Anti-Cortactin Autoantibodies Are Associated With Key Clinical Features in Adult Myositis But Are Rarely Present in Juvenile Myositis.

OBJECTIVE: To define the prevalence and clinical phenotype of anti-cortactin autoantibodies in adult and juvenile myositis. METHODS: In this longitudinal cohort study, anti-cortactin autoantibody titers were assessed by enzyme-linked immunosorbent assay in 670 adult myositis patients and 343 juvenile myositis patients as well as in 202 adult healthy controls and 90 juvenile healthy controls. The prevalence of anti-cortactin autoantibodies was compared among groups. Clinical features of patients with and those without anti-cortactin autoantibodies were also compared. RESULTS: Anti-cortactin autoantibodies were more common in adult dermatomyositis (DM) patients (15%; P = 0.005), particularly those with coexisting anti-Mi-2 autoantibodies (24%; P = 0.03) or anti-NXP-2 autoantibodies (23%; P = 0.04). In adult myositis, anti-cortactin was associated with DM skin involvement (62% of patients with anti-cortactin versus 38% of patients without anti-cortactin; P = 0.03), dysphagia (36% versus 17%; P = 0.02) and coexisting anti-Ro 52 autoantibodies (47% versus 26%; P = 0.001) or anti-NT5c1a autoantibodies (59% versus 33%; P = 0.001). Moreover, the titers of anti-cortactin antibodies were higher in patients with interstitial lung disease (0.15 versus 0.12 arbitrary units; P = 0.03). The prevalence of anti-cortactin autoantibodies was not different in juvenile myositis patients (2%) or in any juvenile myositis subgroup compared to juvenile healthy controls (4%). Nonetheless, juvenile myositis patients with these autoantibodies had a higher prevalence of "mechanic's hands" (25% versus 7%; P = 0.03), a higher number of hospitalizations (2.9 versus 1.3; P = 0.04), and lower peak creatine kinase values (368 versus 818 IU/liter; P = 0.02) than those without anti-cortactin. CONCLUSION: The prevalence of anti-cortactin autoantibodies is increased in adult DM patients with coexisting anti-Mi-2 or anti-NXP-2 autoantibodies. In adults, anti-cortactin autoantibodies are associated with dysphagia and interstitial lung disease.

Role of Src and Cortactin in Pemphigus Skin Blistering.

Autoantibodies against desmoglein (Dsg) 1 and Dsg3 primarily cause blister formation in the autoimmune disease pemphigus vulgaris (PV). Src was proposed to contribute to loss of keratinocyte cohesion. However, the role and underlying mechanisms are unclear and were studied here. In keratinocytes, cell cohesion in response to autoantibodies was reduced in Src-dependent manner by two patient-derived PV-IgG fractions as well as by AK23 but not by a third PV-IgG fraction, although Src was activated by all autoantibodies. Loss of cell cohesion was progredient in a timeframe of 24 h and AK23, similar to PV-IgG, interfered with reconstitution of cell cohesion after Ca2+-switch, indicating that the autoantibodies also interfered with desmosome assembly. Dsg3 co-localized along cell contacts and interacted with the Src substrate cortactin. In keratinocytes isolated from cortactin-deficient mice, cell adhesion was impaired and Src-mediated inhibition of AK23-induced loss of cell cohesion for 24 h was significantly reduced compared to wild-type (wt) cells. Similarly, AK23 impaired reconstitution of cell adhesion was Src-dependent only in the presence of cortactin. Likewise, Src inhibition significantly reduced AK23-induced skin blistering in wt but not cortactin-deficient mice. These data suggest that the Src-mediated long-term effects of AK23 on loss of cell cohesion and skin blistering are dependent on cortactin-mediated desmosome assembly. However, in human epidermis PV-IgG-induced skin blistering and ultrastructural alterations of desmosomes were not affected by Src inhibition, indicating that Src may not be critical for skin blistering in intact human skin, at least when high levels of autoantibodies targeting Dsg1 are present.

Hematopoietic cell-specific lyn substrate (HCLS1 or HS1): A versatile actin-binding protein in leukocytes.

Leukocytes are constantly produced in the bone marrow and released into the circulation. Many different leukocyte subpopulations exist that exert distinct functions. Leukocytes are recruited to sites of inflammation and combat the cause of inflammation via many different effector functions. Virtually all of these processes depend on dynamic actin remodeling allowing leukocytes to adhere, migrate, phagocytose, and release granules. However, actin dynamics are not possible without actin-binding proteins (ABP) that orchestrate the balance between actin polymerization, branching, and depolymerization. The homologue of the ubiquitous ABP cortactin in hematopoietic cells is hematopoietic cell-specific lyn substrate-1, often called hematopoietic cell-specific protein-1 (HCLS1 or HS1). HS1 has been reported in different leukocytes to regulate Arp2/3-dependent migration. However, more evidence is emerging that HS1 functions go far beyond just being a direct actin modulator. For example, HS1 is important for the activation of GTPases and integrins, and mediates signaling downstream of many receptors including BCR, TCR, and CXCR4. In this review, we summarize current knowledge on HS1 functions and discuss them in a pathophysiologic context.

Diagnostic utility of cortactin antibodies in myasthenia gravis.

Patients with myasthenia gravis (MG) without antibodies to the acetylcholine receptor (AChR) or muscle-specific tyrosine kinase (MuSK) have been classified as having double-seronegative myasthenia gravis (dSNMG). We used the sera from six dSNMG patients with positive immunohistochemistry assays in a protein array to screen reactivity with 9000 human proteins. We identified cortactin, an intracellular protein that interacts with agrin/MuSK favoring AChR aggregation, as a new antigen in dSNMG. We then designed an in-house enzyme-linked immunosorbent assay as a screening assay and confirmed these results by western blot. We found that 19.7% of dSNMG patients had anti-cortactin antibodies. In contrast, patients with AChR+ MG or other autoimmune disorders and healthy controls were positive at significantly lower rates. Five percent of healthy controls were positive. In a recent study, we screened sera from 250 patients (AChR+ MG, MuSK+ MG, dSNMG) and 29 healthy controls. Cortactin antibodies were identified in 23.7% of dSNMG and 9.5% AChR+ MG patients (P = 0.02). None of the MuSK+ MG patients, patients with other autoimmune disorders, or healthy controls had antibodies against cortactin. Patients with dSNMG cortactin+ MG were negative for anti-striated muscle and anti-LRP4 antibodies. Patients with dSNMG cortactin+ MG presented ocular or mild generalized MG without bulbar symptoms. We conclude that cortactin autoantibodies are biomarkers of MG that, when present, suggest that the disease will be mild.

Clinical Characteristics of Patients With Double-Seronegative Myasthenia Gravis and Antibodies to Cortactin.

IMPORTANCE: Double-seronegative myasthenia gravis (dSNMG) includes patients with myasthenia gravis (MG) without detectable antibodies to the nicotinic acetylcholine receptor (AChR) or to muscle-specific tyrosine kinase (MuSK). The lack of a biomarker hinders the diagnosis and clinical management in these patients. Cortactin, a protein acting downstream from agrin/low-density lipoprotein receptor-related protein 4 (LRP4)/MuSK, has been described as an antigen in dSNMG. OBJECTIVE: To describe the frequency and clinical features of patients with dSNMG who have cortactin antibodies. DESIGN, SETTING, AND PARTICIPANTS: A retrospective cross-sectional study was conducted at Hospital de la Santa Creu i Sant Pau, an institutional practice referral center in Barcelona, Spain, between May 1, 2015, and November 30, 2015. We included 250 patients with a definitive diagnosis of MG with available serum samples at the time of diagnosis. Descriptive and comparative data analyses were performed. EXPOSURES: Cortactin antibodies were measured by enzyme-linked immunosorbent assay and Western blot; AChR, MuSK, and anti-striated muscle antibodies were detected using a standard method; and LRP4 antibodies were tested using a cell-based assay. MAIN OUTCOMES AND MEASURES: The primary outcome was the frequency of patients with dSNMG who have cortactin antibodies. Secondary outcomes were demographic, clinical, neurophysiological, and laboratory data. RESULTS: Of 250 patients (mean [SD] age at onset, 49.7 [21.2] years; 56% female), 38 (15.2%) had dSNMG, 201 (80.4%) had MG with AChR antibodies, and 11 (4.4%) had MG with MuSK antibodies. Cortactin antibodies were identified in 28 patients with MG: 9 of 38 (23.7%) who had dSNMG, 19 of 201 (9.5%) who had MG with AChR antibodies (significantly lower than those with dSNMG: 9.5% vs 23.7%; P = .02), and 0 of 11 who had MG with MuSK antibodies; 0 of 29 controls had cortactin antibodies. At onset, among the 9 patients with dSNMG and cortactin antibodies, 6 had ocular MG and 3 had Myasthenia Gravis Foundation of America clinical classification IIA. Two patients with ocular MG developed generalized MG. The group with dSNMG and cortactin antibodies, compared with those who had MG with AChR antibodies, more frequently had mild forms at onset (100.0% vs 62.7%; P = .03), had fewer bulbar signs at maximal worsening (0% vs 41.3%; P = .01), and were younger at onset (median [interquartile range], 34.9 [9.5] vs 53.9 [38.5] years; P = .03); the group with dSNMG and cortactin antibodies also more frequently had ocular MG at onset than those with MG and AChR antibodies, although the difference was not statistically significant (66.7% vs 40.8%; P = .17). Of 17 patients with ocular dSNMG, 4 (23.5%) had antibodies to cortactin. CONCLUSIONS AND RELEVANCE: In this study, patients with cortactin antibodies and dSNMG had an ocular or mild generalized phenotype of MG. Including the detection of cortactin antibodies in the routine diagnosis of dSNMG may be helpful in ocular MG.

Endothelial actin-binding proteins and actin dynamics in leukocyte transendothelial migration.

The endothelium is the first barrier that leukocytes have to overcome during recruitment to sites of inflamed tissues. The leukocyte extravasation cascade is a complex multistep process that requires the activation of various adhesion molecules and signaling pathways, as well as actin remodeling, in both leukocytes and endothelial cells. Endothelial adhesion molecules, such as E-selectin or ICAM-1, are connected to the actin cytoskeleton via actin-binding proteins (ABPs). Although the contribution of receptor-ligand interactions to leukocyte extravasation has been studied extensively, the contribution of endothelial ABPs to the regulation of leukocyte adhesion and transendothelial migration remains poorly understood. This review focuses on recently published evidence that endothelial ABPs, such as cortactin, myosin, or α-actinin, regulate leukocyte extravasation by controlling actin dynamics, biomechanical properties of endothelia, and signaling pathways, such as GTPase activation, during inflammation. Thus, ABPs may serve as targets for novel treatment strategies for disorders characterized by excessive leukocyte recruitment.

Identification of a novel myositis-associated antibody directed against cortactin.

OBJECTIVE: The aim of this study is to describe a novel myositis-associated autoantibody (anti-cortactin antibody) and assess related clinical and immunological manifestations and its clinical significance. METHODS: Adult patients with myositis (dermatomyositis, polymyositis, immune-mediated necrotizing myopathy, and inclusion body myositis), as well as patients with other autoimmune diseases and non-inflammatory myopathies were analyzed for the presence of anti-cortactin antibody using in-house developed ELISA and immunoblotting techniques with a commercial source of purified cortactin. The cut-off for positive status was determined in a group of healthy volunteers. RESULTS: Antibody against cortactin was positive in 7/34 (20%) polymyositis patients, 9/117 (7.6%) dermatomyositis, 2/7 (26%) immune-mediated necrotizing myopathy, and none of the 4 patients with inclusion body myositis. The antibody also tested positive in 3/101 patients with other autoimmune diseases (2 systemic sclerosis and 1 systemic lupus erythematosus), and in 1/29 patients with non-inflammatory myopathy. No relevant association with specific clinical features was found in patients with these antibodies. Anti-cortactin antibody was more frequently positive in patients with polymyositis and immune-mediated necrotizing myopathy than in the remaining myositis patients, and was the only myositis autoantibody found in sera of 3 patients from these groups. CONCLUSIONS: Our data indicate that cortactin is a novel target antigen in patients with autoimmune diseases, especially patients with polymyositis or immune-mediated necrotizing myopathy. Anti-cortactin can be considered a new myositis-associated antibody.

Cortactin autoantibodies in myasthenia gravis.

Myasthenia gravis (MG) is an autoimmune disease characterized by muscle weakness, fatigability, and autoantibodies against protein antigens of the muscle endplate. Antibodies against acetylcholine receptor (AChR), and less frequently against muscle-Specific Kinase (MuSK) or lipoprotein related protein 4 (LRP4) occur in patients with seropositive MG (SPMG). However, about 10% of patients do not have detectable autoantibodies despite evidence suggesting that the disorder is immune mediated; this disorder is known as seronegative MG (SNMG). Using a protein array approach we identified cortactin (a protein that acts downstream from agrin/MuSK promoting AChR clustering) as potential new target antigen in SNMG. We set up an ELISA assay and screened sera from patients with SPMG, SNMG, other autoimmune diseases and controls. Results were validated by immunoblot. We found that 19.7% of patients with SNMG had antibodies against cortactin whereas only 4.8% of patients with SPMG were positive. Cortactin antibodies were also found in 12.5% of patients with other autoimmune disorders but only in 5.2% of healthy controls. We conclude that the finding of cortactin antibodies in patients with SNMG, suggests an underlying autoimmune mechanism, supporting the use of immune therapy.

Stratifying fascin and cortactin function in invadopodium formation using inhibitory nanobodies and targeted subcellular delocalization.

Invadopodia are actin-rich protrusions arising through the orchestrated regulation of precursor assembly, stabilization, and maturation, endowing cancer cells with invasive properties. Using nanobodies (antigen-binding domains of Camelid heavy-chain antibodies) as perturbators of intracellular functions and/or protein domains at the level of the endogenous protein, we examined the specific contribution of fascin and cortactin during invadopodium formation in MDA-MB-231 breast and PC-3 prostate cancer cells. A nanobody (K(d)~35 nM, 1:1 stoichiometry) that disrupts fascin F-actin bundling emphasizes the importance of stable actin bundles in invadopodium array organization and turnover, matrix degradation, and cancer cell invasion. Cortactin-SH3 dependent WIP recruitment toward the plasma membrane was specifically inhibited by a cortactin nanobody (K(d)~75 nM, 1:1 stoichiometry). This functional domain is shown to be important for formation of properly organized invadopodia, MMP-9 secretion, matrix degradation, and cancer cell invasion. Notably, using a subcellular delocalization strategy to trigger protein loss of function, we uncovered a fascin-bundling-independent role in MMP-9 secretion. Hence, we demonstrate that nanobodies enable high resolution protein function mapping in cells.

WIP remodeling actin behind the scenes: how WIP reshapes immune and other functions.

Actin polymerization is a fundamental cellular process regulating immune cell functions and the immune response. The Wiskott-Aldrich syndrome protein (WASp) is an actin nucleation promoting factor, which is exclusively expressed in hematopoietic cells, where it plays a key regulatory role in cytoskeletal dynamics. WASp interacting protein (WIP) was first discovered as the binding partner of WASp, through the use of the yeast two hybrid system. WIP was later identified as a chaperone of WASp, necessary for its stability. Mutations occurring at the WASp homology 1 domain (WH1), which serves as the WIP binding site, were found to cause the Wiskott-Aldrich syndrome (WAS) and X-linked thrombocytopenia (XLT). WAS manifests as an immune deficiency characterized by eczema, thrombocytopenia, recurrent infections, and hematopoietic malignancies, demonstrating the importance of WIP for WASp complex formation and for a proper immune response. WIP deficiency was found to lead to different abnormalities in the activity of various lymphocytes, suggesting differential cell-dependent roles for WIP. Additionally, WIP deficiency causes cellular abnormalities not found in WASp-deficient cells, indicating that WIP fulfills roles beyond stabilizing WASp. Indeed, WIP was shown to interact with various binding partners, including the signaling proteins Nck, CrkL and cortactin. Recent studies have demonstrated that WIP also takes part in non immune cellular processes such as cancer invasion and metastasis, in addition to cell subversion by intracellular pathogens. Understanding of numerous functions of WIP can enhance our current understanding of activation and function of immune and other cell types.

Cortactin deficiency is associated with reduced neutrophil recruitment but increased vascular permeability in vivo.

Neutrophil extravasation and the regulation of vascular permeability require dynamic actin rearrangements in the endothelium. In this study, we analyzed in vivo whether these processes require the function of the actin nucleation-promoting factor cortactin. Basal vascular permeability for high molecular weight substances was enhanced in cortactin-deficient mice. Despite this leakiness, neutrophil extravasation in the tumor necrosis factor-stimulated cremaster was inhibited by the loss of cortactin. The permeability defect was caused by reduced levels of activated Rap1 (Ras-related protein 1) in endothelial cells and could be rescued by activating Rap1 via the guanosine triphosphatase (GTPase) exchange factor EPAC (exchange protein directly activated by cAMP). The defect in neutrophil extravasation was caused by enhanced rolling velocity and reduced adhesion in postcapillary venules. Impaired rolling interactions were linked to contributions of ß(2)-integrin ligands, and firm adhesion was compromised by reduced ICAM-1 (intercellular adhesion molecule 1) clustering around neutrophils. A signaling process known to be critical for the formation of ICAM-1-enriched contact areas and for transendothelial migration, the ICAM-1-mediated activation of the GTPase RhoG was blocked in cortactin-deficient endothelial cells. Our results represent the first physiological evidence that cortactin is crucial for orchestrating the molecular events leading to proper endothelial barrier function and leukocyte recruitment in vivo.

Characterization of cortactin as an in vivo protein kinase D substrate: interdependence of sites and potentiation by Src.

Protein Kinase D (PKD) has been implicated in the regulation of actin turnover at the leading edge, invasion and migration. In particular, a complex between cortactin, paxillin and PKD in the invadopodia of invasive breast cancer cells has been described earlier, but so far this complex remained ill defined. Here we have investigated the possible role of PKD as a cortactin kinase. Using a mass spectrometric approach, we found that PKD phosphorylates cortactin on Ser 298 in the 6th cortactin repeat region and on Ser 348, right before the helical-proline rich domain of cortactin. We developed phosphospecific antibodies against these phosphorylated sequences, and used them as tools to follow the in vivo phosphorylation of cortactin by PKD. Examination of cortactin phosphorylation kinetics revealed that Ser 298 serves as a priming site for subsequent phosphorylation of Ser 348. Src, a well-known cortactin kinase, strongly potentiated the in vivo PKD mediated cortactin phosphorylation. This Src effect is neither mediated by pre-phosphorylation of cortactin nor by activation of PKD by Src. Phosphorylation of cortactin by PKD does not affect its subcellular localization, nor does it affect its translocation to podosomes or membrane ruffles. Moreover, there was no effect of PKD mediated cortactin phosphorylation on EGF receptor degradation and LPA induced migration. Taken together, these data establish cortactin as a novel PKD substrate and reveal a novel connection between Src and PKD.