Structural and Mechanistic Insights into Lunatic Fringe from a Kinetic Analysis of Enzyme Mutants

The Notch receptor is critical for proper development where it orchestrates numerous cell fate decisions. The Fringe family of β1,3-N-acetylglucosaminyltransferases are regulators of this pathway. Fringe enzymes add N-acetylglucosamine to O-linked fucose on the epidermal growth factor repeats of Notch. Here we have analyzed the reaction catalyzed by Lunatic Fringe (Lfng) in detail. A mutagenesis strategy for Lfng was guided by a multiple sequence alignment of Fringe proteins and solutions from docking an epidermal growth factor-like O-fucose acceptor substrate onto a homology model of Lfng. We targeted three main areas as follows: residues that could help resolve where the fucose binds, residues in two conserved loops not observed in the published structure of Manic Fringe, and residues predicted to be involved in UDP-N-acetylglucosamine (UDP-GlcNAc) donor specificity. We utilized a kinetic analysis of mutant enzyme activity toward the small molecule acceptor substrate 4-nitrophenyl-α-l-fucopyranoside to judge their effect on Lfng activity. Our results support the positioning of O-fucose in a specific orientation to the catalytic residue. We also found evidence that one loop closes off the active site coincident with, or subsequent to, substrate binding. We propose a mechanism whereby the ordering of this short loop may alter the conformation of the catalytic aspartate. Finally, we identify several residues near the UDP-GlcNAc-binding site, which are specifically permissive toward UDP-GlcNAc utilization.Defects in Notch signaling have been implicated in numerous human diseases, including multiple sclerosis (1), several forms of cancer (2-4), cerebral autosomal dominant arteriopathy with sub-cortical infarcts and leukoencephalopathy (5), and spondylocostal dysostosis (SCD)³ (6-8). The transmembrane Notch signaling receptor is activated by members of the DSL (Delta, Serrate, Lag2) family of ligands (9, 10). In the endoplasmic reticulum, O-linked fucose glycans are added to the epidermal growth factor-like (EGF) repeats of the Notch extracellular domain by protein O-fucosyltransferase 1 (11-13). These O-fucose monosaccharides can be elongated in the Golgi apparatus by three highly conserved β1,3-N-acetylglucosaminyltransferases of the Fringe family (Lunatic (Lfng), Manic (Mfng), and Radical Fringe (Rfng) in mammals) (14-16). The formation of this GlcNAc-β1,3-Fuc-α1, O-serine/threonine disaccharide is necessary and sufficient for subsequent elongation to a tetrasaccharide (15, 19), although elongation past the disaccharide in Drosophila is not yet clear (20, 21). Elongation of O-fucose by Fringe is known to potentiate Notch signaling from Delta ligands and inhibit signaling from Serrate ligands (22). Delta ligands are termed Delta-like (Delta-like1, -2, and -4) in mammals, and the homologs of Serrate are known as Jagged (Jagged1 and -2) in mammals. The effects of Fringe on Drosophila Notch can be recapitulated in Notch ligand in vitro binding assays using purified components, suggesting that the elongation of O-fucose by Fringe alters the binding of Notch to its ligands (21). Although Fringe also appears to alter Notch-ligand interactions in mammals, the effects of elongation of the glycan past the O-fucose monosaccharide is more complicated and appears to be cell type-, receptor-, and ligand-dependent (for a recent review see Ref. 23).The Fringe enzymes catalyze the transfer of GlcNAc from the donor substrate UDP-α-GlcNAc to the acceptor fucose, forming the GlcNAc-β1,3-Fuc disaccharide (14-16). They belong to the GT-A-fold of inverting glycosyltransferases, which includes N-acetylglucosaminyltransferase I and β1,4-galactosyltransferase I (17, 18). The mechanism is presumed to proceed through the abstraction of a proton from the acceptor substrate by a catalytic base (Asp or Glu) in the active site. This creates a nucleophile that attacks the anomeric carbon of the nucleotide-sugar donor, inverting its configuration from α (on the nucleotide sugar) to β (in the product) (24, 25). The enzyme then releases the acceptor substrate modified with a disaccharide and UDP. The Mfng structure (26) leaves little doubt as to the identity of the catalytic residue, which in all likelihood is aspartate 289 in mouse Lfng (we will use numbering for mouse Lunatic Fringe throughout, unless otherwise stated). The structure of Mfng with UDP-GlcNAc soaked into the crystals (26) showed density only for the UDP portion of the nucleotide-sugar donor and no density for two loops flanking either side of the active site. The presence of flexible loops that become ordered upon substrate binding is a common observation with glycosyltransferases in the GT-A fold family (18, 25). Density for the entire donor was observed in the structure of rabbit N-acetylglucosaminyltransferase I (27). In this case, ordering of a previously disordered loop upon UDP-GlcNAc binding may have contributed to increased stability of the donor. In the case of bovine β1,4-galactosyltransferase I, a section of flexible random coil from the apo-structure was observed to change its conformation to α-helical upon donor substrate binding (28). Both loops in Lfng are highly conserved, and we have mutated a number of residues in each to test the hypothesis that they interact with the substrates. The mutagenesis strategy was also guided by docking of an EGF-O-fucose acceptor substrate into the active site of the Lfng model as well as comparison of the Lfng model with a homology model of the β1,3-glucosyltransferase (β3GlcT) that modifies O-fucose on thrombospondin type 1 repeats (29, 30). The β3GlcT is predicted to be a GT-A fold enzyme related to the Fringe family (17, 18, 29).     

The Cholinesterase-like Domain, Essential in Thyroglobulin Trafficking for Thyroid Hormone Synthesis, Is Required for Protein Dimerization

The carboxyl-terminal cholinesterase-like (ChEL) domain of thyroglobulin (Tg) has been identified as critically important in Tg export from the endoplasmic reticulum. In a number of human kindreds suffering from congenital hypothyroidism, and in the cog congenital goiter mouse and rdw rat dwarf models, thyroid hormone synthesis is inhibited because of mutations in the ChEL domain that block protein export from the endoplasmic reticulum. We hypothesize that Tg forms homodimers through noncovalent interactions involving two predicted α-helices in each ChEL domain that are homologous to the dimerization helices of acetylcholinesterase. This has been explored through selective epitope tagging of dimerization partners and by inserting an extra, unpaired Cys residue to create an opportunity for intermolecular disulfide pairing. We show that the ChEL domain is necessary and sufficient for Tg dimerization; specifically, the isolated ChEL domain can dimerize with full-length Tg or with itself. Insertion of an N-linked glycan into the putative upstream dimerization helix inhibits homodimerization of the isolated ChEL domain. However, interestingly, co-expression of upstream Tg domains, either in cis or in trans, overrides the dimerization defect of such a mutant. Thus, although the ChEL domain provides a nidus for Tg dimerization, interactions of upstream Tg regions with the ChEL domain actively stabilizes the Tg dimer complex for intracellular transport.The synthesis of thyroid hormone in the thyroid gland requires secretion of thyroglobulin (Tg)² to the apical luminal cavity of thyroid follicles (1). Once secreted, Tg is iodinated via the activity of thyroid peroxidase (2). A coupling reaction involving a quinol-ether linkage especially engages di-iodinated tyrosyl residues 5 and 130 to form thyroxine within the amino-terminal portion of the Tg polypeptide (3, 4). Preferential iodination of Tg hormonogenic sites is dependent not on the specificity of the peroxidase (5) but upon the native structure of Tg (6, 7). To date, no other thyroidal proteins have been shown to effectively substitute in this role for Tg.The first 80% of the primary structure of Tg (full-length murine Tg: 2,746 amino acids) involves three regions called I-II-III comprised of disulfide-rich repeat domains held together by intradomain disulfide bonds (8, 9). The final 581 amino acids of Tg are strongly homologous to acetylcholinesterase (10–12). Rate-limiting steps in the overall process of Tg secretion involve its structural maturation within the endoplasmic reticulum (ER) (13). Interactions between regions I-II-III and the cholinesterase-like (ChEL) domain have recently been suggested to be important in this process, with ChEL functioning as an intramolecular chaperone and escort for I-II-III (14). In addition, Tg conformational maturation culminates in Tg homodimerization (15, 16) with progression to a cylindrical, and ultimately, a compact ovoid structure (17–19).In human congenital hypothyroidism with deficient Tg, the ChEL domain is a commonly affected site of mutation, including the recently described A2215D (20, 21), R2223H (22), G2300D, R2317Q (23), G2355V, G2356R, and the skipping of exon 45 (which normally encodes 36 amino acids), as well as the Q2638stop mutant (24) (in addition to polymorphisms including P2213L, W2482R, and R2511Q that may be associated with thyroid overgrowth (25)). As best as is currently known, all of the congenital hypothyroidism-inducing Tg mutants are defective for intracellular transport (26). A homozygous G2300R mutation (equivalent to residue 2,298 of mouse Tg) in the ChEL domain is responsible for congenital hypothyroidism in rdw rats (27, 28), whereas we identified the Tg-L2263P point mutation as the cause of hypothyroidism in the cog mouse (29). Such mutations perturb intradomain structure (30), and interestingly, block homodimerization (31). Acquisition of quaternary structure has long been thought to be required for efficient export from the ER (32) as exemplified by authentic acetylcholinesterase (33, 34) in which dimerization enhances protein stability and export (35).Tg comprised only of regions I-II-III (truncated to lack the ChEL domain) is blocked within the ER (30), whereas a secretory version of the isolated ChEL domain of Tg devoid of I-II-III undergoes rapid and efficient intracellular transport and secretion (14). A striking homology positions two predicted α-helices of the ChEL domain to the identical relative positions of the dimerization helices in acetylcholinesterase. This raises the possibility that ChEL may serve as a homodimerization domain for Tg, providing a critical function in maturation for Tg transport to the site of thyroid hormone synthesis (1).In this study, we provide unequivocal evidence for homodimerization of the ChEL domain and “hetero”-dimerization of that domain with full-length Tg, and we provide significant evidence that the predicted ChEL dimerization helices provide a nidus for Tg assembly. On the other hand, our data also suggest that upstream Tg regions known to interact with ChEL (14) actively stabilize the Tg dimer complex. Together, I-II-III and ChEL provide unique contributions to the process of intracellular transport of Tg through the secretory pathway.     

Structural and Biochemical Characterization of the Type II Fructose-1,6-bisphosphatase GlpX from Escherichia coli

Gluconeogenesis is an important metabolic pathway, which produces glucose from noncarbohydrate precursors such as organic acids, fatty acids, amino acids, or glycerol. Fructose-1,6-bisphosphatase, a key enzyme of gluconeogenesis, is found in all organisms, and five different classes of these enzymes have been identified. Here we demonstrate that Escherichia coli has two class II fructose-1,6-bisphosphatases, GlpX and YggF, which show different catalytic properties. We present the first crystal structure of a class II fructose-1,6-bisphosphatase (GlpX) determined in a free state and in the complex with a substrate (fructose 1,6-bisphosphate) or inhibitor (phosphate). The crystal structure of the ligand-free GlpX revealed a compact, globular shape with two α/β-sandwich domains. The core fold of GlpX is structurally similar to that of Li⁺-sensitive phosphatases implying that they have a common evolutionary origin and catalytic mechanism. The structure of the GlpX complex with fructose 1,6-bisphosphate revealed that the active site is located between two domains and accommodates several conserved residues coordinating two metal ions and the substrate. The third metal ion is bound to phosphate 6 of the substrate. Inorganic phosphate strongly inhibited activity of both GlpX and YggF, and the crystal structure of the GlpX complex with phosphate demonstrated that the inhibitor molecule binds to the active site. Alanine replacement mutagenesis of GlpX identified 12 conserved residues important for activity and suggested that Thr⁹⁰ is the primary catalytic residue. Our data provide insight into the molecular mechanisms of the substrate specificity and catalysis of GlpX and other class II fructose-1,6-bisphosphatases.Fructose-1,6-bisphosphatase (FBPase,² EC 3.1.3.11), a key enzyme of gluconeogenesis, catalyzes the hydrolysis of fructose 1,6-bisphosphate to form fructose 6-phosphate and orthophosphate. It is the reverse of the reaction catalyzed by phosphofructokinase in glycolysis, and the product, fructose 6-phosphate, is an important precursor in various biosynthetic pathways (1). In all organisms, gluconeogenesis is an important metabolic pathway that allows the cells to synthesize glucose from noncarbohydrate precursors, such as organic acids, amino acids, and glycerol. FBPases are members of the large superfamily of lithium-sensitive phosphatases, which includes three families of inositol phosphatases and FBPases (the phosphoesterase clan CL0171, 3167 sequences, Pfam data base). These enzymes show metal-dependent and lithium-sensitive phosphomonoesterase activity and include inositol polyphosphate 1-phosphatases, inositol monophosphatases (IMPases), 3′-phosphoadenosine 5′-phosphatases (PAPases), and enzymes acting on both inositol 1,4-bisphosphate and PAP (PIPases) (2). They possess a common structural core with the active site lying between α+β and α/β domains (3). Li⁺-sensitive phosphatases are putative targets for lithium therapy in the treatment of manic depressive patients (4), whereas FBPases are targets for the development of drugs for the treatment of noninsulin-dependent diabetes (5, 6). In addition, FBPase is required for virulence in Mycobacterium tuberculosis and Leishmania major and plays an important role in the production of lysine and glutamate by Corynebacterium glutamicum (7, 8).Presently, five different classes of FBPases have been proposed based on their amino acid sequences (FBPases I to V) (9–11). Eukaryotes contain only the FBPase I-type enzyme, but all five types exist in various prokaryotes. Types I, II, and III are primarily in bacteria, type IV in archaea (a bifunctional FBPase/inositol monophosphatase), and type V in thermophilic prokaryotes from both domains (11). Many organisms have more than one FBPase, mostly the combination of types I + II or II + III, but no bacterial genome has a combination of types I and III FBPases (9). The type I FBPase is the most widely distributed among living organisms and is the primary FBPase in Escherichia coli, most bacteria, a few archaea, and all eukaryotes (9, 11–15). The type II FBPases are represented by the E. coli GlpX and FBPase F-I from Synechocystis PCC6803 (9, 16); type III is represented by the Bacillus subtilis FBPase (17); type IV is represented by the dual activity FBPases/inosine monophosphatases FbpA from Pyrococcus furiosus (18), MJ0109 from Methanococcus jannaschii (19), and AF2372 from Archaeoglobus fulgidus (20); and type V is represented by the FBPases TK2164 from Pyrococcus (Thermococcus) kodakaraensis and ST0318 from Sulfolobus tokodai (10, 21).Three-dimensional structures of the type I (from pig kidney, spinach chloroplasts, and E. coli), type IV (MJ0109 and AF2372), and type V (ST0318) FBPases have been solved (10, 11, 19, 20, 22, 23). FBPases I and IV and inositol monophosphatases share a common sugar phosphatase fold organized in five layered interleaved α-helices and β-sheets (α-β-α-β-α) (2, 19, 24). ST0318 (an FBPase V enzyme) is composed of one domain with a completely different four-layer α-β-β-α fold (10). The FBPases from these three classes (I, IV, and V) require divalent cations for activity (Mg²⁺, Mn²⁺, or Zn²⁺), and their structures have revealed the presence of three or four metal ions in the active site.E. coli has five Li⁺-sensitive phosphatases as follows: CysQ (a PAPase), SuhB (an IMPase), Fbp (a FBPase I enzyme), GlpX (a FBPase II), and YggF (an uncharacterized protein) (see the Pfam data base). CysQ is a 3′-phosphoadenosine 5′-phosphatase involved in the cysteine biosynthesis pathway (25, 26), whereas SuhB is an inositol monophosphatase (IMPase) that is also known as a suppressor of temperature-sensitive growth phenotypes in E. coli (27, 28). Fbp is required for growth on gluconeogenic substrates and probably represents the main gluconeogenic FBPase (12). This enzyme has been characterized both biochemically and structurally and shown to be inhibited by low concentrations of AMP (IC₅₀ 15 μm) (11, 29, 30). The E. coli GlpX, a class II enzyme FBPase, has been shown to possess a Mn²⁺-dependent FBPase activity (9). The increased expression of glpX from a multicopy plasmid complemented the Fbp^- phenotype; however, the glpX knock-out strain grew normally on gluconeogenic substrates (succinate or glycerol) (9).In this study, we present the first structure of a class II FBPase, the E. coli GlpX, in a free state and in the complex with FBP + metals or phosphate. We have demonstrated that the fold of GlpX is similar to that of the lithium-sensitive phosphatases. We have identified the GlpX residues important for activity and proposed a catalytic mechanism. We have also showed that YggF is a third FBPase in E. coli, which has distinct catalytic properties and is more sensitive than GlpX to the inhibition by lithium or phosphate.     

Noonan Syndrome/Leukemia-associated Gain-of-function Mutations in SHP-2 Phosphatase (PTPN11) Enhance Cell Migration and Angiogenesis

Mutations in SHP-2 phosphatase (PTPN11) that cause hyperactivation of its catalytic activity have been identified in Noonan syndrome and various childhood leukemias. Recent studies suggest that the gain-of-function (GOF) mutations of SHP-2 play a causal role in the pathogenesis of these diseases. However, the molecular mechanisms by which GOF mutations of SHP-2 induce these phenotypes are not fully understood. Here, we show that GOF mutations in SHP-2, such as E76K and D61G, drastically increase spreading and migration of various cell types, including hematopoietic cells, endothelial cells, and fibroblasts. More importantly, in vivo angiogenesis in SHP-2 D61G knock-in mice is also enhanced. Mechanistic studies suggest that the increased cell migration is attributed to the enhanced β1 integrin outside-in signaling. In response to β1 integrin cross-linking or fibronectin stimulation, activation of ERK and Akt kinases is greatly increased by SHP-2 GOF mutations. Also, integrin-induced activation of RhoA and Rac1 GTPases is elevated. Interestingly, mutant cells with the SHP-2 GOF mutation (D61G) are more sensitive than wild-type cells to the suppression of cell motility by inhibition of these pathways. Collectively, these studies reaffirm the positive role of SHP-2 phosphatase in cell motility and suggest a new mechanism by which SHP-2 GOF mutations contribute to diseases.SHP-2, a multifunctional SH2 domain-containing protein-tyrosine phosphatase implicated in diverse cell signaling processes (1–3), plays a critical role in cellular function. Homozygous deletion of Exon 2 (4) or Exon 3 (5) of the SHP-2 gene (PTPN11) in mice leads to early embryonic lethality prior to and at midgestation, respectively. SHP-2 null mutant mice die much earlier, at peri-implantation (4). Exon 3 deletion mutation of SHP-2 blocks hematopoietic potential of embryonic stem cells both in vitro and in vivo (6–8), whereas SHP-2 null mutation causes inner cell mass death and diminished trophoblast stem cell survival (4). Recent studies on SHP-2 conditional knock-out or tissue-specific knock-out mice have further revealed an array of important functions of this phosphatase in various physiological processes (9–12). The phenotypes demonstrated by loss of SHP-2 function are apparently attributed to the role of SHP-2 in the cell signaling pathways induced by growth factors/cytokines. SHP-2 generally promotes signal transmission in growth factor/cytokine signaling in both catalytic-dependent and -independent fashion (1–3). The positive role of SHP-2 in the intracellular signaling processes, in particular, the ERK³ and PI3K/Akt kinase pathways, has been well established, although the underlying mechanism remains elusive, in particular, the signaling function of the catalytic activity of SHP-2 in these pathways is poorly understood.In addition to the role of SHP-2 in cell proliferation and differentiation, the phenotypes induced by loss of SHP-2 function may be associated with its role in cell migration. Indeed, dominant negative SHP-2 disrupts Xenopus gastrulation, causing tail truncations (13, 14). Targeted Exon 3 deletion mutation in SHP-2 results in decreased cell spreading, migration (15, 16), and impaired limb development in the chimeric mice (7). The role of SHP-2 in cell adhesion and migration has also been demonstrated by catalytically inactive mutant SHP-2-overexpressing cells (17–20). The molecular mechanisms by which SHP-2 regulates these cellular processes, however, have not been well defined. For example, the role of SHP-2 in the activation of the Rho family small GTPases that is critical for cell motility is still controversial. Both positive (19, 21, 22) and negative roles (18, 23) for SHP-2 in this context have been reported. Part of the reason for this discrepancy might be due to the difference in the cell models used. Catalytically inactive mutant SHP-2 was often used to determine the role of SHP-2 in cell signaling. In the catalytically inactive mutant SHP-2-overexpressing cells, the catalytic activity of endogenous SHP-2 is inhibited. However, as SHP-2 also functions independent of its catalytic activity, overexpression of catalytically deficient SHP-2 may also increase its scaffolding function, generating complex effects.The critical role of SHP-2 in cellular function is further underscored by the identification of SHP-2 mutations in human diseases. Genetic lesions in PTPN11 that cause hyperactivation of SHP-2 catalytic activity have been identified in the developmental disorder Noonan syndrome (24) and various childhood leukemias, including juvenile myelomonocytic leukemia (JMML), B cell acute lymphoblastic leukemia, and acute myeloid leukemia (25, 26). In addition, activating mutations in SHP-2 have been identified in sporadic solid tumors (27). The SHP-2 mutations appear to play a causal role in the development of these diseases as SHP-2 mutations and other JMML-associated Ras or Neurofibromatosis 1 mutations are mutually exclusive in the patients (24–27). Moreover, single SHP-2 gain-of-function (GOF) mutations are sufficient to induce Noonan syndrome, cytokine hypersensitivity in hematopoietic progenitor cells, and JMML-like myeloproliferative disease in mice (28–32). Gain-of-function cell models derived from the newly available SHP-2 GOF mutation (D61G) knock-in mice (28) now provide us with a good opportunity to clarify the role of SHP-2 in cell motility. Unlike the dominant negative approach in which overexpression of mutant forms of SHP-2 generates complex effects, the SHP-2 D61G knock-in model eliminates this possibility as the mutant SHP-2 is expressed at the physiological level (28). Additionally, defining signaling functions of GOF mutant SHP-2 in cell movement can also help elucidate the molecular mechanisms by which SHP-2 mutations contribute to the relevant diseases.     

Major Urinary Protein-1 Increases Energy Expenditure and Improves Glucose Intolerance through Enhancing Mitochondrial Function in Skeletal Muscle of Diabetic Mice

Major urinary protein-1 (MUP-1) is a low molecular weight secreted protein produced predominantly from the liver. Structurally it belongs to the lipocalin family, which carries small hydrophobic ligands such as pheromones. However, the physiological functions of MUP-1 remain poorly understood. Here we provide evidence demonstrating that MUP-1 is an important player in regulating energy expenditure and metabolism in mice. Both microarray and real-time PCR analysis demonstrated that the MUP-1 mRNA abundance in the liver of db/db obese mice was reduced by ∼30-fold compared with their lean littermates, whereas this change was partially reversed by treatment with the insulin-sensitizing drug rosiglitazone. In both dietary and genetic obese mice, the circulating concentrations of MUP-1 were markedly decreased compared with the lean controls. Chronic elevation of circulating MUP-1 in db/db mice, using an osmotic pump-based protein delivery system, increased energy expenditure and locomotor activity, raised core body temperature, and decreased glucose intolerance as well as insulin resistance. At the molecular level, MUP-1-mediated improvement in metabolic profiles was accompanied by increased expression of genes involved in mitochondrial biogenesis, elevated mitochondrial oxidative capacity, decreased triglyceride accumulation, and enhanced insulin-evoked Akt signaling in skeletal muscle but not in liver. Altogether, these findings raise the possibility that MUP-1 deficiency might contribute to the metabolic dysregulation in obese/diabetic mice, and suggest that the beneficial metabolic effects of MUP-1 are attributed in part to its ability in increasing mitochondrial function in skeletal muscle.The liver is the primary organ for carbohydrate and lipid metabolism, including gluconeogenesis, glycogenesis, cholesterol biosynthesis, and lipogenesis (1, 2). These metabolic events in the liver are tightly controlled by several pancreatic hormones including insulin and glucagon. In addition, the liver itself is one of the largest endocrine organs in the body, secreting numerous humoral factors involved in the regulation of systemic glucose and lipid homeostasis. The importance of the liver-derived humoral factors in maintaining glucose metabolism is highlighted by the observation that glucose uptake by skeletal muscle is severely impaired by surgical or pharmacological blockade of hepatic parasympathetic nerves (3). In the past several years, a number of liver-derived humoral metabolic factors, including bone morphogenetic protein-9 (BMP-9) (4), fibroblast growth factor 21 (FGF21) (5–7), retinol-binding protein 4 (RBP4) (8, 9), adropin (10), and angiopoietin-like proteins (Angptl) 3, 4, and 6 (11–13), have been identified, and their roles in glucose and lipid metabolism have been characterized in great detail. Noticeably, BMP-9, FGF21, and Angptl6 exhibit potent insulin-sensitizing and glucose-lowering effects in animal models, and they have been proposed as potential candidates for the treatment of insulin resistance and type II diabetes (4, 6, 7, 13).To search for novel liver-derived secretory factors involved in the regulation of glucose homeostasis, we used microarray analysis as a global screening for systematic identification of genes differentially expressed in the liver of C57BLKS db/db mice (a genetically inherited diabetic mouse model that is characterized by severe insulin resistance and hyperglycemia) and their lean littermates. We found that the mRNA level of mouse major urinary protein-1 (MUP-1)² was markedly down-regulated in db/db mice, and the change was largely normalized upon treatment with the PPARγ agonist rosiglitazone. MUP-1 is a small molecular weight secreted protein abundantly expressed in the liver (14). Its expression in the liver is enhanced by administration of the hepatotoxic agent dimethylnitrosamine (15) but is reduced by interleukin 6-induced acute phase response in mice (16). Like other members of the MUP family, MUP-1 has been proposed to act as a pheromone-binding protein in urine (17), thereby accelerating puberty and promoting aggressive behavior in male mice. However, the precise functions of MUPs have yet to be determined.MUP-1 belongs to the lipocalin superfamily, the members of which share a common tertiary structure with a cup-shaped hydrophobic ligand binding pocket surrounded by an eight-stranded β-barrel (18, 19). This structure confers upon lipocalins the ability to bind and transport a wide variety of small lipophilic substances, including fatty acids, cholesterols, prostaglandins, and pheromones. Noticeably, several members of the lipocalin family, including RBP4, lipocalin-2, and adipocyte fatty acid-binding protein (A-FABP), have recently been shown to be important mediators of obesity-related insulin resistance and glucose intolerance (8, 20–22). Unlike MUP-1, the expression of RBP4, lipocalin-2, and A-FABP are elevated in obesity and diabetes (9, 20, 23).In this study, we investigated the metabolic role of MUP-1 in mice. Our results demonstrated that MUP-1 was abundantly present in the circulation. In genetic and dietary obese mouse models, the serum and urine concentrations of MUP-1 were remarkably decreased. Replenishment of recombinant MUP-1 led to improved glucose tolerance and insulin sensitivity, as well as increased energy expenditure and locomotor activity in db/db diabetic mice. Our data suggest that MUP-1 not only serves as a circulating biomarker, negatively correlated with obesity-related metabolic disorders, but also plays an active role in regulating energy homeostasis and insulin sensitivity in mice.     

Neuroprotective Secreted Amyloid Precursor Protein Acts by Disrupting Amyloid Precursor Protein Dimers

The amyloid precursor protein (APP) is implied both in cell growth and differentiation and in neurodegenerative processes in Alzheimer disease. Regulated proteolysis of APP generates biologically active fragments such as the neuroprotective secreted ectodomain sAPPα and the neurotoxic β-amyloid peptide. Furthermore, it has been suggested that the intact transmembrane APP plays a signaling role, which might be important for both normal synaptic plasticity and neuronal dysfunction in dementia. To understand APP signaling, we tracked single molecules of APP using quantum dots and quantitated APP homodimerization using fluorescence lifetime imaging microscopy for the detection of Förster resonance energy transfer in living neuroblastoma cells. Using selective labeling with synthetic fluorophores, we show that the dimerization of APP is considerably higher at the plasma membrane than in intracellular membranes. Heparan sulfate significantly contributes to the almost complete dimerization of APP at the plasma membrane. Importantly, this technique for the first time structurally defines the initiation of APP signaling by binding of a relevant physiological extracellular ligand; our results indicate APP as receptor for neuroprotective sAPPα, as sAPPα binding disrupts APP dimers, and this disruption of APP dimers by sAPPα is necessary for the protection of neuroblastoma cells against starvation-induced cell death. Only cells expressing reversibly dimerized wild-type, but not covalently dimerized mutant APP are protected by sAPPα. These findings suggest a potentially beneficial effect of increasing sAPPα production or disrupting APP dimers for neuronal survival.The amyloid precursor protein (APP)⁴ is known both for its important role in the development and plasticity of the nervous system (1–6) and for its involvement in Alzheimer disease (AD) (7, 8). Despite intensive research efforts, the initial events that lead to the prevalent sporadic, i.e. non-familial, forms of AD are still unclear. Furthermore, although a higher gene dose of APP (9) or the presence of pathological APP mutations is sufficient to induce familial AD (for review, see Ref. 10), the exact pathological mechanism that is triggered by APP is still under debate.Some fragments of APP, such as the β-amyloid peptide (Aβ), are thought to contribute to synaptic dysfunction and neurotoxicity (11, 12). On the other hand, the α-secretase-derived extracellular fragment of APP (sAPPα), which is present at lower levels in AD patients than in controls (13), has been shown to be beneficial for memory function, to possess neuroprotective properties, and to counteract the effects of Aβ (14–18).Signaling by transmembrane APP may directly contribute to neurodegeneration in AD (19–24); however, the signal transduction pathway for transmembrane APP remains unknown, although several potential regulatory proteins, glycosaminoglycans, and metal ions are known to bind with high affinity to APP and sAPPα (25, 26). The most common form of signal transduction for single-pass transmembrane proteins is the ligand-induced perturbation of a monomer/dimer equilibrium. Indeed, the dimerization of transmembrane APP has been implied several times in the past. Several studies have investigated the effects of presumed dimer-breaking perturbations on biological read-outs, such as the production of Aβ (27, 28), but without directly measuring the APP aggregation state, or have investigated the aggregation state of APP subdomains, often reconstituted in cell-free systems (27–32). Dimerization interfaces in both the extracellular and the transmembrane domain have been suggested.In the studies investigating the aggregation state of full-length APP, most of the employed methods, such as chemical cross-linking and co-immunoprecipitation, do not lend themselves readily to a rigorous quantitative analysis of the abundance of potentially instable dimers (31, 33), whereas in other cases the use of chimeras may have influenced the dimerization potential or precluded the search for a natural stimulus (23, 34). The only previously reported direct observation of APP dimerization by Förster resonance energy transfer (FRET) microscopy uses an assay in which the FRET efficiency varies with the level of overexpression (35). Therefore, a concentration-dependent FRET component due to nonspecific stochastic encounters cannot be excluded in this study.Most importantly, as none of the published procedures permitted the selective detection of APP dimers on the surface of live cells, where they would encounter ligands, they could not differentiate between subpopulations of APP. This may be one reason why no natural ligand of APP has ever been shown to signal via modulation of its monomer/dimer equilibrium.Another elusive goal is the identity of the receptor for neuroprotective sAPPα (36–39). The ligand-dependent dimerization of sAPPα in solution (40) and its origination from transmembrane APP suggest that APP might serve as receptor for sAPPα, but this binding has never been experimentally shown.     

Overexpression of E-cadherin on Melanoma Cells Inhibits Chemokine-promoted Invasion Involving p190RhoGAP/p120ctn-dependent Inactivation of RhoA

Melanoma cells express the chemokine receptor CXCR4 that confers high invasiveness upon binding to its ligand CXCL12. Melanoma cells at initial stages of the disease show reduction or loss of E-cadherin expression, but recovery of its expression is frequently found at advanced phases. We overexpressed E-cadherin in the highly invasive BRO lung metastatic cell melanoma cell line to investigate whether it could influence CXCL12-promoted cell invasion. Overexpression of E-cadherin led to defective invasion of melanoma cells across Matrigel and type I collagen in response to CXCL12. A decrease in individual cell migration directionality toward the chemokine and reduced adhesion accounted for the impaired invasion. A p190RhoGAP-dependent inhibition of RhoA activation was responsible for the impairment in chemokine-stimulated E-cadherin melanoma transfectant invasion. Furthermore, we show that p190RhoGAP and p120ctn associated predominantly on the plasma membrane of cells overexpressing E-cadherin, and that E-cadherin-bound p120ctn contributed to RhoA inactivation by favoring p190RhoGAP-RhoA association. These results suggest that melanoma cells at advanced stages of the disease could have reduced metastatic potency in response to chemotactic stimuli compared with cells lacking E-cadherin, and the results indicate that p190RhoGAP is a central molecule controlling melanoma cell invasion.Cadherins are a family of Ca²⁺-dependent adhesion molecules that mediate cell-cell contacts and are expressed in most solid tissues providing a tight control of morphogenesis (1, 2). Classical cadherins, such as epithelial (E) cadherin, are found in adherens junctions, forming core protein complexes with β-catenin, α-catenin, and p120 catenin (p120ctn). Both β-catenin and p120ctn directly interact with E-cadherin, whereas α-catenin associates with the complex through its binding to β-catenin, providing a link with the actin cytoskeleton (1, 2). E-cadherin is frequently lost or down-regulated in many human tumors, coincident with morphological epithelial to mesenchymal transition and acquisition of invasiveness (3-6).Although melanoma only accounts for 5% of skin cancers, when metastasis starts, it is responsible for 80% of deaths from skin cancers (7). Melanocytes express E-cadherin (8-10), but melanoma cells at early radial growth phase show a large reduction in the expression of this cadherin, and surprisingly, expression has been reported to be partially recovered by vertical growth phase and metastatic melanoma cells (9, 11, 12).Trafficking of cancer cells from primary tumor sites to intravasation into blood circulation and later to extravasation to colonize distant organs requires tightly regulated directional cues and cell migration and invasion that are mediated by chemokines, growth factors, and adhesion molecules (13). Solid tumor cells express chemokine receptors that provide guidance of these cells to organs where their chemokine ligands are expressed, constituting a homing model resembling the one used by immune cells to exert their immune surveillance functions (14). Most solid cancer cells express CXCR4, a receptor for the chemokine CXCL12 (also called SDF-1), which is expressed in lungs, bone marrow, and liver (15). Expression of CXCR4 in human melanoma has been detected in the vertical growth phase and on regional lymph nodes, which correlated with poor prognosis and increased mortality (16, 17). Previous in vivo experiments have provided evidence supporting a crucial role for CXCR4 in the metastasis of melanoma cells (18).Rho GTPases control the dynamics of the actin cytoskeleton during cell migration (19, 20). The activity of Rho GTPases is tightly regulated by guanine-nucleotide exchange factors (GEFs),⁴ which stimulate exchange of bound GDP by GTP, and inhibited by GTPase-activating proteins (GAPs), which promote GTP hydrolysis (21, 22), whereas guanine nucleotide dissociation inhibitors (GDIs) appear to mediate blocking of spontaneous activation (23). Therefore, cell migration is finely regulated by the balance between GEF, GAP, and GDI activities on Rho GTPases. Involvement of Rho GTPases in cancer is well documented (reviewed in Ref. 24), providing control of both cell migration and growth. RhoA and RhoC are highly expressed in colon, breast, and lung carcinoma (25, 26), whereas overexpression of RhoC in melanoma leads to enhancement of cell metastasis (27). CXCL12 activates both RhoA and Rac1 in melanoma cells, and both GTPases play key roles during invasion toward this chemokine (28, 29).Given the importance of the CXCL12-CXCR4 axis in melanoma cell invasion and metastasis, in this study we have addressed the question of whether changes in E-cadherin expression on melanoma cells might affect cell invasiveness. We show here that overexpression of E-cadherin leads to impaired melanoma cell invasion to CXCL12, and we provide mechanistic characterization accounting for the decrease in invasion.     

Engineered Protein Connectivity to Actin Mimics PDZ-dependent Recycling of G Protein-coupled Receptors but Not Its Regulation by Hrs

Many G protein-coupled receptors (GPCRs) recycle after agonist-induced endocytosis by a sequence-dependent mechanism, which is distinct from default membrane flow and remains poorly understood. Efficient recycling of the β2-adrenergic receptor (β2AR) requires a C-terminal PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (PDZbd), an intact actin cytoskeleton, and is regulated by the endosomal protein Hrs (hepatocyte growth factor-regulated substrate). The PDZbd is thought to link receptors to actin through a series of protein interaction modules present in NHERF/EBP50 (Na⁺/H+ exchanger 3 regulatory factor/ezrin-binding phosphoprotein of 50 kDa) family and ERM (ezrin/radixin/moesin) family proteins. It is not known, however, if such actin connectivity is sufficient to recapitulate the natural features of sequence-dependent recycling. We addressed this question using a receptor fusion approach based on the sufficiency of the PDZbd to promote recycling when fused to a distinct GPCR, the δ-opioid receptor, which normally recycles inefficiently in HEK293 cells. Modular domains mediating actin connectivity promoted receptor recycling with similarly high efficiency as the PDZbd itself, and recycling promoted by all of the domains was actin-dependent. Regulation of receptor recycling by Hrs, however, was conferred only by the PDZbd and not by downstream interaction modules. These results suggest that actin connectivity is sufficient to mimic the core recycling activity of a GPCR-linked PDZbd but not its cellular regulation.G protein-coupled receptors (GPCRs)² comprise the largest family of transmembrane signaling receptors expressed in animals and transduce a wide variety of physiological and pharmacological information. While these receptors share a common 7-transmembrane-spanning topology, structural differences between individual GPCR family members confer diverse functional and regulatory properties (1-4). A fundamental mechanism of GPCR regulation involves agonist-induced endocytosis of receptors via clathrin-coated pits (4). Regulated endocytosis can have multiple functional consequences, which are determined in part by the specificity with which internalized receptors traffic via divergent downstream membrane pathways (5-7).Trafficking of internalized GPCRs to lysosomes, a major pathway traversed by the δ-opioid receptor (δOR), contributes to proteolytic down-regulation of receptor number and produces a prolonged attenuation of subsequent cellular responsiveness to agonist (8, 9). Trafficking of internalized GPCRs via a rapid recycling pathway, a major route traversed by the β2-adrenergic receptor (β2AR), restores the complement of functional receptors present on the cell surface and promotes rapid recovery of cellular signaling responsiveness (6, 10, 11). When co-expressed in the same cells, the δOR and β2AR are efficiently sorted between these divergent downstream membrane pathways, highlighting the occurrence of specific molecular sorting of GPCRs after endocytosis (12).Recycling of various integral membrane proteins can occur by default, essentially by bulk membrane flow in the absence of lysosomal sorting determinants (13). There is increasing evidence that various GPCRs, such as the β2AR, require distinct cytoplasmic determinants to recycle efficiently (14). In addition to requiring a cytoplasmic sorting determinant, sequence-dependent recycling of the β2AR differs from default recycling in its dependence on an intact actin cytoskeleton and its regulation by the conserved endosomal sorting protein Hrs (hepatocyte growth factor receptor substrate) (11, 14). Compared with the present knowledge regarding protein complexes that mediate sorting of GPCRs to lysosomes (15, 16), however, relatively little is known about the biochemical basis of sequence-directed recycling or its regulation.The β2AR-derived recycling sequence conforms to a canonical PDZ (PSD-95/Discs Large/ZO-1) protein-binding determinant (henceforth called PDZbd), and PDZ-mediated protein association(s) with this sequence appear to be primarily responsible for its endocytic sorting activity (17-20). Fusion of this sequence to the cytoplasmic tail of the δOR effectively re-routes endocytic trafficking of engineered receptors from lysosomal to recycling pathways, establishing the sufficiency of the PDZbd to function as a transplantable sorting determinant (18). The β2AR-derived PDZbd binds with relatively high specificity to the NHERF/EBP50 family of PDZ proteins (21, 22). A well-established biochemical function of NHERF/EBP50 family proteins is to associate integral membrane proteins with actin-associated cytoskeletal elements. This is achieved through a series of protein-interaction modules linking NHERF/EBP50 family proteins to ERM (ezrin-radixin-moesin) family proteins and, in turn, to actin filaments (23-26). Such indirect actin connectivity is known to mediate other effects on plasma membrane organization and function (23), however, and NHERF/EBP50 family proteins can bind to additional proteins potentially important for endocytic trafficking of receptors (23, 25). Thus it remains unclear if actin connectivity is itself sufficient to promote sequence-directed recycling of GPCRs and, if so, if such connectivity recapitulates the normal cellular regulation of sequence-dependent recycling. In the present study, we took advantage of the modular nature of protein connectivity proposed to mediate β2AR recycling (24, 26), and extended the opioid receptor fusion strategy used successfully for identifying diverse recycling sequences in GPCRs (27-29), to address these fundamental questions.Here we show that the recycling activity of the β2AR-derived PDZbd can be effectively bypassed by linking receptors to ERM family proteins in the absence of the PDZbd itself. Further, we establish that the protein connectivity network can be further simplified by fusing receptors to an interaction module that binds directly to actin filaments. We found that bypassing the PDZ-mediated interaction using either domain is sufficient to mimic the ability of the PDZbd to promote efficient, actin-dependent recycling of receptors. Hrs-dependent regulation, however, which is characteristic of sequence-dependent recycling of wild-type receptors, was recapitulated only by the fused PDZbd and not by the proposed downstream interaction modules. These results support a relatively simple architecture of protein connectivity that is sufficient to mimic the core recycling activity of the β2AR-derived PDZbd, but not its characteristic cellular regulation. Given that an increasing number of GPCRs have been shown to bind PDZ proteins that typically link directly or indirectly to cytoskeletal elements (17, 27, 30-32), the present results also suggest that actin connectivity may represent a common biochemical principle underlying sequence-dependent recycling of various GPCRs.     

Rab1 Guanine Nucleotide Exchange Factor SidM Is a Major Phosphatidylinositol 4-Phosphate-binding Effector Protein of Legionella pneumophila

The causative agent of Legionnaires disease, Legionella pneumophila, forms a replicative vacuole in phagocytes by means of the intracellular multiplication/defective organelle trafficking (Icm/Dot) type IV secretion system and translocated effector proteins, some of which subvert host GTP and phosphoinositide (PI) metabolism. The Icm/Dot substrate SidC anchors to the membrane of Legionella-containing vacuoles (LCVs) by specifically binding to phosphatidylinositol 4-phosphate (PtdIns(4)P). Using a nonbiased screen for novel L. pneumophila PI-binding proteins, we identified the Rab1 guanine nucleotide exchange factor (GEF) SidM/DrrA as the predominant PtdIns(4)P-binding protein. Purified SidM specifically and directly bound to PtdIns(4)P, whereas the SidM-interacting Icm/Dot substrate LidA preferentially bound PtdIns(3)P but also PtdIns(4)P, and the L. pneumophila Arf1 GEF RalF did not bind to any PIs. The PtdIns(4)P-binding domain of SidM was mapped to the 12-kDa C-terminal sequence, termed “P4M” (PtdIns4P binding of SidM/DrrA). The isolated P4M domain is largely helical and displayed higher PtdIns(4)P binding activity in the context of the α-helical, monomeric full-length protein. SidM constructs containing P4M were translocated by Icm/Dot-proficient L. pneumophila and localized to the LCV membrane, indicating that SidM anchors to PtdIns(4)P on LCVs via its P4M domain. An L. pneumophila ΔsidM mutant strain displayed significantly higher amounts of SidC on LCVs, suggesting that SidM and SidC compete for limiting amounts of PtdIns(4)P on the vacuole. Finally, RNA interference revealed that PtdIns(4)P on LCVs is specifically formed by host PtdIns 4-kinase IIIβ. Thus, L. pneumophila exploits PtdIns(4)P produced by PtdIns 4-kinase IIIβ to anchor the effectors SidC and SidM to LCVs.The Gram-negative pathogen Legionella pneumophila is the causative agent of Legionnaires disease, but it evolved as a parasite of various species of environmental predatory protozoa, including the social amoeba Dictyostelium discoideum (1, 2). The human disease is linked to the inhalation of contaminated aerosols, followed by replication in alveolar macrophages. To accommodate the transfer between host cells, L. pneumophila alternates between replicative and transmissive phases, the regulation of which includes an apparent quorum-sensing system (3–5).In macrophages and amoebae, L. pneumophila forms a replicative compartment, the Legionella-containing vacuole (LCV).³ LCVs avoid fusion with lysosomes (6), intercept vesicular traffic at endoplasmic reticulum (ER) exit sites (7), and fuse with the ER (8–10). The uptake of L. pneumophila and formation of LCVs in macrophages and amoebae depends on the Icm/Dot type IV secretion system (T4SS) (11–14). Although more than 100 Icm/Dot substrates (“effector” proteins) have been identified to date, only few are functionally characterized, including effectors that interfere with host cell signal transduction, vesicle trafficking, or apoptotic pathways (15–18).Two Icm/Dot-translocated substrates, SidM/DrrA (19, 20) and RalF (21), have been characterized as guanine nucleotide exchange factors (GEFs) for the Rho subfamily of small GTPases. These bacterial GEFs are recruited to and activate their targets on LCVs. Small GTPases of the Rho subfamily are involved in many eukaryotic signal transduction pathways and in actin cytoskeleton regulation (22). Inactive Rho GTPases bind GDP and a guanine nucleotide dissociation inhibitor (GDI). The GTPases are activated by removal of the GDI and the exchange of GDP with GTP by GEFs, which promotes the interaction with downstream effector proteins, such as protein or lipid kinases and various adaptor proteins. The cycle is closed by hydrolysis of the bound GTP, which is mediated by GTPase-activating proteins.SidM is a GEF for Rab1, which is essential for ER to Golgi vesicle transport, and additionally, SidM acts as a GDI displacement factor (GDF) to activate Rab1 (23, 24). The function of SidM is assisted by the Icm/Dot substrate LidA, which also localizes to LCVs. LidA preferentially binds to activated Rab1, thus supporting the recruitment of early secretory vesicles by SidM (19, 20, 23, 25, 26). Another Icm/Dot substrate, LepB (27), contributes to Rab1-mediated membrane cycling by inactivating Rab1 through its GTPase-activating protein function, thus acting as an antagonist of SidM (24).The Icm/Dot substrate RalF recruits and activates the small GTPase ADP-ribosylation factor 1 (Arf1), which is involved in retrograde vesicle transport from Golgi to ER (21). Dominant negative Arf1 (7, 28) or knockdown of Arf1 by RNA interference (29) impairs the formation of LCVs, as well as the recruitment of the Icm/Dot substrate SidC to the LCV (30).SidC and its paralogue SdcA localize to the LCV membrane (31), where the proteins specifically bind to the host cell lipid phosphatidylinositol 4-phosphate (PtdIns(4)P) (32, 33). Phosphoinositides (PIs) regulate eukaryotic receptor-mediated signal transduction, actin remodeling, and membrane dynamics (34, 35). PtdIns(4)P is present on the cytoplasmic membrane, but localizes preferentially to the trans-Golgi network (TGN), where this PI is produced by an Arf-dependent recruitment of PtdIns(4)P kinase IIIβ (PI4K IIIβ) (36) to promote trafficking along the secretory pathway. Recently, PtdIns(4)P was found to also mediate the export of early secretory vesicles from ER exit sites (37). At present, the L. pneumophila effector proteins that mediate exploitation of host PI signaling remain ill defined.In a nonbiased screen for L. pneumophila PI-binding proteins using different PIs coupled to agarose beads, we identified SidM as a major PtdIns(4)P-binding effector. We mapped its PtdIns(4)P binding activity to a novel P4M domain within a 12-kDa C-terminal sequence. SidM constructs, including the P4M domain, were found to be translocated and bind the LCV membrane, where the levels of PtdIns(4)P are controlled by PI4K IIIβ.     

Wnt11 Promotes Osteoblast Maturation and Mineralization through R-spondin 2

Wnt11 signals through both canonical (β-catenin) and non-canonical pathways and is up-regulated during osteoblast differentiation and fracture healing. In these studies, we evaluated the role of Wnt11 during osteoblastogenesis. Wnt11 overexpression in MC3T3E1 pre-osteoblasts increases β-catenin accumulation and promotes bone morphogenetic protein (BMP)-induced expression of alkaline phosphatase and mineralization. Wnt11 dramatically increases expression of the osteoblast-associated genes Dmp1 (dentin matrix protein 1), Phex (phosphate-regulating endopeptidase homolog), and Bsp (bone sialoprotein). Wnt11 also increases expression of Rspo2 (R-spondin 2), a secreted factor known to enhance Wnt signaling. Overexpression of Rspo2 is sufficient for increasing Dmp1, Phex, and Bsp expression and promotes bone morphogenetic protein-induced mineralization. Knockdown of Rspo2 abrogates Wnt11-mediated osteoblast maturation. Antagonism of T-cell factor (Tcf)/β-catenin signaling with dominant negative Tcf blocks Wnt11-mediated expression of Dmp1, Phex, and Rspo2 and decreases mineralization. However, dominant negative Tcf fails to block the osteogenic effects of Rspo2 overexpression. These studies show that Wnt11 signals through β-catenin, activating Rspo2 expression, which is then required for Wnt11-mediated osteoblast maturation.Wnt signaling is a key regulator of osteoblast differentiation and maturation. In mesenchymal stem cell lines, canonical Wnt signaling by Wnt10b enhances osteoblast differentiation (1). Canonical Wnt signaling through β-catenin has also been shown to enhance the chondroinductive and osteoinductive properties of BMP2² (2, 3). During BMP2-induced osteoblast differentiation of mesenchymal stem cell lines, cross-talk between BMP and Wnt pathways converges through the interaction of Smad4 with β-catenin (2).Canonical Wnt signaling is also critical for skeletal development and homeostasis. During limb development, expression of Wnt3a in the apical ectodermal ridge of limb buds maintains cells in a highly proliferative and undifferentiated state (4, 5). Disruption of canonical Wnt signaling in Lrp5/Lrp6 compound knock-out mice results in limb- and digit-patterning defects (6). Wnt signaling is also involved in the maintenance of post-natal bone mass. Gain of function in the Wnt co-receptor Lrp5 leads to increased bone mass, whereas loss of Lrp5 function is associated with decreased bone mass and osteoporosis pseudoglioma syndrome (7, 8). Mice with increased Wnt10b expression have increased trabecular bone, whereas Wnt10b-deficient mice have reduced trabecular bone (9). Similarly, mice nullizygous for the Wnt antagonist sFrp1 have increased trabecular bone accrual throughout adulthood (10).Although canonical Wnt signaling regulates osteoblastogenesis and bone formation, the profile of endogenous Wnts that play a role in osteoblast differentiation and maturation is not well described. During development, Wnt11 is expressed in the perichondrium and in the axial skeleton and sternum (11). Wnt11 expression is increased during glucocorticoid-induced osteogenesis (12), indicating a potential role for Wnt11 in osteoblast differentiation. Interestingly, Wnt11 activates both β-catenin-dependent as well as β-catenin-independent signaling pathways (13). Targeted disruption of Wnt11 results in late embryonic/early post-natal death because of cardiac dysfunction (14). Although these mice have no reported skeletal developmental abnormalities, early lethality obfuscates a detailed examination of post-natal skeletal modeling and remodeling.In murine development, Wnt11 expression overlaps with the expression of R-spondin 2 (Rspo2) in the apical ectodermal ridge (11, 15). R-spondins are a novel family of proteins that share structural features, including two conserved cysteinerich furin-like domains and a thrombospondin type I repeat (16). The four R-spondin family members can activate canonical Wnt signaling (15, 17–19). Rspo3 interacts with Frizzled 8 and Lrp6 and enhances Wnt ligand signaling. Rspo1 enhances Wnt signaling by interacting with Lrp6 and inhibiting Dkk-mediated receptor internalization (20). Rspo1 was also shown to potentiate Wnt3a-mediated osteoblast differentiation (21). Rspo2 knock-out mice, which die at birth, have limb patterning defects associated with altered β-catenin signaling (22–24). However, the role of Rspo2 in osteoblast differentiation and maturation remains unclear.Herein we report that Wnt11 overexpression in MC3T3E1 pre-osteoblasts activates β-catenin and augments BMP-induced osteoblast maturation and mineralization. Wnt11 increases the expression of Rspo2. Overexpression of Rspo2 in MC3T3E1 is sufficient for augmenting BMP-induced osteoblast maturation and mineralization. Although antagonism of Tcf/β-catenin signaling blocks the osteogenic effects of Wnt11, Rspo2 rescues this block, and knockdown of Rspo2 shows that it is required for Wnt11-mediated osteoblast maturation and mineralization. These studies identify both Wnt11 and Rspo2 as novel mediators of osteoblast maturation and mineralization.     

Bortezomib Overcomes Tumor Necrosis Factor-related Apoptosis-inducing Ligand Resistance in Hepatocellular Carcinoma Cells in Part through the Inhibition of the Phosphatidylinositol 3-Kinase/Akt Pathway

Hepatocellular carcinoma (HCC) is one of the most common and aggressive human malignancies. Recombinant tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising anti-tumor agent. However, many HCC cells show resistance to TRAIL-induced apoptosis. In this study, we showed that bortezomib, a proteasome inhibitor, overcame TRAIL resistance in HCC cells, including Huh-7, Hep3B, and Sk-Hep1. The combination of bortezomib and TRAIL restored the sensitivity of HCC cells to TRAIL-induced apoptosis. Comparing the molecular change in HCC cells treated with these agents, we found that down-regulation of phospho-Akt (P-Akt) played a key role in mediating TRAIL sensitization of bortezomib. The first evidence was that bortezomib down-regulated P-Akt in a dose- and time-dependent manner in TRAIL-treated HCC cells. Second, {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002, a PI3K inhibitor, also sensitized resistant HCC cells to TRAIL-induced apoptosis. Third, knocking down Akt1 by small interference RNA also enhanced TRAIL-induced apoptosis in Huh-7 cells. Finally, ectopic expression of mutant Akt (constitutive active) in HCC cells abolished TRAIL sensitization effect of bortezomib. Moreover, okadaic acid, a protein phosphatase 2A (PP2A) inhibitor, reversed down-regulation of P-Akt in bortezomib-treated cells, and PP2A knockdown by small interference RNA also reduced apoptosis induced by the combination of TRAIL and bortezomib, indicating that PP2A may be important in mediating the effect of bortezomib on TRAIL sensitization. Together, bortezomib overcame TRAIL resistance at clinically achievable concentrations in hepatocellular carcinoma cells, and this effect is mediated at least partly via inhibition of the PI3K/Akt pathway.Hepatocellular carcinoma (HCC)² is currently the fifth most common solid tumor worldwide and the fourth leading cause of cancer-related death. To date, surgery is still the only curative treatment but is only feasible in a small portion of patients (1). Drug treatment is the major therapy for patients with advanced stage disease. Unfortunately, the response rate to traditional chemotherapy for HCC patients is unsatisfactory (1). Novel pharmacological therapy is urgently needed for patients with advanced HCC. In this regard, the approval of sorafenib might open a new era of molecularly targeted therapy in the treatment of HCC patients.Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), a type II transmembrane protein and a member of the TNF family, is a promising anti-tumor agent under clinical investigation (2). TRAIL functions by engaging its receptors expressed on the surface of target cells. Five receptors specific for TRAIL have been identified, including DR4/TRAIL-R1, DR5/TRAIL-R2, DcR1, DcR2, and osteoprotegerin. Among TRAIL receptors, only DR4 and DR5 contain an effective death domain that is essential to formation of death-inducing signaling complex (DISC), a critical step for TRAIL-induced apoptosis. Notably, the trimerization of the death domains recruits an adaptor molecule, Fas-associated protein with death domain (FADD), which subsequently recruits and activates caspase-8. In type I cells, activation of caspase-8 is sufficient to activate caspase-3 to induce apoptosis; however, in another type of cells (type II), the intrinsic mitochondrial pathway is essential for apoptosis characterized by cleavage of Bid and release of cytochrome c from mitochondria, which subsequently activates caspase-9 and caspase-3 (3).Although TRAIL induces apoptosis in malignant cells but sparing normal cells, some tumor cells are resistant to TRAIL-induced apoptosis. Mechanisms responsible for the resistance include receptors and intracellular resistance. Although the cell surface expression of DR4 or DR5 is absolutely required for TRAIL-induced apoptosis, tumor cells expressing these death receptors are not always sensitive to TRAIL due to intracellular mechanisms. For example, the cellular FLICE-inhibitory protein (c-FLIP), a homologue to caspase-8 but without protease activity, has been linked to TRAIL resistance in several studies (4, 5). In addition, inactivation of Bax, a proapoptotic Bcl-2 family protein, resulted in resistance to TRAIL in MMR-deficient tumors (6, 7), and reintroduction of Bax into Bax-deficient cells restored TRAIL sensitivity (8), indicating that the Bcl-2 family plays a critical role in intracellular mechanisms for resistance of TRAIL.Bortezomib, a proteasome inhibitor approved clinically for multiple myeloma and mantle cell lymphoma, has been investigated intensively for many types of cancer (9). Accumulating studies indicate that the combination of bortezomib and TRAIL overcomes the resistance to TRAIL in various types of cancer, including acute myeloid leukemia (4), lymphoma (10–13), prostate (14–17), colon (15, 18, 19), bladder (14, 16), renal cell carcinoma (20), thyroid (21), ovary (22), non-small cell lung (23, 24), sarcoma (25), and HCC (26, 27). Molecular targets responsible for the sensitizing effect of bortezomib on TRAIL-induced cell death include DR4 (14, 27), DR5 (14, 20, 22–23, 28), c-FLIP (4, 11, 21–23, 29), NF-κB (12, 24, 30), p21 (16, 21, 25), and p27 (25). In addition, Bcl-2 family also plays a role in the combinational effect of bortezomib and TRAIL, including Bcl-2 (10, 21), Bax (13, 22), Bak (27), Bcl-xL (21), Bik (18), and Bim (15).Recently, we have reported that Akt signaling is a major molecular determinant in bortezomib-induced apoptosis in HCC cells (31). In this study, we demonstrated that bortezomib overcame TRAIL resistance in HCC cells through inhibition of the PI3K/Akt pathway.     

Effects of Differential Glycosylation of Glycodelins on Lymphocyte Survival

Glycodelin is a human glycoprotein with four reported glycoforms, namely glycodelin-A (GdA), glycodelin-F (GdF), glycodelin-C (GdC), and glycodelin-S (GdS). These glycoforms have the same protein core and appear to differ in their N-glycosylation. The glycosylation of GdA is completely different from that of GdS. GdA inhibits proliferation and induces cell death of T cells. However, the glycosylation and immunomodulating activities of GdF and GdC are not known. This study aimed to use ultra-high sensitivity mass spectrometry to compare the glycomes of GdA, GdC, and GdF and to study the relationship between the immunological activity and glycosylation pattern among glycodelin glycoforms. Using MALDI-TOF strategies, the glycoforms were shown to contain an enormous diversity of bi-, tri-, and tetra-antennary complex-type glycans carrying Galβ1–4GlcNAc (lacNAc) and/or GalNAcβ1–4GlcNAc (lacdiNAc) antennae backbones with varying levels of fucose and sialic acid substitution. Interestingly, they all carried a family of Sda (NeuAcα2–3(GalNAcβ1–4)Gal)-containing glycans, which were not identified in the earlier study because of less sensitive methodologies used. Among the three glycodelins, GdA is the most heavily sialylated. Virtually all the sialic acid on GdC is located on the Sda antennae. With the exception of the Sda epitope, the GdC N-glycome appears to be the asialylated counterpart of the GdA/GdF glycomes. Sialidase activity, which may be responsible for transforming GdA/GdF to GdC, was detected in cumulus cells. Both GdA and GdF inhibited the proliferation, induced cell death, and suppressed interleukin-2 secretion of Jurkat cells and peripheral blood mononuclear cells. In contrast, no immunosuppressive effect was observed for GdS and GdC.Glycodelin is a member of the lipocalin family. It consists of 180 amino acid residues (1) with two sites of N-linked glycosylation. There are four reported glycodelin isoforms, namely glycodelin-A (amniotic fluid isoform, GdA),⁴ glycodelin-F (follicular fluid, GdF), glycodelin-C (cumulus matrix, GdC) and glycodelin-S (seminal plasma, GdS) (2–5). Among the four glycodelin isoforms, only the N-glycan structures of GdA and GdS have been previously determined. This was achieved using fast atom bombardment mass spectrometry (6, 7). The glycan structures of GdA and GdS are completely different. In GdA, the Asn-28 site carries high mannose, hybrid, and complex-type structures, whereas the second Asn-63 site is exclusively occupied by complex-type glycans (6). The major non-reducing epitopes characterized in the complex-type glycans are Galβ1–4GlcNAc (lacNAc), GalNAcβ1–4GlcNAc (lacdiNAc), NeuAcα2–6Galβ1–4GlcNAc (sialylated lacNAc), NeuAcα2–6GalNAcβ1–4GlcNAc (sialylated lacdiNAc), Galβ1–4(Fucα1–3)GlcNAc (Lewis-x), and GalNAcβ1–4(Fucα1–3)GlcNAc (lacdiNAc analog of the blood group substance Lewis-x) (6). Many of these oligosaccharides are rare in other human glycoproteins. GdS glycans are unusually fucose-rich, and the major complex type glycan structures are bi-antennary glycans with Lewis-x and Lewis-y antennae. Glycosylation of GdS is highly site-specific. Asn-28 contains only high mannose structures, whereas Asn-63 contains only complex type glycans. More than 80% of the complex glycans have 3–5 fucose residues/glycan, and none of the glycans is sialylated, which is unusual for a secreted human glycoprotein (7). The glycan structures of GdF and GdC are not known, although they differ in lectin-binding properties and isoelectric point from the other two glycodelin isoforms (5).Glycans are involved in various intracellular, intercellular, and cell-matrix recognition events (8, 9). Glycosylation determines the biological activities of the glycodelin isoforms (2, 10). For example, both GdA and GdF inhibit the spermatozoa-zona pellucida binding (11) via fucosyltransferase-5 (12), but only the latter inhibits progesterone-induced acrosome reaction, thus preventing a premature acrosome reaction of the spermatozoa. There is evidence that cumulus cells can convert exogenous GdA and -F to GdC, the physicochemical properties of which suggest that it is differently glycosylated compared with GdA/F (5). Moreover, GdC stimulated spermatozoa-zona pellucida binding in a dose-dependent manner, and it effectively displaced sperm-bound GdA and -F (4, 5). GdS suppresses capacitation probably via its inhibitory activity on cholesterol efflux from spermatozoa (13).Except for the effects on fertilization, GdA is involved in fetomaternal defense. This glycodelin isoform suppresses proliferation and induces apoptosis of T cells (2) and inhibits natural killer cell (14) and B-cell (15) activities. Glycosylation is involved in the binding of GdA to receptors on T cells (16). The sialic acid of GdA contributes to the apoptotic activity in T cells (17, 18) and binding to CD45, a potential GdA receptor (16). The importance of glycosylation in glycodelin is further shown by the absence of immunosuppressive activities in GdS with different glycosylation (18). The immunomodulating activities of GdF and GdC are unknown.Our previous work showed that glycans are indispensable for the different glycodelins to exhibit their binding activities and biological effects (13, 19, 20). The present study aims to identify the effect of all four glycodelin isoforms on lymphocyte viability, cell death, and interleukin-2 (IL-2) secretion and to correlate these bioactivities with their glycosylation patterns determined by mass spectrometry.