Lack ofl-Iduronic Acid in Heparan Sulfate Affects Interaction with Growth Factors and Cell Signaling

HSEPI (glucuronyl C5-epimerase) catalyzes the conversion of d-glucuronic acid to l-iduronic acid in heparan sulfate (HS) biosynthesis. Disruption of the Hsepi gene in mice yielded a lethal phenotype with selective organ defects but had remarkably little effect on other organ systems. We have approached the underlying mechanisms by examining the course and effects of FGF2 signaling in a mouse embryonic fibroblast (MEF) cell line derived from the Hsepi^−/− mouse. The HS produced by these cells is devoid of l-iduronic acid residues but shows up-regulated N- and 6-O-sulfation compared with wild type (WT) MEF HS. In medium fortified with 10% fetal calf serum, the Hsepi^−/− MEFs proliferated and migrated similarly to WT cells. Under starvation conditions, both cell types showed attenuated proliferation and migration that could be restored by the addition of FGF2 to WT cells, whereas Hsepi^−/− cells were resistant. Moreover, ERK phosphorylation following FGF2 stimulation was delayed in Hsepi^−/− compared with WT cells. Assessment of HS-growth factor interaction by nitrocellulose filter trapping revealed a strikingly aberrant binding property of FGF2 and glia-derived neurotropic factor to Hsepi^−/− but not to WT HS. glia-derived neurotropic factor has a key role in kidney development, defective in Hsepi^−/− mice. By contrast, Hsepi^−/− and WT HS interacted similarly and in conventional mode with FGF10. These findings correlate defective function of growth factors with their mode of HS interaction and may help explain the partly modest organ phenotypes observed after genetic ablation of selected enzymes in HS biosynthesis.Signaling activities of numerous growth factors and morphogens during development involve cell surface receptor systems consisting of a tyrosine kinase-type receptor along with a heparan sulfate proteoglycan (HSPG)2 co-receptor (1, 2). The complex and heterogeneous HSPG macromolecules occur in the extracellular matrix and on the surfaces of virtually all animal cells (3). HS side chains of HSPGs show great structural variability; hence, they interact with a multitude of proteins and influence a variety of biological processes, including growth factor signaling (4).The structural diversity of HS is best envisaged through an account of its biosynthesis (Fig. 1). The process is initiated by glycosylation reactions that generate saccharide sequences composed of alternating GlcA and GlcNAc units covalently bound to a core protein (through a specific “linker” tetrasaccharide sequence). The resulting polymer of (GlcAβ1,4-GlcNAcα1,4-)_n disaccharide repeats is modified through a series of reactions, including N-deacetylation/N-sulfation of GlcNAc residues, C5-epimerization of GlcA to l-iduronic acid (IdoA) units, and O-sulfation at various positions of the hexuronic acid and glucosamine residues (4, 5). Modulation of these modification reactions, through as yet poorly understood mechanisms, yields HS chains of strictly regulated saccharide composition, varying with tissue source and age (6, 7). Interactions between protein ligands and HS involve selective ionic binding of peptide sequences containing basic amino acid residues to saccharide domains with clustered sulfate groups and IdoA residues. The latter components are considered to promote ligand apposition through their conformational flexibility (8).Open in a separate windowFIGURE 1.Schematic display of HS biosynthesis. A precursor structure composed of alternating GlcA and GlcNAc residues linked to a core protein is modified through the series of enzymatic reactions indicated and outlined under“Results.” The modifications yield domains of consecutive N-acetylated or N-sulfated disaccharide units, along with mixed sequences. Wild type HS contains both GlcA and IdoA residues, whereas Hsepi^−/− HS lacks IdoA units but shows increased N- and 6-O-sulfation. In particular, the mutant HS features extended sequences of -GlcA-GlcNS6S- disaccharide units.Targeted disruption of genes encoding HS biosynthesis enzymes demonstrated critical roles for HSPGs in developmental processes (9–11). The observed phenotypes vary dramatically in severity, from gastrulation failure to subtle disturbance of organ development. This variability may reflect redundancy due to the occurrence of isoforms for some of the enzymes. Alternatively, developmental events might be critically dependant on HS involvement but not on the fine structure of the polysaccharide chain, such that even structurally deranged HS would fulfill a functional role (12). The glucuronyl C5-epimerase (HSEPI) that catalyzes the conversion of GlcA to IdoA in HS biosynthesis is encoded by a single gene (13). Targeted disruption of this gene in mice resulted in an abnormal HS structure, completely lacking IdoA residues and with severely distorted sulfation pattern. The Hsepi-deficient mice die shortly after birth with multiple developmental defects, such as skeletal malformations and kidney agenesis. Intriguingly, however, other major organ systems, such as the brain and vascular system, known to depend on HS-supported signaling processes, developed seemingly normally (14).The present study was undertaken to obtain insight into the mechanisms behind the developmental defects in the Hsepi mutant mice, using a model system based on murine embryonic fibroblast (MEF) cell lines. The mutant MEF cells responded poorly to FGF2 in proliferation and migration experiments and showed defective FGF2-dependent intracellular signaling. Moreover, binding studies revealed an aberrant mode of interaction between mutant HS chains and certain growth factors, of potential relevance to growth factor signaling.     

Signaling to a B-cell clone by Ek,but not Ak,does not reflect alteration of A k genes

The mouse B-cell clone, CH12.LX (Ia^k, Ly-1⁺, ⁺, ⁺), can be induced to differentiate and secrete antibody in an antigen-specific, H-2-restricted manner. Induction requires two signals. One must be provided by the binding of specific antigen to the membrane IgM; the other is delivered by the binding of E^k-specific T-cell hybridomas to the E^k molecules of CH12.LX (Bishop and Haughton 1986). Previous studies demonstrated that E^k-specific monoclonal antibodies (mAbs) could substitute for T cells in delivering the second differentiative signal (Bishop and Haughton 1986). Although CH12.LX cells present A^k to A^k-restricted or alloreactive T-helper cells, neither T cells nor mAbs specific for A^k induce differentiation (Bishop and Haughton 1986). However, since the A^kspecifc mAbs tested previously were -chain-specific and the Ia epitope specificity of the T cells used was unknown, it is possible that the differentiative signal delivered to the CH12.LX class 11 molecule is chain-specific. Here we report the effects of ten additional Ia^k-specific mAbs upon the differentiation of CH12.LX. In addition, a cl)NA library was prepared from CHI 2.LX cells, clones corresponding to the and chains of the A^k molecule were isolated, and their nucleotide sequences were determined. Finally, the A^k and E^k molecules of CH12.LX and H-2^k spleen cells were compared by two-dimensional gel electrophoresis to examine possible post-translational differences in the Ia^k molecules of CH12.LX.     

Cytokinin Content of Pea Seeds during their Growth and Development

Seed development in Pisum arvense L. cv. New Zealand Maple has been studied in relation to changes in level of total endogenous cytokinins. Growth of both the whole seed and the embryo is diauxic, having two phases of active growth separated by a lag period. The two maxima in the growth rates of the whole seed and the embryo occur about the 23rd and 31st days after anthesis. The lag period occurs between days 26 and 28. Cell division is thought to have ceased prior to day 19 and the changes in total cytokinin content in pea seeds after this time are believed to be largely independent of cell division. The three maxima in the cylokinin content per gm. fresh weight of seed and per seed were found to be coincident with the maximum volume of endosperm per seed and the two maxima in the growth rates of the whole seed and the embryo.     

The Diageotropica Gene Differentially Affects Auxin and Cytokinin Responses throughout Development in Tomato

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type calli. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways.     

SPYing on GA Signaling and Plant Development

It has been ten years since the SPINDLY (SPY) locus was first identified from a screen of mutagenized wild type Arabidopsis seeds by selecting for germination in the presence of a gibberellin (GA) biosynthesis inhibitor (Jacobsen and Olszewski 1993). Since then research into this novel protein, an O-GlcNAc transferase (OGT), has revealed some fascinating and surprising results. SPY was originally described as a negative regulator specific to the GA signal transduction pathway, but recent research suggests that SPY is involved in additional aspects of plant development. SPY is also being investigated in barley, petunia and rice, adding to the complex story that is SPY.     

Mest but Not MiR-335 Affects Skeletal Muscle Growth and Regeneration

When skeletal muscle fibers are injured, they regenerate and grow until their sizes are adjusted to surrounding muscle fibers and other relevant organs. In this study, we examined whether Mest, one of paternally expressed imprinted genes that regulates body size during development, and miR-335 located in the second intron of the Mest gene play roles in muscle regeneration. We generated miR-335-deficient mice, and found that miR-335 is a paternally expressed imprinted microRNA. Although both Mest and miR-335 are highly expressed during muscle development and regeneration, only Mest^+/- (maternal/paternal) mice show retardation of body growth. In addition to reduced body weight in Mest^+/-; DMD-null mice, decreased muscle growth was observed in Mest^+/- mice during cardiotoxin-induced regeneration, suggesting roles of Mest in muscle regeneration. Moreover, expressions of H19 and Igf2r, maternally expressed imprinted genes were affected in tibialis anterior muscle of Mest^+/-; DMD-null mice compared to DMD-null mice. Thus, Mest likely mediates muscle regeneration through regulation of imprinted gene networks in skeletal muscle.     

The Yin-Yang of Hormones: Cytokinin and Auxin Interactions in Plant Development

The phytohormones auxin and cytokinin interact to regulate many plant growth and developmental processes. Elements involved in the biosynthesis, inactivation, transport, perception, and signaling of these hormones have been elucidated, revealing the variety of mechanisms by which signal output from these pathways can be regulated. Recent studies shed light on how these hormones interact with each other to promote and maintain plant growth and development. In this review, we focus on the interaction of auxin and cytokinin in several developmental contexts, including its role in regulating apical meristems, the patterning of the root, the development of the gynoecium and female gametophyte, and organogenesis and phyllotaxy in the shoot.     

Micromineral Source Affects Intestinal Morphology but not Feather Formation in Pigeons

During 7?weeks, two groups of eight pigeons received Zn, Cu, Mn and Se either as inorganic salts or organically bound to investigate the effect of micromineral source on two fast processes in avian metabolism: feather regrowth and intestinal renewal. Increased plasma Zn with reduced hepatic Cu in the organically bound minerals group suggests improved Zn bio-availability with a net antagonistic effect on Cu. Micromineral source did not affect feather regrowth, organ weight or intestinal pH, but the increased villus height with similar crypt depth pointed to increased absorptive surface.     

Altered Expression of SPINDLY Affects Gibberellin Response and Plant Development

Gibberellins (GAs) are plant hormones with diverse roles in plant growth and development. SPINDLY (SPY) is one of several genes identified in Arabidopsis that are involved in GA response and it is thought to encode an O-GlcNAc transferase. Genetic analysis suggests that SPY negatively regulates GA response. To test the hypothesis that SPY acts specifically as a negatively acting component of GA signal transduction, spy mutants and plants containing a 35S:SPY construct have been examined. A detailed investigation of the spy mutant phenotype suggests that SPY may play a role in plant development beyond its role in GA signaling. Consistent with this suggestion, the analysis of spy er plants suggests that the ERECTA (ER) gene, which has not been implicated as having a role in GA signaling, appears to enhance the non-GA spy mutant phenotypes. Arabidopsis plants containing a 35S:SPY construct possess reduced GA response at seed germination, but also possess phenotypes consistent with increased GA response, although not identical to spy mutants, during later vegetative and reproductive development. Based on these results, the hypothesis that SPY is specific for GA signaling is rejected. Instead, it is proposed that SPY is a negative regulator of GA response that has additional roles in plant development.     

Dysferlin is expressed in human placenta but does not associate with caveolin

A proteomics screen of human placental microvillous syncytiotrophoblasts (STBs) revealed the expression of dysferlin (DYSF), a plasma membrane repair protein associated with certain muscular dystrophies. This was unexpected given that previous studies of DYSF have been restricted to skeletal muscle. Within the placenta, DYSF localized to the STB and, with the exception of variable labeling in the fetal placental endothelium, none of the other cell types expressed detectable levels of DYSF. Such restricted expression was recapitulated using primary trophoblast cell cultures, because the syncytia expressed DYSF, but not the prefusion mononuclear cells. The apical plasma membrane of the STB contained approximately 4-fold more DYSF than the basal membrane, suggesting polarized trafficking. Unlike skeletal muscle, DYSF in the STB is localized to the plasma membrane in the absence of caveolin. DYSF expression in the STB was developmentally regulated, because first-trimester placentas expressed approximately 3-fold more DYSF than term placentas. As the current literature indicates that few cell types express DYSF, it is of interest that the two major syncytial structures in the human body, skeletal muscle and the STB, express this protein.     

Id-1 delays senescence but does not immortalize keratinocytes

Defining the molecular basis responsible for regulating the proliferative potential of keratinocytes has important implications for normal homeostasis and neoplasia of the skin. Under current culture conditions, neonatal foreskin-derived human keratinocytes possess a relatively short replicative lifespan. Recently it was reported that forced overexpression of the helix-loop-helix protein Id-1 was capable of immortalizing keratinocytes, secondary to activation of telomerase activity and suppression of p16/Rb-mediated growth arrest pathways. To investigate the relationship between Id-1, telomerase activity, telomere length, p16, Rb cell cycle regulators, and senescence, whole populations of keratinocytes were infected with a retrovirus to induce overexpression of Id-1. In these unselected cultures, enhanced Id-1 levels clearly extended the lifespan of keratinocytes, but Id-1 did not prevent the onset of replicative senescence. Under these experimental conditions, Id-1 expression did not trigger induction of telomerase activity, and there was progressive shortening of the telomeres that was accompanied by elevated p16 levels and prevalence of active Rb. The ability of Id-1 to postpone, but not prevent, senescence may be related to partial inhibition of p16 expression, as the Id-1-overexpressing cultures displayed a decreased capacity for 12-O-tetradecanoylphorbol-13-acetate-mediated p16 induction. Thus, while no immortalization was observed, Id-1 could delay the onset of replicative senescence in unselected human keratinocyte populations.     

Soil Particle Heterogeneity Affects the Growth of a Rhizomatous Wetland Plant

Soil is commonly composed of particles of different sizes, and soil particle size may greatly affect the growth of plants because it affects soil physical and chemical properties. However, no study has tested the effects of soil particle heterogeneity on the growth of clonal plants. We conducted a greenhouse experiment in which individual ramets of the wetland plant Bolboschoenus planiculmis were grown in three homogeneous soil treatments with uniformly sized quartz particles (small: 0.75 mm, medium: 1.5 mm, or large: 3 mm), one homogeneous treatment with an even mixture of large and medium particles, and two heterogeneous treatments consisting of 16 or 4 patches of large and medium particles. Biomass, ramet number, rhizome length and spacer length were significantly greater in the treatment with only medium particles than in the one with only large particles. Biomass, ramet number, rhizome length and tuber number in the patchy treatments were greater in patches of medium than of large particles; this difference was more pronounced when patches were small than when they were large. Soil particle size and soil particle heterogeneity can greatly affect the growth of clonal plants. Thus, studies to test the effects of soil heterogeneity on clonal plants should distinguish the effects of nutrient heterogeneity from those of particle heterogeneity.