Expression Of Talin1 Research Articles

Cyanidin-3-glucoside binds to talin and modulates colon cancer cell adhesions and 3D growth.

Cyanidin-3-glucoside (C3G) is a natural pigment, found in many colorful fruits and vegetables. It has many health benefits, including anti-inflammation, cancer prevention, and anti-diabetes. Although C3G is assumed to be an antioxidant, it has been reported to affect cell-matrix adhesions. However, the underlying molecular mechanism is unknown. Here, we show that the expression of talin1, a key regulator of integrins and cell adhesions, negatively correlated with the survival rate of colon cancer patients and that depletion of talin1 inhibited 3D spheroid growth in colon cancer cells. Interestingly, C3G bound to talin and promoted the interaction of talin with β1A-integrin. Molecular docking analysis shows that C3G binds to the interface of the talin-β-integrin complex, acting as an allosteric regulator and altering the interaction between talin and integrin. Moreover, C3G promoted colon cancer cell attachment to fibronectin. While C3G had no significant effect on colon cancer cell proliferation, it significantly inhibited 3D spheroid growth in fibrin gel assays. Since C3G has no or very low toxicity, it could be potentially used for colon cancer prevention or therapy.

Regulation of Embryonic Signal on Talin1 in Mouse Endometrium.

Embryonic signals can affect the spatiotemporal-specific expression of the uterus to establish a successful pregnancy. Our previous study has found that talin1 underwent dynamic changes in the mouse endometrium during peri-implantation period. However, whether talin1 is affected by the embryo signals is not clear. In order to investigate the effect of embryonic signals, especially human chorionic gonadotropin (HCG) on talin1, we have designed mouse models of pseudopregnancy, delayed implantation and activation, and HCG treatment. Using these models, the expression of talin1 in the mouse endometrium was determined by immunohistochemistry and Western blotting. In the pseudopregnancy model, an increased expression of talin1 was found from day 3 to day 5, whereas the talin1 protein was decreased on day 5 in the normal pregnant mice. In the delayed implantation model, a strong cytoplasmic staining of talin1 was found, especially in stromal cells. However, after activation of the implantation, the expression of talin1 decreased (P < .05). Furthermore, a significantly lower expression of talin1 was found at the implantation site when compared to the interimplantation sites (P < .05). In the HCG treatment model, an intrauterine perfusion of 10u HCG significantly reduced the expression of talin1 in both stromal and epithelial cells in pseudopregnant mice (P < .05), although further increase in the HCG concentration did not have additional effect on expression of talin1. Taken together, our data suggest that the presence of embryos can affect expression of talin1 in the mouse endometrium, and a certain concentration of HCG can regulate its expression.

Changes in expression of Talin1 and F-actin during serum-induced proliferation of pulmonary artery smooth muscle cells of rats with hepatopulmonary syndrome

Objective To evaluate the changes in the expression of Talin1 and F-actin during serum-induced proliferation of pulmonary artery smooth muscle cells (PASMCs) of rats with hepatopulmonary syndrome (HPS). Methods Twenty healthy male Sprague-Dawley rats, weighing 220-250 g, were used for producing HPS by chronic ligation of the common bile duct.Blood samples from the abdominal aorta were collected to prepare serum.Primarily cultured PASMCs obtained from rats were seed in 6- or 96-well plates and divided into 2 groups using a random number table method: control group (group C) and HPS group (group HPS), with 24 wells in each group (for 6-well plates) or with 30 wells in each group (for 96-well plates). In C and HPS groups, normal rat serum or HPS rat serum were added, respectively, with the final concentration of 5%.At 24, 48 and 72 h of incubation, the expression of Talin1, F-actin and G-actin was determined by Western blot, the F-actin/G-actin ratio was calculated, and the proliferation of PASMCs was measured by 3H-TdR incorporation and CCK-8 assays. Results Compared with group C, the proliferation of PASMCs was significantly enhanced, the expression of Talin1 was up-regulated, and the F-actin/G-actin ratio was increased in group HPS (P<0.05). The proliferation of PASMCs was gradually enhanced, the expression of Talin1 was gradually up-regulated, and the F-actin/G-actin ratio was gradually increased with the prolonged incubation time in group HPS (P<0.05). Conclusion The mechanism by which the HPS rat serum induces proliferation of PASMCs may be related to up-regulating the expression of Talin1 and F-actin. Key words: Hepatopulmonary syndrome; Myocytes, smooth muscle; Pulmonary artery; Cell proliferation; Talin; Actins

IL-1β Induces Pathologically Activated Osteoclasts Bearing Extremely High Levels of Resorbing Activity: A Possible Pathological Subpopulation of Osteoclasts, Accompanied by Suppressed Expression of Kindlin-3 and Talin-1.

As osteoclasts have the central roles in normal bone remodeling, it is ideal to regulate only the osteoclasts performing pathological bone destruction without affecting normal osteoclasts. Based on a hypothesis that pathological osteoclasts form under the pathological microenvironment of the bone tissues, we here set up optimum culture conditions to examine the entity of pathologically activated osteoclasts (PAOCs). Through searching various inflammatory cytokines and their combinations, we found the highest resorbing activity of osteoclasts when osteoclasts were formed in the presence of M-CSF, receptor activator of NF-κB ligand, and IL-1β. We have postulated that these osteoclasts are PAOCs. Analysis using confocal laser microscopy revealed that PAOCs showed extremely high proton secretion detected by the acid-sensitive fluorescence probe Rh-PM and bone resorption activity compared with normal osteoclasts. PAOCs showed unique morphology bearing high thickness and high motility with motile cellular processes in comparison with normal osteoclasts. We further examined the expression of Kindlin-3 and Talin-1, essential molecules for activating integrin β-chains. Although normal osteoclasts express high levels of Kindlin-3 and Talin-1, expression of these molecules was markedly suppressed in PAOCs, suggesting the abnormality in the adhesion property. When whole membrane surface of mature osteoclasts was biotinylated and analyzed, the IL-1β-induced cell surface protein was detected. PAOCs could form a subpopulation of osteoclasts possibly different from normal osteoclasts. PAOC-specific molecules could be an ideal target for regulating pathological bone destruction.

Downregulation of Talin-1 expression associates with increased proliferation and migration of vascular smooth muscle cells in aortic dissection

BackgroundThis study aimed to assessed whether Talin-1 is involved in the pathogenesis of aortic dissection via regulating vascular smooth muscle cell (VSMC) biological function.MethodsHuman aortic samples were obtained from organ donors who died from nonvascular diseases as normal controls and from patients undergoing surgical repair of thoracic aortic dissection. The expression level and distribution of Talin-1 were detected using westernblot analysis and immunohistochemistry in each sample. We inhibited the expression of Talin-1 via RNA interference in VSMCs. VSMC proliferation was detected by Cell-counting Kit-8 analyses. Scratch test and flow cytometry were used to identify the migration and apoptosis ability. Antibody microarray analysis and qRT-PCR were used to detect some protein and mRNA changes which were induced by Talin-1 downregulation.ResultsTalin-1 was significantly downregulated in the media of aortic dissection samples compared with controls (P < 0.05). Talin-1 knockdown significantly induced VSMC proliferation and migration in vitro. Proteins which involved in cell cycle can be regulated by downregulating Talin-1. Down regulation of Talin-1 can significanly increased the expression of anaphase-promoting complex subunit 2 (APC2) and decreased p19 alternative reading frame (p19ARF), Cullin-3, and beta actin’s expression.ConclusionsTalin-1 induces VSMCs proliferation and migration. It downregulated in aortic dissection, which might play a potential role in the development of aortic dissection.

Downregulation of Talin1 promotes hepatocellular carcinoma progression through activation of the ERK1/2 pathway.

Talin1 is an adaptor protein that conjugates integrins to the cytoskeleton and regulates integrins and focal adhesion signaling. Several studies have found that Talin1 is overexpressed in several tumor types and promotes tumor progression. However, the explicit role of Talin1 in hepatocellular carcinoma (HCC) progression is still unclear and its functional mechanism remains largely unknown. In this study, we showed a trend of gradually decreasing expression of Talin1 from normal liver tissues to hepatocirrhosis, liver hyperplasia, the corresponding adjacent non‐tumor, primary HCC, and eventually metastatic foci, indicating that Talin1 may correlate with HCC initiation to progression. Talin1 was significantly downregulated in HCC tissues compared with adjacent non‐tumor tissues and low Talin1 expression was associated with HCC progression and poor prognosis. Furthermore, Talin1 knockdown induced epithelial–mesenchymal transition and promoted migration and invasion in SK‐Hep‐1 cells and HepG2 cells. Mechanistically, we found that the ERK pathway was responsible for these promoting effects of Talin1 knockdown in HCC cells. The promoting effects of Talin1 knockdown on epithelial–mesenchymal transition, migration, and invasion were reversed by U0126, a specific ERK1/2 inhibitor. Taken together, our results suggested that Talin1 might serve as a tumor suppressor in HCC and a potential prognostic biomarker for HCC patients.

Curdione attenuates thrombin-induced human platelet activation: β1-tubulin as a potential therapeutic target

Rhizoma Curcumae, the dry rhizomes derived from Curcuma aromatica Salisb., are a classical Chinese medicinal herb used to activate blood circulation, remove blood stasis and alleviate pain. Our previous study proved that curdione, a sesquiterpene compound isolated from the essential oil of Curcuma aromatica Salisb. can inhibit platelet activation suggesting its significant anticoagulant and antithrombotic effects. However, the underlying mechanism of curdione mediated anti-platelet effect has not been fully elucidated. Platelet proteins extracted from washed human platelets, including normal group (treated with normal saline), thrombin group and curdione group were digested and analysed by nano ESI-LC-MS/MS. UniProt database and SIEVE software were employed to identify and reveal the differentially expressed proteins. Furthermore, possible mechanisms involved were explored by Ingenuity Pathway Analysis (IPA) Software and validated by western blot experiments. Twenty-two differentially expressed proteins between the normal and thrombin group were identified. Compared with the thrombin group, the curdione treatment was significantly down-regulated only 2 proteins (Talin1 and β1-tubulin). Bioinformatics analysis showed that Talin1 and β1-tubulin could be involved in the integrin signal pathway. The results of western blot analysis were consistent with that of the proteomics data. Vinculin, identified in IPA database was involved in the formation of cell cytoskeletal. The down-regulation of β1-tubulin facilitated the decrease in vinculin/Talin1. Curdione regulated the expression of vinculin and Talin1 by β1-tubulin affecting the integrin signalling pathway and eventually inhibiting platelet activation. The β1-tubulin may be a potential target of curdione, which attenuates thrombin-induced human platelet activation.

The L-Arginine Transporter Solute Carrier Family 7 Member 2 Mediates the Immunopathogenesis of Attaching and Effacing Bacteria.

Solute carrier family 7 member 2 (SLC7A2) is an inducible transporter of the semi-essential amino acid L-arginine (L-Arg), which has been implicated in immune responses to pathogens. We assessed the role of SLC7A2 in murine infection with Citrobacter rodentium, an attaching and effacing enteric pathogen that causes colitis. Induction of SLC7A2 was upregulated in colitis tissues, and localized predominantly to colonic epithelial cells. Compared to wild-type mice, Slc7a2 –/–mice infected with C. rodentium had improved survival and decreased weight loss, colon weight, and histologic injury; this was associated with decreased colonic macrophages, dendritic cells, granulocytes, and Th1 and Th17 cells. In infected Slc7a2 –/–mice, there were decreased levels of the proinflammatory cytokines G-CSF, TNF-α, IL-1α, IL-1β, and the chemokines CXCL1, CCL2, CCL3, CCL4, CXCL2, and CCL5. In bone marrow chimeras, the recipient genotype drove the colitis phenotype, indicative of the importance of epithelial, rather than myeloid SLC7A2. Mice lacking Slc7a2 exhibited reduced adherence of C. rodentium to the colonic epithelium and decreased expression of Talin-1, a focal adhesion protein involved in the attachment of the bacterium. The importance of SLC7A2 and Talin-1 in the intimate attachment of C. rodentium and induction of inflammatory response was confirmed in vitro, using conditionally-immortalized young adult mouse colon (YAMC) cells with shRNA knockdown of Slc7a2 or Tln1. Inhibition of L-Arg uptake with the competitive inhibitor, L-lysine (L-Lys), also prevented attachment of C. rodentium and chemokine expression. L-Lys and siRNA knockdown confirmed the role of L-Arg and SLC7A2 in human Caco-2 cells co-cultured with enteropathogenic Escherichia coli. Overexpression of SLC7A2 in human embryonic kidney cells increased bacterial adherence and chemokine expression. Taken together, our data indicate that C. rodentium enhances its own pathogenicity by inducing the expression of SLC7A2 to favor its attachment to the epithelium and thus create its ecological niche.

The Integrin LFA-1 Controls T Follicular Helper Cell Generation and Maintenance

T follicular helper (Tfh) cells are a CD4+ Tcell subset critical for long-lived humoral immunity. We hypothesized that integrins play a decisive role in Tfh cell biology. Here we show that Tfh cells expressed a highly active form of leukocyte function-associated antigen-1 (LFA-1) that was required for their survival within the germinal center niche. In addition, LFA-1 promoted expression of Bcl-6, a transcriptional repressor critical for Tfh cell differentiation, and inhibition of LFA-1 abolished Tfh cell generation and prevented protective humoral immunity to intestinal helminth infection. Furthermore, we demonstrated that expression of Talin-1, an adaptor protein that regulates LFA-1 affinity, dictated Tfh versus Th2 effector cell differentiation. Collectively, our results define unique functions for LFA-1 in the Tfh cell effector program and suggest that integrin activity is important in lineage decision-making events in the adaptive immune system.

Integrin activation controls regulatory T cell identity and stability

Abstract Maintenance of the regulatory T (Treg) cell pool is essential for peripheral tolerance and prevention of autoimmunity. Integrins, heterodimeric transmembrane proteins consisting of α and β subunits that mediate cell-cell and cell-extracellular matrix interactions, have been shown to play an important role in facilitating cell contact-mediated suppression by Treg cells. Here we show that integrin activation plays an essential, previously unappreciated role in maintaining the stability of the Treg cell pool. Treg cell-specific loss of talin1, a β integrin-binding protein, or expression of talin1(L325R), a mutant that selectively abrogates integrin activation, resulted in dysregulation of Treg cell identity and lethal systemic autoimmunity. Moreover, the absence of sustained interactions between the integrin LFA-1 on Treg cells and its ligand ICAM-1 on dendritic cells reduced the expression of Foxp3 and caused Treg cells to adopt an effector CD4+ T cell-like phenotype. Taken together, these results reveal a critical role for tonic, integrin-mediated signals in controlling peripheral tolerance by virtue of maintaining Treg cell identity and stability.

Increased expression of Talin1 in the eutopic and ectopic endometria of women with adenomyosis

Adenomyosis is a prevalent gynecologic benign disease in women. Despite its significance, there is only a limited understanding of its pathological mechanisms. Talin1, a cytoskeletal protein, plays an important role in cell survival, proliferation, invasion and migration. The objective of this study was to investigate the mRNA and protein expression of talin1 in both the eutopic and ectopic endometria of women with adenomyosis. Higher talin1 mRNA levels were observed in both ectopic and eutopic endometria from the adenomyosis subjects compared with the eutopic endometria from women without adenomyosis. Immunohistochemistry revealed increased epithelial expression of talin1 in the ectopic and eutopic endometria from patients with adenomyosis compared with those without adenomyosis. When tests were performed on matched samples of eutopic and ectopic endometria of adenomyosis subjects, the mRNA and protein expression of talin1 was much higher in the ectopic endometria than in the eutopic endometria. The results reveal that the expression pattern of talin1 in the eutopic and ectopic endometria is enhanced in women with adenomyosis. Increased talin1 expression may play a role in the pathogenesis and development of adenomyosis.

Extracellular rigidity sensing by talin isoform-specific mechanical linkages.

The ability of cells to adhere and sense differences in tissue stiffness is crucial for organ development and function. The central mechanisms by which adherent cells detect extracellular matrix compliance, however, are still unknown. Using two single-molecule–calibrated biosensors that allow the analysis of a previously inaccessible but physiologically highly relevant force regime in cells, we demonstrate that the integrin activator talin establishes mechanical linkages upon cell adhesion, which are indispensable for cells to probe tissue stiffness. Talin linkages are exposed to a range of piconewton (pN) forces and bear, on average, 7–10 pN during cell adhesion depending on their association with f-actin and vinculin. Disruption of talin’s mechanical engagement does not impair integrin activation and initial cell adhesion but prevents focal adhesion reinforcement and thus extracellular rigidity sensing. Intriguingly, talin mechanics are isoform-specific so that expression of either talin-1 or talin-2 modulates extracellular rigidity sensing.

High expression of Talin-1 is associated with poor prognosis in patients with nasopharyngeal carcinoma.

BackgroundTalin-1 is a cytoskeletal protein that plays an important role in tumourgenesis, migration and metastasis in several malignant tumors. The aim of this study was to evaluate the expression and prognostic value of Talin-1 in nasopharyngeal carcinoma (NPC).MethodsTalin-1 mRNA and protein expression were examined in NPC cell lines and clinical nasopharyngeal tissues by quantitative RT-PCR, agarose gel electrophoresis and western blotting. The expression of Talin-1 was analyzed by immunohistochemical staining in 233 paraffin-embedded NPC specimens with clinical follow-up data and cox regression analysis was used to identify independent prognostic factors. The functional role of Talin-1 in NPC cell lines was evaluated by small interfering RNA-mediated depletion of the protein followed by the wound healing and transwell invasion assays.ResultsThe expression of Talin-1 was significantly upregulated in most NPC cell lines and clinical tissues at both the mRNA and protein levels. High expression of Talin-1 was significantly associated with distant metastasis (P = 0.001) and patient death (P = 0.001). In addition, high expression of Talin-1 was associated with significantly poorer overall survival (OS: HR, 2.15; 95% CI, 1.28-3.63; P = 0.003) and poorer distant metastasis-free survival (DMFS: HR, 2.39; 95% CI, 1.38-4.15; P = 0.001). Cox regression analysis indicated that high expression of Talin-1 and TNM stage were independent prognostic indicators (both P < 0.05). Stratified analysis demonstrated that high expression of Talin-1 was associated with significantly poorer survival in patients with advanced stage disease (stage III-IV, HR, 1.91; 95% CI, 1.09-3.35; P = 0.02 for OS and HR, 2.22; 95% CI, 1.24-3.99; P = 0.006 for DMFS). Furthermore, the depletion of Talin-1 suppressed the migratory and invasive ability of NPC cells in vitro.ConclusionsOur data demonstrate that high expression of Talin-1 is associated with significantly poorer OS and poorer DMFS in NPC and depletion of Talin-1 expression inhibited NPC cell migration and invasion. Talin-1 may serve as novel prognostic biomarker in NPC.

Talin-1 correlates with reduced invasion and migration in human hepatocellular carcinoma cells.

Talin-1 is a cytoskeleton protein that participates in cell migration and plays a role in tumor formation, migration, and metastasis in different types of cancer. Chinese investigators have observed that the levels of Talin-1 protein and mRNA expression in HCC tissues are significantly lower than in the adjacent non-cancerous tissue. However, Japanese investigators have reported that Talin-1 is upregulated in HCC. Tln2 as homologous gene of Tln-1, which encodes a very similar protein, but the role of Talin-2 is very little known in primary liver cancer (PLC). We investigated whether the expression of Talin-1 in PLC may be associated with the histological subtype as well as the role of Talin-1 in tumor cell invasion and migration using human hepatocellular carcinoma cell lines. We measured the mRNA expression levels of Talin-1 and Talin-2 in five human liver cancer cell lines and normal human liver cell (LO2 cell line) by real-time PCR and the protein expression levels of Talin-1 by Western blot. Migration and invasion of the cells were assessed using transwell assays and cell scratch experiments, respectively, and proliferation was assessed by soft AGAR colony formation. Talin-1 and Talin-2 expression differed significantly between the five human liver cancer cell lines and LO2 cell line (p<0.05). Compared with the LO2 cell line, the invasion and migration capabilities of the five cancer cell lines differed significantly (p<0.05). Similarly, the colony-forming ability differed (p<0.05). High levels of Talin-1 expression are correlated with reduced invasion and migration as well as decreased malignancy in human liver cancer cell lines; the suppression of Talin-1 promotes invasion and migration. In addition, Talin-2 may be correlated with invasion and migration in human hepatocellular carcinoma.

Agonist Stimulation, Talin-1, and Kindlin-3 Are Crucial for α¶bβ3 activation in a Human Megakaryoblastic Cell Line, CMK

Abstract 1137Integrin αIIbβ3 plays a central role in platelet aggregation, by changing its conformation from low-affinity to high-affinity state for ligand via inside-out signaling (integrin activation). However, detailed mechanism of αIIbβ3 activation remains to be determined. We have developed a powerful system to analyze the mechanism of physiological agonist-induced αIIbβ3 activation, by employing a human megakaryoblastic leukemia cell line, CMK. In contrast to exogenously expressed αIIbβ3 in CHO cells, endogenously expressed αIIbβ3 in GPIb-positive CMK cells can be activated by physiological agonists such as thrombin and protease-activated receptor-actibvating peptide (PAR-AP). An activated state of αIIbβ3 induced by agonists was only transient and the activated αIIbβ3 gradually returned inactive state. A robust αIIbβ3 activation was detected by an initial velocity analysis for FITC-PAC-1 binding. In this study, we performed direct genetic manipulation to examine an effect of talin-1 and kindlin-3 on αIIbβ3 activation in CMK. We measured the initial PAC-1 binding velocity by incubating FITC-PAC-1 and PAR1-TRAP with CMK cells only for 2 minutes. Over-expression of full length talin-1 (FL-TLN), talin head domain (THD) or kindlin-3 significantly augmented PAC-1 binding after 50 μM PAR1-TRAP stimulation. In sharp contrast to the CHO system, αIIbβ3 activation was not induced without any agonist stimulation in CMK system even if THD was over-expressed.Overexpression of FL-TLN mutant in critical residues for its head-rod interaction (FL-TLN-K318A or FL-TLN-M319A) more strongly augmented αIIbβ3 activation, as compared with wild-type FL-TLN. In contrast, overexpression of THD with the same mutation did not augment it, as compared with wild-type THD. These findings confirmed an autoinhibition mechanism for talin-1. FL-TLN mutants or THD mutants in critical residues for the interaction with β3 cytoplasmic tail (W359A, L325R, S365D, S379R, Q381V or K324D) or in the critical residue for the interaction with membrane (K322D) failed to augnent αIIbβ3 activation. In order to gain stable talin-1 or kindlin-3 knocked-down cell line, we used lentivirus mediated RNAi technology. In brief, lentiviral particles containing short-hairpin RNAs targeting talin-1 mRNA were integrated in CMK, and DsRed2 which is expressed with shRNA was used as transfection marker. Stable clones were obtained by limitting dilution. In these clones the expression of endogenous talin-1 or kindlin-3 was reduced to approximately half of non-silencing cells. Knock-down each molecule resulted in a robust decrease in PAC-1 binding induced by PAR1-TRAP. The impaired αIIbβ3 activation in kindlin-3 knocked down cells was rescued by exogenously expressed kindlin-3. Interestingly, the impaired αIIbβ3 activation in kindlin-3 knocked down CMK cells was also rescued by overexpression of THD. However, the increase in αIIbβ3 activation by PAR1-TRAP after over-expression of THD in kindlin-3 knocked-down cells was still smaller, as compared with that in THD overexpressed wild-type cells. Those results suggest that talin-1 and kindlin-3 play a crucial role in αIIbβ3 activation co-operatively. Taken together, this experimental system with CMK using RNAi and overexpression technology provides a newly alternative way to investigate molecular mechanism for the αIIbβ3 activation induced by physiological agonists. Disclosures:No relevant conflicts of interest to declare.

Talin1 Promotes Tumor Invasion and Metastasis via Focal Adhesion Signaling and Anoikis Resistance

Talin1 is a focal adhesion complex protein that regulates integrin interactions with ECM. This study investigated the significance of talin1 in prostate cancer progression to metastasis in vitro and in vivo. Talin1 overexpression enhanced prostate cancer cell adhesion, migration, and invasion by activating survival signals and conferring resistance to anoikis. ShRNA-mediated talin1 loss led to a significant suppression of prostate cancer cell migration and transendothelial invasion in vitro and a significant inhibition of prostate cancer metastasis in vivo. Talin1-regulated cell survival signals via phosphorylation of focal adhesion complex proteins, such as focal adhesion kinase and Src, and downstream activation of AKT. Targeting AKT activation led to a significant reduction of talin1-mediated prostate cancer cell invasion. Furthermore, talin1 immunoreactivity directly correlated with prostate tumor progression to metastasis in the transgenic adenocarcinoma mouse prostate mouse model. Talin1 profiling in human prostate specimens revealed a significantly higher expression of cytoplasmic talin1 in metastatic tissue compared with primary prostate tumors (P < 0.0001). These findings suggest (a) a therapeutic significance of disrupting talin1 signaling/focal adhesion interactions in targeting metastatic prostate cancer and (b) a potential value for talin1 as a marker of tumor progression to metastasis.

Proteomic analysis of PBMCs: characterization of potential HIV-associated proteins

BackgroundThe human immunodeficiency virus type 1 (HIV-1) pandemic has continued unabated for nearly 30 years. To better understand the influence of virus on host cells, we performed the differential proteome research of peripheral blood mononuclear cells (PBMCs) from HIV-positive patients and healthy controls.Results26 protein spots with more than 1.5-fold difference were detected in two dimensional electrophoresis (2DE) gels. 12 unique up-regulated and one down-regulated proteins were identified in HIV-positive patients compared with healthy donors. The mRNA expression of 10 genes was analyzed by real time RT-PCR. It shows that the mRNA expression of talin-1, vinculin and coronin-1C were up-regulated in HIV positive patients and consistent with protein expression. Western blotting analysis confirmed the induction of fragments of vinculin, talin-1 and filamin-A in pooled and most part of individual HIV-positive clinical samples. Bioinformatic analysis showed that a wide host protein network was disrupted in HIV-positive patients.ConclusionsTogether, this work provided useful information to facilitate further investigation of the underlying mechanism of HIV and host cell protein interactions, and discovered novel potential biomarkers such as fragment of vinculin, filamin-A and talin-1 for anti-HIV research.

Platelets Derived From Patients with Myelodysplastic Syndromes (MDS) Show Defective Platelet Aggregation and Integrin aIIbβ3 Receptor (GPIIb/IIIa) Signaling Caused by a Reduced Expression of Proteins Required for Inside-out Signaling.

Abstract 598Thrombocytopenia is prevalent in up to 65% of patients with myelodysplastic syndrome (MDS) at the time of diagnosis and thrombocytopenic hemorrhage is a significant clinical problem that is often complicated by platelet aggregation defects. Little is known about the pathophysiology of this insufficient platelet function. Here, we delineate a reduced expression of critical platelet aggregation-related proteins by analyzing the platelet proteome of 7 patients with MDS and 7 normal donors. Patients' median platelet count was 60 × 10E9/L (range 37–109 × 109/L) and none of the patients examined had received prior anticoagulant treatment, chemotherapy or platelet transfusions. Differential 2-dimensional in-gel electrophoresis coupled with a time-of-flight Ultraflex-Tof/Tof mass spectrometer enabled the discovery of 120 differential protein spots. Among these, we identified a total of 35 proteins including 26 proteins that are integral part of the integrin aIIbβ3 receptor (GPIIb/IIIa, Fibrinogen receptor) signaling such as Talin-1 and Vinculin. In resting platelets the integrin aIIbβ3 receptor exhibits a low-affinity (inactive) state which is shifted to a high-affinity (active) state following inside-out activation. Talin-1 expression has been shown to be essential for this inside-out activation of the integrin aIIbβ3 receptor and consecutive platelet aggregation in an in-vivo model. We hypothesized that the reduced expression of Talin-1 and its co-factor Vinculin may inhibit activation of the integrin aIIbβ3 receptor and thereby contribute to the platelet aggregation defect seen in patients with MDS. Therefore, we performed further functional studies on integrin aIIbβ3 receptor activation and platelet spreading/aggregation with platelets derived from an independent cohort of 7 patients with MDS and 7 normal donors. In this new cohort, patients' median platelet count was 94 × 109/L (range 60–120 × 109/L) and again all patients had never received prior platelet transfusions or anti-coagulant treatment. When we looked at the surface expression of the integrin aIIbβ3 receptor on resting platelets by means of flow-cytometry, we did not detect any differences between platelets from patients with MDS and normal donors. Then, we activated platelets from normal donors and patients with MDS with 0.01U/μl and 0.001U/μl thrombin and measured binding of PAC-1, which is highly specific for detection of the active form of the integrin aIIbβ3 receptor. Here, we found a significantly lower shift from the inactive to the active form in platelets derived from patients with MDS dropping from 92.15% and 91.46% in normal donors to 41.97% and 48.45% (p = 0.01 and p = 0.006), respectively. We confirmed this suggested lack of adhesion and aggregation capacities in MDS platelets by confocal microscopy and single platelet imaging of washed platelets stimulated with 0.01U/μl thrombin which were adhered to immobilized fibrinogen. Consecutive platelet aggregation assays also revealed an insufficient response to stimuli like Collagen and ADP. Our findings provide for the first-time insight into the molecular pathology of defective platelet aggregation in MDS and suggest a mechanism of defective inside-out signaling caused by a reduced expression of proteins required for integrin aIIbβ3 receptor activation. Disclosures:No relevant conflicts of interest to declare.

Talin1 Promotes Prostate Cancer Invasion and Metastasis via AKT Signaling and Anoikis Resistance

AbstractTalin1 is an integrin regulatory protein that mediates integrin interactions with the extracellular matrix (ECM). This study investigated the significance of talin1 in prostate cancer progression to metastasis in vitro and in vivo. Talin1 overexpression enhanced prostate cancer cell adhesion, migration and invasion by activating survival signals and anoikis resistance. ShRNA-mediated talin1 loss led to a significant suppression of prostate cancer cell migration and transendothelial invasion in vitro and a significant inhibition of prostate cancer metastasis in vivo. Talin1 regulates cell survival signals via phosphorylation of focal adhesion kinase (FAK) and AKT. Targeting AKT activation led to a significant reduction of talin1-mediated prostate cancer cell invasion. Furthermore, talin1 expression was determined by immunostaining in prostate tissue from the TRAMP mouse model and in human prostate cancer specimens. Talin1 levels directly correlated with prostate tumor progression to metastasis in TRAMP mice. Talin1 profiling in human prostate specimens revealed a significantly higher expression of cytoplasmic talin1 in metastatic tissue compared to primary prostate tumors and benign prostate tissue (P&amp;lt;0.0001). This evidence suggests a potential value for talin1 as a marker of prostate cancer metastasis and implies that disrupting talin1 mediated signaling may have therapeutic significance in the treatment of metastatic disease.

Talin2 is induced during striated muscle differentiation and is targeted to stable adhesion complexes in mature muscle

The cytoskeletal protein talin serves as an essential link between integrins and the actin cytoskeleton in several similar, but functionally distinct, adhesion complexes, including focal adhesions, costameres, and intercalated disks. Vertebrates contain two talin genes, TLN1 and TLN2, but the different roles of Talin1 and Talin2 in cell adhesion are unclear. In this report we have analyzed Talin1 and Talin2 in striated muscle. Using isoform-specific antibodies, we found that Talin2 is highly expressed in mature striated muscle. Using mouse C2C12 cells and primary human skeletal muscle myoblasts as models of muscle differentiation, we show that Talin1 is expressed in undifferentiated myoblasts and that Talin2 expression is upregulated during muscle differentiation at both the mRNA and protein levels. We have also identified regulatory sequences that may be responsible for the differential expression of Talin1 and Talin2. Using GFP-tagged Talin1 and Talin2 constructs, we found that GFP-Talin1 targets to focal adhesions while GFP-Talin2 targets to abnormally large adhesions in myoblasts. We also found that ectopic expression of Talin2 in myoblasts, which do not contain appreciable levels of Talin2, dysregulates the actin cytoskeleton. Finally we demonstrate that Talin2, but not Talin1, localizes to costameres and intercalated disks, which are stable adhesions required for the assembly of mature striated muscle. Our results suggest that Talin1 is the primary link between integrins and actin in dynamic focal adhesions in undifferentiated, motile cells, but that Talin2 may serve as the link between integrins and the sarcomeric cytoskeletonin stable adhesion complexes in mature striated muscle.