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Cell Surface Heparan Sulfate Proteoglycans: Selective Regulators of Ligand-Receptor Encounters

Cell surface heparan sulfate proteoglycans (HSPGs), substantially more abundant than most receptors, modulate encounters of extracellular protein ligands with their receptors by forming HSprotein complexes. Two gene families account for most cell surface HSPGs. Both consist of discrete core proteins covalently attached to two or three chains of HS, an Nand O-sulfated linear polysaccharide of repeating disaccharides containing N-acetylglucosamine (GlcNAc) and uronic acid (glucuronic acid (GlcA) or iduronic acid (IdoUA)). The syndecan family was the first discovered, which in mammals contains four gene products with distinctive extracellular domains (ectodomains) and highly conserved short cytoplasmic domains. These apparently extended proteins place the HS chains distal from the plasma membrane (1, 2). The syndecan family contrasts with the glypican family, which in mammals contains six gene products that are covalently linked to plasma membrane lipid by glycosylphosphatidylinositol anchor (1, 3). The glypican core proteins contain six invariant disulfide bonds, are likely to be globular, and place HS chains adjacent to the plasma membrane. Expression of both the syndecans and glypicans is extensively regulated during mouse embryogenesis and results in discrete adult expression patterns for each HSPG such that every adherent cell exhibits a distinct repertoire of cell surface HSPGs. Binding to HS chains is remarkably widespread among extracellular proteins, especially matrix proteins, proteases and their inhibitors, lipases, lipoproteins, growth factors and their binding proteins, cytokines, chemokines, collectins, and antimicrobial peptides. These proteins are involved in morphogenesis, tissue repair, energy balance, and host defense (Fig. 1). Additionally, numerous pathogens (e.g. herpes simplex virus, Neisseria, Plasmodium) bind to the cell surface via HS (4). Importantly, many of these ligand-HS interactions are essentially identical from Drosophila to the mouse, including those involved in generation of the basic metazoan body plan, e.g. dpp (bone morphogenetic proteins 2–4), wg (Wnt-1), and sog (chordin). Formation of the complexes can enhance or reduce receptor activation, often depending on the concentrations of ligand, receptor, and HSPG. The HS chains catalyze encounters between ligand and signaling receptor by bringing them together. Because binding to the HS chain reduces the dimensionality of this interaction from three (when the ligand is soluble) to two (when the ligand is bound to the HS chain), interaction could result from a localized increase in ligand concentration at optimal HS concentrations (5). However, at HS levels lower or higher than optimal, the effective ligand concentration for engaging the receptor will fall, potentially accounting for the bell-shaped activity curve typically seen experimentally when HSPG (or heparin) concentrations are varied. The curve may be concave or convex depending on whether ligand binding to the HSPG is inhibitory or stimulatory (6). Furthermore, the cytoplasmic domains of the syndecans also form complexes with cytoplasmic enzymes and scaffolding proteins, adding to the modulating influence of these proteoglycans (7). The HS chains are structurally diverse by virtue of their biosynthesis. A non-sulfated repeating disaccharide precursor is generated while attached to a core protein and is then sequentially modified by a variety of enzymes in reactions that do not go to completion. The details of this biosynthetic scheme have been recently reviewed in this series (8). Three major characteristics of this scheme produce HS chains with selectivity for protein binding. First, the process yields an extraordinary variety of saccharide sequences. Second, clustering of the modifications along the HS chain yields highly N-sulfated domains (NS domains) of approximately 12–20 residues that alternate with typically larger sized, relatively unmodified N-acetyl-rich domains (NA domains). The NS domains are rich in IdoUA, which can assume several different conformations and thus influence the orientation of the sulfate residues in space. This domain organization places relatively flexible NA domains adjacent to relatively rigid NS domains, thus facilitating protein interactions with the sulfate residues. Finally, this microsequence diversity and macro-organization are cell typespecific and do not appear to be core protein-specific (e.g. HS chains of syndecan-1 and -4 from mammary epithelia are indistinguishable), presumably the result of cell type-specific repertoires of the HS chain-modifying enzymes. Distinct oligosaccharide sequences in HS chains are recognized by the various proteins whose function depends on this interaction. The best characterized of these interactions is the recognition of a specific pentasaccharide sequence by antithrombin III (9). FGF-2 binds most tightly to a specific hexasaccharide sequence, but an additional 4–6 sugar residues are required to activate the receptor (10). Specific oligosaccharide binding sequences are known for multiple ligands (11–14); however, there is no universal consensus amino acid sequence for protein binding to HS chains. Most studies suggest that multiple arginine and/or lysine residues aligned on the protein surface accommodate a distinctive array of anionic sites on the HS chain (14).

Transgenic expression of syndecan-1 uncovers a physiological control of feeding behavior by syndecan-3.

Transgenic expression in the hypothalamus of syndecan-1, a cell surface heparan sulfate proteoglycan (HSPG) and modulator of ligand-receptor encounters, produces mice with hyperphagia and maturity-onset obesity resembling mice with reduced action of alpha melanocyte stimulating hormone (alphaMSH). Via their HS chains, syndecans potentiate the action of agouti-related protein and agouti signaling protein, endogenous inhibitors of alphaMSH. In wild-type mice, syndecan-3, the predominantly neural syndecan, is expressed in hypothalamic regions that control energy balance. Food deprivation increases hypothalamic syndecan-3 levels several-fold. Syndecan-3 null mice, otherwise apparently normal, respond to food deprivation with markedly reduced reflex hyperphagia. We propose that oscillation of hypothalamic syndecan-3 levels physiologically modulates feeding behavior.

Peripheral, but Not Central, CB1 Antagonism Provides Food Intake–Independent Metabolic Benefits in Diet-Induced Obese Rats

OBJECTIVE—Blockade of the CB1 receptor is one of the promising strategies for the treatment of obesity. Although antagonists suppress food intake and reduce body weight, the role of central versus peripheral CB1 activation on weight loss and related metabolic parameters remains to be elucidated. We therefore specifically assessed and compared the respective potential relevance of central nervous system (CNS) versus peripheral CB1 receptors in the regulation of energy homeostasis and lipid and glucose metabolism in diet-induced obese (DIO) rats. RESEARCH DESIGN AND METHODS—Both lean and DIO rats were used for our experiments. The expression of key enzymes involved in lipid metabolism was measured by real-time PCR, and euglycemic-hyperinsulinemic clamps were used for insulin sensitivity and glucose metabolism studies. RESULTS—Specific CNS-CB1 blockade decreased body weight and food intake but, independent of those effects, had no beneficial influence on peripheral lipid and glucose metabolism. Peripheral treatment with CB1 antagonist (Rimonabant) also reduced food intake and body weight but, in addition, independently triggered lipid mobilization pathways in white adipose tissue and cellular glucose uptake. Insulin sensitivity and skeletal muscle glucose uptake were enhanced, while hepatic glucose production was decreased during peripheral infusion of the CB1 antagonist. However, these effects depended on the antagonist-elicited reduction of food intake. CONCLUSIONS—Several relevant metabolic processes appear to independently benefit from peripheral blockade of CB1, while CNS-CB1 blockade alone predominantly affects food intake and body weight.

Leptin deficiency suppresses MMTV-Wnt-1 mammary tumor growth in obese mice and abrogates tumor initiating cell survival

Obesity increases both the risk and mortality associated with many types of cancer including that of the breast. In mice, obesity increases both incidence of spontaneous tumors and burden of transplanted tumors. Our findings identify leptin, an adipose secreted cytokine, in promoting increased mammary tumor burden in obese mice and provide a link between this adipokine and cancer. Using a transplantable tumor that develops spontaneously in the murine mammary tumor virus-Wnt-1 transgenic mice, we show that tumors transplanted into obese leptin receptor (LepRb)-deficient (db/db) mice grow to eight times the volume of tumors transplanted into lean wild-type (WT) mice. However, tumor outgrowth and overall tumor burden is reduced in obese, leptin-deficient (ob/ob) mice. The residual tumors in ob/ob mice contain fewer undifferentiated tumor cells (keratin 6 immunopositive) compared with WT or db/db mice. Furthermore, tumors in ob/ob mice contain fewer cells expressing phosphorylated Akt, a growth promoting kinase activated by the LepRb, compared with WT and db/db mice. In vivo limiting dilution analysis of residual tumors from ob/ob mice indicated reduced tumor initiating activity suggesting fewer cancer stem cells (CSCs). The tumor cell populations reduced by leptin deficiency were identified by fluorescence-activated cell sorting and found to express LepRb. Finally, LepRb expressing tumor cells exhibit stem cell characteristics based on the ability to form tumorspheres in vitro and leptin promotes their survival. These studies provide critical new insight on the role of leptin in tumor growth and implicate LepRb as a CSC target.

Mice lacking the syndecan-3 gene are resistant to diet-induced obesity

The accurate matching of caloric intake to caloric expenditure involves a complex system of peripheral signals and numerous CNS neurotransmitter systems. Syndecans are a family of membrane-bound heparan sulfate proteoglycans that modulate ligand-receptor interactions. Syndecan-3 is heavily expressed in several areas of the brain, including hypothalamic nuclei, which are known to regulate energy balance. In particular, syndecans have been implicated in modulation of the activity of the melanocortin system, which potently regulates energy intake, energy expenditure, and peripheral glucose metabolism. Our data demonstrate that syndecan-3-null mice have reduced adipose content compared with wild-type mice. On a high-fat diet, syndecan-3-null male and female mice exhibited a partial resistance to obesity due to reduced food intake in males and increased energy expenditure in females relative to that of wild-type mice. As a result, syndecan-3-null mice on a high-fat diet accumulated less adipose mass and showed improved glucose tolerance compared with wild-type controls. The data implicate syndecan-3 in the regulation of body weight and suggest that inhibition of syndecan-3 may provide a therapeutic approach for the treatment of obesity resulting from exposure to high-fat diets.

Myosin II isoform switching mediates invasiveness after TGF-β–induced epithelial–mesenchymal transition

Despite functional significance of nonmuscle myosin II in cell migration and invasion, its role in epithelial–mesenchymal transition (EMT) or TGF-β signaling is unknown. Analysis of normal mammary gland expression revealed that myosin IIC is expressed in luminal cells, whereas myosin IIB expression is up-regulated in myoepithelial cells that have more mesenchymal characteristics. Furthermore, TGF-β induction of EMT in nontransformed murine mammary gland epithelial cells results in an isoform switch from myosin IIC to myosin IIB and increased phosphorylation of myosin heavy chain (MHC) IIA on target sites known to regulate filament dynamics (S1916, S1943). These expression and phosphorylation changes are downstream of heterogeneous nuclear ribonucleoprotein-E1 (E1), an effector of TGF-β signaling. E1 knockdown drives cells into a migratory, invasive mesenchymal state and concomitantly up-regulates MHC IIB expression and MHC IIA phosphorylation. Abrogation of myosin IIB expression in the E1 knockdown cells has no effect on 2D migration but significantly reduced transmigration and macrophage-stimulated collagen invasion. These studies indicate that transition between myosin IIC/myosin IIB expression is a critical feature of EMT that contributes to increases in invasive behavior.

Syndecan‐3 Modulates Food Intake by Interacting with the Melanocortin/AgRP Pathway

Abstract: Syndecan‐3, expressed in the developing nervous system and adult brain, alters feeding behavior through its interaction with the CNS melanocortin system, which provides critical tonic inhibition of both food intake and body adipose stores. A variety of both in vitro and transgenic data supports the hypothesis that syndecan‐3 modulates melanocortin activity via syndecan‐3 facilitation of agouti‐related protein (AgRP), a competitive antagonist of α‐melanocyte‐stimulating hormone (α‐MSH) at the melanocortin‐3 and ‐4 receptors. Consistent with this hypothesis, mice lacking syndecan‐3, which therefore would be predicted to have less effective AgRP, are more sensitive to inhibition of food intake by the melanocortin agonist MTII. Additionally, we took advantage of the fact that syndecan‐3 facilitation of AgRP is limited to when it is bound to the cell membrane. Pharmacologic inhibition of the enzyme that cleaves syndecan‐3 from the cell membrane leads to increased food intake in fasted rats, which have elevated levels of AgRP. Furthermore, the shedding process appears to be regulated under physiologic conditions, because a putative inhibitor of the shedding process, tissue inhibitor of metalloprotease‐3 (TIMP‐3), is increased by food deprivation. These observations contribute to the hypothesis that syndecan‐3 regulation of melanocortin signaling contributes to the normal control of energy balance. Collectively, the data suggest that the modulation of melanocortin regulation of energy balance by syndecan‐3 is modulated by the action of a TIMP‐3‐sensitive metalloprotease.

Leptin receptor maintains cancer stem-like properties in triple negative breast cancer cells

Despite new therapies, breast cancer continues to be the second leading cause of cancer mortality in women, a consequence of recurrence and metastasis. In recent years, a population of cancer cells has been identified, called cancer stem cells (CSCs) with self-renewal capacity, proposed to underlie tumor recurrence and metastasis. We previously showed that the adipose tissue cytokine LEPTIN, increased in obesity, promotes the survival of CSCs in vivo. Here, we tested the hypothesis that the leptin receptor (LEPR), expressed in mammary cancer cells, is necessary for maintaining CSC-like and metastatic properties. We silenced LEPR via shRNA lentivirus transduction and determined that the expression of stem cell self-renewal transcription factors NANOG, SOX2, and OCT4 (POU5F1) is inhibited. LEPR-NANOG signaling pathway is conserved between species because we can rescue NANOG expression in human LEPR-silenced cells with the mouse LepR. Using a NANOG promoter GFP reporter, we showed that LEPR is enriched in NANOG promoter active (GFP+) cells. In lineage tracing studies, we showed that the GFP+ cells divide in a symmetric and asymmetric manner. LEPR-silenced MDA-MB-231 cells exhibit a mesenchymal to epithelial transition morphologically, increased E-CADHERIN and decreased VIMENTIN expression compared with control cells. Finally, LEPR-silenced cells exhibit reduced cell proliferation, self-renewal in tumor sphere assays, and tumor outgrowth in xenotransplant studies. Given the emergence of NANOG as a pro-carcinogenic protein in multiple cancers, these studies suggest that inhibition of LEPR may be a promising therapeutic approach to inhibit NANOG and thereby neutralize CSC functions.

Differential Expression of Proteoglycans at Central and Peripheral Nodes of Ranvier

The nodes of Ranvier are regularly spaced gaps between myelin sheaths that are markedly enriched in voltage‐gated sodium channels and associated proteins. Myelinating glia play a key role in promoting node formation, although the requisite glial signals remain poorly understood. In this study, we have examined the expression of glial proteoglycans in the peripheral and central nodes. We report that the heparan sulfate proteoglycan, syndecan‐3, becomes highly enriched with PNS node formation; its ligand, collagen V, is also concentrated at the PNS nodes and at lower levels along the abaxonal membrane. The V1 isoform of versican, a chondroitin sulfate proteoglycan, is also present in the nodal gap. By contrast, CNS nodes are enriched in versican isoform V2, but not syndecan‐3. We have examined the molecular composition of the PNS nodes in syndecan‐3 knockout mice. Nodal components are normally expressed in mice deficient in syndecan‐3, suggesting that it has a nonessential role in the organization of nodes in the adult. These results indicate that the molecular composition and extracellular environment of the PNS and CNS nodes of Ranvier are significantly distinct.

Diet-induced Obese Mice Are Leptin Insufficient After Weight Reduction

Behavioral therapies aimed at reducing excess body fat result in limited fat loss after dieting. To understand the causes for maintenance of adiposity, high‐fat (HF) diet–induced obese (DIO) mice were switched to a low‐fat chow diet, and the effects of chow on histological and molecular alterations of adipose tissue and metabolic parameters were examined. DIO mice reduced and stabilized their body weights after being switched to chow (HF‐chow), but retained a greater amount of adiposity than chow‐fed mice. Reduction in adipocyte volume, not number, caused a decrease in fat mass. HF‐chow mice showed normalized circulating insulin and leptin levels, improved glucose tolerance, and reduced inflammatory status in white adipose tissue (WAT). Circulating leptin levels corrected for fat mass were lower in HF‐chow mice. Leptin administration was used to test whether reduced leptin level of HF‐chow mice inhibited further fat loss. Leptin treatment led to an additional reduction in adiposity. Finally, HF‐HF mice had lower mRNA levels of β3 adrenergic receptor (β3‐AR) in epididymal WAT (EWAT) compared to chow‐fed mice, and diet change led to an increase in the WAT β3‐AR mRNA levels that were similar to the levels of chow‐fed mice, suggesting an elevation in sympathetic activation of WAT during diet switch relative to HF‐HF mice leading to the reduced leptin level and proinflammatory cytokine content. In summary, HF‐chow mice were resistant to further fat loss due to leptin insufficiency. Diet alteration from HF to low fat improved metabolic state of DIO mice, although their adiposity was defended at a higher level.