) is a bioactive lipid known to control cell growth that was recently shown to act as a trophic factor for skeletal muscle, reducing the progress of denervation atrophy. The aim of this work was to investigate whether S1P is involved in skeletal muscle fiber recovery (regeneration) after myotoxic injury induced by bupivacaine. The postnatal ability of skeletal muscle to grow and regenerate is dependent on resident stem cells called satellite cells. Immunofluorescence analysis demonstrated that S1P-specific receptors S1P1 and S1P3 are expressed by quiescent satellite cells. Soleus muscles undergoing regeneration following injury induced by intramuscular injection of bupivacaine exhibited enhanced expression of S1P1 receptor, while S1P3 expression progressively decreased to adult levels. S1P 2 receptor was absent in quiescent cells but was transiently expressed in the early regenerating phases only. Administration of S1P (50 M) at the moment of myotoxic injury caused a significant increase of the mean crosssectional area of regenerating fibers in both rat and mouse. In separate experiments designed to test the trophic effects of S1P, neutralization of endogenous circulating S1P by intraperitoneal administration of anti-S1P antibody attenuated fiber growth. Use of selective modulators of S1P receptors indicated that S1P1 receptor negatively and S1P3 receptor positively modulate the early phases of regeneration, whereas S1P2 receptor appears to be less important. The present results show that S1P signaling participates in the regenerative processes of skeletal muscle. sphingosine 1-phosphate receptors; satellite cells SPHINGOSINE 1-PHOSPHATE (S1P) is a bioactive lysolipid known to regulate many critical biological processes, such as cell proliferation, survival, migration, and angiogenesis (15, 42). The extracellular action of S1P is exerted by binding to five specific cell surface G protein-coupled S1P receptors, S1P 1 -S1P 5 (4). S1P 1 , S1P 2 , and S1P 3 are expressed in all mammalian tissues, whereas S1P 4 and S1P 5 are more tissue specific. Genetic deletion of S1P 1 receptor is fatal to embryos (19), and the simultaneous deletion of both S1P 2 and S1P 3 receptors produces perinatal lethality, while S1P 2 -deficient mice are apparently healthy (16). The distinctive combination of individual S1P receptors, differentially coupled to heterotrimeric G proteins, determines in a given cell the specific biological response produced by S1P (42). S1P receptor-dependent signaling has been demonstrated in skeletal muscle cells. The mRNAs of S1P 1 -S1P 3 receptors are detectable in the myogenic C2C12 cell line derived from mouse satellite cells, with S1P 1 expression being the highest (25, 36). The relative expression of S1P receptors changes during myogenic differentiation of C2C12 cells, particularly that of S1P 2 , which progressively diminishes during differentiation and becomes almost absent by the time myotubes are formed (22). Consistently in rat adult skeletal muscle RT-PCR and Western blot data demonstrated the exp...

Sarcoglycanopathies are progressive muscle-wasting disorders caused by genetic defects of four proteins, ␣-, ␤-, ␥-, and ␦-sarcoglycan, which are elements of a key transmembrane complex of striated muscle. The proper assembly of the sarcoglycan complex represents a critical issue of sarcoglycanopathies, as several mutations severely perturb tetramer formation. Misfolded proteins are generally degraded through the cell's quality-control system; however, this can also lead to the removal of some functional polypeptides. To explore whether it is possible to rescue sarcoglycan mutants by preventing their degradation, we generated a heterologous cell system, based on human embryonic kidney (HEK) 293 cells, constitutively expressing three (␤, ␥, and ␦) of the four sarcoglycans. In these ␤␥␦-HEK cells, the lack of ␣-sarcoglycan prevented complex formation and cell surface localization, wheras the presence of ␣-sarcoglycan allowed maturation and targeting of the tetramer. As in muscles of sarcoglycanopathy patients, transfection of ␤␥␦-HEK cells with disease-causing ␣-sarcoglycan mutants led to dramatic reduction of the mutated proteins and the absence of the complex from the cell surface. Proteasomal inhibition reduced the degradation of mutants and facilitated the assembly and targeting of the sarcoglycan complex to the plasma membrane. These data provide important insights for the potential development of pharmacological therapies for sarcoglycanopathies.

Specific surface proteins of Neisseria meningitidis have been proposed to stimulate leukocytes during tissue invasion and septic shock. In this study, we demonstrate that the adhesin N. meningitidis Adhesin A (NadA) involved in the colonization of the respiratory epithelium by hypervirulent N. meningitidis B strains also binds to and activates human monocytes/macrophages. Expression of NadA on the surface on Escherichia coli does not increase bacterial-monocyte association, but a NadA-positive strain induced a significantly higher amount of TNF-alpha and IL-8 compared with the parental NadA-negative strain, suggesting that NadA has an intrinsic stimulatory action on these cells. Consistently, highly pure, soluble NadA(Delta351-405), a proposed component of an antimeningococcal vaccine, efficiently stimulates monocytes/macrophages to secrete a selected pattern of cytokines and chemotactic factors characterized by high levels of IL-8, IL-6, MCP-1, and MIP-1alpha and low levels of the main vasoactive mediators TNF-alpha and IL-1. NadA(Delta351-405) also inhibited monocyte apoptosis and determined its differentiation into a macrophage-like phenotype.

Hypervirulent MenB causing fatal human infections frequently display the oligomeric-coiled coil adhesin NadA, a 45-kDa intrinsic outer membrane protein implicated in binding to and invasion of respiratory epithelial cells. A recombinant soluble mutant lacking the 10-kDa COOH terminal membrane domain (NadA(Delta351-405)) also activates human monocytes/macrophages/DCs. As NadA is physiologically released during sepsis as part of OMVs, in this study, we tested the hypothesis that NadA(+) OMVs have an enhanced or modified proinflammatory/proimmune action compared with NadA(-) OMVs. To do this we investigated the activity of purified free NadA(Delta351-405) and of OMVs from MenB and Escherichia coli strains, expressing or not full-length NadA. NadA(Delta351-405) stimulated monocytes and macrophages to secrete cytokines (IL-1beta, TNF-alpha, IL-6, IL-12p40, IL-12p70, IL-10) and chemokines (IL-8, MIP-1alpha, MCP-1, RANTES), and full-length NadA improved MenB OMV activity, preferentially on macrophages, and only increased cytokine release. NadA(Delta351-405) induced the lymphocyte costimulant CD80 in monocytes and macrophages, and NadA(+) OMVs induced a wider set of molecules supporting antigen presentation (CD80, CD86, HLA-DR, and ICAM-1) more efficiently than NadA(-) OMVs only in macrophages. Moreover, membrane NadA effects, unlike NadA(Delta351-405) ones, were much less IFN-gamma-sensitive. The activity of NadA-positive E. coli OMVs was similar to that of control OMVs. NadA in MenB OMVs acted at adhesin concentrations approximately 10(6) times lower than those required to stimulate cells with free NadA(Delta351-405).

NadA is a trimeric autotransporter protein of Neisseria meningitidis belonging to the group of oligomeric coiled-coil adhesins. It is implicated in the colonization of the human upper respiratory tract by hypervirulent serogroup B N. meningitidis strains and is part of a multiantigen anti-serogroup B vaccine. Structure prediction indicates that NadA is made by a COOH-terminal membrane anchor (also necessary for autotranslocation to the bacterial surface), an intermediate elongated coiled-coil-rich stalk, and an NH 2 -terminal region involved in cell interaction. Electron microscopy analysis and structure prediction suggest that the apical region of NadA forms a compact and globular domain. Deletion studies proved that the NH 2 -terminal sequence (residues 24 to 87) is necessary for cell adhesion. In this study, to better define the NadA cell binding site, we exploited (i) a panel of NadA mutants lacking sequences along the coiled-coil stalk and (ii) several oligoclonal rabbit antibodies, and their relative Fab fragments, directed to linear epitopes distributed along the NadA ectodomain. We identified two critical regions for the NadA-cell receptor interaction with Chang cells: the NH 2 globular head domain and the NH 2 dimeric intrachain coiled-coil ␣-helices stemming from the stalk. This raises the importance of different modules within the predicted NadA structure. The identification of linear epitopes involved in receptor binding that are able to induce interfering antibodies reinforces the importance of NadA as a vaccine antigen.