Cleavage of talin by calpain promotes platelet-mediated fibrin clot contraction.

Blood clot contraction is driven by traction forces generated by the platelet cytoskeleton that are transmitted to fibrin fibers via the integrin αIIbß3. Here we show that clot contraction is impaired by inhibitors of the platelet cytosolic protease calpain. We used subtiligase-mediated labeling of amino termini and mass spectrometry to identify proteolytically cleaved platelet proteins involved in clot contraction. Of 32 calpain-cleaved proteins after TRAP stimulation, 14 were cytoskeletal, most prominently talin and vinculin. A complex of talin and vinculin constitutes a mechanosensitive clutch connecting integrins bound to the extracellular matrix with the actin cytoskeleton. Accordingly, we focused on talin and vinculin. Talin is composed of an N-terminal head domain and a C-terminal rod domain organized into a series of 4- and 5-helix bundles. The bundles contain 11 vinculin binding sites (VBSs), each of which is an α-helix packed into a bundle interior and requiring structural rearrangement to initiate vinculin binding. We detected 8 calpain-mediated cleavages in talin, 2 previously identified in unstructured regions and 6 in α-helical regions in proximity to a VBS. There is evidence in vitro that applying mechanical force across talin enables vinculin binding to the talin rod. However, we found that inhibiting platelet cytoskeletal contraction had no effect on talin cleavage, indicating that talin cleavage by calpain in platelets does not require cytoskeleton-generated tensile force. Therefore, it is likely that calpain acts in the later stages of clot retraction through focal adhesion disassembly.

Trypanosoma cruzi down-regulates mechanosensitive proteins in cardiomyocytes.

BACKGROUND: Cardiac physiology depends on coupling and electrical and mechanical coordination through the intercalated disc. Focal adhesions offer mechanical support and signal transduction events during heart contraction-relaxation processes. Talin links integrins to the actin cytoskeleton and serves as a scaffold for the recruitment of other proteins, such as paxillin in focal adhesion formation and regulation. Chagasic cardiomyopathy is caused by infection by Trypanosoma cruzi and is a debilitating condition comprising extensive fibrosis, inflammation, cardiac hypertrophy and electrical alterations that culminate in heart failure. OBJECTIVES: Since mechanotransduction coordinates heart function, we evaluated the underlying mechanism implicated in the mechanical changes, focusing especially in mechanosensitive proteins and related signalling pathways during infection of cardiac cells by T. cruzi. METHODS: We investigated the effect of T. cruzi infection on the expression and distribution of talin/paxillin and associated proteins in mouse cardiomyocytes in vitro by western blotting, immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). FINDINGS: Talin and paxillin spatial distribution in T. cruzi-infected cardiomyocytes in vitro were altered associated with a downregulation of these proteins and mRNAs levels at 72 h post-infection (hpi). Additionally, we observed an increase in the activation of the focal adhesion kinase (FAK) concomitant with increase in ß-1-integrin at 24 hpi. Finally, we detected a decrease in the activation of FAK at 72 hpi in T. cruzi-infected cultures. MAIN CONCLUSION: The results suggest that these changes may contribute to the mechanotransduction disturbance evidenced in chagasic cardiomyopathy.

Parental perinatal exposure to bisphenol A reduces the threshold to disrupt blastocyst implantation via decreasing talin, occudin and E-cadherin levels.

The aim was to evaluate the effect of perinatal BPA exposure of one or both parents on the implantation index and expression of talin, occludin and E-cadherin in the uterine epithelial cells (UEC) of the offspring. Pregnant Wistar dams (F0) received BPA or vehicle from gestational day (GD) 6 to lactation day 21. F1 animals were mated forming four groups: Control dam-Control sire (C♀-C♂), BPA dam -Control sire (B♀-C♂), Control dam -BPA sire (C♀-B♂), BPA dam -BPA sire (B♀-B♂). F1 dams were sacrificed at GD 6. Significantly decreased number of implantation sites was observed in the B♀-B♂ group as compared to the C♀-C♂ group, which correlated with decreased talin apical/basal expression ratio, occludin apical expression, and E-cadherin apical/lateral expression ratio in the UEC. Furthermore, decreased E-cadherin expression in the blastocyst was observed. Our data suggest that reduced protein expressions in F1 BPA offspring could result from decreased progesterone serum levels.

Calpain2 mediates Rab5-driven focal adhesion disassembly and cell migration.

The early endosome protein Rab5 was recently shown to promote cell migration by enhancing focal adhesion disassembly through mechanisms that remain elusive. Focal adhesion disassembly is associated to proteolysis of talin, in a process that requires calpain2. Since calpain2 has been found at vesicles and endosomal compartments, we hypothesized that Rab5 stimulates calpain2 activity, leading to enhanced focal adhesion disassembly in migrating cells. We observed that calpain2 co-localizes with EEA1-positive early endosomes and co-immunoprecipitates with EEA1 and Rab5 in A549 lung carcinoma cells undergoing spreading, whereas Rab5 knock-down decreased the accumulation of calpain2 at early endosomal-enriched fractions. In addition, Rab5 silencing decreased calpain2 activity, as shown by cleavage of the fluorogenic substrate tBOC-LM-CMAC and the endogenous substrate talin. Accordingly, Rab5 promoted focal adhesion disassembly in a calpain2-dependent manner, as expression of GFP-Rab5 accelerated focal adhesion disassembly in nocodazole-synchronized cells, whereas pharmacological inhibition of calpain2 with N-acetyl-Leu-Leu-Met prevented both focal adhesion disassembly and cell migration induced by Rab5. In summary, these data uncover Rab5 as a novel regulator of calpain2 activity and focal adhesion proteolysis leading to cell migration.

Adhesion molecules affected by treatment of lung cancer cells with epidermal growth factor.

Lung cancer is one of the leading causes of death in the world. Some tumor events are attributed to an important group of molecules (cadherins and integrins). We evaluated the interactions of cell adhesion molecules in cell lines from lung cancer. Two lung cancer cell lines were nonmetastatic (H358 and H441) and two were metastatic (H1299 and H292). All cell lines were treated with epidermal growth factor (EGF), and Western blot analysis was performed to assess the interactions between these proteins. The bronchoalveolar cells H358 showed the three analyzed proteins: E-cadherin, ß-catenin, and p120 catenin. The adenocarcinoma cells H441 did not present p120 catenin, and carcinoma cells did not show E-cadherin (H1299) or p120 catenin (H292). FAK (pTyr925) was dephosphorylated in adenocarcinoma cells H441, absent in carcinoma cells H1299, and upregulated in the other carcinoma cells H292. p130Cas showed no difference when the cell lines were treated with EGF for 30 min; it was absent in the metastatic carcinoma cells H1299. Paxillin was dephosphorylated in adenocarcinoma cells H441 and also absent in other metastatic carcinoma cells H292. Vinculin showed the same results, and talin was downregulated in adenocarcinoma cells H441 when the cells were treated with EGF. Rap1 was downregulated and PYK2 was upregulated in the same cell line. Our data help to comprehend the mechanism involved in cell migration to the blood and metastasis generation. In conclusion, the expression patterns of cell-cell adhesion were not affected by EGF treatment but it affected cell-extracellular matrix adhesion.

Bradykinin modulates focal adhesions and induces stress fiber remodeling in renal papillary collecting duct cells.

Focal adhesions (FAs) are specialized regions of cell attachment to the extracellular matrix. Previous works have suggested that bradykinin (BK) can modulate cell-matrix interaction. In the present study, we used a physiological cellular model to evaluate the potential role of BK in modulating FAs and stress fibers. We performed a quantitative morphometric analysis of FAs in primary cultured rat renal papillary collecting duct cells, which included size, axial ratio (shape), and average length. After 1, 5, or 10 min of incubation with BK, cultured cells were immunostained and analyzed by confocal microscopy. Although the shape of FAs was not altered, BK induced a decrease in the number of vinculin-stained FAs per cell, and a decrease in both their size and their average length, but not in talin-containing FAs, thus suggesting that BK could be inducing a restructuring of FAs. BK also induced a remodeling of the actin filament assemblies rather than their dissipation. Since we have previously demonstrated that BK stimulates activation of PLCbeta in rat renal papillae, we attempted to determine whether BK can modulate FA restructuring by this mechanism, by pretreating cultured cells with the PLCbeta inhibitor U73122. The present study, performed under physiological conditions with cells that were not genetically manipulated, provides new experimental evidence supporting the notion that the intrarenal hormone BK modulates FAs and actin cytoskeleton organization through a mechanism that involves the activation of PLCbeta. We propose this finding as a novel mechanism for BK modulation of tubular collecting duct function.

Potential role for insulin-like growth factor II and vitronectin in the endothelial-mesenchymal transition process.

Endothelial-to-mesenchymal transition (EndoMT) is a process through which certain subsets of endothelial cells lose endothelial characteristics and transform into mesenchymal or smooth muscle-like cells. Emerging evidence suggests that this process plays an important role during vascular development and in many vascular pathologies. As in epithelial-mesenchymal transition, EndoMT seems to progress through a series of important steps whose interdependence and order are not clear, and that some of them are regulated by soluble growth factors. Insulin-like growth factor II (IGFII), apart from being considered important in cancer, angiogenesis, and atherosclerotic lesions, is also considered as essential to embryonic development. Here, we report that addition of IGFII promoted the EndoMT process in the presence of very low amounts of chicken serum to arrested primary embryonic aortic chicken endothelial cells attached to fibronectin (FN), gelatin, or native type I collagen. This was demonstrated by cell spreading, loss of cell-cell contacts, detachment, migration, and transformation. These cellular events also occurred when IGFII was added to medium containing vitronectin (VN). Additionally, we demonstrated that these proteins were present in the spontaneous intimal thickenings that are observed at day 11-13 of chicken embryo development. We also show that alterations in the distribution of VE-cadherin and beta-catenin occur after IGFII and serum or VN stimulation, and propose that the via VN IGFII effects may be facilitated by interaction of the mannose-6-phosphate/IGFII receptor (M6P/IGFIIR) with the urokinase-type plasminogen activator receptor (uPAR) and its ligand (uPA). Collectively, these findings provide the first evidence for a potential role of the IGFII-VN complex during the EndoMT process. From our observations and previous studies, we postulate a working hypothesis supporting a fundamental role for these molecules during EndoMT.