Affixin activates Rac1 via betaPIX in C2C12 myoblast.

Affixin/beta-parvin is an integrin-linked kinase (ILK)-binding focal adhesion protein highly expressed in skeletal muscle and heart. To elucidate the possible role of affixin in skeletal muscle, we established stable C2C12 cell line expressing T7-tagged human affixin (C2C12-affixin cells). Exogenous expression of affixin promotes lamellipodium formation where affixin, ILK alphap21-activated kinase (PAK)-interactive exchange factor (PIX) and betaPIX accumulate. The association of affixin and betaPIX was confirmed by immunoprecipitation and pull down assay. In C2C12-affixin cells, an increased level of activated Rac1 but not Cdc42 was observed, and mutant betaPIX lacking guanine nucleotide exchange factor activity inhibited lamellipodium formation. These results suggest that affixin is involved in reorganization of subsarcolemmal cytoskeletal actin by activation of Rac1 through alpha and betaPIXs in skeletal muscle.

The gamma-parvin-integrin-linked kinase complex is critically involved in leukocyte-substrate interaction.

Leukocyte extravasation is an important step of inflammation, in which integrins have been demonstrated to play an essential role by mediating the interaction of leukocytes with the vascular endothelium and the subendothelial extracellular matrix. Previously, we identified an integrin-linked kinase (ILK)-binding protein affixin (beta-parvin), which links initial integrin signals to rapid actin reorganization, and thus plays critical roles in fibroblast migration. In this study, we demonstrate that gamma-parvin, one of three mammalian parvin family members, is specifically expressed in several lymphoid and monocytic cell lines in a complementary manner to affixin. Like affixin, gamma-parvin directly associates with ILK through its CH2 domain and colocalizes with ILK at focal adhesions as well as the leading edge of PMA-stimulated U937 cells plated on fibronectin. The overexpression of the C-terminal fragment containing CH2 domain or the depletion of gamma-parvin by RNA interference inhibits the substrate adhesion of MCP-1-stimulated U937 cells and the spreading of PMA-stimulated U937 cells on fibronectin. Interestingly, the overexpression of the CH2 fragment or the gamma-parvin RNA interference also disrupts the asymmetric distribution of PTEN and F-actin observed at the very early stage of cell spreading, suggesting that the ILK-gamma-parvin complex is essential for the establishment of cell polarity required for leukocyte migration. Taken together with the results that gamma-parvin could form a complex with some important cytoskeletal proteins, such as alphaPIX, alpha-actinin, and paxillin as demonstrated for affixin and actopaxin (alpha-parvin), the results in this study suggest that the ILK-gamma-parvin complex is critically involved in the initial integrin signaling for leukocyte migration.

[A case of cT0N2M0 small cell lung cancer with Lambert-Eaton myasthenic syndrome].

We encountered a very rare case of cT0N2M0 small cell lung cancer (SCLC) with Lambert-Eaton myasthenic syndrome (LEMS). A 69-year-old man with a complaint of muscle weakness was admitted to our hospital. Although his chest radiograph on admission showed no abnormal findings, CT scanning detected a mediastinal lymphadenopathy. Also, 2-[18F]-2-fluorodeoxy-D-glucose position emission tomography (FDG-PET) revealed increased accumulation in the same portion in the mediastinum. A diagnosis of LEMS was made from the distinctive electromyogram (EMG) findings (waning and waxing phenomenon in response to low-and high-frequency repetitive stimulation, respectively) in combination with the increased serum level of a P/Q-type anti-voltage-gated calcium channel (VGCC) antibody. Subsequent histopathological diagnosis by mediastinoscopic resection of a paraaortic lymph node was small cell carcinoma. No distant metastasis was detected by MRI of the brain, abdominal CT scan or an FDG-PET. Eight courses of chemotherapy (carboplatin + etoposide) with radiotherapy of the mediastinum (for a total dose of 45 Gy) was performed. A decreased serum level of P/Q-type anti-VGCC antibody titers followed by marked improvement of neurological dysfunction (muscle weakness, gait disturbance and scanning speech) and of an EMG finding (a loss of waning phenomenon) was observed. A close relationship between reduction of the antibody titers and improvement of neurological symptoms after the therapy was noticed. It was suggested that monitoring the level of a P/Q-type anti-VGCC antibody titer in the serum is important for evaluating the efficacy of chemotherapy for LEMS associated with SCLC.

Affixin interacts with alpha-actinin and mediates integrin signaling for reorganization of F-actin induced by initial cell-substrate interaction.

The linking of integrin to cytoskeleton is a critical event for an effective cell migration. Previously, we have reported that a novel integrin-linked kinase (ILK)-binding protein, affixin, is closely involved in the linkage between integrin and cytoskeleton in combination with ILK. In the present work, we demonstrated that the second calponin homology domain of affixin directly interacts with alpha-actinin in an ILK kinase activity-dependent manner, suggesting that integrin-ILK signaling evoked by substrate adhesion induces affixin-alpha-actinin interaction. The overexpression of a peptide corresponding to the alpha-actinin-binding site of affixin as well as the knockdown of endogenous affixin by small interference RNA resulted in the blockade of cell spreading. Time-lapse observation revealed that in both experiments cells were round with small peripheral blebs and failed to develop lamellipodia, suggesting that the ILK-affixin complex serves as an integrin-anchoring site for alpha-actinin and thereby mediates integrin signaling to alpha-actinin, which has been shown to play a critical role in actin polymerization at focal adhesions.

The first CH domain of affixin activates Cdc42 and Rac1 through alphaPIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor.

Rho GTPases, Cdc42 and Rac1, play pivotal roles in cell migration by efficiently integrating cell-substrate adhesion and actin polymerization. Although it has been suggested that integrins stimulate these Rho GTPases via some of integrin binding proteins such as focal adhesion kinase (FAK) and paxillin, the precise molecular mechanism is largely unknown. In this study, we showed that the over-expression of RP1 corresponding to the first CH domain (CH1) of affixin, an integrin-linked kinase (ILK)-binding protein, induced a significant actin reorganization in MDCK cells by activating Cdc42/Rac1. Affixin full length and RP1 co-immunoprecipitated with alphaPIX, a Cdc42/Rac1-specific guanine nucleotide exchanging factor (GEF), and they co-localized at the tips of lamellipodia in motile cells. The involvement of alphaPIX in the RP1-induced Cdc42 activation was demonstrated by the significant dominant negative effect of a point mutant of alphaPIX, alphaPIX (L383R, L384S), lacking GEF activity. Our data strongly support that ILK and affixin provide a novel signalling pathway that links integrin signalling to Cdc42/Rac1 activation.

[Hypogammaglobulinemia associated with thymoma (Good syndrome) similar to diffuse panbronchiolitis].

A 65-year-old woman complained of dyspnea and a productive cough after surgical treatment and irradiation therapy for thymoma. Chest radiography and high-resolution computed tomography showed small nodules in centrilobular lesions in all of both lung fields, but predominantly in the lower fields. In addition, blood tests showed hypogammaglobulinemia. Chronic sinusitis, mild hypoxemia, severe obstructive impairment and the pathological findings of bronchiolitis led to a diagnosis of sinobronchial syndrome caused by Good syndrome. Treatment with oral erythromycin 600 mg/day was started. After 6 months, the patient improved both clinically and radiologically. Low-dose, long-term treatment with erythromycin was effective against sinobronchial syndrome caused by Good syndrome.

Possible role of ILK-affixin complex in integrin-cytoskeleton linkage during platelet aggregation.

Integrin-mediated adhesion induces the formation of focal adhesions that link the extracellular matrix and intracellular actin cytoskeletal networks. We previously showed that integrin-linked kinase (ILK), which can interact with beta1 and beta3 integrins, and its interacting protein, affixin, play an essential role in the initial assembly of focal adhesion structures and actin stress fibers. Although the relevant structures are also observed in integrin alphaIIbbeta3 in platelets, the precise underlying molecular mechanism remains unclarified. Here, we found that ILK stably forms a complex with ss-affixin in platelets. Thrombin stimulation induces their association with integrin beta3, which is followed by their incorporation into the Triton-insoluble membrane-cytoskeletal fraction. During the course of thrombin-induced platelet aggregation, ILK activity was enhanced within 90s to 2.1-fold of the basal level, independent of phosphatidylinositol 3-kinase. Taken together with the observation that the treatment with an anti-integrin beta3 antibody stimulates ILK activity without inducing platelet aggregation, these results suggest that the outside-in signaling induced by fibrinogen binding to integrin enhances ILK activity and results in the initial phase to reorganize the actin cytoskeleton.