Syk Tyrosine 317 Negatively Regulates Osteoclast Function via the Ubiquitin-Protein Isopeptide Ligase Activity of Cbl

Cytoskeletal organization of the osteoclast (OC), which is central to the capacity of the cell to resorb bone, is induced by occupancy of the αvβ3 integrin or the macrophage colony-stimulating factor (M-CSF) receptor c-Fms. In both circumstances, the tyrosine kinase Syk is an essential signaling intermediary. We demonstrate that Cbl negatively regulates OC function by interacting with Syk^Y317. Expression of nonphosphorylatable Syk^Y317F in primary Syk^−/− OCs enhances M-CSF- and αvβ3-induced phosphorylation of the cytoskeleton-organizing molecules, SLP76, Vav3, and PLCγ2, to levels greater than wild type, thereby accelerating the resorptive capacity of the cell. Syk^Y317 suppresses cytoskeletal organization and function while binding the ubiquitin-protein isopeptide ligase Cbl. Consequently, Syk^Y317F abolishes M-CSF- and integrin-stimulated Syk ubiquitination. Thus, Cbl/Syk^Y317 association negatively regulates OC function and therefore is essential for maintenance of skeletal homeostasis.OCs² are multinucleated cells generated by fusion of mononuclear progenitors of the monocyte/macrophage family under the aegis of M-CSF and receptor activator of nuclear factor κB ligand (RANKL) (1). Upon mineralized matrix recognition, the OC polarizes its fibrillar actin, eventuating in the formation of an acidified extracellular microenvironment that degrades bone. Failure to undergo this polarization event results in OC hypo-function and consequently in varying degrees of osteopetrosis (2).Integrins are transmembrane α/β heterodimers that mediate cell-cell and cell-matrix interactions and generate intracellular signals when occupied by ligands (3). The integrin, αvβ3, is expressed by OCs, and binding of this complex to bone is pivotal to the resorptive process (4).M-CSF recognizes its transmembrane receptor tyrosine kinase, c-Fms, and induces receptor autophosphorylation at seven tyrosine residues within the cytoplasmic domain (5). Several Src homology-2 domain-containing molecules are recruited to the phosphotyrosine residues upon M-CSF binding and initiate signaling cascades that lead to cytoskeletal organization, survival, and proliferation of OC lineage cells (5–7). Both the αvβ3 integrin and M-CSF are important regulators of OC actin remodeling (4, 6, 8).Syk is a 72-kDa nonreceptor tyrosine kinase, which mediates αvβ3- and c-Fms-induced OC cytoskeletal organization and function in a phosphorylation-dependent manner via a process involving activation of associated adaptor proteins, such as SLP-76 and Vav3 (9, 10). A number of Syk tyrosine residues undergo phosphorylation following engagement of the integrin and Fcγ receptor in immune (11) and mast cells (12). Three conserved tyrosine residues in the Syk linker region, namely Tyr³¹⁷, Tyr³⁴², and Tyr³⁴⁶, lie within consensus sequences for recognition by Src homology 2 domains, suggesting they transduce signals. Although phospho-Syk^Y342 and phospho-Syk^Y346 may serve as positive signaling regulators (12–14), phosphorylation of Syk^Y317 creates a binding site for c-Cbl, an E3 ubiquitin ligase proposed to prompt ubiquitination and subsequent degradation of Syk (15, 16). Hence, Syk^Y317 is a candidate negative regulatory site, but its role in OC function and/or differentiation is unknown.Cbl is a 120-kDa protein that is tyrosine-phosphorylated following activation by growth factors, cytokines, and integrins. It has two distinct but related activities, serving both as an adaptor protein (17, 18) and E3 ubiquitin ligase (19, 20). Cbl functions principally as an adaptor in OCs by participating in signaling complexes that are important in the assembly and remodeling of the actin cytoskeleton (18, 21). In other cell types, Cbl is also a negative regulator of receptor and nonreceptor tyrosine kinases, as it promotes their degradation (22). OCs and their precursors express c-Cbl and another family member Cbl-b that compensates for the absence of c-Cbl (23, 24). As combined deletion of both isoforms eventuates in early embryonic lethality (24), it is not clear if c-Cbl functions as an E3 ubiquitin ligase in OCs. We establish that c-Cbl, recognizing Syk^Y317, prompts the ubiquitination of the kinases thereby arresting activation of cytoskeleton-organizing molecules and thus OC function. The Cbl-Syk^Y317 complex is therefore important in maintenance of normal skeletal mass.     

Granulysin, a T cell product, kills bacteria by altering membrane permeability

Granulysin, a protein located in the acidic granules of human NK cells and cytotoxic T cells, has antimicrobial activity against a broad spectrum of microbial pathogens. A predicted model generated from the nuclear magnetic resonance structure of a related protein, NK lysin, suggested that granulysin contains a four alpha helical bundle motif, with the alpha helices enriched for positively charged amino acids, including arginine and lysine residues. Denaturation of the polypeptide reduced the alpha helical content from 49 to 18% resulted in complete inhibition of antimicrobial activity. Chemical modification of the arginine, but not the lysine, residues also blocked the antimicrobial activity and interfered with the ability of granulysin to adhere to Escherichia coli and Mycobacterium tuberculosis. Granulysin increased the permeability of bacterial membranes, as judged by its ability to allow access of cytosolic ss-galactosidase to its impermeant substrate. By electron microscopy, granulysin triggered fluid accumulation in the periplasm of M. tuberculosis, consistent with osmotic perturbation. These data suggest that the ability of granulysin to kill microbial pathogens is dependent on direct interaction with the microbial cell wall and/or membrane, leading to increased permeability and lysis.     

Interleukin-4 reversibly inhibits osteoclastogenesis via inhibition of NF-kappa B and mitogen-activated protein kinase signaling.

To define the molecular mechanism(s) by which interleukin (IL)-4 reversibly inhibits formation of osteoclasts (OCs) from bone marrow macrophages (BMMs), we examined the capacity of this T cell-derived cytokine to impact signals known to modulate osteoclastogenesis, which include those initiated by macrophage colony-stimulating factor (M-CSF), receptor for activation of NF-kappa B ligand (RANKL), tumor necrosis factor (TNF), and IL-1. We find that although pretreatment of BMMs with IL-4 does not alter M-CSF signaling, it reversibly blocks RANKL-dependent activation of the NF-kappa B, JNK, p38, and ERK signals. IL-4 also selectively inhibits TNF signaling, while enhancing that of IL-1. Contrary to previous reports, we find that MEK inhibitors dose-dependently inhibit OC differentiation. To identify more proximal signals mediating inhibition of OC formation by IL-4, we used mice lacking STAT6 or SHIP1, two adapter proteins that bind the IL-4 receptor. IL-4 fails to inhibit RANKL/M-CSF-induced osteoclastogenesis by BMMs derived from STAT6-, but not SHIP1-, knockout mice. Consistent with this observation, the inhibitory effects of IL-4 on RANKL-induced NF-kappa B and mitogen-activated protein kinase activation are STAT6-dependent. We conclude that IL-4 reversibly arrests osteoclastogenesis in a STAT6-dependent manner by 1) preventing I kappa B phosphorylation and thus NF-kappa B activation, and 2) blockade of the JNK, p38, and ERK mitogen-activated protein kinase pathways.     

Podosome expression in osteoclasts: influence of high extracellular calcium concentration

In this study the effect of high extracellular calcium concentration has been evaluated, by immunofluorescence, on podosome expression in chicken osteoclasts. Cells were cultured in presence of 0.2 and 4 mM calcium for 90 minutes and microfilaments were detected, after fixation and permeabilization, by decoration with rodhamine conjugated phalloidin. Results showed that increased extracellular calcium concentration induces the inhibition of podosome expression indicating that these close-contact areas are capable of calcium-mediated regulation.     

Interleukin 1 stimulates proliferation of a nontransformed T lymphocyte line in the absence of a co-mitogen

Although interleukin (IL) 2-responsive T cell lines provide an opportunity to study the cellular effects of this lymphokine on homogeneous T lymphocyte populations, T cell clones which proliferate in response to IL-1 alone have not been available. We have isolated from cultures of the nontransformed murine T helper cell line, D10 . G4 . 1, a variant (MD10 cells) which proliferates (no lectin or antigen needed) in response to IL-1 alone. The MD10 cells are markedly sensitive to either murine or human recombinant IL-alpha (HrIL-1 alpha) with half-maximal responses observed at monokine concentrations as low as 0.4 X 10(-12) M or 0.8 U/ml, respectively. MD10 cells show the maximal IL-1 effect at 72 hr where the response exceeds the base line by 100-fold (approximately 3,000----300,000 cpm of [3H]thymidine). Whereas both HrIL-2 and purified murine B cell-stimulatory factor 1 (MpBSF-1) induce MD10 proliferation, the maximal response to either is much lower (HrIL-2: 50X baseline; MpBSF-1: less than 20X base line) than to IL-1. Conditioned media from control, concanavalin A-, or IL-1-treated MD10 cells fail to stimulate CTLL or HT-2 cell proliferation alone or inhibit CTLL mitogenesis in the presence of added HrIL-2. Furthermore, monoclonal antibodies to BSF-1 fail to inhibit IL-1-stimulated MD10 replication, and neither HT-2 nor CTLL cells proliferate despite direct cell-to-cell contact with IL-1-treated MD10 cells. When combined, IL-1 (10(-13), 10(-12) M) and IL-2 (10(-13) to 10(-10) M) act synergistically in their MD10 cell growth-promoting effects. MD10 proliferation induced by either IL-1 or IL-2 is relatively resistant to cyclosporine A, with the ID50 of cyclosporine for both IL-1- and IL-2-exposed MD10 cells (ID50 5000 ng/ml) exceeding that for concanavalin A-activated splenocytes (ID50 20 ng/ml) by 2 to 3 orders of magnitude. Finally, MD10 cells bear the L3T4 antigen, IL-2 receptors, and the same clonotypic antigen receptor as the parent clone as recognized by monoclonal antibody 3D3. These data suggest that, in respect to this particular T cell line, IL-1 is directly growth-promoting or, alternatively, induces the production of undetectable, intermediate growth factor(s) resistant to inhibition by cyclosporine A.     

1H-NMR studies on substance P hexapeptide analogs

¹H-nmr studies of [pGlu⁶]SP_6–11, [gpGlu⁶,mPhe⁷]SP_6–11, and [pGlu⁶,N-CH₃Phe⁷]SP_6–11 in DMSO-d₆ reveal characteristic chemical shifts, ³J_NH-αCH, temperature dependence, as well as deuterium exchange half-times. Marked similarities are revealed for the two first analogs, whereas the N-methylated analog is clearly different. Possible conformations are considered.