Reduction in SNAP-23 Alters Microfilament Organization in Myofibrobastic Hepatic Stellate Cells.

Hepatic stellate cells (HSC) are critical effector cells of liver fibrosis. In the injured liver, HSC differentiate into a myofibrobastic phenotype. A critical feature distinguishing myofibroblastic from quiescent HSC is cytoskeletal reorganization. Soluble NSF attachment receptor (SNARE) proteins are important in trafficking of newly synthesized proteins to the plasma membrane for release into the extracellular environment. The goals of this project were to determine the expression of specific SNARE proteins in myofibroblastic HSC and to test whether their alteration changed the HSC phenotype in vitro and progression of liver fibrosis in vivo. We found that HSC lack the t-SNARE protein, SNAP-25, but express a homologous protein, SNAP-23. Downregulation of SNAP-23 in HSC induced reduction in polymerization and disorganization of the actin cytoskeleton associated with loss of cell movement. In contrast, reduction in SNAP-23 in mice by monogenic deletion delayed but did not prevent progression of liver fibrosis to cirrhosis. Taken together, these findings suggest that SNAP-23 is an important regular of actin dynamics in myofibroblastic HSC, but that the role of SNAP-23 in the progression of liver fibrosis in vivo is unclear.

LIMK/cofilin pathway and Slingshot are implicated in human colorectal cancer progression and chemoresistance.

Cofilin phospho-regulation is important for actin filament turnover and is implicated in cancer. Phosphorylation of cofilin is mediated by LIM kinases (LIMKs) and dephosphorylation by Slingshot phosphatases (SSH). LIMKs and SSH promote cancer cell invasion and metastasis and represent novel anti-cancer targets. However, little is known regarding LIMK/cofilin and SSH in human colorectal cancer (CRC). In this study, we aimed to address their expression and significance in human CRC. We evaluated expression of non-phosphorylated (active) and phosphorylated cofilin, LIMK1, LIMK2, and SSH1 by immunohistochemistry in 143 human CRC samples in relation to clinicopathologic parameters, response of metastatic disease to chemotherapy, and epithelial-mesenchymal transition (EMT) markers ß-catenin, E-cadherin, and ZEB. We show that active cofilin, LIMK1, LIMK2, and SSH1 are overexpressed in human CRC and are associated with tumor progression parameters. SSH1 is an independent predictor of lymph node metastasis by multivariate analysis. LIMK1 and SSH1 expression is also higher in non-responders to chemotherapy, and SSH1 is shown by multivariate analysis to independently predict response of metastatic disease to chemotherapy. Active cofilin, LIMK1, LIMK2, and SSH1 also correlated with the EMT markers examined. In addition, immunofluorescence analysis showed increased expression of active cofilin, LIMK1, LIMK2, and SSH1 in HT29 colon cancer cells resistant to 5-fluorouracil compared to parental HT29 cells. Our results suggest that F-actin regulators LIMK/cofilin pathway and SSH1 are associated with CRC progression and chemoresistance representing promising tumor biomarkers and therapeutic targets in CRC.

Effect of HSP27 and Cofilin in the injury of hypoxia/reoxygenation on hepatocyte membrane F-actin microfilaments.

Hypoxia-reoxygenation (H/R) injury hepatocyte models were established to simulate the ischemia/reperfusion injury of transplanted organ. Through the study of the molecular mechanism of H/R on the F-actin damage of the liver cytomembrane, the mechanism of F-actin damage induced by ischemia and reperfusion was studied from the level of cell and molecule.The hypoxic environment of cells in vitro was simulated by chemical hypoxia agent CoCl2. Liver cells were detected by MTT, H/R group was subdivided into 3 subgroups: H/R 2, 4, and 6 h. Changes of cell shape and the growth state, apoptosis, ultrastructural changes, and the changes in F-actin microfilament content were observed. Heat shock protein 27 (HSP27), Cofilin, and F-actin gene and protein levels were determined by real-time polymerase chain reaction and western blot assay, respectively.Cells showed circular adherence growth under normal circumstances, while the spindle cells and shedding cells were significantly increased in H/R groups. Apoptosis cells in H/R group were increased significantly with the extension of hypoxia time. The number of endoplasmic reticulum was decreased significantly in the H/R group, the mitochondrion hydropic was degenerated and the glycogen was disappeared. The F-actin fibers in the H/R group were disordered, the morphology of the fibers was obviously decreased, and the fluorescence staining decreased obviously (P < .05). The transcription and expression levels of HSP27, Cofilin, and F-actin were significantly lower than those in the control group (P < .05).These results demonstrate that H/R can affect the correct assembly of F-actin microfilaments and weakens the normal cycle of F-actin microfilaments through inhibiting the protein expression and gene transcription of HSP27 and Cofilin in hepatocytes, thereby changing the skeleton of F-actin microfilaments.

Targeted proteomics driven verification of biomarker candidates associated with breast cancer aggressiveness.

Breast cancer is the most common and molecularly relatively well characterized malignant disease in women, however, its progression to metastatic cancer remains lethal for 78% of patients 5years after diagnosis. Novel markers could identify the high risk patients and their verification using quantitative methods is essential to overcome genetic, inter-tumor and intra-tumor variability and translate novel findings into cancer diagnosis and treatment. We recently identified 13 proteins associated with estrogen receptor, tumor grade and lymph node status, the key factors of breast cancer aggressiveness, using untargeted proteomics. Here we verified these findings in the same set of 96 tumors using targeted proteomics based on selected reaction monitoring with mTRAQ labeling (mTRAQ-SRM), transcriptomics and immunohistochemistry and validated in 5 independent sets of 715 patients using transcriptomics. We confirmed: (i) positive association of anterior gradient protein 2 homolog (AGR2) and periostin (POSTN) and negative association of annexin A1 (ANXA1) with estrogen receptor status; (ii) positive association of stathmin (STMN1), cofilin-1 (COF1), plasminogen activator inhibitor 1 RNA-binding protein (PAIRBP1) and negative associations of thrombospondin-2 (TSP2) and POSTN levels with tumor grade; and (iii) positive association of POSTN, alpha-actinin-4 (ACTN4) and STMN1 with lymph node status. This study highlights a panel of gene products that can contribute to breast cancer aggressiveness and metastasis, the understanding of which is important for development of more precise breast cancer treatment.

Functional consequences of the over-expression of TRPC6 channels in HEK cells: impact on the homeostasis of zinc.

The canonical transient receptor potential 6 (TRPC6) protein is a non-selective cation channel able to transport essential trace elements like iron (Fe) and zinc (Zn) through the plasma membrane. Its over-expression in HEK-293 cells causes an intracellular accumulation of Zn, indicating that it could be involved in Zn transport. This finding prompted us to better understand the role played by TRPC6 in Zn homeostasis. Experiments done using the fluorescent probe FluoZin-3 showed that HEK cells possess an intracellular pool of mobilisable Zn present in compartments sensitive to the vesicular proton pump inhibitor Baf-A, which affects endo/lysosomes. TRPC6 over-expression facilitates the basal uptake of Zn and enhances the size of the pool of Zn sensitive to Baf-A. Quantitative RT-PCR experiments showed that TRPC6 over-expression does not affect the mRNA expression of Zn transporters (ZnT-1, ZnT-5, ZnT-6, ZnT-7, ZnT-9, Zip1, Zip6, Zip7, and Zip14); however it up-regulates the mRNA expression of metallothionein-I and -II. This alters the Zn buffering capacities of the cells as illustrated by the experiments done using the Zn ionophore Na pyrithione. In addition, HEK cells over-expressing TRPC6 grow slower than their parental HEK cells. This feature can be mimicked by growing HEK cells in a culture medium supplemented with 5 µM of Zn acetate. Finally, a proteomic analysis revealed that TRPC6 up-regulates the expression of the actin-associated proteins ezrin and cofilin-1, and changes the organisation of the actin cytoskeleton without changing the cellular actin content. Altogether, these data indicate that TRPC6 is participating in the transport of Zn and influences the Zn storage and buffering capacities of the cells.

Arabidopsis Actin-Depolymerizing Factor-4 links pathogen perception, defense activation and transcription to cytoskeletal dynamics.

The primary role of Actin-Depolymerizing Factors (ADFs) is to sever filamentous actin, generating pointed ends, which in turn are incorporated into newly formed filaments, thus supporting stochastic actin dynamics. Arabidopsis ADF4 was recently shown to be required for the activation of resistance in Arabidopsis following infection with the phytopathogenic bacterium Pseudomonas syringae pv. tomato DC3000 (Pst) expressing the effector protein AvrPphB. Herein, we demonstrate that the expression of RPS5, the cognate resistance protein of AvrPphB, was dramatically reduced in the adf4 mutant, suggesting a link between actin cytoskeletal dynamics and the transcriptional regulation of R-protein activation. By examining the PTI (PAMP Triggered Immunity) response in the adf4 mutant when challenged with Pst expressing AvrPphB, we observed a significant reduction in the expression of the PTI-specific target gene FRK1 (Flg22-Induced Receptor Kinase 1). These data are in agreement with recent observations demonstrating a requirement for RPS5 in PTI-signaling in the presence of AvrPphB. Furthermore, MAPK (Mitogen-Activated Protein Kinase)-signaling was significantly reduced in the adf4 mutant, while no such reduction was observed in the rps5-1 point mutation under similar conditions. Isoelectric focusing confirmed phosphorylation of ADF4 at serine-6, and additional in planta analyses of ADF4's role in immune signaling demonstrates that nuclear localization is phosphorylation independent, while localization to the actin cytoskeleton is linked to ADF4 phosphorylation. Taken together, these data suggest a novel role for ADF4 in controlling gene-for-gene resistance activation, as well as MAPK-signaling, via the coordinated regulation of actin cytoskeletal dynamics and R-gene transcription.

Cofilin overexpression affects actin cytoskeleton organization and migration of human colon adenocarcinoma cells.

The dynamic reorganization of actin cytoskeleton is regulated by a large number of actin-binding proteins. Among them, the interaction of ADF/cofilin with monomeric and filamentous actin is very important, since it severs actin filaments. It also positively influences actin treadmilling. The activity of ADF/cofilin is reversibly regulated by phosphorylation and dephosphorylation at Ser-3, with the phosphorylated form (P-cofilin) being inactive. Here, we studied the effects of overexpression of cofilin and two cofilin variants in the human colon adenocarcinoma LS180 cell line. We have generated the LS180 cells expressing three different cofilin variants: WT (wild type), Ser 3 Ala (S3A) (constitutively active) or Ser 3 Asp (S3D) (constitutively inactive cofilin). The cells expressing WT cofilin were characterized by abundant cell spreading and colocalization of cofilin with the submembranous F-actin. Similar effects were observed in cells expressing S3A cofilin. In contrast, LS180 cells expressing S3D cofilin remained longitudinal in morphology and cofilin was equally distributed within the cell body. Furthermore, the migration ability of LS180 cells expressing different cofilin mutants was analyzed. In comparison to control cells, we have noticed a significant, approximately fourfold increase in the migration factor value of cells overexpressing WT type cofilin. The overexpression of S3D cofilin resulted in an almost complete inhibition of cell motility. The estimation of actin pool in the cytosol of LS180 cells expressing S3A cofilin has shown a significantly lower level of total actin in reference to control cells. The opposite effect was observed in LS180 cells overexpressing S3D cofilin. In summary, the results of our experiments indicate that phosphorylation "status" of cofilin is a factor affecting the actin cytoskeleton organization and migration abilities of colon adenocarcinoma LS180 cells.

[Fibrillar actin in the nuclear apparatus of the ciliate Paramecium caudatum].

Due to their nuclear dualism, ciliates provide a good model for studying the role of actin in spatial organization and transcription activity of the nucleus. The actin in the nuclear apparatus of the ciliate Paramecium caudatum was studied using fluorescently labeled phalloiodin and indirect immunocytochemistry. Fibrillar actin was demonstrated in both of the nuclei. Actin was revealed in the chromatin areas, and was often associated with the periphery of the amplified nucleoli in the macronucleus. Redistribution of actin was observed depending on different physiological state of the cells. Stable infection of the macronulear with the intranuclear endobionts Holospora obtuse led to the loss of nuclear actin accompanied by significant nuclear fragility and redistribution of the phosphorylated form of the actin-binding protein cofilin. Spherical bodies resembling karyosphere were found in the macronuclear anlagen.

[Effects of cofilin phosphorylation on the actin cytoskeleton reorganization induced by shear stress].

OBJECTIVE: To explore the effects of cofilin on the actin cytoskeleton reorganization in osteoblasts induced by fluid shear stress. METHODS: Fluid shear stress (1.2 Pa) was applied to osteoblasts for 0 (control group), 15, 30, 45, 60, 120 min in vitro. Cells were stained with fluorescein isothiocyanate (FITC)-phalloidin for fiber-actin, and confocal laser scanning microscope(CLSM) was used to observe the fluorescence of fiber-actin. Western blotting was used to detect the expression of the cofilin and the phospho-cofilin. RESULTS: Actin filaments became organized into stress fibers that were thicker and more abundant than those in non-flowed cells. The fluorescence intensity (38.00 ± 6.88) of fiber-actin after 120 min (42.93 ± 6.41) loading it was 2.8 times as much as that in control group (15.41 ± 3.60, P < 0.05). Additionally, the level of phospho-cofilin protein was dramatically elevated after loading. Fluid shear stress induced an initial decrease of cofilin at 60 min. However, at 120 min cofilin (0.254 ± 0.026) increased to 1.5 times as much as that at 60 min (0.162 ± 0.004). CONCLUSIONS: The results indicate that cofilin phosphorylation mediates fiber-actin reorganization in the osteoblasts induced by fluid shear stress.