Src‐like adaptor protein regulates osteoclast generation and survival

Src‐like adaptor protein (SLAP) is a hematopoietic adaptor containing Src homology (SH)3 and SH2 motifs and a unique carboxy terminus. Unlike c‐Src, SLAP lacks a tyrosine kinase domain. We investigated the role of SLAP in osteoclast development and resorptive function. Employing SLAP‐deficient mice, we find lack of the adaptor enhances in vitro proliferation of osteoclast precursors in the form of bone marrow macrophages (BMMs), without altering their survival. Furthermore, osteoclastogenic markers appear more rapidly in SLAP?/? BMMs exposed to RANK ligand (RANKL). The accelerated proliferation of M‐CSF‐treated, SLAP‐deficient precursors is associated with enhanced ERK activation. SLAP's role as a mediator of M‐CSF signaling, in osteoclastic cells, is buttressed by complexing of the adaptor protein and c‐Fms in lipid rafts. Unlike c‐Src, SLAP does not impact resorptive function of mature osteoclasts but induces their early apoptosis. Thus, SLAP negatively regulates differentiation of osteoclasts and proliferation of their precursors. Conversely, SLAP decreases osteoclast death by inhibiting activation of caspase 3. These counterbalancing events yield indistinguishable bones of WT and SLAP?/? mice which contain equal numbers of osteoclasts in basal and stimulated conditions. J. Cell. Biochem. 110: 201–209, 2010. © 2010 Wiley‐Liss, Inc.     

Fyn positively regulates the activation of DAP12 and FcRγ-mediated costimulatory signals by RANKL during osteoclastogenesis

Osteoclasts (OCs) are the only bone-resorbing cells and are critically involved in various bone-associated diseases, including osteoporosis and rheumatoid arthritis. Differentiation of OCs from bone marrow macrophage cells (BMMs) is regulated by RANK and the adaptor protein (DAP12/FcRγ)-mediated costimulatory signals. However, it is unknown how RANKL/RANK signal stimulates phosphorylation of DAP12/FcRγ to initiate the costimulatory signals. As reported here, we found that OC differentiation and acquisition of bone resorption capacity were suppressed in RANKL-stimulated Fyn(-/-) or Fyn-siRNA-transfected BMMs, but could be restored by overexpression of Fyn kinase in Fyn(-/-) BMMs. However, the RANKL-stimulated proliferation of BMMs was unaffected by the absence of Fyn. In addition, RANKL-stimulated Fyn(-/-) BMMs no longer exhibited the optimal induction of typical OC markers such as NFATc1, c-Fos, c-Src, TRAF6, and cathepsin K or costimulatory signals such as the activating phosphorylations of Syk, PLCγ2, and Gab2. These were restored by overexpression of Fyn in Fyn(-/-) BMMs. Immunoprecipitation studies also indicated that the adaptor proteins DAP12/FcRγ and Syk interacted with RANK during RANKL stimulation in BMMs in a Fyn-dependent manner. Phosphorylation of the DAP12/FcRγ and the recruitment of Syk by DAP12/FcRγ were suppressed in Fyn(-/-) BMMs. This is the first demonstration that Fyn relays the initial RANK/RANKL signal to the ITAM-containing adaptors DAP12/FcRγ for OC differentiation.     

Paracrine-mediated differentiation and activation of human haematopoietic osteoclast precursor cells by skin and gingival fibroblasts

Objective: Fibroblasts appear to modulate osteoclastogenesis, but their precise role in this process remains unclear. In this work, paracrine‐mediated osteoclastogenic potential of different human fibroblasts was assessed. Materials and methods: Fibroblast‐conditioned media (CM) from foetal skin (CM1), adult skin (CM2) and adult gingiva (CM3) were used to promote osteoclastogenesis of osteoclast precursor cells. Cultures supplemented with macrophage‐colony stimulating factor (M‐CSF) and receptor activator of nuclear factor‐κB ligand (RANKL) were used as controls. Results: All fibroblast cultures expressed FSP‐1, M‐CSF and RANKL and produced osteoprotegerin (OPG); gingival fibroblasts presented lowest expression of osteoclastogenic genes and higher production of OPG. All fibroblast CM were able to induce osteoclastogenesis. CM1 showed behaviour similar to positive controls, and slightly higher osteoclastogenic potential than CM, from adult ones. Gingival fibroblasts revealed lowest osteoclastogenic ability. Presence of anti‐MCSF or anti‐RANKL partially inhibited osteoclastogenesis promoted by CM, although the former antibody revealed higher inhibitory response. Differences among the osteoclastogenic effect of CM were noted, mainly in expression of genes involved in differentiation and activation of osteoclast precursor cells, c‐myc and c‐src, and less regarding functional related parameters. Conclusions: Fibroblasts are able to induce osteoclastogenesis by paracrine mechanisms, and age and anatomical location affect this ability. Other factors produced by fibroblasts, in addition to M‐CSF and RANKL, appear to contribute to observed osteoclastogenic potential.     

Osteoclast differentiation gene expression profiling reveals chemokine CCL4 mediates RANKL‐induced osteoclast migration and invasion via PI3K pathway

The migration of osteoclasts (OCs) from circulation and bone marrow into bone surface plays a critical role in the pathogenesis of some bone resorptive diseases, such as rheumatoid arthritis and osteoporosis. To date, how the migration of OCs remains unclear. We investigated gene expression profiling in osteoclastic differentiation of bone marrow–derived macrophages (BMMs) into OCs by microarray analysis. We identified 387 genes overexpressed in osteoclastic differentiation of BMMs. Among them, chemokine CCL4 showed a robust up‐regulation signal. High expression of CCL4 was validated in primary BMMs and OC precursor cell line RAW264.7 during differentiation into OCs. The CCL4 neutralization decreased RANKL‐induced OC precursor cell migration and invasion in Matrigel‐coated transwell membranes assay and in vitro wound healing assay. However, CCL4 inhibition did not affect OCs differentiation and differentiation associated gene expression. The CCL4 inhibition promoted the PI3K phosphorylation at 45 to 60 minutes after RANKL stimulation in RAW264.7. This study indicated that chemokine CCL4 is an important regulator for OCs migration via PI3K pathway, providing a novel therapy target for bone resorptive diseases.     

Characterisation of the osteoclastogenic potential of human osteoblastic and fibroblastic conditioned media

Although M‐CSF and RANKL are sufficient to promote in vitro osteoclastogenesis, in vivo this is a complex process which requires the action of many signalling molecules and cellular crosstalks. In this work, isolated or combined conditioned media, obtained from human adult skin fibroblast and bone marrow cells, were tested for their osteoclastogenic potential, through an indirect co‐culture system, in the absence of recombinant M‐CSF and RANKL. Osteoclastogenesis was assessed on human peripheral blood mononuclear cells (PBMC) and CD14+ cell cultures by quantification of total protein content, tartrate‐resistant acid phosphatase (TRAP) activity, presence of multinucleated cells positive for TRAP, RT‐PCR of TRAP, CATK, CA2, c‐myc and c‐src and presence of multinucleated cells displaying actin rings, vitronectin and calcitonin receptors. Cultures supplemented with M‐CSF and RANKL were used as positive controls. It was observed that the conditioned medium from dexamethasone osteogenic‐induced bone marrow cell cultures displayed the highest osteoclastogenic potential, with similar behaviour to that observed in the presence of both M‐CSF and RANKL. Comparatively, fibroblastic conditioned medium elicited a slightly lower osteoclastogenic response. Combination of both conditioned media resulted in a significant increase of TRAP activity. On the other hand, conditioned medium from non‐osteogenic‐induced bone marrow cell cultures presented the lowest osteoclastogenic potential. These results were observed for both PBMC and CD14+ cell cultures, suggesting that fibroblast and osteoblast cells are able to modulate osteoclastogenesis in the absence of physical cell–cell interactions. In addition, osteoclastogenic potential of bone marrow cells increases with their osteoblastic differentiation. J. Cell. Biochem. 109: 205–216, 2010. © 2009 Wiley‐Liss, Inc.     

Conformational insights into the inhibitory mechanism of phyto-compounds against Src kinase family members implicated in psoriasis

Abstract

Psoriasis is a chronic immune mediated disorder of the skin. There is growing evidence that the Src family tyrosine kinases (SFK) are highly upregulated in psoriasis. The SFK are the key components of the signaling pathways triggering cell growth and differentiation in addition to the immune cascades. In the current work, the interactions between SFK and selective phyto-compounds were studied using molecular docking approach. Based on the results of docking and binding energy calculations quercetin was identified as potential lead compound. To get a deeper insight into the binding of quercetin with the SFK, a combined molecular dynamics and binding free energy calculations were performed. The binding of quercetin disrupted the intra-molecular contacts making the SFK compact except Src kinase. The MM/PBSA free energy decomposition analysis highlighted the significance of hydrophobic and polar residues which are involved in the binding of quercetin. An experimental validation was carried out against the activated forms of Fyn, Lyn and Src kinases, the top three proteins which showed high preference for quercetin. The flow cytometry analysis showed that the expression levels of Fyn, Lyn and Src kinases were dramatically increased in HaCaT cells. However, the treatment of quercetin at the concentration of 51.65 µM for 24?h markedly decreased their expression in HaCaT cells. Besides, similar results were also observed when the HaCaT cells were treated with the kinase inhibitor Ponitinib (1.43 µM) for 24?h.

Communicated by Ramaswamy H. Sarma     

Glycosaminoglycans modulate RANKL‐induced osteoclastogenesis

Skeletal integrity is tightly regulated by the activity of osteoblasts and osteoclasts that are both under the control of extracellular glycosaminoglycans (GAGs) through their interactions with endogenous growth factors and differentiation‐promoting ligands. Receptor activator of NF‐kappa‐B ligand (RANKL), which is a tumor necrosis factor (TNF)‐related protein that is critical for osteoclast formation, is produced by osteoblasts and further modulated by certain types of GAGs. Using unfractionated osteoblast‐derived GAGs that reflect the complex tissue microenvironment within which osteoclasts reside, we demonstrate that these GAGs block the osteoclastogenic activity of RANKL. Furthermore, RANKL significantly reduces extracellular signal‐regulated protein kinase (ERK) activity, a putative suppressor of osteoclastogenesis, but osteoblast‐derived GAGs eliminate the inhibitory effects of RANKL on ERK activity. Notably, while imposing an anti‐osteoclastic effect, these GAGs also enhanced the proliferation of osteoblasts. Thus, the osteoblast microenvironment is a potent source of GAGs that promote bone anabolic activities. The anti‐osteoclastogenic and osteoblast‐related mitogenic activities of these GAGs together may provide a key starting point for the development of selective sugar‐based therapeutic compounds for the treatment of osteopenic disorders. J. Cell. Biochem. 109: 1222–1231, 2010. © 2010 Wiley‐Liss, Inc.     

HIV-1 Nef selectively activates Src family kinases Hck, Lyn, and c-Src through direct SH3 domain interaction

Nef is an HIV-1 virulence factor that promotes viral pathogenicity by altering host cell signaling pathways. Nef binds several members of the Src kinase family, and these interactions have been implicated in the pathogenesis of HIV/AIDS. However, the direct effect of Nef interaction on Src family kinase (SFK) regulation and activity has not been systematically addressed. We explored this issue using Saccharomyces cerevisiae, a well defined model system for the study of SFK regulation. Previous studies have shown that ectopic expression of c-Src arrests yeast cell growth in a kinase-dependent manner. We expressed Fgr, Fyn, Hck, Lck, Lyn, and Yes as well as c-Src in yeast and found that each kinase was active and induced growth suppression. Co-expression of the negative regulatory kinase Csk suppressed SFK activity and reversed the growth-inhibitory effect. We then co-expressed each SFK with HIV-1 Nef in the presence of Csk. Nef strongly activated Hck, Lyn, and c-Src but did not detectably affect Fgr, Fyn, Lck, or Yes. Mutagenesis of the Nef PXXP motif essential for SH3 domain binding greatly reduced the effect of Nef on Hck, Lyn, and c-Src, suggesting that Nef activates these Src family members through allosteric displacement of intramolecular SH3-linker interactions. These data show that Nef selectively activates Hck, Lyn, and c-Src among SFKs, identifying these kinases as proximal effectors of Nef signaling and potential targets for anti-HIV drug discovery.     

A CD19-dependent signaling pathway regulates autoimmunity in Lyn-deficient mice

CD19 and the Src family protein tyrosine kinases (PTKs) are important regulators of intrinsic signaling thresholds in B cells. Regulation is achieved by cross-talk between Src family PTKs and CD19; Lyn is essential for CD19 phosphorylation, while CD19 establishes an Src family PTK activation loop that amplifies kinase activity. However, CD19-deficient (CD19(-/-)) B cells are hyporesponsive to transmembrane signals, while Lyn-deficient (Lyn(-/-)) B cells exhibit a hyper-responsive phenotype resulting in autoimmunity. To identify the outcome of interactions between CD19 and Src family PTKs in vivo, B cell function was examined in mice deficient for CD19 and Lyn (CD19/Lyn(-/-)). Remarkably, CD19 deficiency suppressed the hyper-responsive phenotype of Lyn(-/-) B cells and autoimmunity characterized by serum autoantibodies and immune complex-mediated glomerulonephritis in Lyn(-/-) mice. Consistent with Lyn and CD19 each regulating conventional B cell development, B1 cell development was markedly reduced by Lyn deficiency, with further reductions in the absence of CD19 expression. Tyrosine phosphorylation of Fyn and other cellular proteins induced following B cell Ag receptor ligation was dramatically reduced in CD19/Lyn(-/-) B cells relative to Lyn(-/-) B cells, while Syk phosphorylation was normal. In addition, the enhanced intracellular Ca(2+) responses following B cell Ag receptor ligation that typify Lyn deficiency were delayed by the loss of CD19 expression. BCR-induced proliferation and humoral immune responses were also markedly inhibited by CD19/Lyn deficiency. These findings demonstrate that while the CD19/Lyn amplification loop is a major regulator of signal transduction thresholds in B lymphocytes, CD19 regulation of other Src family PTKs also influences B cell function and the development of autoimmunity.     

Osteoclastogenic activity and RANKL expression are inhibited in osteoblastic cells expressing constitutively active Gα12 or constitutively active RhoA

Gα₁₂–RhoA signaling is a parathyroid hormone (PTH)‐stimulated pathway that mediates effects in bone and may influence genetic susceptibility to osteoporosis. To further elucidate effects of the pathway in osteoblasts, UMR‐106 osteoblastic cells were stably transfected with constitutively active (ca) Gα₁₂ or caRhoA or dominant negative (dn) RhoA and co‐cultured with RAW 264.7 cells to determine effects on hormone‐stimulated osteoclastogenesis. Whereas PTH and calcitriol‐stimulated osteoclastogenesis in co‐cultures with UMR‐106 cells expressing pcDNA or dominant negative RhoA, the osteoclastogenic effects of PTH and calcitriol were significantly attenuated when the UMR‐106 cells expressed either caRhoA or caGα₁₂. These inhibitory effects were partially reversed by the Rho kinase inhibitor Y27632. None of the constructs affected osteoclastogenesis in untreated co‐cultures, and the constructs did not inhibit the osteoclastogenic responses to receptor activator of NFκB ligand (RANKL). To investigate the mechanism of the inhibitory effects of caGα₁₂ and caRhoA, expression of RANKL, osteoprotegerin (OPG), osteopontin (OPN), and intercellular adhesion molecule‐1 (ICAM) in response to PTH or calcitriol was examined in the UMR‐106 cells. In the cells expressing pcDNA or dnRhoA, PTH and calcitriol increased RANKL mRNA and decreased OPG mRNA, whereas these effects were absent in the cells expressing caGα₁₂ or caRhoA. Basal expression of RANKL and OPG was unaffected by the constructs. The results suggest that Gα₁₂–RhoA signaling can inhibit hormone‐stimulated osteoclastogenesis by effects on expression of RANKL and OPG. Since PTH can stimulate the Gα₁₂–RhoA pathway, the current findings could represent a homeostatic mechanism for regulating osteoclastogenic action. J. Cell. Biochem. 111: 1531–1536, 2010. © 2010 Wiley‐Liss, Inc.     

Integrins direct Src family kinases to regulate distinct phases of oligodendrocyte development

Specific integrins expressed on oligodendrocytes, the myelin-forming cells of the central nervous system, promote either differentiation and survival or proliferation by amplification of growth factor signaling. Here, we report that the Src family kinases (SFKs) Fyn and Lyn regulate each of these distinct integrin-driven behaviors. Fyn associates with alpha6beta1 and is required to amplify platelet-derived growth factor survival signaling, to promote myelin membrane formation, and to switch neuregulin signaling from a phosphatidylinositol 3-kinase to a mitogen-activated protein kinase pathway (thereby changing the response from proliferation to differentiation). However, earlier in the lineage Lyn, not Fyn, is required to drive alphaVbeta3-dependent progenitor proliferation. The two SFKs respond to integrin ligation by different mechanisms: Lyn, by increased autophosphorylation of a catalytic tyrosine; and Fyn, by reduced Csk phosphorylation of the inhibitory COOH-terminal tyrosine. These findings illustrate how different SFKs can act as effectors for specific cell responses during development within a single cell lineage, and, furthermore, provide a molecular mechanism to explain similar region-specific hypomyelination in laminin- and Fyn-deficient mice.     

Tumor necrosis factor stimulates osteoclastogenesis from human bone marrow cells under hypoxic conditions

Bone homeostasis is maintained by the balance between osteoblastic bone formation and osteoclastic bone resorption. In this study, we used human bone marrow cells (BMCs) to investigate the role of hypoxic exposure on human osteoclast (OC) formation in the presence of tumor necrosis factor (TNF). Exposing the BMCs to 3%, 5%, or 10% O₂ in the presence of receptor activator of NF-κB ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) generated tartrate-resistant acid phosphatase (TRAP)-positive multinuclear cells, consistent with OCs. The addition of TNF under hypoxic conditions generated significantly greater numbers of mature OCs with more nuclei than OCs generated under normoxic conditions. Longer initial hypoxic exposure increased the number of OC precursor cells and facilitated the differentiation of OC precursor cells into multinucleated OCs. Quantitative RT-PCR analysis revealed that RANKL and TNFR1 were expressed at higher levels in non-OC cells from BMCs under hypoxic conditions than under normoxic conditions. Furthermore, to confirm the involvement of TNF-induced signaling, we examined the effects of blocking antibodies against TNFR1 and TNFR2 on OC formation under hypoxic conditions. The TNFR1 antibody was observed to significantly suppress OC formation. These results suggest that hypoxic exposure plays an important role in TNF-induced osteoclastogenesis from human BMCs.     

Activated invariant NKT cells regulate osteoclast development and function

Invariant NKT (iNKT) cells modulate innate and adaptive immune responses through activation of myeloid dendritic cells and macrophages and via enhanced clonogenicity, differentiation, and egress of their shared myeloid progenitors. Because these same progenitors give rise to osteoclasts (OCs), which also mediate the egress of hematopoietic progenitors and orchestrate bone remodeling, we hypothesized that iNKT cells would extend their myeloid cell regulatory role to the development and function of OCs. In this study, we report that selective activation of iNKT cells by α-galactosylceramide causes myeloid cell egress, enhances OC progenitor and precursor development, modifies the intramedullary kinetics of mature OCs, and enhances their resorptive activity. OC progenitor activity is positively regulated by TNF-α and negatively regulated by IFN-γ, but is IL-4 and IL-17 independent. These data demonstrate a novel role of iNKT cells that couples osteoclastogenesis with myeloid cell egress in conditions of immune activation.     

Cbl-mediated degradation of Lyn and Fyn induced by constitutive fibroblast growth factor receptor-2 activation supports osteoblast differentiation

Fibroblast growth factors (FGFs) play an important regulatory role in skeletal development and bone formation. However, the FGF signaling mechanisms controlling osteoblast function are poorly understood. Here, we identified a role for the Src family members Lyn and Fyn in osteoblast differentiation promoted by constitutive activation of FGF receptor-2 (FGFR2). We show that the overactive FGFR2 S252W mutation induced decreased Src family kinase tyrosine phosphorylation and activity associated with decreased Lyn and Fyn protein expression in human osteoblasts. Pharmacological stimulation of Src family kinases or transfection with Lyn or Fyn vectors repressed alkaline phosphatase (ALP) up-regulation induced by overactive FGFR2. Inhibition of proteasome activity restored normal Lyn and Fyn expression and ALP activity in FGFR2 mutant osteoblasts. Immunoprecipitation studies showed that Lyn, Fyn, and FGFR2 interacted with the ubiquitin ligase c-Cbl and ubiquitin. Transfection with c-Cbl in which the RING finger was disrupted or with c-Cbl with a point mutation that abolishes the binding ability of the Cbl phosphotyrosine-binding domain restored Src kinase activity and Lyn, Fyn, and FGFR2 levels and reduced ALP up-regulation in mutant osteoblasts. Thus, constitutive FGFR2 activation induces c-Cbl-dependent Lyn and Fyn proteasome degradation, resulting in reduced Lyn and Fyn kinase activity, increased ALP expression, and FGFR2 down-regulation. This reveals a common Cbl-mediated negative feedback mechanism controlling Lyn, Fyn, and FGFR2 degradation in response to overactive FGFR2 and indicates a role for Cbl-dependent down-regulation of Lyn and Fyn in osteoblast differentiation induced by constitutive FGFR2 activation.     

Src Family Kinases Are Required for Prolactin Induction of Cell Proliferation

Prolactin (PRL) is a pleiotropic cytokine promoting cellular proliferation and differentiation. Because PRL activates the Src family of tyrosine kinases (SFK), we have studied the role of these kinases in PRL cell proliferation signaling. PRL induced [(3)H]thymidine incorporation upon transient transfection of BaF-3 cells with the PRL receptor. This effect was inhibited by cotransfection with the dominant negative mutant of c-Src (K>A295/Y>F527, SrcDM). The role of SFK in PRL-induced proliferation was confirmed in the BaF-3 PRL receptor-stable transfectant, W53 cells, where PRL induced Fyn and Lyn activation. The SFK-selective inhibitors PP1/PP2 and herbimycin A blocked PRL-dependent cell proliferation by arresting the W53 cells in G1, with no evident apoptosis. In parallel, PP1/PP2 inhibited PRL induction of cell growth-related genes c-fos, c-jun, c-myc, and odc. These inhibitors have no effect on PRL-mediated activation of Ras/Mapk and Jak/Start pathways. In contrast, they inhibited the PRL-dependent stimulation of the SFKs substrate Sam68, the phosphorylation of the tyrosine phosphatase Shp2, and the PI3K-dependent Akt and p70S6k serine kinases. Consistently, transient expression of SrcDM in W53 cells also blocked PRL activation of Akt. These results demonstrate that activation of SFKs is required for cell proliferation induced by PRL.     

NIK stabilization in osteoclasts results in osteoporosis and enhanced inflammatory osteolysis

Background

Maintenance of healthy bone requires the balanced activities of osteoclasts (OCs), which resorb bone, and osteoblasts, which build bone. Disproportionate action of OCs is responsible for the bone loss associated with postmenopausal osteoporosis and rheumatoid arthritis. NF-κB inducing kinase (NIK) controls activation of the alternative NF-κB pathway, a critical pathway for OC differentiation. Under basal conditions, TRAF3-mediated NIK degradation prevents downstream signaling, and disruption of the NIK:TRAF3 interaction stabilizes NIK leading to constitutive activation of the alternative NF-κB pathway.

Methodology/Principal Findings

Using transgenic mice with OC-lineage expression of NIK lacking its TRAF3 binding domain (NT3), we now find that alternative NF-κB activation enhances not only OC differentiation but also OC function. Activating NT3 with either lysozyme M Cre or cathepsinK Cre causes high turnover osteoporosis with increased activity of OCs and osteoblasts. In vitro, NT3-expressing precursors form OCs more quickly and at lower doses of RANKL. When cultured on bone, they exhibit larger actin rings and increased resorptive activity. OC-specific NT3 transgenic mice also have an exaggerated osteolytic response to the serum transfer model of arthritis.

Conclusions

Constitutive activation of NIK drives enhanced osteoclastogenesis and bone resorption, both in basal conditions and in response to inflammatory stimuli.     

Src family kinases are required for integrin but not PDGFR signal transduction

Src family kinases (SFKs) have been implicated as important regulators of ligand-induced cellular responses including proliferation, survival, adhesion and migration. Analysis of SFK function has been impeded by extensive redundancy between family members. We have generated mouse embryos harboring functional null mutations of the ubiquitously expressed SFKs Src, Yes and Fyn. This triple mutation leads to severe developmental defects and lethality by E9.5. To elucidate the molecular mechanisms underlying this phenotype, SYF cells (deficient for Src, Yes and Fyn) were derived and tested for their ability to respond to growth factors or plating on extracellular matrix. Our studies reveal that while Src, Yes and Fyn are largely dispensable for platelet-derived growth factor (PDGF)-induced signaling, they are absolutely required to mediate specific functions regulated by extracellular matrix proteins. Fibronectin-induced tyrosine phosphorylation of focal adhesion proteins, including the focal adhesion kinase FAK, was nearly eliminated in the absence of Src, Yes and Fyn. Furthermore, consistent with previous reports demonstrating the importance of FAK for cell migration, SYF cells displayed reduced motility in vitro. These results demonstrate that SFK activity is essential during embryogenesis and suggest that defects observed in SYF triple mutant embryos may be linked to deficiencies in signaling by extracellular matrix-coupled receptors.     

Lyn inhibits osteoclast differentiation by interfering with PLCγ1-mediated Ca signaling

Osteoclasts differentiate from macrophage-lineage cells to become specialized for bone resorption function. By a proteomics approach, we found that Lyn was down-regulated by the osteoclast differentiation factor, receptor activator of NF-κB ligand (RANKL). The forced reduction of Lyn caused a striking increase in the RANKL-induced PLCγ1, Ca²⁺, and NFATc1 responses during differentiation. These data suggest that Lyn plays a negative role in osteoclastogenesis by interfering with the PLCγ1-mediated Ca²⁺ signaling that leads to NFATc1 activation. Consistent with the in vitro results, in vivo injection of Lyn specific siRNA into mice calvariae provoked a fulminant bone resorption. Our study provides the first evidence of the involvement of Lyn in the negative regulation of osteoclastogenesis by RANKL.