The TGF-β signaling pathway is involved in numerous cellular processes, and its deregulation may result in cancer development. One of the key processes in tumor progression and metastasis is epithelial to mesenchymal transition (EMT), in which TGF-β signaling plays important roles. Recently, AGR2 was identified as a crucial component of the cellular machinery responsible for maintaining the epithelial phenotype, thereby interfering with the induction of mesenchymal phenotype cells by TGF-β effects in cancer. Here, we performed transcriptomic profiling of A549 lung cancer cells with CRISPR-Cas9 mediated AGR2 knockout with and without TGF-β treatment. We identified significant changes in transcripts associated with focal adhesion and eicosanoid production, in particular arachidonic acid metabolism. Changes in transcripts associated with the focal adhesion pathway were validated by RT-qPCR of COL4A1, COL4A2, FLNA, VAV3, VEGFA, and VINC mRNAs. In addition, immunofluorescence showed the formation of stress fibers and vinculin foci in cells without AGR2 and in response to TGF-β treatment, with synergistic effects observed. These findings imply that both AGR2 downregulation and TGF-β have a role in focal adhesion formation and cancer cell migration and invasion. Transcripts associated with arachidonic acid metabolism were downregulated after both AGR2 knockout and TGF-β treatment and were validated by RT-qPCR of GPX2, PTGS2, and PLA2G4A. Since PGE2 is a product of arachidonic acid metabolism, its lowered concentration in media from AGR2-knockout cells was confirmed by ELISA. Together, our results demonstrate that AGR2 downregulation and TGF-β have an essential role in focal adhesion formation; moreover, we have identified AGR2 as an important component of the arachidonic acid metabolic pathway.

• MeSH
• Cyclooxygenase 2 genetics   MeSH
• Epithelial-Mesenchymal Transition * genetics   MeSH
• Arachidonic Acid MeSH
• Cell Line, Tumor MeSH
• Cell Movement genetics   MeSH
• Prostaglandins E MeSH
• Gene Expression Regulation, Neoplastic * MeSH
• Transforming Growth Factor beta genetics   MeSH
• Vinculin genetics   MeSH
• Publication type
• Journal Article MeSH

Diamond-like carbon (DLC) is a biocompatible material that has many potential biomedical applications, including in orthopaedics. DLC layers doped with Cr at atomic percent (at.%) of 0, 0.9, 1.8, 7.3, and 7.7 at.% were evaluated with reference to their osteoinductivity with human bone marrow mesenchymal stromal cells (hMSCs), immune activation potential with RAW 264.7 macrophage-like cells, and their effect on apoptosis in Saos-2 human osteoblast-like cells and neonatal human dermal fibroblasts (NHDFs). At mRNA level, hMSCs on DLC doped with 0.9 and 7.7 at.% of Cr reached higher maximum values of both RUNX2 and alkaline phosphatase. An earlier onset of mRNA production of type I collagen and osteocalcin was also observed on these samples; they also supported the production of both type I collagen and osteocalcin. RAW 264.7 macrophages were screened using a RayBio™ Human Cytokine Array for cytokine production. 10 cytokines were at a concentration more than 2 × as high as the concentration of a positive control, but the values for the DLC samples were only moderately higher than the values on glass. NHDF cells, but not Saos-2 cells, had a higher expression of pro-apoptotic markers Bax and Bim and a lower expression of anti-apoptotic factor BCL-XL in proportion to the Cr content. Increased apoptosis was also proven by annexin V staining. These results show that a Cr-doped DLC layer with a lower Cr content can act as an osteoinductive material with relatively low immunogenicity, but that a higher Cr content can induce cell apoptosis.

• MeSH
• Actins metabolism   MeSH
• Alkaline Phosphatase genetics   metabolism   MeSH
• Apoptosis drug effects   immunology   MeSH
• Cell Adhesion drug effects   MeSH
• Cell Differentiation drug effects   immunology   MeSH
• Cell Line MeSH
• Chromium pharmacology   MeSH
• Cytokines metabolism   MeSH
• Diamond pharmacology   MeSH
• Fibroblasts cytology   drug effects   MeSH
• Collagen Type I genetics   metabolism   MeSH
• Humans MeSH
• Macrophages drug effects   metabolism   MeSH
• Mesenchymal Stem Cells cytology   drug effects   immunology   metabolism   MeSH
• Mice MeSH
• Osteogenesis drug effects   MeSH
• Osteocalcin genetics   metabolism   MeSH
• Cell Count MeSH
• Cell Proliferation drug effects   MeSH
• Core Binding Factor Alpha 1 Subunit genetics   metabolism   MeSH
• Gene Expression Regulation drug effects   MeSH
• RNA metabolism   MeSH
• Cell Shape drug effects   MeSH
• Calcium metabolism   MeSH
• Vinculin metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

During meiosis, homologous chromosomes undergo a dramatic movement in order to correctly align. This is a critical meiotic event but the molecular properties of this 'chromosomal dance' still remainunclear. We identified DEB-1 - an orthologue of mammalian vinculin - as a new component of the mechanistic modules responsible for attaching the chromosomes to the nuclear envelope as apart of the LINC complex. In early meiotic nuclei of C. elegans, DEB-1 is localized to the nuclear periphery and alongside the synaptonemal complex of paired homologues. Upon DEB-1 depletion, chromosomes attached to SUN-1 foci remain highly motile until late pachytene. Although the initiation of homologue pairing started normally, irregularities in the formation of the synaptonemal complex occur, and these results in meiotic defects such as increased number of univalents at diakinesis and high embryonic lethality. Our data identify DEB-1 as a new player regulating chromosome dynamics and pairing during meiotic prophase I.

• MeSH
• Caenorhabditis elegans genetics   MeSH
• Chromosomes genetics   MeSH
• Meiosis genetics   MeSH
• Chromosome Pairing genetics   MeSH
• Vinculin genetics   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Diamond-like carbon (DLC) thin films are promising for use in coating orthopaedic, dental and cardiovascular implants. The problem of DLC layers lies in their weak layer adhesion to metal implants. Chromium is used as a dopant for improving the adhesion of DLC films. Cr-DLC layers were prepared by a hybrid technology, using a combination of pulsed laser deposition (PLD) from a graphite target and magnetron sputtering. Depending on the deposition conditions, the concentration of Cr in the DLC layers moved from zero to 10.0 at.%. The effect of DLC layers with 0.0, 0.9, 1.8, 7.3, 7.7 and 10.0 at.% Cr content on the adhesion and osteogenic differentiation of human osteoblast-like Saos-2 cells was assessed in vitro. The DLC samples that contained 7.7 and 10.0 at.% of Cr supported cell spreading on day 1 after seeding. On day three after seeding, the most apparent vinculin-containing focal adhesion plaques were also found on samples with higher concentrations of chromium. On the other hand, the expression of type I collagen and alkaline phosphatase at the mRNA and protein level was the highest on Cr-DLC samples with a lower concentration of Cr (0-1.8 at.%). We can conclude that higher concentrations of chromium supported cell adhesion; however DLC and DLC doped with a lower concentration of chromium supported osteogenic cell differentiation.

• MeSH
• Alkaline Phosphatase metabolism   MeSH
• Coated Materials, Biocompatible MeSH
• Cell Adhesion * MeSH
• Cell Differentiation * MeSH
• Cell Line MeSH
• Chromium chemistry   MeSH
• Diamond chemistry   MeSH
• Focal Adhesions MeSH
• Collagen Type I metabolism   MeSH
• Metals chemistry   MeSH
• Lasers MeSH
• Humans MeSH
• RNA, Messenger metabolism   MeSH
• Osteoblasts cytology   MeSH
• Osteogenesis MeSH
• Surface Properties MeSH
• Gene Expression Profiling MeSH
• Talin chemistry   MeSH
• Carbon chemistry   MeSH
• Vinculin metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Nanotubes with diameters ranging from 40 to 60nm were prepared by electrochemical oxidation of the Ti-6Al-4V alloy in electrolyte containing ammonium sulphate and ammonium fluoride. The nanotubes were further modified with calcium and phosphate ions or were heat treated. Polished Ti-6Al-4V alloy served as a reference sample. The spreading of human osteoblast-like cells was similar on all nanotube samples but lower than on polished samples. The number of initially adhered cells was higher on non-modified nanotubes, but the final cell number was the highest on Ca-enriched nanotubes and the lowest on heat-treated nanotubes. However, these differences were relatively small and less pronounced than the differences in the concentration of specific molecular markers of cell adhesion and differentiation, estimated by their intensity of immunofluorescence staining. The concentration of vinculin, i.e. a protein of focal adhesion plaques, was the lowest on nanotubes modified with calcium. Collagen I, an early marker of osteogenic cell differentiation, was also the lowest on samples modified with calcium and was highest on polished samples. Alkaline phosphatase, a middle marker of osteogenic differentiation, was observed in lowest concentration on nanotubes modified with phosphorus and the highest on heat-treated samples. Osteocalcin concentrations, a late marker of osteogenic cell differentiation, were similar on all tested samples, although they tended to be the highest on heat-treated samples. Thus, osteogenic differentiation can be modulated by various additional treatments of nanotube coatings on Ti-6Al-4V implants.

• MeSH
• Cell Adhesion drug effects   MeSH
• Cell Differentiation drug effects   MeSH
• Microscopy, Fluorescence MeSH
• Collagen Type I metabolism   MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Nanotubes chemistry   MeSH
• Osteoblasts cytology   metabolism   MeSH
• Osteocalcin metabolism   MeSH
• Titanium chemistry   pharmacology   MeSH
• Calcium chemistry   MeSH
• Vinculin chemistry   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Ti-6Al-4V-based nanotubes were prepared on a Ti-6Al-4V surface by anodic oxidation on 10 V, 20 V, and 30 V samples. The 10 V, 20 V, and 30 V samples and a control smooth Ti-6Al-4V sample were evaluated in terms of their chemical composition, diameter distribution, and cellular response. The surfaces of the 10 V, 20 V, and 30 V samples consisted of nanotubes of a relatively wide range of diameters that increased with the voltage. Saos-2 cells had a similar initial adhesion on all nanotube samples to the control Ti-6Al-4V sample, but it was lower than on glass. On day 3, the highest concentrations of both vinculin and talin measured by enzyme-linked immunosorbent assay and intensity of immunofluorescence staining were on 30 V nanotubes. On the other hand, the highest concentrations of ALP, type I collagen, and osteopontin were found on 10 V and 20 V samples. The final cellular densities on 10 V, 20 V, and 30 V samples were higher than on glass. Therefore, the controlled anodization of Ti-6Al-4V seems to be a useful tool for preparing nanostructured materials with desirable biological properties.

• MeSH
• Actins metabolism   MeSH
• Biomarkers metabolism   MeSH
• Cell Adhesion drug effects   MeSH
• Cell Differentiation drug effects   MeSH
• Fluorescent Antibody Technique MeSH
• Photoelectron Spectroscopy MeSH
• Humans MeSH
• Cell Line, Tumor MeSH
• Nanotubes chemistry   MeSH
• Osteoblasts cytology   drug effects   MeSH
• Osteogenesis drug effects   MeSH
• Surface Properties MeSH
• Cell Proliferation drug effects   MeSH
• Titanium pharmacology   MeSH
• Particle Size * MeSH
• Cell Survival drug effects   MeSH
• Vinculin metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Protein-repulsive surfaces modified with ligands for cell adhesion receptors have been widely developed for controlling the cell adhesion and growth in tissue engineering. However, the question of matrix production and deposition by cells on these surfaces has rarely been addressed. In this study, protein-repulsive polydopamine-poly(ethylene oxide) (PDA-PEO) surfaces were functionalized with an RGD-containing peptide (RGD), with a collagen-derived peptide binding fibronectin (Col), or by a combination of these peptides (RGD + Col, ratio 1:1) in concentrations of 90 fmol/cm(2) and 700 fmol/cm(2) for each peptide type. When seeded with vascular endothelial CPAE cells, the PDA-PEO surfaces proved to be completely non-adhesive for cells. On surfaces with lower peptide concentrations and from days 1 to 3 after seeding, cell adhesion and growth was restored practically only on the RGD-modified surface. However, from days 3 to 7, cell adhesion and growth was improved on surfaces modified with Col and with RGD + Col. At higher peptide concentrations, the cell adhesion and growth was markedly improved on all peptide-modified surfaces in both culture intervals. However, the collagen-derived peptide did not increase the expression of fibronectin in the cells. The deposition of fibronectin on the material surface was generally very low and similar on all peptide-modified surfaces. Nevertheless, the RGD + Col surfaces exhibited the highest cell adhesion stability under a dynamic load, which correlated with the highest expression of talin and vinculin in the cells on these surfaces. A combination of RGD + Col therefore seems to be the most promising for surface modification of biomaterials, e.g. vascular prostheses.

• MeSH
• Adsorption MeSH
• Biomimetics * MeSH
• Cell Adhesion * MeSH
• Gene Expression MeSH
• Fibronectins chemistry   genetics   MeSH
• Indoles chemistry   MeSH
• Cells, Cultured MeSH
• Humans MeSH
• Molecular Sequence Data MeSH
• Oligopeptides chemistry   MeSH
• Polyethylene Glycols chemistry   MeSH
• Polymers chemistry   MeSH
• Surface Properties MeSH
• Amino Acid Sequence MeSH
• Talin genetics   MeSH
• Vinculin genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

Focal adhesions are cellular structures through which both mechanical forces and regulatory signals are transmitted. Two focal adhesion-associated proteins, Crk-associated substrate (CAS) and vinculin, were both independently shown to be crucial for the ability of cells to transmit mechanical forces and to regulate cytoskeletal tension. Here, we identify a novel, direct binding interaction between CAS and vinculin. This interaction is mediated by the CAS SRC homology 3 domain and a proline-rich sequence in the hinge region of vinculin. We show that CAS localization in focal adhesions is partially dependent on vinculin, and that CAS-vinculin coupling is required for stretch-induced activation of CAS at the Y410 phosphorylation site. Moreover, CAS-vinculin binding significantly affects the dynamics of CAS and vinculin within focal adhesions as well as the size of focal adhesions. Finally, disruption of CAS binding to vinculin reduces cell stiffness and traction force generation. Taken together, these findings strongly implicate a crucial role of CAS-vinculin interaction in mechanosensing and focal adhesion dynamics.

• MeSH
• Amino Acid Motifs MeSH
• Biomechanical Phenomena MeSH
• Cell Adhesion MeSH
• Cell Line MeSH
• Fibroblasts cytology   metabolism   MeSH
• Focal Adhesions metabolism   ultrastructure   MeSH
• Focal Adhesion Protein-Tyrosine Kinases metabolism   MeSH
• Phosphorylation MeSH
• Protein Interaction Maps MeSH
• Mice MeSH
• Peptides chemistry   metabolism   MeSH
• src Homology Domains MeSH
• Crk-Associated Substrate Protein analysis   metabolism   MeSH
• Protein Binding MeSH
• Vinculin analysis   metabolism   MeSH
• Animals MeSH
• Check Tag
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

The modification of implant surface situated in the area of peri-implant sulcus has important role in bacterial and cell adhesion. Six different chemically and physically modified titanium discs were prepared: glazed (Tis-MALP), unglazed (Tis-O), unglazed and alkali-etched (Tis-OA), unglazed and coated with ZrN (Tis-OZ), unglazed, sand blasted, and acid etched (Tis-OPAE), and unglazed, sand blasted, acid, and alkali etched (Tis-OPAAE). Analysis of surface topography was determined using scanning electron microscopy and atomic force microscopy (AFM). Biocompatibility of gingival fibroblasts was characterized by the production of tumor necrosis factor alpha, collagen I, matrix metalloproteinase 2 (MMP-2) after 24 and 72 h and expression of α3 β1 integrin and vinculin using enzyme-linked immunosorbent assay (ELISA) or modified ELISA after 6 and 24 h. Microorganism adhesion (five bacterial strains) and biofilm formation was also evaluated. The adhesion of bacteria and gingival fibroblasts was significantly higher on titanium disc Tis-OPAAE and biofilm formation on the same surface for Streptococcus mutans, Streptococcus gordonii, and Streptococcus intermedius. The gingival fibroblasts on Tis-OPAAE disc had also significantly lower production of MMP-2. The collagen production was significantly lower on all surfaces with roughness higher than 0.2 μm. This study confirmed that the titanium disc with the surface roughness 3.39 μm (Tis-OPAAE) supported the adhesion of bacterial strains as well as gingival fibroblasts.

• MeSH
• Bacterial Adhesion drug effects   MeSH
• Biofilms drug effects   growth & development   MeSH
• Biocompatible Materials pharmacology   MeSH
• Cell Adhesion drug effects   MeSH
• Fibroblasts cytology   drug effects   microbiology   MeSH
• Focal Adhesions drug effects   metabolism   MeSH
• Gingiva cytology   MeSH
• Integrin alpha3beta1 metabolism   MeSH
• Collagen Type I biosynthesis   MeSH
• Humans MeSH
• Matrix Metalloproteinase 2 biosynthesis   MeSH
• Microscopy, Atomic Force MeSH
• Microscopy, Electron, Scanning MeSH
• Spectrometry, X-Ray Emission MeSH
• Streptococcus cytology   drug effects   physiology   MeSH
• Materials Testing * MeSH
• Titanium pharmacology   MeSH
• Tumor Necrosis Factor-alpha biosynthesis   MeSH
• Vinculin metabolism   MeSH
• Dental Implants microbiology   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Comparative Study MeSH

Preparation of soft poly(amino acid) hydrogels containing biomimetic cell-adhesive peptides was investigated. Covalently crosslinked gels were formed by radical co-polymerization of methacryloylated macromonomer poly[N(5)-(2-hydroxyethyl)-L-glutamine-stat-L-alanine-stat-methacryloyllysine] with 2-hydroxyethyl methacrylate (HEMA) as minor co-monomer. Hydrogels carrying biomimetic peptides were prepared by using methacryloylated peptides, such as methacryloyl-GGGRGDSG-OH and methacryloyl-GGGYIGSR-OH, as additional monomers in the polymerization mixture. Mechanical stability and swelling in water of the hydrogels obtained for different solid:water and polypeptide:HEMA ratios were evaluated. The microporosity of gels (5-20 microm), dependent on the polyHEMA phase separation in water, was followed by low-vacuum SEM. The effect of biomimetic modification of hydrogels with RGDS and YIGSR peptides on the seeding efficiency of porcine mesenchymal stem cells (MSCs) was studied in vitro. While unmodified hydrogels showed very low cell adhesion, due to their highly hydrophilic nature, the incorporation of adhesive peptides significantly improved the adhesion and viability of seeded cells.

• MeSH
• Actins metabolism   MeSH
• Amino Acids pharmacology   MeSH
• Biocompatible Materials chemistry   pharmacology   MeSH
• Cell Adhesion drug effects   MeSH
• Fluorescence MeSH
• Hydrogels chemistry   pharmacology   MeSH
• Cells, Cultured MeSH
• Mesenchymal Stem Cells cytology   metabolism   drug effects   MeSH
• Polymers chemistry   pharmacology   MeSH
• Porosity drug effects   MeSH
• Surface Properties drug effects   MeSH
• Proteins chemistry   pharmacology   MeSH
• Sus scrofa MeSH
• Tissue Engineering methods   MeSH
• Tubulin metabolism   MeSH
• Cell Shape drug effects   MeSH
• Cell Survival drug effects   MeSH
• Vinculin metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH