The immunomodulatory adapter proteins DAP12 and Fc receptor gamma-chain (FcRgamma) regulate development of functional osteoclasts through the Syk tyrosine kinase

Osteoclasts, the only bone-resorbing cells, are central to the pathogenesis of osteoporosis, yet their development and regulation are incompletely understood. Multiple receptors of the immune system use a common signaling paradigm whereby phosphorylated immunoreceptor tyrosine-based activation motifs (ITAMs) within receptor-associated adapter proteins recruit the Syk tyrosine kinase. Here we demonstrate that a similar mechanism is required for development of functional osteoclasts. Mice lacking two ITAM-bearing adapters, DAP12 and the Fc receptor gamma-chain (FcRgamma), are severely osteopetrotic. DAP12(-/-)FcRgamma(-/-) bone marrow cells fail to differentiate into multinucleated osteoclasts or resorb bone in vitro and show impaired phosphorylation of the Syk tyrosine kinase. syk(-/-) progenitors are similarly defective in osteoclast development and bone resorption. Intact SH2-domains of Syk, introduced by retroviral transduction, are required for functional reconstitution of syk(-/-) osteoclasts, whereas intact ITAM-domains on DAP12 are required for reconstitution of DAP12(-/-) FcRgamma(-/-) cells. These data indicate that recruitment of Syk to phosphorylated ITAMs is critical for osteoclastogenesis. Although DAP12 appears to be primarily responsible for osteoclast differentiation in cultures directly stimulated with macrophage-colony stimulating factor and receptor activator of NF-kappaB ligand cytokines, DAP12 and FcRgamma have overlapping roles in supporting osteoclast development in osteoblast-osteoclast cocultures, which mirrors their overlapping functions in vivo. These results provide new insight into the biology of osteoclasts and suggest novel therapeutic targets in diseases of bony remodeling.     

Presentation of ovalbumin internalized via the immunoglobulin-A Fc receptor is enhanced through Fc receptor gamma-chain signaling

The mechanism of enhanced presentation of ovalbumin (OVA)internalized as immunoglobulin A (IgA)-OVA via the IgA Fc receptor (FcR) was analyzed by focusing on the role of the FcR-associated  chain. Comparison of B-cell transfectants expressing FcR plus wild-type (WT)  chain or  chain in which theimmunoreceptor tyrosine-based activation motif (ITAM) was altered bytyrosine mutation or substitution with the ITAM of FcRIIA showedthat signaling-competent ITAM was not required for endocytosis ofIgA-OVA. However, antigen presentation was impaired by ITAM changes.Signaling-competent -chain ITAM appeared necessary for transport ofligated FcR to a lamp-1⁺ late endocytic compartment forremodeling and/or activation of that compartment and also for efficientdegradation of IgA complexes. Moreover, FcR ligation also activatedefficient processing of nonreceptor-targeted antigen. Theresults suggest that -chain signaling activates the antigenprocessing compartment.     

Nerve growth factor prevents apoptosis of rat peritoneal mast cells through the trk proto-oncogene receptor

We investigated the inhibitory activity of nerve growth factor (NGF) on apoptosis of rat peritoneal mast cells (PMCs) and compared it with that of recombinant stem cell factor (rSCF), which is a mast cell growth factor. When PMCs were incubated up to 72 hours in the presence of control medium, internucleosomal fragmentation of DNA indicating apoptosis was detected by agarose gel electrophoresis and flow cytometry. The aged PMCs showed morphological changes typical for apoptosis, such as chromatin condensation and loss of microvilli of the cell membrane. Addition of NGF or rSCF prevented development of the characteristic DNA fragmentation and decreased the proportion of apoptotic cells with low DNA content values in a dose-dependent manner. Polyclonal antibody to NGF completely abolished the inhibitory activity of NGF but not of rSCF. NGF-induced PMCs were in the G0/G1 phase of the cell cycle, but rSCF transited them from the G0/G1 phase to the S/G2M phase, suggesting that NGF, unlike rSCF, may have no proliferation activity to PMCs. By flow cytometric analysis with antibodies to NGF receptors p75LNGFR and p140trk, we defined that PMCs expressed p140trk but not p75LNGFR. Addition of herbimycin A or K-252a, tyrosine kinase inhibitors, to NGF resulted in blockage of the NGF-induced p140trk phosphorylation and restriction of the inhibitory activity of NGF on apoptosis of PMCs. These results indicated that NGF suppressed apoptosis of rat PMCs through the p140trk tyrosine phosphorylation and possessed no proliferative activity. Thus, NGF may act as a key factor to promote survival of connective tissue-type mast cells.     

Signalling through the insulin receptor and the insulin-like growth factor-I receptor

The insulin receptor and the insulin-like growth factor I receptor belong to the family of tyrosine kinase receptors. Both receptors appear as a disulphide-linked dimer; each half of the dimer consisting of a 130 k M_r -subunit linked to a 90 k M_r -subunit. Both halves of the dimer are linked together by disulphide bonds to form an _{2 2} structure. The insulin receptor functions as an allosteric enzyme in which the binding of the hormone to the -subunit leads to a series of conformational changes resulting in activation of the -subunit tyrosine kinase. Upon multisite autophosphorylation the latter becomes competent to phosphorylate cellular substrates resulting in the biological responses of insulin. Recent findings have recognized the mitogen activated protein kinase cascade as a central signalling circuitry linking cell surface receptors, such as the insulin receptor, to the nucleus, and playing a role in regulation of metabolism, growth and differentiation.     

Signalling through the insulin receptor and the insulin-like growth factor-I receptor

Diabetologia - The insulin receptor and the insulin-like growth factor I receptor belong to the family of tyrosine kinase receptors. Both receptors appear as a disulphide-linked dimer; each half of...     

IgA and IgG antineutrophil cytoplasmic antibody engagement of Fc receptor genetic variants influences granulomatosis with polyangiitis

Granulomatosis with polyangiitis (Wegener's) is a rare autoimmune neutrophil-mediated vasculitis that can cause renal disease and mucosal manifestations. Antineutrophil cytoplasmic antibodies (ANCA) are present in many patients, vary in level over time, and induce neutrophil activation through engagement with Fc receptors (FcRs). Given roles for FcRs in ANCA-mediated neutrophil activation and IgA antibodies in mucosal immunity, we hypothesized that FcR genetics and previously unappreciated IgA ANCA affect clinical presentation. We assembled a total of 673 patients and 413 controls from two multicenter cohorts, performed ELISA and immunofluorescence assays to determine IgA and IgG ANCA positivity, and used Illumina, TaqMan, or Pyrosequencing to genotype eight haplotype-tagging SNPs in the IgA FcR (FCAR) and to determine NA1/NA2 genotype of FCGR3B, the most prevalent neutrophil IgG FcR. We evaluated neutrophil activation by measuring degranulation marker CD11b with flow cytometry or neutrophil extracellcular trap formation with confocal microscopy. Functional polymorphisms in FCGR3B and FCAR differed between patient groups stratified by renal involvement. IgA ANCA were found in ～30% of patients and were less common in patients with severe renal disease. Neutrophil stimulation by IgA or IgG ANCA led to degranulation and neutrophil extracellcular trap formation in a FcR allele-specific manner (IgA:FCAR P = 0.008; IgG:FCGR3B P = 0.003). When stimulated with IgA and IgG ANCA together, IgG ANCA induced neutrophil activation was reduced (P = 0.0001). FcR genotypes, IgA ANCA, and IgG ANCA are potential prognostic and therapeutic targets for understanding the pathogenesis and presentation of granulomatosis with polyangiitis (Wegener's).     

Fc receptor function and circulating immune complexes in gluten sensitive enteropathy--possible significance of serum IgA.

The capacity to clear IgG containing immune complexes from the circulation was studied in patients with coeliac disease (n = 13), dermatitis herpetiformis (n = 8), and coeliac disease with concomitant serum IgA deficiency (n = 4). A small group of patients with active ulcerative colitis (n = 4) was included as a bowel disease control group. Clearance was estimated by measuring the disappearance rate of a bolus dose of intravenously injected IgG coated autologous erythrocytes. The mean T1/2 of clearance was prolonged in both coeliac disease (86 (24) minutes) and dermatitis herpetiformis (111 (35) minutes), compared with healthy subjects (20 (5) minutes) and coeliac patients with concomitant serum IgA deficiency (T1/2 = 17 (6) minutes). Patients with ulcerative colitis had a prolonged clearance, with a T1/2 of 195 (63) minutes. Values of circulating immune complexes were measured by four assays; C1q binding and C3, IgG, and IgA containing immune complexes. C1q binding immune complexes were detected only in IgA deficient gluten sensitive enteropathy. Patients with coeliac disease and dermatitis herpetiformis had higher values of C3, IgG, and IgA containing immune complexes than control subjects and serum IgA deficient patients with coeliac disease. The clearance rate was inversely correlated to the amount of immune complexes for the subgroups of gluten sensitive enteropathy.     

Platelet activation by cross-linking HLA class I molecules and Fc receptor.

The effect on platelet activation of monoclonal antibodies directed against common determinants of the HLA class I heavy chain molecule was studied. Cross-linking W6/32, an anti-HLA class I of IgG2a subclass, led to platelet activation. Two other antibodies of the same subclass did not have this effect on platelets. The lack of activity of the F(ab')2 fragments suggests that the activation signal is mediated by the platelet Fc receptor (Fc gamma RII). Indeed, except for a higher sensitivity of W6/32 to aspirin and apyrase, activations by cross-linking IV-3 (an anti-Fc gamma RII) and W6/32 are similar at the level of InsP3 formation, calcium mobilization, pH modifications, and activation of protein kinase C and myosin kinase. When HLA class I molecules and Fc gamma RII are cross-linked together, platelet activation occurs. This is not observed when a control IgG2a is substituted for W6/32 or when CD9 and Fc receptor are cross-linked together. This suggests that HLA class I molecules and Fc gamma RII synergize to activate platelets.     

Cysteine proteinases in tumor cell growth and apoptosis

During their evolution tumor cells acquire and mobilize various mechanisms that crucially affect their capability of proliferation, invasiveness and metastasis. Recent findings provide evidence that tumor cell associated cysteine proteinases such as some lysosomal cathepsins and apoptotic caspases are fundamentally involved in specific developmental traits of tumor cell populations. Tumor cell exterior-associated cysteine cathepsins B and L promote tumor growth, invasion and metastasis through degradation of extracellular connective matrices and through endothelial cell growth-directed activities. On the other hand, caspases -3, -7 and -6, generated in tumor cell cytoplasm via a robust activation of their zymogens, suppress tumor cell growth, invasion and metastasis through proteolytic devitalizing and remodeling of tumor cells into readily phagocytable apoptotic corpses. Tumor cell variants that are deficient in expression of effector caspase zymogens or are capable to suppress the extrinsic and intrinsic activation mechanisms of effector caspase zymogens and the activity of effector caspases have a significant survival advantage in environments of various death stimuli. Advancements in pharmacological targeting of tumor associated pathogenic lysosomal cysteine cathepsins and in apoptotic caspases-oriented conditioning of tumor cells may substantially contribute to therapeutic control of tumor diseases.     

A subunit common to an IgG Fc receptor and the T-cell receptor mediates assembly through different interactions.

The zeta subunit is a component of the Fc gamma receptor of natural killer cells (Fc gamma RIII or CD16), as well as the multimeric T-cell receptor/CD3 complex, and is required for assembly of both native receptors. The role of the zeta subunit in human Fc gamma RIIIA assembly differs from its role in T-cell receptor/CD3 complex assembly. The transmembrane domain of the Fc gamma RIIIA alpha subunit forms noncovalent interactions with the comparable domain of the zeta subunit and is sufficient for surface expression of the Fc gamma RIIIA complex. In the absence of these interactions, sequences in the transmembrane domain of the Fc gamma RIIIA alpha subunit signal its degradation. Leu-46, present in the transmembrane domain of the human zeta subunit, is important for assembly with the Fc gamma RIIIA alpha subunit. Substitution of this leucine with an isoleucine, as found in the mouse zeta subunit, significantly reduces this interaction. In contrast, the mouse and human zeta subunits interact with the pentameric T-cell receptor/CD3 complex, resulting in surface expression of this receptor.     

Effects of receptor dimerization on the interaction between the class I major histocompatibility complex-related Fc receptor and IgG.

The neonatal Fc receptor (FcRn) transports maternal IgG from ingested milk in the gut to the bloodstream of newborn mammals. An FcRn dimer was observed in crystals of the receptor alone and of an FcRn-Fc complex, but its biological relevance was unknown. Here we use surface plasmon resonance-based biosensor assays to assess the role of FcRn dimerization in IgG binding. We find high-affinity IgG binding when FcRn is immobilized on a biosensor chip in an orientation facilitating dimerization but not when its orientation disrupts dimerization. This result supports a model in which IgG-induced dimerization of FcRn is relevant for signaling the cell to initiate endocytosis of the IgG-FcRn complex.     

C/EBPalpha functionally and physically interacts with GABP to activate the human myeloid IgA Fc receptor (Fc alphaR, CD89) gene promoter

Human Fcalpha receptor (Fc alphaR; CD89), the receptor for the crystallizable fragment (Fc) of immunoglobulin A (IgA), is expressed exclusively in myeloid cells, including granulocytes and monocytes/macrophages, and is considered to define a crucial role of these cells in immune and inflammatory responses. A 259-base pair fragment of the FCAR promoter is sufficient to direct myeloid expression of a reporter gene and contains functionally important binding sites for CCAAT/enhancer-binding protein alpha (C/EBPalpha) (CE1, CE2, and CE3) and an unidentified Ets-like nuclear protein. Here, we show that the Ets-binding site is bound by a heterodimer composed of GA-binding protein alpha (GABPalpha), an Ets-related factor, and GABPbeta, a Notch-related protein. Cotransfection of GABP increased FCAR promoter activity 3.7-fold through the Ets-binding site. GABP and C/EBPalpha synergistically activated the FCAR promoter 280-fold. Consistent with these observations, in vitro binding analyses revealed a physical interaction between the GABPalpha subunit and C/EBPalpha. This is the first report demonstrating both physical and functional interactions between GABP and C/EBPalpha and will provide new insights into the molecular basis of myeloid gene expression.     

Growth inhibition by keratinocyte growth factor receptor of human salivary adenocarcinoma cells through induction of differentiation and apoptosis

We have reported that normal human salivary gland-derived epithelial cells exclusively express keratinocyte growth factor receptor (KGFR). In the process of malignant transformation of human salivary gland tumors, KGFR gene expression disappeared concomitantly with the de novo expression of the fibroblast growth factor receptor 1 (FGFR1) and FGFR4 genes. In the present study, we introduced wild-type KGFR cDNA or chimeric KGFR/FGFR1 cDNA, which encoded the extracellular domain of KGFR and the intracellular domain of FGFR1, into the HSY human salivary adenocarcinoma cell line. The KGFR tyrosine kinase suppressed the activity of FGF receptor substrate 2 (FRS2) and inhibited the growth of HSY by inducing differentiation and apoptosis in vitro and in vivo. Our results provided significant insight into the mechanism of KGFR tumor suppression and suggest that KGFR gene therapy might be a viable method of inhibiting human salivary adenocarcinoma growth.     

IL-17A通过增强肿瘤血管生成和侵袭促进肿瘤发展

目的探讨IL-17A在非小细胞肺癌血管形成和侵袭中的作用。方法Lewis肺癌细胞（LLC）在不同浓度IL-17A（0、25、50、100和200μg／L）条件下行增殖实验,LLC在50μg／L IL-17A和0μg／LIL-17A刺激下行侵袭实验。LLC在IL-17A刺激不同时间点下用实时定量PCR（RT—PCR）测定血管内皮生长因子-A（VEGF—A）、血管生成素-2（Ang-2）、基质金属蛋白酶-2（MMP-2）、MMP-9mRNA表达。C57BL／6小鼠接种LLC后随机分为3组,每组6只,分别予以瘤内注射PBS、对照腺病毒（Ad—NC）和干扰腺病毒（Ad—si—IL-17a,IL-17a基因沉默）,16d后处死小鼠,提取肿瘤组织RNA,RT-PCR检测VEGF-A、Ang-2、MMP-2和MMP-9mRNA表达。结果体外培养条件下IL-17A对肿瘤细胞的增殖能力没有影响,与无IL-17A组相比,50μg／L浓度的IL-17A组的肿瘤细胞VEGF-A、Ang-2、MMP-2和MMP-9mRNA表达水平明显升高,且肿瘤细胞侵袭能力增强（P〈0．01）。Ad—si—IL-17a组小鼠在13d（P〈0．05）和16d（P〈0．01）时肿瘤明显小于Ad-NC组和PBS组。Ad-si-IL-17a组肿瘤VEGF-A、Ang-2、MMP-2和MMP-9ITIRNA表达水平低于Ad—NC组和PBS组。结论靶向IL-17A的治疗可能为肿瘤治疗提供新思路。     

The role of insulin-like growth factor I and its receptor in cell growth, transformation, apoptosis, and chemoresistance in solid tumors

Insulin-like growth factor I (IGF-I) exerts pleiotropic effects on mammalian cells via stimulation of its receptor (IGF-IR), a receptor tyrosine kinase. In vivo, IGF-I acts both as a local tissue growth factor and as a circulating hormone. In oncological research, IGF-I has received increased attention as the activated IGF-I/IGF-IR system displays mitogeneic, transforming, and anti-apoptotic properties in various cell types by stimulating distinct intracellular signaling pathways. Recent data suggest that the anti-apoptotic effect of IGF-I may mediate decreased sensitivity to chemotherapeutic drugs in vitro and in vivo. Thus, targeting the IGF-I/IGF-IR system could serve as an approach to overcome clinical drug resistance in certain tumors. Received: 15 December 1998 / Accepted: 4 January 1999