The mechano-sensitive response of ß1 integrin promotes SRC-positive late endosome recycling and activation of Yes-associated protein.

Yes-associated protein (YAP) signaling has emerged as a crucial pathway in several normal and pathological processes. Although the main upstream effectors that regulate its activity have been extensively studied, the role of the endosomal system has been far less characterized. Here, we identified the late endosomal/lysosomal adaptor MAPK and mTOR activator (LAMTOR) complex as an important regulator of YAP signaling in a preosteoblast cell line. We found that p18/LAMTOR1-mediated peripheral positioning of late endosomes allows delivery of SRC proto-oncogene, nonreceptor tyrosine kinase (SRC) to the plasma membrane and promotes activation of an SRC-dependent signaling cascade that controls YAP nuclear shuttling. Moreover, ß1 integrin engagement and mechano-sensitive cues, such as external stiffness and related cell contractility, controlled LAMTOR targeting to the cell periphery and thereby late endosome recycling and had a major impact on YAP signaling. Our findings identify the late endosome recycling pathway as a key mechanism that controls YAP activity and explains YAP mechano-sensitivity.

Src deficient mice demonstrate behavioral and electrophysiological alterations relevant to psychiatric and developmental disease.

Much evidence suggests that hypofunction of the N-methyl-d-aspartate glutamate receptor (NMDAR) may contribute broadly towards a subset of molecular, cognitive and behavioral abnormalities common among psychiatric and developmental diseases. However, little is known about the specific molecular changes that lead to NMDAR dysfunction. As such, personalized approaches to remediating NMDAR dysfunction based on a specific etiology remains a challenge. Sarcoma tyrosine kinase (Src) serves as a hub for multiple signaling mechanisms affecting GluN2 phosphorylation and can be disrupted by convergent alterations of various signaling pathways. We recently showed reduced Src signaling in post mortem tissue from schizophrenia patients, despite increased MK-801 binding and NMDA receptor complex expression in the postsynaptic density (PSD). These data suggest that Src dysregulation may be an important underlying mechanism responsible for reduced glutamate signaling. Despite this evidence for a central role of Src in NMDAR signaling, little is known about how reductions in Src activity might regulate phenotypic changes in cognition and behavior. As such, the current study sought to characterize behavioral and electrophysiological phenotypes in mice heterozygous for the Src Acl gene (Src+/- mice). Src+/- mice demonstrated decreased sociability and working memory relative to Src+/+ (WT) mice while no significant differences were seen on locomotive activity and anxiety-related behavior. In relation to WT mice, Src+/- mice showed decreased mid-latency P20 auditory event related potential (aERP) amplitudes, decreased mismatch negativity (MMN) and decreased evoked gamma power, which was only present in males. These data indicate that Src+/- mice are a promising new model to help understand the pathophysiology of these electrophysiological, behavioral and cognitive changes. As such, we propose that Src+/- mice can be used in the future to evaluate potential therapeutic approaches by targeting increased Src activity as a common final pathway for multiple etiologies of SCZ and other diseases characterized by reduced glutamate function.

Signalling through Src family kinase isoforms is not redundant in models of thrombo-inflammatory vascular disease.

The Src family kinases (SFK) are a group of signalling molecules with important regulatory functions in inflammation and haemostasis. Leucocytes and platelets express multiple isoforms of the SFKs. Previous studies used broad-spectrum pharmacological inhibitors, or murine models deficient in multiple SFK isoforms, to demonstrate the functional consequences of deficiencies in SFK signalling. Here, we hypothesized that individual SFK operate in a non-redundant fashion in the thrombo-inflammatory recruitment of monocyte during atherosclerosis. Using in vitro adhesion assays and single SFK knockout mice crossed with the ApoE-/- model of atherosclerosis, we find that SFK signalling regulates platelet-dependent recruitment of monocytes. However, loss of a single SFK, Fgr or Lyn, reduced platelet-mediated monocyte recruitment in vitro. This translated into a significant reduction in the burden of atherosclerotic disease in Fgr-/- /ApoE-/- or Lyn-/- /ApoE-/- animals. SFK signalling is not redundant in thrombo-inflammatory vascular disease and individual SFK may represent targets for therapeutic intervention.

Src Promotes Metastasis of Human Non-Small Cell Lung Cancer Cells through Fn14-Mediated NF-κB Signaling.

BACKGROUND Src and Fn14 are implicated in the aggressiveness of non-small cell lung cancer (NSCLC) cells, yet the molecular mechanism is not fully understood. MATERIAL AND METHODS The proliferation, migration, and invasion of HCC827 cells with Src knockdown were examined in vitro. The expression of Fn14 and the activation of NF-κB signaling pathway in Src-silenced HCC827 cells were detected by western blot. The role of Fn14 in Src-regulated cell migration/invasion and activation of NF-κB signaling was investigated by overexpressing Fn14 in Src knockdown NSCLC cells. Furthermore, the pro-metastatic role of Src was validated in a NSCLC metastasis mouse model. RESULTS Knockdown of Src inhibited the proliferation, migration, and invasion of HCC827 cells, which was associated with reduced levels of Fn14, p-IκBα, p-IKKß, and nuclear NF-κB p65. Overexpression of Fn14 restored the potential of migration and invasion as well as the activation of NF-κB signaling in Src-silenced NSCLC cells. In addition, silencing of Src suppressed lung metastasis of HCC827 cells in mice, and inhibited the expression of Fn14 and nuclear translocation of NF-κB p65 in vivo. CONCLUSIONS The data demonstrated that the Src/Fn14/NF-κB axis plays a critical role in NSCLC metastasis.

Serum CTX levels and histomorphometric analysis in Src versus RANKL knockout mice.

Src knockout (KO) and RANKL KO mice both exhibit near complete osteopetrosis in terms of 3D-bone volume (BV) fraction by micro-CT, whereas the serum CTX concentration of Src KO is apparently normal and that of RANKL KO is 30% of wild-type (WT) despite the fact that they lack osteoclasts. By histomorphometry we found that, whereas eroded surface (ES) and osteoid surface (OS) are zero values in RANKL KO, they are indistinguishable from WT in Src KO; because of marked increase in bone surface (BS), ES/BS and OS/BS of Src KO are 30-40% of WT. While RANKL KO lack both osteoclasts and osteoblasts, Src KO reveal increased numbers of osteoclasts and indistinguishable numbers of osteoblasts compared with WT; again, on the basis of BS, N.Oc/BS is comparable to WT and N.Ob/BS is markedly decreased in Src KO. The apparently increased number of total osteoclasts may be due to increased expression of RANKL found in Src KO bone in vivo. Src has a gene dosage-dependent effect on osteoclast function in vitro, with Src-/- osteoclasts completely lacking bone-resorbing function as determined by CTX release on dentin. Thus, Src KO osteoclasts retain some bone-resorbing function in vivo. The number of osteocytes is proportionally increased in RANKL KO, while Src KO mice have relative osteocyte deficiency, raising the possibility that RANKL and Src has an unrecognized role in osteocyte survival.

Nintedanib reduces ventilation-augmented bleomycin-induced epithelial-mesenchymal transition and lung fibrosis through suppression of the Src pathway.

Mechanical ventilation (MV) used in patients with acute respiratory distress syndrome (ARDS) can increase lung inflammation and pulmonary fibrogenesis. Src is crucial in mediating the transforming growth factor (TGF)-ß1-induced epithelial-mesenchymal transition (EMT) during the fibroproliferative phase of ARDS. Nintedanib, a multitargeted tyrosine kinase inhibitor that directly blocks Src, has been approved for the treatment of idiopathic pulmonary fibrosis. The mechanisms regulating interactions among MV, EMT and Src remain unclear. In this study, we suggested hypothesized that nintedanib can suppress MV-augmented bleomycin-induced EMT and pulmonary fibrosis by inhibiting the Src pathway. Five days after administrating bleomycin to mimic acute lung injury (ALI), C57BL/6 mice, either wild-type or Src-deficient were exposed to low tidal volume (VT ) (6 ml/kg) or high VT (30 ml/kg) MV with room air for 5 hrs. Oral nintedanib was administered once daily in doses of 30, 60 and 100 mg/kg for 5 days before MV. Non-ventilated mice were used as control groups. Following bleomycin exposure in wild-type mice, high VT MV induced substantial increases in microvascular permeability, TGF-ß1, malondialdehyde, Masson's trichrome staining, collagen 1a1 gene expression, EMT (identified by colocalization of increased staining of α-smooth muscle actin and decreased staining of E-cadherin) and alveolar epithelial apoptosis (P < 0.05). Oral nintedanib, which simulated genetic downregulation of Src signalling using Src-deficient mice, dampened the MV-augmented profibrotic mediators, EMT profile, epithelial apoptotic cell death and pathologic fibrotic scores (P < 0.05). Our data indicate that nintedanib reduces high VT MV-augmented EMT and pulmonary fibrosis after bleomycin-induced ALI, partly by inhibiting the Src pathway.

Histochemical assessment for osteoblastic activity coupled with dysfunctional osteoclasts in c-src deficient mice.

Since osteoblastic activities are believed to be coupled with osteoclasts, we have attempted to histologically verify which of the distinct cellular circumstances, the presence of osteoclasts themselves or bone resorption by osteoclasts, is essential for coupled osteoblastic activity, by examining c-fos-/- or c-src-/- mice. Osteopetrotic c-fos deficient (c-fos-/-) mice have no osteoclasts, while c-src deficient (c-src-/-) mice, another osteopetrotic model, develop dysfunctional osteoclasts due to a lack of ruffled borders. c-fos-/- mice possessed no tartrate-resistant acid phosphatase (TRAPase)-reactive osteoclasts, and showed very weak tissue nonspecific alkaline phosphatase (TNALPase)-reactive mature osteoblasts. In contrast, c-src-/- mice had many TNALPase-positive osteoblasts and TRAPase-reactive osteoclasts. Interestingly, the parallel layers of TRAPase-reactive/osteopontin-positive cement lines were observed in the superficial region of c-src-/- bone matrix. This indicates the possibility that in c-src-/- mice, osteoblasts were activated to deposit new bone matrices on the surfaces that osteoclasts previously passed along, even without bone resorption. Transmission electron microscopy demonstrated cell-to-cell contacts between mature osteoblasts and neighboring ruffled border-less osteoclasts, and osteoid including many mineralized nodules in c-src-/- mice. Thus, it seems likely that osteoblastic activities would be maintained in the presence of osteoclasts, even if they are dysfunctional.

Hck/Fgr Kinase Deficiency Reduces Plaque Growth and Stability by Blunting Monocyte Recruitment and Intraplaque Motility.

BACKGROUND: Leukocyte migration is critical for the infiltration of monocytes and accumulation of monocyte-derived macrophages in inflammation. Considering that Hck and Fgr are instrumental in this process, their impact on atherosclerosis and on lesion inflammation and stability was evaluated. METHODS AND RESULTS: Hematopoietic Hck/Fgr-deficient, LDLr(-/-) chimeras, obtained by bone marrow transplantation, had smaller but, paradoxically, less stable lesions with reduced macrophage content, overt cap thinning, and necrotic core expansion as the most prominent features. Despite a Ly6C(high)-skewed proinflammatory monocyte phenotype, Hck/Fgr deficiency led to disrupted adhesion of myeloid cells to and transmigration across endothelial monolayers in vitro and atherosclerotic plaques in vivo, as assessed by intravital microscopy, flow cytometry, and histological examination of atherosclerotic arteries. Moreover, Hck/Fgr-deficient macrophages showed blunted podosome formation and mesenchymal migration capacity. In consequence, transmigrated double-knockout macrophages were seen to accumulate in the fibrous cap, potentially promoting its focal erosion, as observed for double-knockout chimeras. CONCLUSIONS: The hematopoietic deficiency of Hck and Fgr led to attenuated atherosclerotic plaque formation by abrogating endothelial adhesion and transmigration; paradoxically, it also promoted plaque instability by causing monocyte subset imbalance and subendothelial accumulation, raising a note of caution regarding src kinase-targeted intervention in plaque inflammation.

Lyn deficiency leads to increased microbiota-dependent intestinal inflammation and susceptibility to enteric pathogens.

The Lyn tyrosine kinase governs the development and function of various immune cells, and its dysregulation has been linked to malignancy and autoimmunity. Using models of chemically induced colitis and enteric infection, we show that Lyn plays a critical role in regulating the intestinal microbiota and inflammatory responses as well as protection from enteric pathogens. Lyn(-/-) mice were highly susceptible to dextran sulfate sodium (DSS) colitis, characterized by significant wasting, rectal bleeding, colonic pathology, and enhanced barrier permeability. Increased DSS susceptibility in Lyn(-/-) mice required the presence of T but not B cells and correlated with dysbiosis and increased IFN-γ(+) and/or IL-17(+) colonic T cells. This dysbiosis was characterized by an expansion of segmented filamentous bacteria, associated with altered intestinal production of IL-22 and IgA, and was transmissible to wild-type mice, resulting in increased susceptibility to DSS. Lyn deficiency also resulted in an inability to control infection by the enteric pathogens Salmonella enterica serovar Typhimurium and Citrobacter rodentium. Lyn(-/-) mice exhibited profound cecal inflammation, bacterial dissemination, and morbidity following S. Typhimurium challenge and greater colonic inflammation throughout the course of C. rodentium infection. These results identify Lyn as a key regulator of the mucosal immune system, governing pathophysiology in multiple models of intestinal disease.

Induced pluripotent stem cell therapy ameliorates hyperoxia-augmented ventilator-induced lung injury through suppressing the Src pathway.

BACKGROUND: High tidal volume (VT) mechanical ventilation (MV) can induce the recruitment of neutrophils, release of inflammatory cytokines and free radicals, and disruption of alveolar epithelial and endothelial barriers. It is proposed to be the triggering factor that initiates ventilator-induced lung injury (VILI) and concomitant hyperoxia further aggravates the progression of VILI. The Src protein tyrosine kinase (PTK) family is one of the most critical families to intracellular signal transduction related to acute inflammatory responses. The anti-inflammatory abilities of induced pluripotent stem cells (iPSCs) have been shown to improve acute lung injuries (ALIs); however, the mechanisms regulating the interactions between MV, hyperoxia, and iPSCs have not been fully elucidated. In this study, we hypothesize that Src PTK plays a critical role in the regulation of oxidants and inflammation-induced VILI during hyperoxia. iPSC therapy can ameliorate acute hyperoxic VILI by suppressing the Src pathway. METHODS: Male C57BL/6 mice, either wild-type or Src-deficient, aged between 2 and 3 months were exposed to high VT (30 mL/kg) ventilation with or without hyperoxia for 1 to 4 h after the administration of Oct4/Sox2/Parp1 iPSCs at a dose of 5×10(7) cells/kg of mouse. Nonventilated mice were used for the control groups. RESULTS: High VT ventilation during hyperoxia further aggravated VILI, as demonstrated by the increases in microvascular permeability, neutrophil infiltration, macrophage inflammatory protein-2 (MIP-2) and plasminogen activator inhibitor-1 (PAI-1) production, Src activation, nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity, and malaldehyde (MDA) level. Administering iPSCs attenuated ALI induced by MV during hyperoxia, which benefited from the suppression of Src activation, oxidative stress, acute inflammation, and apoptosis, as indicated by the Src-deficient mice. CONCLUSION: The data suggest that iPSC-based therapy is capable of partially suppressing acute inflammatory and oxidant responses that occur during hyperoxia-augmented VILI through the inhibition of Src-dependent signaling pathway.

A balance between B cell receptor and inhibitory receptor signaling controls plasma cell differentiation by maintaining optimal Ets1 levels.

Signaling through the BCR can drive B cell activation and contribute to B cell differentiation into Ab-secreting plasma cells. The positive BCR signal is counterbalanced by a number of membrane-localized inhibitory receptors that limit B cell activation and plasma cell differentiation. Deficiencies in these negative signaling pathways may cause autoantibody generation and autoimmune disease in both animal models and human patients. We have previously shown that the transcription factor Ets1 can restrain B cell differentiation into plasma cells. In this study, we tested the roles of the BCR and inhibitory receptors in controlling the expression of Ets1 in mouse B cells. We found that Ets1 is downregulated in B cells by BCR or TLR signaling through a pathway dependent on PI3K, Btk, IKK2, and JNK. Deficiencies in inhibitory pathways, such as a loss of the tyrosine kinase Lyn, the phosphatase Src homology region 2 domain-containing phosphatase 1 (SHP1) or membrane receptors CD22 and/or Siglec-G, result in enhanced BCR signaling and decreased Ets1 expression. Restoring Ets1 expression in Lyn- or SHP1-deficient B cells inhibits their enhanced plasma cell differentiation. Our findings indicate that downregulation of Ets1 occurs in response to B cell activation via either BCR or TLR signaling, thereby allowing B cell differentiation and that the maintenance of Ets1 expression is an important function of the inhibitory Lyn → CD22/SiglecG → SHP1 pathway in B cells.

Roles of raft-anchored adaptor Cbp/PAG1 in spatial regulation of c-Src kinase.

The tyrosine kinase c-Src is upregulated in numerous human cancers, implying a role for c-Src in cancer progression. Previously, we have shown that sequestration of activated c-Src into lipid rafts via a transmembrane adaptor, Cbp/PAG1, efficiently suppresses c-Src-induced cell transformation in Csk-deficient cells, suggesting that the transforming activity of c-Src is spatially regulated via Cbp in lipid rafts. To dissect the molecular mechanisms of the Cbp-mediated regulation of c-Src, a combined analysis was performed that included mathematical modeling and in vitro experiments in a c-Src- or Cbp-inducible system. c-Src activity was first determined as a function of c-Src or Cbp levels, using focal adhesion kinase (FAK) as a crucial c-Src substrate. Based on these experimental data, two mathematical models were constructed, the sequestration model and the ternary model. The computational analysis showed that both models supported our proposal that raft localization of Cbp is crucial for the suppression of c-Src function, but the ternary model, which includes a ternary complex consisting of Cbp, c-Src, and FAK, also predicted that c-Src function is dependent on the lipid-raft volume. Experimental analysis revealed that c-Src activity is elevated when lipid rafts are disrupted and the ternary complex forms in non-raft membranes, indicating that the ternary model accurately represents the system. Moreover, the ternary model predicted that, if Cbp enhances the interaction between c-Src and FAK, Cbp could promote c-Src function when lipid rafts are disrupted. These findings underscore the crucial role of lipid rafts in the Cbp-mediated negative regulation of c-Src-transforming activity, and explain the positive role of Cbp in c-Src regulation under particular conditions where lipid rafts are perturbed.

B cell-specific loss of Lyn kinase leads to autoimmunity.

The Lyn tyrosine kinase regulates inhibitory signaling in B and myeloid cells: loss of Lyn results in a lupus-like autoimmune disease with hyperactive B cells and myeloproliferation. We have characterized the relative contribution of Lyn-regulated signaling pathways in B cells specifically to the development of autoimmunity by crossing the novel lyn(flox/flox) animals with mice carrying the Cre recombinase under the control of the Cd79a promoter, resulting in deletion of Lyn in B cells. The specific deletion of Lyn in B cells is sufficient for the development of immune complex-mediated glomerulonephritis. The B cell-specific Lyn-deficient mice have no defects in early bone marrow B cell development but have reduced numbers of mature B cells with poor germinal centers, as well as increased numbers of plasma and B1a cells, similar to the lyn(-/-) animals. Within 8 mo of life, B cell-specific Lyn mutant mice develop high titers of IgG anti-Smith Ag ribonucleoprotein and anti-dsDNA autoantibodies, which deposit in their kidneys, resulting in glomerulonephritis. B cell-specific Lyn mutant mice also develop myeloproliferation, similar to the lyn(-/-) animals. The additional deletion of MyD88 in B cells, achieved by crossing lyn(flox/flox)Cd79a-cre mice with myd88(flox/flox) animals, reversed the autoimmune phenotype observed in B cell-specific Lyn-deficient mice by blocking production of class-switched pathogenic IgG autoantibodies. Our results demonstrate that B cell-intrinsic Lyn-dependent signaling pathways regulate B cell homeostasis and activation, which in concert with B cell-specific MyD88 signaling pathways can drive the development of autoimmune disease.

Interleukin-6 trans-signaling exacerbates inflammation and renal pathology in lupus-prone mice.

OBJECTIVE: Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease that is characterized by the production of antinuclear antibodies (ANAs) and leads to immune complex deposition in the kidneys and nephritis. Lyn tyrosine kinase is a regulator of antibody-mediated autoimmune disease, as evidenced by studies in gene-targeted mice and as suggested in genome-wide association studies in SLE. Like SLE patients, Lyn-deficient mice have increased levels of interleukin-6 (IL-6). Deletion of IL-6 from Lyn-deficient mice abrogates levels of inflammation, pathogenic autoantibodies, and nephritis. The purpose of this study was to assess the role of IL-6 trans-signaling in autoimmune disease by overexpressing soluble gp130Fc (sgp130Fc) in a mouse model. METHODS: The effect of overexpression of sgp130Fc on immune cell phenotypes was determined by flow cytometry in young and aged mice with lupus, and ANAs were measured by enzyme-linked immunosorbent assay. Glomerulonephritis was assessed by histopathologic analysis, by measuring the glomerular area and the blood urea nitrogen concentration, and by immunohistochemistry. Immunofluorescence defined renal immune complex and complement deposition. The acute-phase response was determined by quantitative real-time polymerase chain reaction. RESULTS: In contrast to removing IL-6, impaired IL-6 trans-signaling had little effect on many immune cell abnormalities in Lyn-/- mice. Pathogenic ANAs and kidney deposition of immune complexes were also unaltered by sgp130Fc. However, sgp130Fc overexpression led to diminished macrophage expansion, reduced glomerular leukocyte infiltration, reduced complement fixation, significantly attenuated glomerulonephritis, and improved renal function in Lyn-deficient mice. CONCLUSION: Our results reveal key roles of leukocytes, complement, and the innate immune system in mediating glomerulonephritis, and they implicate IL-6 trans-signaling in this process. We suggest that targeting this pathway may be an effective adjunct to B cell depletion in SLE treatment.

Src mediates the mechanical activation of myogenesis by activating TNFα-converting enzyme.

Mechanical stimulation affects many biological aspects in living cells through mechanotransduction. In myogenic precursor cells (MPCs), mechanical stimulation activates p38 mitogen-activated protein kinase (MAPK), a key regulator of myogenesis, via activating TNFα-converting enzyme (TACE, also known as ADAM17), to release autocrine TNFα. However, the signaling mechanism of mechanical activation of TACE is unknown. Because TACE possesses the structural features of substrates of the non-receptor tyrosine kinase Src, we tested the hypothesis that Src mediates mechanical activation of TACE in MPCs. We observed that mechanical stretch of C2C12 or primary rat myoblasts rapidly activates Src, which in turn interacts and colocalizes with TACE, resulting in tyrosine phosphorylation and activation of TACE. Particularly, Src activates TACE via the phosphorylation of amino acid residue Tyr702 in the intracellular tail of TACE, resulting in increased TNFα release and p38 activation. Src inhibition or deficiency blocks stretch activation of the TACE-p38-MAPK signaling, resulting in impaired myogenic gene expression. In response to functional overloading, Src and TACE are activated in mouse soleus muscle. Further, overloading-induced myogenesis and regeneration are impaired in the soleus of Src(+/-) mice. Therefore, Src mediates mechano-activation of TACE and myogenesis.

Lyn signaling to upregulate GANP is critical for the survival of high-affinity B cells in germinal centers of lymphoid organs.

Signals through BCR and costimulatory molecules play essential roles in selecting high-affinity B cells with Ig V-region mutations in the germinal centers (GCs) of peripheral lymphoid organs. Lyn-deficient (lyn(-/-)) mice show impaired BCR signal triggering for cell proliferation and GC formation, causing hyper-IgM, and display autoimmunity after aging. In this study, we demonstrate that Lyn-mediated signaling to upregulate GANP is essential for the survival of mature GC-like (mGC) B cells with high-affinity type BCR mutations upon Ag immunization. Transgenic ganp expression into lyn(-/-) mice did not recover the Lyn-deficient phenotype with regard to B cell differentiation, serum Igs, and impaired GC formation in spleens after immunization with nitrophenyl-chicken γ-globulin, but it markedly rescued cell survival of mGC B cells by suppressing DNA damage, thereby increasing the frequency of the Trp(33)-to-Leu mutation in the IgV(H)-186.2 region and affinity maturation of nitrophenyl-binding B cells. GANP may play a critical role in Lyn-mediated signaling for the selection of high-affinity B cells in peripheral lymphoid organs.

Experimental Pneumocystis lung infection promotes M2a alveolar macrophage-derived MMP12 production.

Among several bacterial and viral pathogens, the atypical fungal organism Pneumocystis jirovecii has been implicated as a contributor to the pathogenesis of chronic obstructive pulmonary disease (COPD). In a previous study, we reported that Pneumocystis-colonized HIV-positive subjects had worse obstruction of airways and higher sputum levels of macrophage elastase/matrix metalloproteinase 12 (MMP12), a protease strongly associated with the development of COPD. Here, we examined parameters of Pneumocystis-induced MMP12 in the lungs of mice and its role in the lung immune response to murine Pneumocystis. Initial studies demonstrated that P. murina exposure induced Mmp12 mRNA expression in whole lungs and alveolar macrophages (AMs), which was dependent on the presence of CD4+ T cells as well as signal transducer and activator of transcription 6. Mmp12 mRNA expression was upregulated in AMs by interleukin (IL)-4 treatment, but downregulated by interferon (IFN)-γ, indicating preferential expression in alternatively activated (M2a) macrophages. IL-4 treatment induced the 54-kDa proenzyme form of MMP12 and the 22-kDa fully processed and active form, whereas IFN-γ failed to induce either. Despite a reported antimicrobial role in macrophage phagolysosomes, mice deficient in MMP12 were not found to be more susceptible to lung infection with P. murina. Collectively, our data indicate that MMP12 induction is a component of the P. murina-induced M2 response and thus provides insight into the link between Pneumocystis colonization/infection and exacerbations in COPD.

A computational model for early events in B cell antigen receptor signaling: analysis of the roles of Lyn and Fyn.

BCR signaling regulates the activities and fates of B cells. BCR signaling encompasses two feedback loops emanating from Lyn and Fyn, which are Src family protein tyrosine kinases (SFKs). Positive feedback arises from SFK-mediated trans phosphorylation of BCR and receptor-bound Lyn and Fyn, which increases the kinase activities of Lyn and Fyn. Negative feedback arises from SFK-mediated cis phosphorylation of the transmembrane adapter protein PAG1, which recruits the cytosolic protein tyrosine kinase Csk to the plasma membrane, where it acts to decrease the kinase activities of Lyn and Fyn. To study the effects of the positive and negative feedback loops on the dynamical stability of BCR signaling and the relative contributions of Lyn and Fyn to BCR signaling, we consider in this study a rule-based model for early events in BCR signaling that encompasses membrane-proximal interactions of six proteins, as follows: BCR, Lyn, Fyn, Csk, PAG1, and Syk, a cytosolic protein tyrosine kinase that is activated as a result of SFK-mediated phosphorylation of BCR. The model is consistent with known effects of Lyn and Fyn deletions. We find that BCR signaling can generate a single pulse or oscillations of Syk activation depending on the strength of Ag signal and the relative levels of Lyn and Fyn. We also show that bistability can arise in Lyn- or Csk-deficient cells.

Distinct and overlapping functional roles of Src family kinases in mouse platelets.

BACKGROUND AND OBJECTIVES: Src family kinases (SFKs) play a critical role in initiating and propagating signals in platelets. The aims of this study were to quantitate SFK members present in platelets and to analyze their contribution to platelet regulation using glycoprotein VI (GPVI) and intregrin αIIbß3, and in vivo. METHODS AND RESULTS: Mouse platelets express four SFKs, Fgr, Fyn, Lyn and Src, with Lyn expressed at a considerably higher level than the others. Using mutant mouse models, we demonstrate that platelet activation by collagen-related peptide (CRP) is delayed and then potentiated in the absence of Lyn, but only marginally reduced in the absence of Fyn or Fgr, and unaltered in the absence of Src. Compound deletions of Lyn/Src or Fyn/Lyn, but not of Fyn/Src or Fgr/Lyn, exhibit a greater delay in activation relative to Lyn-deficient platelets. Fibrinogen-adherent platelets show reduced spreading in the absence of Src, potentiation in the absence of Lyn, but no change in the absence of Fyn or Fgr. In mice double-deficient in Lyn/Src or Fgr/Lyn, the inhibitory role of Lyn on spreading on fibrinogen is lost. Lyn is the major SFK-mediating platelet aggregation on collagen at arterial shear and its absence leads to a reduction in thrombus size in a laser injury model. CONCLUSION: These results demonstrate that SFKs share individual and overlapping roles in regulating platelet activation, with Lyn having a dual role in regulating GPVI signaling and an inhibitory role downstream of αIIbß3, which requires prior signaling through Src.

Attenuation of phosphoinositide 3-kinase δ signaling restrains autoimmune disease.

Systemic lupus erythematosus (SLE) is a heterogeneous autoimmune disease characterized by the production of autoantibodies against nuclear components. Lyn-deficient mice are an excellent animal model of SLE manifesting clinical, pathological and biochemical features seen in the human disease. They develop autoreactive antibodies, glomerulonephritis and show generalized inflammation, and their B cells have a hyperactive phenotype. Since loss of Lyn confers hyper-activation of phosphoinositide 3-kinase (PI3K) signaling, we studied the effect of down-modulating PI3K in Lyn-deficient mice. We found that heterozygous inactivation of the p110δ isoform of PI3K was sufficient to restrain disease in Lyn-deficient mice, leading to significantly decreased autoantibody development and autoimmune-mediated kidney pathology, and improved survival. Intriguingly, haploinsufficiency of p110δ did not dampen signaling in Lyn-deficient B cells. However, plasma cell numbers, serum immunoglobulin titers, inflammation and T cell signaling and activation were significantly moderated in Lyn(-/-)p110δ(+/KD) mice. Importantly, we have shown that haploinsufficiency of p110δ has minor effects on the B cell compartment per se but leads to significant defects in T cell activation and B cell class-switching. These studies suggest that agents targeting p110δ PI3K need not achieve full blockade of the enzyme to be of great benefit in the treatment of SLE.