis in fly and vertebrate embryos showed that heparan sulfate proteoglycans (HSPGs) are required for Hh transport and signaling. To further understand how HSPGs regulate Sonic hedgehog (Shh), we performed experiments using cell culture and biochemical assays. When the synthesis of HSPGs was reduced, a decrease in Shh activity was observed. Contrary to that, addition of a peptide that competes the binding of Shh to HSPGs resulted in augmentation of Shh activity. From these results, we concluded that HSPGs exert positive and negative effects in Shh activity. This dual effect correlates with the finding that Shh interacts preferentially with two HSPGs. The current model for the role of HSPGs in Shh diffusion is discussed in view of our findings.     

Selective inhibition of ventral temporal but not dorsal nasal neurites from mouse retinal explants during contact with chondroitin sulphate

We have determined whether chondroitin sulphate (CS) glycosaminoglycans are sufficient to direct a selective inhibition of neurite growth from ventral temporal (VT) but not from dorsal nasal (DN) retina in mouse embryos; this may underlie the formation of axon divergence in the optic chiasm. Explants from the retinal region of embryonic day-14 mouse were grown on a laminin–polylysine substrate near to a circular spot coated with CS. In control cultures, in which no CS was added to the spot, both VT and DN retinal neurites grew extensively into the coated territory. When presented with spots coated with 10 mg/ml CS, neurite growth from the VT retina into the CS territory was dramatically reduced but that from the DN retina was not significantly affected. The selective inhibition to VT neurites was completely abolished by treatment with chondroitinase ABC, indicating a specific contribution of CS glycosaminoglycan in this regionally specific behaviour. This differential behaviour was not observed in explants presented with a lower or higher concentration of CS or in explants grown on substrate coated with a different laminin concentration. Thus, a critical ratio of CS to laminin seems to be essential to induce this differential behaviour in retinal neurites towards contact with CS. Furthermore, this behavior was not observed in explants cultured directly on a CS-rich substrate, suggesting that contact with growth-promoting molecules is necessary for the selective responses of retinal neurites during subsequent contact with CS. We concluded that CS glycosaminoglycan is sufficient to drive selective inhibition of VT but not DN neurites and that, together with a critical combination of growth-promoting factors, it may control the axon divergence process at the mouse optic chiasm.This project is partially supported by a grant from the Research Grant Council of the Hong Kong Special Administrative Region (project no. CUHK 4417/03M) and a Direct Grant from the Chinese University of HK (project no. 2004.1.051).     

Zippers Make Signals: NCAM-mediated Molecular Interactions and Signal Transduction

The neural cell adhesion molecule, NCAM, is involved in multiple cis- and trans-homophilic interactions (NCAM binding to NCAM) thereby facilitating cell–cell adhesion through the formation of zipper-like NCAM-complexes. NCAM is also involved in heterophilic interactions with a number of proteins and extracellular matrix molecules. Some of these heterophilic interactions are mutually exclusive, and some interfere with or are dependent on homophilic NCAM interactions. Furthermore, both homo- and heterophilic interactions are modulated by posttranslational modifications of NCAM. Heterophilic NCAM-interactions initiate several intracellular signal transduction pathways ultimately leading to biological responses involving cellular differentiation, proliferation, migration and survival. Both homo- and heterophilic NCAM-interactions can be mimicked by synthetic peptides, which can induce NCAM-like signalling, and in vitroand in vivo studies suggest that such NCAM mimetics may be used for the treatment of neurodegenerative disorders.Special issue dedicated to Lawrence F. Eng.     

Influence of collagen gel substrata on certain biochemical activities of hepatocytes in primary culture

In order to study the influence of cell shape as modulated by the extracellular matrix on the cellular activity, hepatocytes isolated from liver were maintained on collagen I coated plastic substrata and collage I gel substrata and certain hepatocyte specific functions were investigated. The incorporation of³[H]-leucine into total proteins and albumin secreted by cells maintained on collagen gel was found to be significantly higher compared to those maintained on a collagen coated plastic substrata, indicating that hepatocytes on collagen gel have an enhanced albumin synthesizing capacity. Increased incorporation of³⁵[S]-sulphate into total proteoglycans (PG) and a relatively higher fraction of the³⁵[S]-PG in the extracellular space showed an increased rate of synthesis and secretion of sulphated PGs by cells maintained on collagen gels. But in contrast to the above results, the incorporation of³[H]-leucine into cytokeratins C₈, C₁₈ and actin were significantly low in cells maintained on collagen gel. The tyrosine amino transferase activity exhibited by hepatocytes preincubated with dexamethasone on collagen gel was also significantly low. The different forms of collagen substrata appeared to have no effect on the amino acid transport by hepatocytes, further suggesting that the various hepatocyte specific functions are not uniformly altered when hepatocytes are maintained on three-dimensional collagen gel substrata. These results indicate that the shape of the cell as determined by the nature of the matrix substratum influences the synthetic activity of secretory proteins and those remaining intracellularly, differently.     

A “coupled” subchondral bone-articular cartilage tissue culture system for the study of cartilage proteoglycan metabolism

Summary Current evidence suggests that interactions between the subchondral bone and the articular cartilage of mammalian diarthrodial joints may occur through the action of bone-associated peptide factors. However, there is no suitable organ culture model for studying these interactions. This study defines a long-term tissue culture system where the articular cartilage is coupled to the adjacent subchondral bone obtained from the proximal ends of bovine metacarpals. Autoradiography done over 3 mo., by utilizing [³⁵S]SO₄ incorporation into cartilage proteoglycan (PG) and a procedure for cutting non-decalcified bone, demonstrated similar numbers of silver grains over chondrocytes in all cartilage zones, including the bone-cartilage interface. Newly synthesized PG (NSPG) from the cartilage of the “coupled” system over a 3-wk period was primarily of large hydrodynamic size (Kav of 0.34). Comparable bovine articular and nasal cartilage slice systems, incubated for short periods of time, yielded similar and somewhat larger NSPG, respectively. Labeled chondroitin sulphate PG accumulating in the medium of primary chondrocyte monolayer cultures, derived from the cartilage of the coupled system at 0, 1, 2, and 3 wk, revealed two polydisperse subpopulations (Kav of 0.30 to 0.38 and 0.51 to 0.68). We conclude that this coupled bone-cartilage system is viable for prolonged periods, is suitable for studies on the metabolism of articular cartilage PGs, and seems to have some advantages over the cultured articular cartilage slice system.