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.     

Syk, c-Src, the alphavbeta3 integrin, and ITAM immunoreceptors, in concert, regulate osteoclastic bone resorption

In this study, we establish that the tyrosine kinase Syk is essential for osteoclast function in vitro and in vivo. Syk^−/− osteoclasts fail to organize their cytoskeleton, and, as such, their bone-resorptive capacity is arrested. This defect results in increased skeletal mass in Syk^−/− embryos and dampened basal and stimulated bone resorption in chimeric mice whose osteoclasts lack the kinase. The skeletal impact of Syk deficiency reflects diminished activity of the mature osteoclast and not impaired differentiation. Syk regulates bone resorption by its inclusion with the αvβ3 integrin and c-Src in a signaling complex, which is generated only when αvβ3 is activated. Upon integrin occupancy, c-Src phosphorylates Syk. αvβ3-induced phosphorylation of Syk and the latter's capacity to associate with c-Src is mediated by the immunoreceptor tyrosine-based activation motif (ITAM) proteins Dap12 and FcRγ. Thus, in conjunction with ITAM-bearing proteins, Syk, c-Src, and αvβ3 represent an essential signaling complex in the bone-resorbing osteoclast, and, therefore, each is a candidate therapeutic target.     

Fyn promotes proliferation,differentiation, survival and function of osteoclast lineage cells

c‐Src and Lyn are the only Src family kinases (SFKs) with established activity in osteoclasts (OCs). c‐Src promotes function via cytoskeletal organization of the mature resorptive cell while Lyn is a negative regulator of osteoclastogenesis. We establish that Fyn, another SFK, also impacts the OC, but in a manner distinctly different than c‐Src and Lyn. Fyn deficiency principally alters cells throughout the osteoclastogenic process, resulting in diminished numbers of resorptive polykaryons. Arrested OC formation in the face of insufficient Fyn reflects reduced proliferation of precursors, in response to M‐CSF and retarded RANK ligand (RANKL)‐induced differentiation, attended by suppressed activation of the osteoclastogenic signaling molecules, c‐Jun, and NF‐κB. The anti‐apoptotic properties of RANKL are also compromised in cells deleted of Fyn, an event mediated by increased Bim expression and failed activation of Akt. The defective osteoclastogenesis of Fyn?/? OCs dampens bone resorption, in vitro. Finally, while Fyn deficiency does not regulate basal osteoclastogenesis, in vivo, it reduces that stimulated by RANKL by ～2/3. Thus, Fyn is a pro‐resorptive SFK, which exerts its effects by prompting proliferation and differentiation while attenuating apoptosis of OC lineage cells. J. Cell. Biochem. 111: 1107–1113, 2010. © 2010 Wiley‐Liss, Inc.     

Integrin-Mediated Signal Transduction in Human Endothelial Cells: Analysis of Tyrosine Phosphorylation Events

Adhesion of human umbilical endothelial cells to fibronectin resulted in increased tyrosine phosphorylation of a group of proteins with molecular mass ranging from 100 to 130 kDa and of a 70 kDa protein. This pattern of tyrosine phosphorylation was also observed when endothelial cells adhered to vitronectin, collagen IV, collagen I and laminin or to culture dishes coated with antibodies directed to either βl, α3, α5, α6 or β3 integrin subunits. Increased phosphorylation of the 100–130 kDa proteins was detectable as early as 30 sec after adhesion, reached maximal level after 15 min, and remained high as long as the cells adhere to culture dishes. The 70 kDa protein was phosphorylated with a slower kinetics and its phosphorylation increased over a period of 3 h. Using specific monoclonal antibodies, the major component of the 100–130 kDa complex was identified as the focal adhesion tyrosine kinase p125^FAK. The phosphorylation of the pl25^FAK was also observed by inducing βl integrin clustering in rum adherent HEC, indicating that this is a primary signalling event induced by integrins. Using tyrosine kinase inhibitors, we show a direct correlation between integrin-stimulated tyrosine kinases and assembly of focal adhesions and actin fibres.     

IL‐1α and IL‐1β have different effects on formation and activity of large osteoclasts

Interleukin 1 (IL‐1) is a proinflammatory cytokine upregulated in conditions such as rheumatoid arthritis and periodontal disease. Both isoforms, IL‐1α and IL‐1β, have been shown to activate osteoclasts (OCs), the cells responsible for resorbing bone. Inflammatory conditions are also characterized by increased bone loss and by the presence of large OCs (10+ nuclei). We and others have previously shown that large OCs are more likely to be resorbing compared to small OCs (2–5 nuclei). Moreover, large OCs express higher levels of the IL‐1 activating receptor IL‐1RI, integrins αv and β3, RANK, and TNFR1, while small OCs have higher levels of the decoy receptor IL‐1RII. We hypothesized that IL‐1 would have different effects on large and small OCs due to these distinct receptor expression patterns. To test this hypothesis, RAW 264.7 cells were differentiated into populations of small and large OCs and treated with IL‐1α or IL‐1β (1 and 10 ng/ml). In the presence of sRANKL, both IL‐1α and IL‐1β increased total OC number and resorptive activity of large OCs. IL‐1α stimulated formation of large OCs and increased the number of resorption pits, while IL‐1β changed the morphology of large OCs and integrin‐β3 phosphorylation. No effects were seen in small OCs in response to either IL‐1 isoform. These results demonstrate that IL‐1 predominantly affects large OCs. The dissimilarity of responses to IL‐1α and IL‐1β suggests that these isoforms activate different signaling pathways within the two OC populations. J. Cell. Biochem. 109: 975–982, 2010. © 2010 Wiley‐Liss, Inc.     

Vinculin Regulates Osteoclast Function

Osteoclastic bone resorption depends upon the cell''s ability to organize its cytoskeleton. Because vinculin (VCL) is an actin-binding protein, we asked whether it participates in skeletal degradation. Thus, we mated VCL^fl/fl mice with those expressing cathepsin K-Cre (CtsK-VCL) to delete the gene in mature osteoclasts or lysozyme M-Cre (LysM-VCL) to target all osteoclast lineage cells. VCL-deficient osteoclasts differentiate normally but, reflecting cytoskeletal disorganization, form small actin rings and fail to effectively resorb bone. In keeping with inhibited resorptive function, CtsK-VCL and LysM-VCL mice exhibit a doubling of bone mass. Despite cytoskeletal disorganization, the capacity of VCL^−/− osteoclastic cells to normally phosphorylate c-Src in response to αvβ3 integrin ligand is intact. Thus, integrin-activated signals are unrelated to the means by which VCL organizes the osteoclast cytoskeleton. WT VCL completely rescues actin ring formation and bone resorption, as does VCL^P878A, which is incapable of interacting with Arp2/3. As expected, deletion of the VCL tail domain (VCL^1–880), which binds actin, does not normalize VCL^−/− osteoclasts. The same is true regarding VCL^I997A, which also prevents VCL/actin binding, and VCL^A50I and VCL^811–1066, both of which arrest talin association. Thus, VCL binding talin, but not Arp2/3, is critical for osteoclast function, and its selective inhibition retards physiological bone loss.     

The N-terminal SH2 domains of Syk and ZAP-70 mediate phosphotyrosine-independent binding to integrin beta cytoplasmic domains

Syk and ZAP-70 form a subfamily of nonreceptor tyrosine kinases that contain tandem SH2 domains at their N termini. Engagement of these SH2 domains by tyrosine-phosphorylated immunoreceptor tyrosine-based activation motifs leads to kinase activation and downstream signaling. These kinases are also regulated by beta3 integrin-dependent cell adhesion via a phosphorylation-independent interaction with the beta3 integrin cytoplasmic domain. Here, we report that the interaction of integrins with Syk and ZAP-70 depends on the N-terminal SH2 domain and the interdomain A region of the kinase. The N-terminal SH2 domain alone is sufficient for weak binding, and this interaction is independent of tyrosine phosphorylation of the integrin tail. Indeed, phosphorylation of tyrosines within the two conserved NXXY motifs in the integrin beta3 cytoplasmic domain blocks Syk binding. The tandem SH2 domains of these kinases bind to multiple integrin beta cytoplasmic domains with varying affinities (beta3 (Kd = 24 nm) > beta2 (Kd = 38 nm) > beta1 (Kd = 71 nm)) as judged by both affinity chromatography and surface plasmon resonance. Thus, the binding of Syk and ZAP-70 to integrin beta cytoplasmic domains represents a novel phosphotyrosine-independent interaction mediated by their N-terminal SH2 domains.     

Activation of Syk protein tyrosine kinase through interaction with integrin beta cytoplasmic domains.

Syk protein tyrosine kinase is essential for immune system development and function [1]and for the maintenance of vascular integrity [2,3]. In leukocytes, Syk is activated by binding to diphosphorylated immune receptor tyrosine-based activation motifs (pITAMs)[1]. Syk can also be activated by integrin adhesion receptors [4,5], but the mechanism of its activation is unknown. Here we report a novel mechanism for Syk's recruitment and activation, which requires that Syk bind to the integrin beta3 cytoplasmic tail. We found that both Syk and the related kinase ZAP-70 bound the beta3 cytoplasmic tail through their tandem SH2 domains. However, unlike Syk binding to pITAMs, this interaction was independent of tyrosine phosphorylation and of the phosphotyrosine binding function of Syk's tandem SH2 domains. Deletion of the four C-terminal residues of the beta3 cytoplasmic tail [beta3(759X)] decreased Syk binding and disrupted its physical association with integrin alphaIIbbeta3. Furthermore, cells expressing alphaIIbbeta3(759X) failed to exhibit Syk activation or lamellipodia formation upon cell adhesion to the alphaIIbbeta3 ligand, fibrinogen. In contrast, FAK phosphorylation and focal adhesion formation were unimpaired by this mutation. Thus, the direct binding of Syk kinase to the integrin beta3 cytoplasmic tail is a novel and functionally significant mechanism for the regulation of this important non-receptor tyrosine kinase.     

The molecular adapter SLP-76 relays signals from platelet integrin alphaIIbbeta3 to the actin cytoskeleton

Platelet adhesion to fibrinogen through integrin alpha(IIb)beta(3) triggers actin rearrangements and cell spreading. Mice deficient in the SLP-76 adapter molecule bleed excessively, and their platelets spread poorly on fibrinogen. Here we used human platelets and a Chinese hamster ovary (CHO) cell expression system to better define the role of SLP-76 in alpha(IIb)beta(3) signaling. CHO cell adhesion to fibrinogen required alpha(IIb)beta(3) and stimulated tyrosine phosphorylation of SLP-76. SLP-76 phosphorylation required coexpression of Syk tyrosine kinase and stimulated association of SLP-76 with the adapter, Nck, and with the Rac exchange factor, Vav1. SLP-76 expression increased lamellipodia formation induced by Syk and Vav1 in adherent CHO cells (p < 0.001). Although lamellipodia formation requires Rac, SLP-76 functioned downstream of Rac by potentiating adhesion-dependent activation of PAK kinase (p < 0.001), a Rac effector that associates with Nck. In platelets, adhesion to fibrinogen stimulated the association of SLP-76 with the SLAP-130 adapter and with VASP, a SLAP-130 binding partner implicated in actin reorganization. Furthermore, SLAP-130 colocalized with VASP at the periphery of spread platelets. Thus, SLP-76 functions to relay signals from alpha(IIb)beta(3) to effectors of cytoskeletal reorganization. Therefore, deficient recruitment of specific adapters and effectors to sites of adhesion may explain the integrin phenotype of SLP-76(-/-) platelets.     

PI3K is involved in L‐selectin‐ and PSGL‐1‐mediated neutrophil rolling on E‐selectin via F‐actin redistribution and assembly

L‐selectin and P‐selectin glycoprotein ligand‐1 (PSGL‐1) are adhesion molecules that play critical roles in neutrophil rolling during inflammation and lymphocyte homing. On the other hand they also function as signaling receptors to induce cytoskeleton changes. The present study is to investigate the signaling kinases responsible for the F‐actin changes mediated by L‐selectin and PSGL‐1 during neutrophil rolling on E‐selectin. Western blot analysis demonstrated that PI3K activation, peaking within 5 min, was induced by ligation of L‐selectin and PSGL‐1 with E‐selectin, and that Vav1 (the pivotal downstream effector of PI3K signaling pathway involved in cytoskeleton regulation) was recruited to the membrane and tyrosine‐phosphorylated, depending on PI3K. Furthermore, the F‐actin redistribution and assembly mediated by ligation with E‐selectin were blocked by LY294002, a PI3K specific inhibitor. Additional experiments showed that PI3K activity was involved in neutrophil rolling on E‐selectin. However, Syk/Zap70, the well‐known upstream kinase of PI3K, was not involved in this event. These data suggest that PI3K is required for the F‐actin‐based cytoskeleton changes during neutrophil rolling on E‐selectin, which may consequently regulate the rolling event. J. Cell. Biochem. 110: 910–919, 2010. © 2010 Wiley‐Liss, Inc.     

CD19 and BAFF‐R can signal to promote B‐cell survival in the absence of Syk

The development and function of B lymphocytes is regulated by numerous signaling pathways, some emanating from the B‐cell antigen receptor (BCR). The spleen tyrosine kinase (Syk) plays a central role in the activation of the BCR, but less is known about its contribution to the survival and maintenance of mature B cells. We generated mice with an inducible and B‐cell‐specific deletion of the Syk gene and found that a considerable fraction of mature Syk‐negative B cells can survive in the periphery for an extended time. Syk‐negative B cells are defective in BCR, RP105 and CD38 signaling but still respond to an IL‐4, anti‐CD40, CpG or LPS stimulus. Our in vivo experiments show that Syk‐deficient B cells require BAFF receptor and CD19/PI3K signaling for their long‐term survival. These studies also shed a new light on the signals regulating the maintenance of the normal mature murine B‐cell pool.     

Cutting edge: dexamethasone negatively regulates Syk in mast cells by up-regulating SRC-like adaptor protein

We have identified Src-like adaptor protein (SLAP) as one of several dexamethasone-inducible inhibitory regulators in mast cells. SLAP is a known inhibitor of T cell signaling and interacts with the tyrosine kinase, Zap70. Exposure of RBL-2H3 mast cells to dexamethasone markedly increased expression of SLAP. Cells so exposed or made to overexpress SLAP exhibited reduced Ag-stimulated phosphorylation of Syk (a cognate of Zap70), linker for activation of T cells, phospholipase Cgamma, and ERK. Ca(2+) mobilization, Ca(2+)-dependent degranulation, and ERK-dependent release of arachidonic acid were suppressed as well. Small interfering RNA directed against SLAP blocked the induction of SLAP and reversed the inhibitory effects of dexamethasone on phosphorylation of Syk, linker for activation of T cells, and phospholipase Cgamma, but not downstream events, which are likely suppressed by up-regulation of downstream of tyrosine kinase-1 and MAPK phosphatase-1. The induction of these inhibitory regulators may contribute to the immunosuppressive activity of dexamethasone in mast cells.     

Vav3 regulates osteoclast function and bone mass

Osteoporosis, a leading cause of morbidity in the elderly, is characterized by progressive loss of bone mass resulting from excess osteoclastic bone resorption relative to osteoblastic bone formation. Here we identify Vav3, a Rho family guanine nucleotide exchange factor, as essential for stimulated osteoclast activation and bone density in vivo. Vav3-deficient osteoclasts show defective actin cytoskeleton organization, polarization, spreading and resorptive activity resulting from impaired signaling downstream of the M-CSF receptor and alpha(v)beta3 integrin. Vav3-deficient mice have increased bone mass and are protected from bone loss induced by systemic bone resorption stimuli such as parathyroid hormone or RANKL. Moreover, we provide genetic and biochemical evidence for the role of Syk tyrosine kinase as a crucial upstream regulator of Vav3 in osteoclasts. Thus, Vav3 is a potential new target for antiosteoporosis therapy.     

The Syk Protein Tyrosine Kinase Is Essential for Fcγ Receptor Signaling in Macrophages and Neutrophils

The cytoplasmic protein tyrosine kinase Syk has two amino-terminal SH2 domains that engage phosphorylated immunoreceptor tyrosine-based activation motifs in the signaling subunits of immunoreceptors. Syk, in conjunction with Src family kinases, has been implicated in immunoreceptor signaling in both lymphoid and myeloid cells. We have investigated the role of Syk in Fcγ receptor (FcγR)-dependent and -independent responses in bone marrow-derived macrophages and neutrophils by using mouse radiation chimeras reconstituted with fetal liver cells from Syk^−/− embryos. Chimeric mice developed an abdominal hemorrhage starting 2 to 3 months after transplantation that was ultimately lethal. Syk-deficient neutrophils derived from the bone marrow were incapable of generating reactive oxygen intermediates in response to FcγR engagement but responded normally to tetradecanoyl phorbol acetate stimulation. Syk-deficient macrophages were defective in phagocytosis induced by FcγR but showed normal phagocytosis in response to complement. The tyrosine phosphorylation of multiple cellular polypeptides, including the FcγR γ chain, as well as Erk2 activation, was compromised in Syk^−/− macrophages after FcγR stimulation. In contrast, the induction of nitric oxide synthase in macrophages stimulated with lipopolysaccharide and gamma interferon was not dependent on Syk. Surprisingly, Syk-deficient macrophages were impaired in the ability to survive or proliferate on plastic petri dishes. Taken together, these results suggest that Syk has specific physiological roles in signaling from FcγRs in neutrophils and macrophages and raise the possibility that in vivo, Syk is involved in signaling events other than those mediated by immunoreceptors.     

Coordinate interactions of Csk, Src, and Syk kinases with [alpha]IIb[beta]3 initiate integrin signaling to the cytoskeleton

Integrins regulate cell adhesion and motility through tyrosine kinases, but initiation of this process is poorly understood. We find here that Src associates constitutively with integrin alphaIIbbeta3 in platelets. Platelet adhesion to fibrinogen caused a rapid increase in alphaIIbbeta3-associated Src activity, and active Src localized to filopodia and cell edges. Csk, which negatively regulates Src by phosphorylating Tyr-529, was also constitutively associated with alphaIIbbeta3. However, fibrinogen binding caused Csk to dissociate from alphaIIbbeta3, concomitant with dephosphorylation of Src Tyr-529 and phosphorylation of Src activation loop Tyr-418. In contrast to the behavior of Src and Csk, Syk was associated with alphaIIbbeta3 only after fibrinogen binding. Platelets multiply deficient in Src, Hck, Fgr, and Lyn, or normal platelets treated with Src kinase inhibitors failed to spread on fibrinogen. Inhibition of Src kinases blocked Syk activation and inhibited phosphorylation of Syk substrates (Vav1, Vav3, SLP-76) implicated in cytoskeletal regulation. Syk-deficient platelets exhibited Src activation upon adhesion to fibrinogen, but no spreading or phosphorylation of Vav1, Vav3, and SLP-76. These studies establish that platelet spreading on fibrinogen requires sequential activation of Src and Syk in proximity to alphaIIbbeta3, thus providing a paradigm for initiation of integrin signaling to the actin cytoskeleton.     

TULA‐family proteins: A new class of cellular regulators

Proteins of the UBASH3/STS/TULA family recently emerged as potent regulators of cellular functions. They are characterized by a unique architecture, featuring at least three functional domains. One of them is a histidine phosphatase domain, which mediates the protein tyrosine phosphatase activity of these proteins. Recent studies demonstrated that UBASH3/STS/TULA‐family proteins play a key role in down‐regulating receptor‐mediated signal transduction and physiologic responses of T cells and platelets in vitro and in vivo. The Syk‐family protein tyrosine kinases Syk and Zap‐70 were identified as major targets of TULA‐2 in full agreement with the suppressive effect of this phosphatase in systems where Syk and Zap‐70 carry out the essential early steps of signal transduction. In spite of significant similarity between TULA and TULA‐2, there are also considerable functional differences between them. Thus, TULA‐2 is expressed ubiquitously in mammalian tissues and exhibits high phosphatase activity, whereas TULA is expressed specifically in lymphocytes and exhibits low phosphatase activity. However, TULA also functions as a down‐regulator of cellular responses, and therefore its role may be mediated by dephosphorylation of yet‐unknown substrates or by promoting T‐cell apoptosis (the latter activity is unique for this UBASH3/STS/TULA family member). The down‐regulatory role of TULA and TULA‐2 revealed in experimental systems is consistent with the recently discovered association of several autoimmune diseases with certain risk alleles encoding for these proteins. J. Cell. Physiol. 228: 43–49, 2013. © 2012 Wiley Periodicals, Inc.     

Proximal, selective, and dynamic interactions between integrin alphaIIbbeta3 and protein tyrosine kinases in living cells

Stable platelet aggregation, adhesion, and spreading during hemostasis are promoted by outside-in alphaIIbbeta3 signals that feature rapid activation of c-Src and Syk, delayed activation of FAK, and cytoskeletal reorganization. To evaluate these alphaIIbbeta3-tyrosine kinase interactions at nanometer proximity in living cells, we monitored bioluminescence resonance energy transfer between GFP and Renilla luciferase chimeras and bimolecular fluorescence complementation between YFP half-molecule chimeras. These techniques revealed that alphaIIbbeta3 interacts with c-Src at the periphery of nonadherent CHO cells. After plating cells on fibrinogen, complexes of alphaIIbbeta3-c-Src, alphaIIbbeta3-Syk, and c-Src-Syk are observed in membrane ruffles and focal complexes, and the interactions involving Syk require Src activity. In contrast, FAK interacts with alphaIIbbeta3 and c-Src, but not with Syk, in focal complexes and adhesions. All of these interactions require the integrin beta3 cytoplasmic tail. Thus, alphaIIbbeta3 interacts proximally, if not directly, with tyrosine kinases in a coordinated, selective, and dynamic manner during sequential phases of alphaIIbbeta3 signaling to the actin cytoskeleton.     

α11β1 integrin‐mediated MMP‐13‐dependent collagen lattice contraction by fibroblasts: Evidence for integrin‐coordinated collagen proteolysis

We have previously determined that integrin α11β1 is required on mouse periodontal ligament (PDL) fibroblasts to generate the force needed for incisor eruption. As part of the phenotype of α11^?/? mice, the incisor PDL (iPDL) is thickened, due to disturbed matrix remodeling. To determine the molecular mechanism behind the disturbed matrix dynamics in the PDL we crossed α11^?/? mice with the Immortomouse and isolated immortalized iPDL cells. Microarray analysis of iPDL cells cultured inside a 3D collagen gel demonstrated downregulated expression of a number of genes in α11‐deficient iPDL cells, including matrix metalloproteinase‐13 (MMP‐13) and cathepsin K. α11^?/? iPDL cells in vitro displayed disturbed interactions with collagen I during contraction of attached and floating collagen lattices and furthermore displayed reduced MMP‐13 protein expression levels. The MMP‐13 specific inhibitor WAY 170523 and the Cathepsin K Inhibitor II both blocked part of the α11 integrin‐mediated collagen remodeling. In summary, our data demonstrate that in iPDL fibroblasts the mechanical strain generated by α11β1 integrin regulates molecules involved in collagen matrix dynamics. The positive regulation of α11β1‐dependent matrix remodeling, involving MMP‐13 and cathepsin K, might also occur in other types of fibroblasts and be an important regulatory mechanism for coordinated extracellular and intracellular collagen turnover in tissue homeostasis. J. Cell. Physiol. © 2012 Wiley Periodicals, Inc.     

Integrin function: molecular hierarchies of cytoskeletal and signaling molecules

Integrin receptors play important roles in organizing the actin- containing cytoskeleton and in signal transduction from the extracellular matrix. The initial steps in integrin function can be analyzed experimentally using beads coated with ligands or anti- integrin antibodies to trigger rapid focal transmembrane responses. A hierarchy of transmembrane actions was identified in this study. Simple integrin aggregation triggered localized transmembrane accumulation of 20 signal transduction molecules, including RhoA, Rac1, Ras, Raf, MEK, ERK, and JNK. In contrast, out of eight cytoskeletal molecules tested, only tensin coaccumulated. Integrin aggregation alone was also sufficient to induce rapid activation of the JNK pathway, with kinetics of activation different from those of ERK. The tyrosine kinase inhibitors herbimycin A or genistein blocked both the accumulation of 19 out of 20 signal transduction molecules and JNK- and ERK-mediated signaling. Cytochalasin D had identical effects, whereas three other tyrosine kinase inhibitors did not. The sole exception among signaling molecules was the kinase pp125FAK which continued to coaggregate with alpha 5 beta 1 integrins even in the presence of these inhibitors. Tyrosine kinase inhibition also failed to block the ability of ligand occupancy plus integrin aggregation to trigger transmembrane accumulation of the three cytoskeletal molecules talin, alpha-actinin, and vinculin; these molecules accumulated even in the presence of cytochalasin D. However, it was necessary to fulfill all four conditions, i.e., integrin aggregation, integrin occupancy, tyrosine kinase activity, and actin cytoskeletal integrity, to achieve integrin- mediated focal accumulation of other cytoskeletal molecules including F- actin and paxillin. Integrins therefore mediate a transmembrane hierarchy of molecular responses.     

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.