SLAP deficiency decreases dsDNA autoantibody production.

Src-like adaptor protein (SLAP) adapts c-Cbl, an E3 ubiquitin ligase, to activated components of the BCR signaling complex regulating BCR levels and signaling in developing B cells. Based on this function, we asked whether SLAP deficiency could decrease the threshold for tolerance and eliminate development of autoreactive B cells in two models of autoantibody production. First, we sensitized mice with a dsDNA mimetope that causes an anti-dsDNA response. Despite equivalent production of anti-peptide antibodies compared to BALB/c controls, SLAP(-/-) mice did not produce anti-dsDNA. Second, we used the 56R tolerance model. SLAP(-/-) 56R mice had decreased levels of dsDNA-reactive antibodies compared to 56R mice due to skewed light chain usage. Thus, SLAP is a critical regulator of B-cell development and function and its deficiency leads to decreased autoreactive B cells that are otherwise maintained by inefficient receptor editing or failed negative selection.

SLAP deficiency increases TCR avidity leading to altered repertoire and negative selection of cognate antigen-specific CD8+ T cells.

How T cell receptor (TCR) avidity influences CD8(+) T cell development and repertoire selection is not yet fully understood. To fill this gap, we utilized Src-like adaptor protein (SLAP)-deficient mice as a tool to increase TCR avidity on double positive (DP) thymocytes. We generated SLAP(-/-) mice with the transgenic MHC class I-restricted TCR (OT-1) and SLAP(-/-) Vß5 mice, expressing only the ß-chain of the TCR OT-1 transgene, to examine the effects of increased TCR surface levels on CD8(+) T cell development and repertoire selection. In comparing SLAP(-/-) OT-1 and Vß5 mice with wild-type controls, we performed compositional analysis and assessed thymocyte signaling by measuring CD5 levels. In addition, we performed tetramer and compositional staining to measure affinity for the cognate antigen, ovalbumin (OVA) peptide, presented by MHC. Furthermore, we quantified differences in α-chain repertoire in SLAP(-/-) Vß5 mice. We have found that SLAP(-/-) OT-1 mice have fewer CD8(+) thymocytes but have increased CD5 expression. SLAP(-/-) OT-1 mice have fewer DP thymocytes expressing Vα2, signifying increased endogenous α-chain rearrangement, and more non-OVA-specific CD8(+) splenocytes upon tetramer staining. Our data demonstrate that SLAP(-/-) Vß5 mice also have fewer OVA-specific cells and increased Vα2 usage in the peripheral Vß5 CD8(+) T cells that were non-OVA-specific, demonstrating differences in α-chain repertoire. These studies provide direct evidence that increased TCR avidity in DP thymocytes enhances CD8(+) T cell negative selection deleting thymocytes with specificity for cognate antigen, an antigen the mature T cells may never encounter. Collectively, these studies provide new insights into how TCR avidity during CD8(+) T cell development influences repertoire selection.

SLAP deficiency enhances number and function of regulatory T cells preventing chronic autoimmune arthritis in SKG mice.

To test if manipulating TCR complex-mediated signaling (TCR signaling) could treat autoimmune disease, we generated the double SKG Src-like adapter protein (SLAP) knockout (DSSKO) mouse model. The SKG mutation in ZAP70 and SLAP have opposing functions on the regulation of TCR signaling. The combination of these two mutations alters TCR signaling in the context of a defined genetic background, uniform environmental conditions, and a well-characterized signaling disruption. In contrast to SKG mice, DSSKO mice do not develop zymosan-induced chronic autoimmune arthritis. This arthritis prevention is not due to significant alterations in thymocyte development or repertoire selection but instead enhanced numbers of regulatory T cells (Tregs) and decreased numbers of Th17 cells skewing the ratio of Tregs to autoreactive effector T cells. Treg depletion and/or functional blockade led to the development of arthritis in DSSKO mice. In vitro suppression of effector T cell proliferation was also enhanced, demonstrating that DSSKO mice have increased numbers of Tregs with increased function. Understanding how TCR signals influence development, expansion, and function of Tregs in DSSKO mice could advance our ability to manipulate Treg biology to treat ultimately autoimmune disease.

The Src-like adaptor protein regulates GM-CSFR signaling and monocytic dendritic cell maturation.

GM-CSF is an important cytokine involved in myeloid differentiation and inflammatory processes. Signaling through the GM-CSFR also plays a critical role in the generation of monocyte-derived dendritic cells (DC). In this article, we report that the Src-like adaptor protein (SLAP) functions as a negative regulator of the GM-CSFR. In bone marrow-derived DC (BM-DC) lacking SLAP and the closely related SLAP2, downregulation of GM-CSFRß is impaired, leading to enhanced phosphorylation of Jak2 and prolonged activation of Akt and Erk1/2 in response to GM-CSF stimulation. Compared with wild-type bone marrow, SLAP/SLAP2(-/-) bone marrow gave rise to similar numbers of CD11c(+) and CD11b(+) DC, but SLAP/SLAP2(-/-) BM-DC failed to acquire high levels of MHC class II, CD80, and CD86, indicating an impairment in maturation. Furthermore, MHC class II expression in SLAP/SLAP2(-/-) BM-DC was rescued by decreasing GM-CSF concentration, suggesting that enhanced GM-CSF signaling mediates the block in maturation. In addition, SLAP/SLAP2(-/-) BM-DC produced less IL-12 and TNF-α in response to LPS compared with controls and failed to stimulate T cells in an MLR. Ag-specific T cell activation assays showed that SLAP/SLAP2(-/-) BM-DC were less robust at inducing IFN-γ secretion by DO11.10 T cells. These results indicated that SLAP-mediated GM-CSFR regulation is important for the generation of functionally mature monocytic DC.

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.

An immunohistochemical and ultrastructural study of the pericellular matrix of uneroded hypertrophic chondrocytes in the mandibular condyle of aged c-src-deficient mice.

OBJECTIVE: Previous studies indicate that hypertrophic chondrocytes can transdifferentiate or dedifferentiate and redifferentiate into bone cells during the endochondral bone formation. Mandibular condyle in aged c-src-deficient mice has incremental line-like striations consisting of cartilaginous and non-cartilaginous layers, and the former contains intact hypertrophic chondrocytes in uneroded lacunae. The purpose of this study is to determine the phenotype changes of uneroded hypertrophic chondrocytes. DESIGN: Immunohistochemical and ultrastructural examinations of the pericellular matrix of hypertrophic chondrocytes in the upper, middle, and lower regions of the mandibular condyle were conducted in aged c-src-deficient mice, using several antibodies of cartilage/bone marker proteins. RESULTS: Co-localisation of aggrecan, type I collagen, and dentin matrix protein-1 (DMP-1) or matrix extracellular phosphoprotein (MEPE) was detected in the pericellular matrix of the middle region. Ultrastructurally, granular substances in the pericellular matrix of the middle region were the remains of upper region chondrocytes, which were mixed with thick collagen fibrils. In the lower region, the width of the pericellular matrix and the amount of collagen fibrils were increased. Versican, type I collagen, DMP-1, and MEPE were detected in the osteocyte lacunae. Additionally, DMP-1 and MEPE were detected in the pericellular matrix of uneroded hypertrophic chondrocytes located in the lower, peripheral region of the mandibular condyle in younger c-src-deficient mice, but not in the aged wild-type mice. CONCLUSIONS: These results indicate that long-term survived, uneroded hypertrophic chondrocytes, at least in a part, acquire osteocytic characteristics.

c-Src is required for tropomyosin receptor kinase C (TrkC)-induced activation of the phosphatidylinositol 3-kinase (PI3K)-AKT pathway.

TrkC mediates many aspects of growth and development in the central nervous system. TrkC is expressed in a variety of non-neuronal tissues as well as human cancers. TrkC overexpression may drive tumorigenesis, invasion, and metastatic capability in cancer cells. However, relatively little is known about whether TrkC activity is also essential to maintain the malignant properties in human tumors. TrkC expression leads to the constitutive activation of two major effector pathways, namely the Ras-MAP kinase (MAPK) mitogenic pathway and the phosphatidylinositol 3-kinase (PI3K)-AKT pathway mediating cell survival. However, it remains unclear how TrkC activates Ras-Erk1/2 and/or PI3K-Akt cascades. Here we define some aspects of the molecular mechanisms regulating TrkC-dependent Ras-Erk1/2 and PI3K/Akt activation. We show that endogenous TrkC associated with c-Src in human and mouse cancer cells which express TrkC. TrkC-c-Src complexes were also detected in primary human breast cancer tissues. Suppression of c-Src by RNA interference in highly metastatic 4T1 mammary cancer cells, which express endogenous TrkC, resulted in markedly decreased expression of cyclin D1 and suppression of activation of Ras-Erk1/2 and PI3K-Akt. Moreover, inhibition of c-Src expression almost completely blocks colony formation of 4T1 cells in soft agar. Furthermore, in c-Src-deficient SYF cells, TrkC failed to activate the PI3K-Atk pathway, but not the Ras-Erk1/2 pathway. Therefore these data indicate that TrkC induces the PI3K-Akt cascade through the activation of c-Src.

Diverse effects of c-src deficiency on molar tooth development and eruption in mice.

C-src deficiency is characterized by osteopetrosis due to impaired bone resorption by hypofunctional osteoclasts and the resultant failure of tooth eruption. In preliminary observations, we frequently encountered erupted molars in c-src deficient mice unlike in other osteopetrotic animals. Here we examine the effects of c-src deficiency on the development of molar teeth with an emphasis on the spatial relation of growing teeth with the surrounding bones. In c-src deficient mice, the magnitude of tooth impaction differed considerably among the types of molars; all maxillary 1st molars were totally impacted deep in the alveolar sockets, whereas most mandibular 1st molars fully erupted into oral cavity. Distribution of osteoclasts in the alveolar bone was identical among all types of molars, and electron microscopy revealed signs of bone resorbing activity in these osteoclasts despite the absence of a ruffled border. From early development, the alveolar space was much narrower in the upper molar tooth germs than in the lower ones in both wild type and homozygous animals, and particularly so in the upper 1st molars. Current observations thus indicate a significant contribution of "hypofunctional osteoclasts" in c-src deficient mice in molar tooth development except for the upper 1st molars, which appear to require highly functional osteoclasts to gain sufficient space for them to grow normally. Taken together, these findings on the seemingly tooth-type specific effects of c-src deficiency on the development and eruption of molar teeth in c-src deficient mice can be attributed to the given differential spatial relation of the respective tooth germs with the surrounding bones in the presence of hypofunctional osteoclasts.

Src-like adaptor protein (SLAP) regulates B cell receptor levels in a c-Cbl-dependent manner.

Src-like adaptor protein (SLAP) and c-Cbl recently have been shown to cooperate in regulating T cell receptor (TCR) levels in developing T cells. SLAP also is expressed in developing B cells, and its deficiency leads to alterations in B cell receptor (BCR) levels and B cell development. Hence, we hypothesized that SLAP and c-Cbl may cooperate during B cell development to regulate BCR levels. In mice deficient in both SLAP and c-Cbl, we found that B cell development is altered, suggesting that they function through intersecting pathways. To study the mechanism by which SLAP and c-Cbl alter BCR levels, we coexpressed them in a mature mouse B cell line (Bal-17). First we determined that SLAP associates with proximal components of the BCR complex after stimulation and internalization. Coexpression of SLAP and c-Cbl in Bal-17 led to decreased surface and total BCR levels. This decrease in BCR levels depended on intact Src homology 2 (SH2) and C-terminal domains of SLAP. In addition, a mutation in the SH2 domain of SLAP blocked its colocalization with c-Cbl and the BCR complex, whereas deletion of the C terminus did not affect its localization. Last, coexpression of SLAP and c-Cbl altered BCR complex recycling. This alteration in BCR complex recycling depended on enzymatically active c-Cbl and Src family kinases, as well as the intact SH2 and C-terminal domains of SLAP. These data suggest that SLAP has a conserved function in B and T cells by adapting c-Cbl to the antigen-receptor complex and targeting it for degradation.

Identification of the nonreceptor tyrosine kinase MATK/CHK as an essential regulator of immune cells using Matk/CHK-deficient mice.

Matk/CHK knockout mice were reported to show no apparent phenotypic abnormalities. This was thought to be due to the homologous kinase Csk that compensates for Matk/CHK. Here, we present the first evidence that the nonreceptor tyrosine kinase, Matk/CHK, is an important modulator of immune cell signaling. We found that the frequency of primitive hematopoietic cells, the side population c-kit(+) Lin(-) Sca-1(+) (SPKLS) cells, in Matk/CHK(-/-) mice was increased 2.2-fold compared with the control mice. Moreover, Matk/CHK deficiency led to significantly higher pre-B cell colony formation following IL-7 stimulation. Interestingly, when mice received the in vivo antigen challenge of TNP-ovalbumin followed by restimulation, the Matk/CHK(-/-) lymph node and spleen cells produced significantly lower IFN-gamma levels compared with the respective wild-type cells. Our study indicates that Matk/CHK is not functionally redundant with Csk, and that this tyrosine kinase plays an important role as a regulator of immunologic responses.

SRC-like adaptor protein regulates B cell development and function.

The avidity of BCRs and TCRs influences signal strength during processes of lymphocyte development. Avidity is determined by both the intrinsic affinity for Ag and surface levels of the Ag receptor. The Src-like adaptor protein (SLAP) is a regulator of TCR levels on thymocytes, and its deficiency alters thymocyte development. We hypothesized that SLAP, which is expressed in B cells, also is important in regulating BCR levels, signal strength, and B cell development. To test this hypothesis, we analyzed the B cell compartment in SLAP-deficient mice. We found increased splenic B cell numbers and decreased surface IgM levels on mature, splenic B cells deficient in SLAP. Immature bone marrow and splenic B cells from BCR-transgenic, SLAP-deficient mice were found to express higher surface levels of IgM. In contrast, mature splenic B cells from BCR-transgenic mice expressed decreased levels of surface BCR associated with decreased calcium flux and activation-induced markers, compared with controls. These data suggest that SLAP regulates BCR levels and signal strength during lymphocyte development.

Botrocetin/VWF-induced signaling through GPIb-IX-V produces TxA2 in an alphaIIbbeta3- and aggregation-independent manner.

Binding of von Willebrand factor (VWF) to the platelet membrane glycoprotein (GP) Ib-IX-V complex initiates a signaling cascade that causes alphaIIbbeta3 activation and platelet aggregation. Previous work demonstrated that botrocetin (bt)/VWF-mediated agglutination activates alphaIIbbeta3 and elicits adenosine triphosphate (ATP) secretion in a thromboxane A2 (TxA2)- and Ca2+-dependent manner. This agglutination-elicited TxA2 production occurs in the absence of ATP secretion. However, the signaling components and signaling network or pathway activated by GPIb-mediated agglutination to cause TxA2 production have not been identified. Therefore, the focus of this study was to elucidate at least part of the signal transduction network or pathway activated by GPIb-mediated agglutination to cause TxA2 production. The phosphatidylinositol 3-kinase (PI3K) selective inhibitor wortmannin, and mouse platelets deficient in Lyn, Src, Syk, Src homology 2 (SH2) domain-containing leukocyte protein 76 (SLP-76), phospholipase Cgamma2 (PLCgamma2), linker for activation of T cells (LAT), or Fc receptor gamma-chain (FcRgamma-chain) were used for these studies. LAT and FcRgamma-chain were found not to be required for agglutination-driven TxA2 production or activation of alphaIIbbeta3, but were required for granule secretion and aggregation. The results also clearly demonstrate that bt/VWF-mediated agglutination-induced TxA2 production is dependent on signaling apparently initiated by Lyn, enhanced by Src, and propagated through Syk, SLP-76, PI3K, PLCgamma2, and protein kinase C (PKC).

Aldosterone activates vascular p38MAP kinase and NADPH oxidase via c-Src.

Increasing evidence indicates that aldosterone elicits vascular effects through nongenomic signaling pathways. We tested the hypothesis that aldosterone induces activation of vascular mitogen-activated protein (MAP) kinases and NADPH oxidase via c-Src-dependent mechanisms in vascular smooth muscle cells (VSMCs). Aldosterone effects on activation of c-Src, p38MAP kinase, and NADPH oxidase, and incorporation of [3H]proline, an index of collagen synthesis, were assessed in cultured rat VSMCs. Studies were performed in the absence and presence of eplerenone, a selective mineralocorticoid receptor blocker, PP2, a selective Src inhibitor, and SB212190, a selective p38MAPK inhibitor. Phosphorylation of c-Src was dose-dependently increased by aldosterone, with maximal responses obtained at 10(-7) mol/L. Aldosterone increased p38MAP kinase phosphorylation, NAD(P)H oxidase activation, and [3H]proline incorporation. These responses were abrogated by eplerenone and almost abolished by PP2. Aldosterone-stimulated incorporation of [3H]proline was significantly reduced by SB212190, indicating that p38MAP kinase plays a role in profibrotic actions of aldosterone. To unambiguously demonstrate the importance of aldosterone in c-Src signaling, VSMCs from c-Src+/+ and c-Src+/- mice were also studied. Aldosterone increased phosphorylation of c-Src, p38MAP kinase, and cortactin, a Src-specific substrate, in c-Src+/+ VSMCs, but not in c-Src-deficient cells. Taken together, our findings demonstrate that nongenomic signaling by aldosterone occurs through c-Src-dependent pathways. These processes may play an important role in profibrotic actions of aldosterone.

Probing the communication between the regulatory and catalytic domains of a protein tyrosine kinase, Csk.

Protein tyrosine kinases (PTKs) are important regulators of mammalian cell function and their own activities are tightly regulated. Underlying their tight regulation, all PTKs contain multiple regulatory domains in addition to a catalytic domain. C-terminal Src kinase (Csk) contains a catalytic domain and a regulatory region, consisting of an SH3 and an SH2 domain. In this study, we probed the communication between the regulatory and catalytic domains of Csk. First, kinetic characterization of SH3 and SH2 domain deletion mutants demonstrated that the SH3 and SH2 domains were crucial in maintaining the full activity of Csk, but were not directly involved in Csk recognition of its physiological substrate, Src. Second, highly conserved Trp188, corresponding to a key residue in domain-domain communication in other PTKs, was found to be important for maintaining the active structure of Csk by the presence of the regulatory region, but not required for Csk activation triggered by a phosphopeptide binding to the SH2 domain. Third, structural alignment indicated that the presence of the regulatory domains modulated the conformation of multiple substructures in the catalytic domain, some directly and others remotely. Mutagenic and kinetic studies supported this assignment. This report extended previous studies of Csk domain-domain communication, and provided a foundation for further detailed investigation of this communication.

Distinct roles of p130Cas and c-Cbl in adhesion-induced or macrophage colony-stimulating factor-mediated signaling pathways in prefusion osteoclasts.

Both p130Cas and c-Cbl have been reported to play critical roles in osteoclast function as downstream targets of c-Src kinase. The purpose of this study was to examine adhesion- and macrophage colony-stimulating factor (M-CSF)-induced tyrosine phosphorylation of these two molecules in prefusion osteoclasts (pOCs) derived from either Src+/? or Src-/- mice and to directly compare the roles of p130Cas and c-Cbl in osteoclast function. Cell attachment of normal pOCs to vitronectin induces tyrosine phosphorylation of p130Cas and, to a much lesser extent, of c-Cbl. Treatment with M-CSF results in further tyrosine phosphorylation of both p130Cas and c-Cbl, suggesting cooperation between alpha v beta 3 integrin and the M-CSF receptor, c-Fms, in osteoclasts. However, M-CSF induces tyrosine phosphorylation of c-Cbl, but not p130Cas in pOCs in suspension, confirming the role of c-Cbl as a downstream effector of c-Fms. This observation also suggests that M-CSF-mediated p130Cas phosphorylation requires ligand engagement of alpha v beta 3 integrin. In Src-deficient pOCs plated on vitronectin, although M-CSF highly induces Cbl phosphorylation, it does not affect p130Cas phosphorylation. These results suggest that in osteoclasts 1) tyrosine phosphorylation of p130Cas depends on alpha v beta 3 integrin-mediated cell adhesion, even in the presence of M-CSF; 2) on the other hand, c-Cbl phosphorylation is predominantly activated by M-CSF and is independent of cell adhesion; 3) lastly, although c-Src is essential for both adhesion- and M-CSF-mediated phosphorylation of p130Cas, it is clearly not required for c-Cbl phosphorylation in M-CSF-treated pOCs. Taken together, p130Cas and c-Cbl play distinct roles in the signal transduction pathways that mediate cytoskeletal organization in osteoclasts.

Role of c-Src in regulation of endothelial nitric oxide synthase expression during exercise training.

We have shown that c-Src plays a role in shear stress stimulation of endothelial nitric oxide synthase (eNOS) expression in cultured cells. To examine the role of c-Src in vivo, we exercised C57Blk/6 and c-Src heterozygous (c-Src(+/-)) mice on a treadmill for 3 wk. Western analysis demonstrated that c-Src(+/-) mice express less than one-half the normal amount of c-Src. Exercise increased heart rate and blood pressure to identical levels in both strains as determined using radiotelemetry. Exercise training increased eNOS protein >2-fold in the aorta and 1.7-fold in the heart in C57Blk/6 mice but had no effect on eNOS protein levels in c-Src(+/-) mice. In contrast to exercise, treatment of mice with mevastatin, which stimulates expression of eNOS posttranscriptionally, increased eNOS protein in both strains. Training also increased aortic extracellular superoxide dismutase protein expression, which is regulated by nitric oxide, in C57Blk/6 mice but not in c-Src(+/-)mice. These data indicate that c-Src has an important role in modulating vascular adaptations to exercise training, in particular increasing eNOS and extracellular superoxide dismutase protein expression.

Analysis of difference in vivo effects of TCDD between c-src +/+ mice, c-src deficient, -/+ and -/- B6, 129-Src(tm 1 sor) mice and their wild-type littermates.

Toxicities of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) were tested on two strains of c-src deficient B6, 129-Src(tm 1 sor) mice, c-src -/+ and c-src -/- and their matched wild-type littermates c-src +/+ mice along with another c-src +/+ mice, from the same genetic lineage, B6, 129-Fos(tm 1 Pa) mice. The most conspicuous effect of c-src deficiency on the toxicity of TCDD appears to be the reduced hepatotoxicity. TCDD-treated c-src deficient mice show only modest degrees of hepatomegaly and triglycerides accumulation as compared to treated wild-type mice.

G-protein-coupled receptors act via protein kinase C and Src to regulate NMDA receptors.

The N-methyl-D-aspartate (NMDA) receptor contributes to synaptic plasticity in the central nervous system and is both serine-threonine and tyrosine phosphorylated. In CA1 pyramidal neurons of the hippocampus, activators of protein kinase C (PKC) as well as the G-protein-coupled receptor ligands muscarine and lysophosphatidic acid enhanced NMDA-evoked currents. Unexpectedly, this effect was blocked by inhibitors of tyrosine kinases, including a Src required sequence and an antibody selective for Src itself. In neurons from mice lacking c-Src, PKC-dependent upregulation was absent. Thus, G-protein-coupled receptors can regulate NMDA receptor function indirectly through a PKC-dependent activation of the non-receptor tyrosine kinase (Src) signaling cascade.

Colony-stimulating factor-1 induces cytoskeletal reorganization and c-src-dependent tyrosine phosphorylation of selected cellular proteins in rodent osteoclasts.

Colony-stimulating factor-1 (CSF-1) stimulates motility and cytoplasmic spreading in mature osteoclasts. Therefore, we examined the cellular events and intracellular signaling pathways that accompany CSF-1-induced spreading in normal osteoclasts. To explore the role c-src plays in these processes, we also studied osteoclasts prepared from animals with targeted disruption of the src gene. In normal osteoclasts, CSF-1 treatment induces rapid cytoplasmic spreading, with redistribution of F-actin from a well-delineated central attachment ring to the periphery of the cell. CSF-1 increases membrane phosphotyrosine staining in osteoclasts and induces the phosphorylation of several cellular proteins in cultured, osteoclast-like cells, including c-fms, c-src, and an 85-kD Grb2-binding protein. Src kinase activity is increased threefold after CSF-1 treatment. In src- cells, no attachment ring is present, and CSF-1 fails to induce spreading or a change in the pattern of F-actin distribution. Although c-fms becomes phosphorylated after CSF-1 treatment, the 85-kD protein is significantly less phosphorylated in src- osteoclast-like cells. These results indicate that c-src is critical for the normal cytoskeletal architecture of the osteoclast, and, in its absence, the spreading response induced by CSF-1 is abrogated, and downstream signaling from c-fms is altered.

Rescue of osteoclast function by transgenic expression of kinase-deficient Src in src-/- mutant mice.

The Src tyrosine kinase has been implicated in a wide variety of signal transduction pathways, yet despite the nearly ubiquitous expression of c-src, src-/- mice show only one major phenotype-osteopetrosis caused by an intrinsic defect in osteoclasts, the cells responsible for resorbing bone. To explore further the role of Src both in osteoclasts and other cell types, we have generated transgenic mice that express the wild-type and mutated versions of the chicken c-src proto-oncogene from the promoter of tartrate resistant acid phosphatase (TRAP), a gene that is expressed highly in osteoclasts. We demonstrate here that expression of a wild-type transgene in only a limited number of tissues can fully rescue the src-/- phenotype. Surprisingly, expression of kinase-defective alleles of c-src also reduces osteopetrosis in src-/- animals and partially rescues a defect in cytoskeletal organization observed in src-/- osteoclasts. These results suggest that there are essential kinase-independent functions for Src in vivo. Biochemical examination of osteoclasts from these mice suggest that Src may function in part by recruiting or activating other tyrosine kinases.