Protein Paxillin Research Articles

Clinical Importance of Focal Adhesion Kinase (FAK)-Src and Paxillin Expression in Renal Cell Carcinoma.

The complex focal adhesion kinase (FAK)/Src and paxillin seem to play a key role in the pathogenesis and progression of cancer. The aim of this study is to evaluate the expression of these proteins in renal cell carcinomas (RCCs), considering the immunoreactive score (IRS), the positivity and the intensity, and to find any association with patients' clinical characteristics, histologic type and other pathological features that imply a possible pathophysiological or prognostic role of FAK/Src and paxillin in RCC. Patients with RCC who had undergone partial or radical nephrectomy from January 2009 to September 2010 were eligible for this retrospective cross-sectional study. The immunohistochemical expression of FAK, Src and paxillin proteins in formalin-fixed paraffin-embedded tumour tissue was analysed in association with various clinicopathological features. Out of ninety patients, 58 had clear cell renal carcinoma, 15 had papillary, 11 had chromophobe and six had unclassified RCC. FAK, Src and paxillin were expressed in 55.6%, 32.2% and 18.9% of all cases, respectively.In univariate analysis, FAK positivity and IRS were more likely in patients with papillary and chromophobe histologic type versus clear cell RCC (p<0.005), Src positivity and IRS presented more frequently in stage T3 versus T1 (p<0.005) and paxillin positivity was more likely in patients with stage T3 versus T2 (p=0.021) and grades 3-4 versus grade 2 (p=0.013). Paxillin-IRS was not associated with any clinicopathological features. The same associations were also reproduced in the multifactorial analysis for the FAK and Src positivity and IRS, while it was found that paxillin positivity and IRS were associated with the female gender (p=0.052, p=0.024), and were higher in grades 3-4 versus grade 2 (p=0.022, p=0.020). Our study suggests that RCC shows immunohistochemical expression of FAK, Src and paxillin proteins, and this expression varies in relation to the histologic type, the stage and the stage/grade/gender, respectively. These findings imply a possible involvement of the FAK/Src signalling pathway in the pathogenesis and progression of cancer in RCC, providing future perspectives for targeted therapies with inhibitors.

Evaluation of Paxillin Expression in Odontogenic Cysts and Tumors.

Paxillin (PXN) is one of the proteins involved in cell adhesion. PXN and integrins constitute a key site for the focal adhesion between the cell and extracellular matrix. Several studies have shown that PXN is a factor in tumor formation, progression, invasion, and metastasis. This study evaluated PXN expression in four types of odontogenic lesions with different aggressive behaviors. In this retrospective cross-sectional study, PXN expression was immunohistochemically assessed in 68 paraffin-embedded tissue samples from patients with the confirmed diagnosis of four types of odontogenic lesions, including 14 dentigerous cysts (DC), 20 odontogenic keratocyst (OKC), 16 unicystic ameloblastoma, and 18 solid ameloblastoma. The PXN expression in these samples were scored based on the percentage and intensity of immunoreactivity, and compared among the groups by Chi-square test. The PXN marker was detected in the cytoplasm of tumor cells (unicystic and solid ameloblastoma) and the epithelial layer of cysts (DC and OKC). The intensively stained marker of PXN was observed in 9 cases (64.3%) of the DC, 14 cases (70%) of OKC, 12 cases (75%) of unicystic ameloblastoma, and 13 cases (72.2%) of solid ameloblastoma. However, there was not statistical difference of PXN protein expression between DC and OKC (p Value = 0.51) and unicystic and solid ameloblastoma (p = 0.58), also the same was true for cysts and tumors (p = 0.37). The expression of PXN is not related to the biological behaviors of odontogenic lesions.

Abstract 193: Melanoma cytoskeletal dynamics, membrane remodeling, and motility are regulated by TRIM9

Abstract Cell shape change and motility are essential in metastasis and involve remodeling of the actin cytoskeleton, cell adhesions, and plasma membrane. How these cytoskeletal and membrane remodeling are regulated and coordinated remains largely unknown. We previously identified TRIM9 as a regulator of actin dynamics and exocytosis in developing neurons. Deletion of murine Trim9 impairs neuronal migration, netrin-induced axon turning, and axonal and dendritic branching, and increased exocytosis and filopodial stability. In addition, we have shown TRIM9 alters dynamics of the actin polymerase VASP at filopodia tips via non-degrative mono-ubiquitination. TRIM9 is expressed in other motile cells, but the non-neuronal role of TRIM9 remains unknown. TRIM9 was identified as a possible prognostic biomarker in melanoma and high expression correlates with low patient survival. Melanomas undergo phenotype switching, where three distinct phenotypes exist that are associated with differences in gene expression. The three phenotypes consist of a melanocytic proliferative state, a neural crest-like invasive state, an intermediate state. Single cell RNAseq (scRNASeq) data from patient-derived melanoma indicate TRIM9 is highly expressed in the proliferative melanocytic state, which correlates with poor prognosis. We hypothesize that TRIM9 coordinates actin dynamics, adhesion, and exocytosis in melanoma. We show that TRIM9 protein is enriched in several human melanoma lines. Here we examine the role of TRIM9 in regulation of focal adhesions, exocytosis, migration, and invasion in two of these lines. Genetic loss of TRIM9 increased random migration velocity but reduced directional persistence. Interestingly, we find that TRIM9 plays a role in promoting bleb-like morphology and inhibits the ability to durotax on shallow gradients on soft substrate. Fluorescence recovery after photobleaching (FRAP), Total internal reflection fluorescence microscopy (TIRF), and widefield microscopy revealed that loss of TRIM9 results in increased focal adhesions, cell size, and altered dynamics of focal adhesion proteins VASP, zyxin, and paxillin dynamics. In addition, TRIM9 knockout cells exhibit reduced filopodial length and density and altered filopodial localization of VASP. In-gel zymography indicates that TRIM9 knockout cells also display an increased degradative capacity. Current studies are investigating how loss of TRIM9 alters parameters of cell contractility, lamellipodia, invadopodia, and exocytosis to define the role of TRIM9 in melanoma motility. Together these findings suggest TRIM9 reduces adhesion and migration, increases proliferation and blebbing and may be an important regulator of phenotype switching in melanoma. Citation Format: Kimberly Lukasik, Jordan Brooks, Sam Redinbo, James Bear, Stephanie Gupton. Melanoma cytoskeletal dynamics, membrane remodeling, and motility are regulated by TRIM9 [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 193.

Progerin Inhibits the Proliferation and Migration of Melanoma Cells by Regulating the Expression of Paxillin.

Progerin, the underlying cause of Hutchinson-Gilford Progeria Syndrome (HGPS), has been extensively studied for its impact on normal cells and premature aging patients. However, there is a lack of research on its specific effects on tumor cells. Melanoma is one of the most common malignant tumors with high morbidity and mortality. This study aimed to elucidate the potential therapeutic role of progerin in melanoma. We constructed the melanoma A375 cell line and M14 cell line with stable expression of progerin. The expression of progerin, paxillin, and epithelial-mesenchymal transition (EMT) marker proteins in each cell group was measured using Western blot. The migration, proliferation, and cell cycle of cancer cells were assessed using the transwell assay, wound healing assay, colony formation assay, CCK 8 assay, and flow cytometry. RT-qPCR technology was used to examine the impact of progerin overexpression on microRNA expression. Finally, we transfected paxillin into the progerin overexpression cell group to verify whether progerin regulates the phenotype of tumor cells through paxillin. Our study demonstrated that overexpression of progerin leads to decreased expression of paxillin and inhibits cancer cell migration, proliferation, EMT process and cell cycle progression. Additionally, rescue experiments revealed that the migration, proliferation ability, and EMT marker protein expression in progerin overexpressing cancer cells could be partially restored by transfecting a plasmid containing the paxillin gene. Mechanistic investigations further revealed that progerin achieves this inhibition of paxillin expression by upregulating miR-212. This study reveals that progerin may inhibit the migration and proliferation of melanoma cells through the miR-212/paxillin axis, which provides a new approach for the future treatment of this disease.

Alteration of actin cytoskeletal organisation in fetal akinesia deformation sequence

Fetal akinesia deformation sequence (FADS) represents the severest form of congenital myasthenic syndrome (CMS), a diverse group of inherited disorders characterised by impaired neuromuscular transmission. Most CMS originate from defects in the muscle nicotinic acetylcholine receptor, but the underlying molecular pathogenesis is only poorly understood. Here we show that RNAi-mediated silencing of FADS-related proteins rapsyn and NUP88 in foetal fibroblasts alters organisation of the actin cytoskeleton. We show that fibroblasts from two independent FADS individuals have enhanced and shorter actin stress fibre bundles, alongside with an increased number and size of focal adhesions, with an otherwise normal overall connectivity and integrity of the actin-myosin cytoskeleton network. By proximity ligation assays and bimolecular fluorescence complementation, we show that rapsyn and NUP88 localise nearby adhesion plaques and that they interact with the focal adhesion protein paxillin. Based on these findings we propose that a respective deficiency in rapsyn and NUP88 in FADS alters the regulation of actin dynamics at focal adhesions, and thereby may also plausibly dictate myofibril contraction in skeletal muscle of FADS individuals.

HERPUD1 governs tumor cell mitochondrial function via inositol 1,4,5-trisphosphate receptor-mediated calcium signaling

The intricate relationship between calcium (Ca2+) homeostasis and mitochondrial function is crucial for cellular metabolic adaptation in tumor cells. Ca2+-initiated signaling maintains mitochondrial respiratory capacity and ATP synthesis, influencing critical cellular processes in cancer development. Previous studies by our group have shown that the homocysteine-inducible ER Protein with Ubiquitin-Like Domain 1 (HERPUD1) regulates inositol 1,4,5-trisphosphate receptor (ITPR3) levels and intracellular Ca2+ signals in tumor cells. This study explores the role of HERPUD1 in regulating mitochondrial function and tumor cell migration by controlling ITPR3-dependent Ca2+ signals.We found HERPUD1 levels correlated with mitochondrial function in tumor cells, with HERPUD1 deficiency leading to enhanced mitochondrial activity. HERPUD1 knockdown increased intracellular Ca2+ release and mitochondrial Ca2+ influx, which was prevented using the ITPR3 antagonist xestospongin C or the Ca2+ chelator BAPTA-AM. Furthermore, HERPUD1 expression reduced tumor cell migration by controlling ITPR3-mediated Ca2+ signals. HERPUD1-deficient cells exhibited increased migratory capacity, which was attenuated by treatment with xestospongin C or BAPTA-AM. Additionally, HERPUD1 deficiency led to reactive oxygen species-dependent activation of paxillin and FAK proteins, which are associated with enhanced cell migration.Our findings highlight the pivotal role of HERPUD1 in regulating mitochondrial function and cell migration by controlling intracellular Ca2+ signals mediated by ITPR3. Understanding the interplay between HERPUD1 and mitochondrial Ca2+ regulation provides insights into potential therapeutic targets for cancer treatment and other pathologies involving altered energy metabolism.

Phenotype screens of murine pancreatic cancer identify a Tgf-α-Ccl2-paxillin axis driving human-like neural invasion.

Solid cancers like pancreatic ductal adenocarcinoma (PDAC), a type of pancreatic cancer, frequently exploit nerves for rapid dissemination. This neural invasion (NI) is an independent prognostic factor in PDAC, but insufficiently modeled in genetically engineered mouse models (GEMM) of PDAC. Here, we systematically screened for human-like NI in Europe's largest repository of GEMM of PDAC, comprising 295 different genotypes. This phenotype screen uncovered 2 GEMMs of PDAC with human-like NI, which are both characterized by pancreas-specific overexpression of transforming growth factor α (TGF-α) and conditional depletion of p53. Mechanistically, cancer-cell-derived TGF-α upregulated CCL2 secretion from sensory neurons, which induced hyperphosphorylation of the cytoskeletal protein paxillin via CCR4 on cancer cells. This activated the cancer migration machinery and filopodia formation toward neurons. Disrupting CCR4 or paxillin activity limited NI and dampened tumor size and tumor innervation. In human PDAC, phospho-paxillin and TGF-α-expression constituted strong prognostic factors. Therefore, we believe that the TGF-α-CCL2-CCR4-p-paxillin axis is a clinically actionable target for constraining NI and tumor progression in PDAC.

Estrogen receptors differentially modifies lamellipodial and focal adhesion dynamics in airway smooth muscle cell migration

Sex-steroid signaling, especially estrogen, has a paradoxical impact on regulating airway remodeling. In our previous studies, we demonstrated differential effects of 17β-estradiol (E2) towards estrogen receptors (ERs: α and β) in regulating airway smooth muscle (ASM) cell proliferation and extracellular matrix (ECM) production. However, the role of ERs and their signaling on ASM migration is still unexplored. In this study, we examined how ERα versus ERβ affects the mitogen (Platelet-derived growth factor, PDGF)-induced human ASM cell migration as well as the underlying mechanisms involved. We used Lionheart-FX automated microscopy and transwell assays to measure cell migration and found that activating specific ERs had differential effects on PDGF-induced ASM cell migration. Pharmacological activation of ERβ or shRNA mediated knockdown of ERα and specific activation of ERβ blunted PDGF-induced cell migration. Furthermore, specific ERβ activation showed inhibition of actin polymerization by reducing the F/G-actin ratio. Using Zeiss confocal microscopy coupled with three-dimensional algorithmic ZEN-image analysis showed an ERβ-mediated reduction in PDGF-induced expressions of neural Wiskott-Aldrich syndrome protein (N-WASP) and actin-related proteins-2/3 (Arp2/3) complex, thereby inhibiting actin-branching and lamellipodia. In addition, ERβ activation also reduces the clustering of actin-binding proteins (vinculin and paxillin) at the leading edge of ASM cells. However, cells treated with E2 or ERα agonists do not show significant changes in actin/lamellipodial dynamics. Overall, these findings unveil the significance of ERβ activation in regulating lamellipodial and focal adhesion dynamics to regulate ASM cell migration and could be a novel target to blunt airway remodeling.

Fibrin Coating Contributes to the Retention of the Endothelial Layer in Pulsating Flow

The presence of a modifying coating based on extracellular matrix proteins on the inner surface of vascular prostheses is known to enhance endothelial cell adhesion and prevent detachment under pulsating flow conditions. This coating effectively reduces the risk of thrombosis and plays a critical role in determining implantation outcomes. Although proteins like collagen, fibrin, and fibrinogen are commonly used as coatings to improve cell adhesion, their relative effectiveness remains uncertain. Objective: This study aims to identify the optimal coating, based on extracellular matrix proteins, that preserves prosthesis functionality and maintains endothelial layer integrity under pulsating flow conditions. Methods: Scaffolds and vascular prostheses were fabricated using poly(3-hydroxybutyrate-co-3-hydroxyvalerate) and poly(ε-caprolactonone) through an electrospinning process. These structures were then modified with collagen I, fibronectin, or fibrin. Endothelial colony-forming cells (ECFCs) were seeded onto the protein-modified electrospun samples and cultured under both static and dynamic conditions. After a 3-day incubation period under static conditions, cell viability, metabolic and proliferative activity, as well as adhesive properties, were evaluated. Adhesive properties were assessed by analyzing the area occupied by the focal adhesion protein paxillin. Cell retention was determined by comparing cell density on the inner surface of 4 mm diameter vascular prostheses after a 7-day incubation period, both under pulsating flow conditions and static conditions. Results: Cell metabolic activity, viability, number, proliferation, and the area occupied by the focal adhesion protein paxillin were found to be significantly higher in samples coated with fibrin compared to those coated with collagen I and fibrinogen. The cell density (cells/cm2) of ECFCs on the inner surface of fibrin-coated prostheses showed no significant difference between dynamic and static conditions. In contrast, collagen and fibronectin coatings resulted in approximately half the cell density under pulsating flow conditions compared to static conditions. Conclusion: The fibrin coating demonstrated superior biological activity, adhesive properties, and preservation of the endothelial layer under both static and pulsating flow conditions, as compared to collagen I and fibronectin coatings. Consequently, the utilization of fibrin coating emerges as a promising option for modifying the inner surface of vascular prostheses.

Engineered Biomimetic Fibrillar Fibronectin Matrices Regulate Cell Adhesion Initiation, Migration, and Proliferation via α5β1 Integrin and Syndecan-4 Crosstalk.

Cells regulate adhesion to the fibrillar extracellular matrix (ECM) of which fibronectin is an essential component. However, most studies characterize cell adhesion to globular fibronectin substrates at time scales long after cells polarize and migrate. To overcome this limitation, a simple and scalable method to engineer biomimetic 3D fibrillar fibronectin matrices is introduced and how they are sensed by fibroblasts from the onset of attachment is characterized. Compared to globular fibronectin substrates, fibroblasts accelerate adhesion initiation and strengthening within seconds to fibrillar fibronectin matrices via α5β1 integrin and syndecan-4. This regulation, which additionally accelerates on stiffened fibrillar matrices, involves actin polymerization, actomyosin contraction, and the cytoplasmic proteins paxillin, focal adhesion kinase, and phosphoinositide 3-kinase. Furthermore, this immediate sensing and adhesion of fibroblast to fibrillar fibronectin guides migration speed, persistency, and proliferation range from hours to weeks. The findings highlight that fibrillar fibronectin matrices, compared to widely-used globular fibronectin, trigger short- and long-term cell decisions very differently and urge the use of such matrices to better understand in vivo interactions of cells and ECMs. The engineered fibronectin matrices, which can be printed onto non-biological surfaces without loss of function, open avenues for various cell biological, tissue engineering and medical applications.

Enhanced hydrophilicity of one-step electrosprayed red blood cell-like PLGA microparticles by block polymer PLGA-PEG-PLGA with excellent magnetic-luminescent bifunction and affinity to HUVECs

The incidence of cancer and other diseases continues to increase, posing a serious threat to human health. Drug delivery systems using bionic red blood cell (RBC)-like microparticles (MPs) as drug carriers have been extensively studied, as they are considered ideal carriers due to the unique shape. However, most methods of preparing multifunctional RBC-like MPs are complicated, and the cell affinity and therapeutic effect of MPs prepared using simple electrospray are unsatisfactory. In this context,hydrophilicity-enhanced and magnetic-luminescent bifunctional RBCs-like Janus MPs [Fe3O4@poly(lactic-co-glycolic acid) (PLGA)-poly(ethylene glycol) (PEG)-PLGA/PLGA]//[NaYF4:Eu3+@PLGA-PEG-PLGA/PLGA] were successfully prepared by one-step double-needle electrospray method. The physical and chemical properties, such as morphology, particle size, structure, and thermal stability of the MPs, were studied. The addition of block polymer PLGA-PEG-PLGA was found to enhance the hydrophilicity of the MPs, promote cell spreading and adhesion proteins (focal adhesion kinase and Paxillin) mRNA expressions of human umbilical vein endothelial cells, significantly improving the cell affinity. The results also demonstrated that RBC-like MPs exhibited excellent magnetic and enhanced luminescent properties, making them a promising candidate for various biomedical applications, including drug delivery systems, magnetic targeting, bio-imaging, and fluorescent tracing.

Podocyte protease activated receptor 1 stimulation in mice produces focal segmental glomerulosclerosis mirroring human disease signaling events

About 30% of patients who have a kidney transplant with underlying nephrotic syndrome (NS) experience rapid relapse of disease in their new graft. This is speculated to be due to a host-derived circulating factor acting on podocytes, the target cells in the kidney, leading to focal segmental glomerulosclerosis (FSGS). Our previous work suggests that podocyte membrane protease receptor 1 (PAR-1) is activated by a circulating factor in relapsing FSGS. Here, the role of PAR-1 was studied in human podocytes invitro, and using a mouse model with developmental or inducible expression of podocyte-specific constitutively active PAR-1, and using biopsies from patients with nephrotic syndrome. Invitro podocyte PAR-1 activation caused a pro-migratory phenotype with phosphorylation of the kinase JNK, VASP protein and docking protein Paxillin. This signaling was mirrored in podocytes exposed to patient relapse-derived NS plasma and in patient disease biopsies. Both developmental and inducible activation of transgenic PAR-1 (NPHS2 Cre PAR-1Active+/-) caused early severe nephrotic syndrome, FSGS, kidney failure and, in the developmental model, premature death. We found that the non-selective cation channel protein TRPC6 could be a key modulator of PAR-1 signaling and TRPC6 knockout in our mouse model significantly improved proteinuria and extended lifespan. Thus, our work implicates podocyte PAR-1 activation as a key initiator of human NS circulating factor and that the PAR-1 signaling effects were partly modulated through TRPC6.

Focal adhesion formation of primary human gingival fibroblast on hydrothermally and in-sol-made TiO2 -coated titanium.

Optimal cell adhesion of the gingival fibroblasts to dental implants is important for maintaining good implant integration. The aim of this study was to discover, if the nanoporous TiO2 -coating on titanium alloy substrates is able to increase the cell adhesion of the human gingival fibroblasts (HGF). The study consisted of three differently produced titanium groups: hydrothermally produced TiO2 -coating (HT), novel TiO2 -coating made in sol (SOL), and noncoated control group. Primary HGF cells were initiated from gingival biopsies from patients having a third molar extraction. HGF were cultivated on titanium discs for 2 and 24 h to determine the initial attachment with confocal microscope. The cell spreading and adhesion protein signals were measured. In addition, expression of adhesion proteins vinculin, paxillin, and focal adhesion kinase (FAK) were measured after 3 days of cultivation by using Western Blotting. Higher protein levels of paxillin, vinculin, and FAK were induced on both coated discs compared to noncoated discs. The difference was statistically significant (p < 0.05) concerning expression of paxillin. The cell spreading was significantly larger on SOL discs after 2 and 24 h when comparing to noncoated controls. The confocal microscope analyses revealed significantly higher adhesion protein signals on both HT- and SOL-coated titanium compared to control group. This study showed, that both methods to produce TiO2 -coatings are able to increase HGF adhesion protein expression and cell spreading on titanium surface. Accordingly, the coatings can potentially improve the gingival attachment to titanium implant surfaces.

Programmed death ligand 1 intracellular interactions with STAT3 and focal adhesion protein Paxillin facilitate lymphatic endothelial cell remodeling

Lymphatic endothelial cells (LECs) comprise lymphatic capillaries and vessels that guide immune cells to lymph nodes (LNs) and form the subcapsular sinus and cortical and medullary lymphatic structures of the LN. During an active immune response, the lymphatics remodel to accommodate the influx of immune cells from the tissue, but factors involved in remodeling are unclear. Here, we determined that a TSS motif within the cytoplasmic domain of programmed death ligand 1 (PD-L1), expressed by LECs in the LN, participates in lymphatic remodeling. Mutation of the TSS motif to AAA does not affect surface expression of PD-L1, but instead causes defects in LN cortical and medullary lymphatic organization following immunostimulant, Poly I:C, administration invivo. Supporting this observation, invitro treatment of the LEC cell line, SVEC4-10, with cytokines TNFα and IFNα significantly impeded SVEC4-10 movement in the presence of the TSS-AAA cytoplasmic mutation. The cellular movement defects coincided with reduced F-actin polymerization, consistent with differences previously found in dendritic cells. Here, in addition to loss of actin polymerization, we define STAT3 and Paxillin as important PD-L1 binding partners. STAT3 and Paxillin were previously demonstrated to be important at focal adhesions for cellular motility. We further demonstrate the PD-L1 TSS-AAA motif mutation reduced the amount of pSTAT3 and Paxillin bound to PD-L1 both before and after exposure to TNFα and IFNα. Together, these findings highlight PD-L1 as an important component of a membrane complex that is involved in cellular motility, which leads to defects in lymphatic organization.

β1-Integrin plays a major role in resveratrol-mediated anti-invasion effects in the CRC microenvironment.

Background: Tumor microenvironment (TME) is one of the most important factors in tumor aggressiveness, with an active exchange between tumor and other TME-associated cells that promotes metastasis. The tumor-inhibitory effect of resveratrol on colorectal cancer (CRC) cells has been frequently reported. However, whether resveratrol can specifically suppress TME-induced CRC invasion via β1-integrin receptors has not been fully elucidated yet. Methods: Two CRC cell lines (HCT116, RKO) were cultured in multicellular, pro-inflammatory 3D-alginate TME cultures (containing fibroblasts, T-lymphocytes) to investigate the role of β1-integrin receptors in the anti-invasive and anti-metastatic effect of resveratrol by antisense oligonucleotides (ASO). Results: Our results show that resveratrol dose-dependently suppressed the migration-promoting adhesion adapter protein paxillin and simultaneously enhanced the expression of E-cadherin associated with the phenotype change of CRC cells, and their invasion. Moreover, resveratrol blocked TME-induced phosphorylation and nuclear translocation of p65-NF-κB, which was associated with changes in the expression pattern of epithelial-mesenchymal-transition-related biomarkers (slug, vimentin, E-cadherin), metastasis-related factors (CXCR4, MMP-9, FAK), and apoptosis (caspase-3). Finally, transient transfection of β1-integrin, in contrast to knockdown of NF-κB, abrogated most anti-invasive, anti-metastatic effects as well as downstream signaling of resveratrol, resulting in a concomitant increase in CRC cell invasion, indicating a central role of β1-integrin receptors in the anti-invasive function of resveratrol. Conclusion: These results demonstrate for the first time that silencing β1-integrins may suppress, at least in part the inhibitory effects of resveratrol on invasion and migration of CRC cells, underscoring the crucial homeostatic role of β1-integrin receptors.

Folding and Binding Mechanisms of the SH2 Domain from Crkl.

SH2 domains are structural modules specialized in the recognition and binding of target sequences containing a phosphorylated tyrosine residue. They are mostly incorporated in the 3D structure of scaffolding proteins that represent fundamental regulators of several signaling pathways. Among those, Crkl plays key roles in cell physiology by mediating signals from a wide range of stimuli, and its overexpression is associated with several types of cancers. In myeloid cells expressing the oncogene BCR/ABL, one interactor of Crkl-SH2 is the focal adhesion protein Paxillin, and this interaction is crucial in leukemic transformation. In this work, we analyze both the folding pathway of Crkl-SH2 and its binding reaction with a peptide mimicking Paxillin, under different ionic strength and pH conditions, by using means of fluorescence spectroscopy. From a folding perspective, we demonstrate the presence of an intermediate along the reaction. Moreover, we underline the importance of the electrostatic interactions in the early event of recognition, occurring between the phosphorylated tyrosine of the Paxillin peptide and the charge residues of Crkl-SH2. Finally, we highlight a pivotal role of a highly conserved histidine residue in the stabilization of the binding complex. The experimental results are discussed in light of previous works on other SH2 domains.

Opposite roles of Kindlin orthologs in cell survival and proliferation.

ObjectiveIt is unclear why adhesion‐dependent cells such as epithelium undergo anoikis without anchorage, while adhesion‐independent blood cells thrive in suspension. The adhesive machinery of these cells is similar, with the exception of Kindlin orthologs, Kindlin 2 (K2) and Kindlin 3 (K3). Here we address how Kindlins control cell survival and proliferation in anchorage‐dependent and independent cells.Material and MethodsTo demonstrate the opposite roles of Kindlin's in cell survival we utilized in vivo and in vitro models and K3 and K2 knockdown and knockin cells. We used human lymphocytes from the K3 deficient patients in tumour model, K3 knockout and knockin macrophages and K2 knockout and knockin MEF cells for experiments in under conditions of adhesion and in suspension.ResultsDepletion of K3 promotes cell proliferation and survival of anchorage‐independent cells regardless of cell attachment. In contrast, the absence of K2 in anchorage‐dependent cells accelerates apoptosis and limits proliferation. K3 deficiency promotes human lymphoma growth and survival in vivo. Kindlins' interaction with paxillin, is critical for their differential roles in cell anchorage. While disruption of K2‐paxillin binding leads to increased apoptosis, the lack of K3‐paxillin binding has an opposite effect in adhesion‐independent cells.ConclusionKindlin ortologs and their interaction to cytoskeletal protein paxillin define the mechanisms of anchorage dependence. Our study identifies the key elements of the cell adhesion machinery in cell survival and tumour metastasis, proposing possible targets for tumour treatment.

Impact of Polypyridyl Ru Complexes on Angiogenesis-Contribution to Their Antimetastatic Activity.

The use of polypyridyl Ru complexes to inhibit metastasis is a novel approach, and recent studies have shown promising results. We have reported recently that Ru (II) complexes gathering two 4,7-diphenyl-1,10-phenanthroline (dip) ligands and the one being 2,2′-bipyridine (bpy) or its derivative with a 4-[3-(2-nitro-1H-imidazol-1-yl)propyl (bpy-NitroIm) or 5-(4-{4′-methyl-[2,2′-bipyridine]-4-yl}but-1-yn-1-yl)pyridine-2-carbaldehyde semicarbazone (bpy-SC) moieties can alter the metastatic cascade, among others, by modulating cell adhesion properties. In this work, we show further studies of this group of complexes by evaluating their effect on HMEC-1 endothelial cells. While all the tested complexes significantly inhibited the endothelial cell migration, Ru-bpy additionally interrupted the pseudovessels formation. Functional changes in endothelial cells might arise from the impact of the studied compounds on cell elasticity and expression of proteins (vinculin and paxillin) involved in focal adhesions. Furthermore, molecular studies showed that complexes modulate the expression of cell adhesion molecules, which has been suggested to be one of the factors that mediate the activation of angiogenesis. Based on the performed studies, we can conclude that the investigated polypyridyl Ru (II) complexes can deregulate the functionality of endothelial cells which may lead to the inhibition of angiogenesis.

Epithelial cell attachment and adhesion protein expression on novel in sol TiO2 coated zirconia and titanium alloy surfaces.

An adequate mucosal attachment is important when it comes to preventing peri‐implant inflammation. The aim of this study was to compare epithelial cell adhesion and adhesion protein expression on in sol TiO2‐coated and non‐coated zirconia and titanium alloy surfaces. Fifty‐six zirconia and titanium discs were cut, and half of them were coated with bioactive TiO2‐coating. To study the epithelial cell attachment, human gingival keratinocytes were cultivated on discs for 1, 3, 6, and 24 h. The cell proliferation was detected by cultivating cells for 1, 3, and 7 days. In addition, the levels of adhesion proteins laminin y2, integrin α6, β4, vinculin, and paxillin were detected with Western Blot method. Furthermore, high‐resolution imaging of the actin cytoskeleton and focal adhesion proteins was established. Longer‐term cell culture (1–7 days) revealed higher cell numbers on the coated zirconia and titanium discs compared to non‐coated discs. The difference was statistically significant (p < .05) after 24 h on coated zirconia and after 3 and 7 days on coated titanium discs compared to non‐coated discs. Clear induction in the protein levels of laminin y2 and integrin α6 were detected on both coated samples, meanwhile integrin β4 were clearly induced on coated titanium alloy. The microscope evaluation showed significantly increased cell spreading on the coated discs. According to this study, the in sol induced TiO2‐coating increases keratinocyte attachment and the expression of adhesion proteins on coated zirconia and titanium in vitro. Consequently, the coating has potential to enhance the mucosal attachment on implant surfaces.

Mechanism of integrin activation by talin and its cooperation with kindlin

Talin-induced integrin binding to extracellular matrix ligands (integrin activation) is the key step to trigger many fundamental cellular processes including cell adhesion, cell migration, and spreading. Talin is widely known to use its N-terminal head domain (talin-H) to bind and activate integrin, but how talin-H operates in the context of full-length talin and its surrounding remains unknown. Here we show that while being capable of inducing integrin activation, talin-H alone exhibits unexpectedly low potency versus a constitutively activated full-length talin. We find that the large C-terminal rod domain of talin (talin-R), which otherwise masks the integrin binding site on talin-H in inactive talin, dramatically enhances the talin-H potency by dimerizing activated talin and bridging it to the integrin co-activator kindlin-2 via the adaptor protein paxillin. These data provide crucial insight into the mechanism of talin and its cooperation with kindlin to promote potent integrin activation, cell adhesion, and signaling.