Decreased expression of p53 is associated with down expression of zyxin in breast cancer.

Background and Aims: Breast cancer (BC) is considered one of the most common malignant tumors leading to death in women, and genetic factors have a crucial role in BC pathogenesis. Zyxin (ZYX) is one of these factors that may be important in p53 level and function. Thus, the present work aimed to investigate the ZYX gene and protein expression in tumor tissue and matched margin tissue and its correlation with the p53 expression. Methods: In a present case-control study, 30 tumors and 30 matched margin tissues were obtained from Iran Tumor Bank/Tehran University of Medical Sciences. Real-time polymerase chain reaction and western blot analysis techniques were applied to evaluate the genes and protein expression, respectively. Results: The data showed that expression of the ZYX gene in tumor tissues significantly decreased (p = 0.0274) compared to matched margin tissues. In contrast, the p53 gene expression in tumor tissues had no significant difference with matched margin tissues. Additionally, we observed that ZYX and p53 genes expression in tumor tissues of estrogen receptor-positive patients had significant elevation than estrogen receptor-negative patients (p < 0.001, p < 0.001, respectively). The data of the western blot analysis technique showed that protein expression of ZYX (p = 0.0024) and P53 protein (p = 0.0218) in tumor tissues was significantly reduced compared to matched margin tissues. Additionally, our analysis showed a direct and significant correlation between the expression of ZYX and p53 proteins (r = 0.7797, p = 0.0126) and expression of ZYX and p53 genes (r = 0.3079, p = 0.0187). Conclusion: Based on our observation, ZYX might have a tumor suppressor role and is associated with p53.

Leveraging biotin-based proximity labeling to identify cellular factors governing early alphaherpesvirus infection.

Neurotropic alphaherpesviruses, including herpes simplex virus type 1 and pseudorabies virus, establish a lifelong presence within the peripheral nervous system of their mammalian hosts. Upon entering cells, two conserved tegument proteins, pUL36 and pUL37, traffic DNA-containing capsids to nuclei. These proteins support long-distance retrograde axonal transport and invasion of the nervous system in vivo. To better understand how pUL36 and pUL37 function, recombinant viral particles carrying BioID2 fused to these proteins were produced to biotinylate cellular proteins in their proximity (<10 nm) during infection. Eighty-six high-confidence host proteins were identified by mass spectrometry and subsequently targeted by CRISPR-Cas9 gene editing to assess their contributions to early infection. Proteins were identified that both supported and antagonized infection in immortalized human epithelial cells. The latter included zyxin, a protein that localizes to focal adhesions and regulates actin cytoskeletal dynamics. Zyxin knockout cells were hyper-permissive to infection and could be rescued with even modest expression of GFP-zyxin. These results provide a resource for studies of the virus-cell interface and identify zyxin as a novel deterrent to alphaherpesvirus infection.IMPORTANCENeuroinvasive alphaherpesviruses are highly prevalent with many members found across mammals [e.g., herpes simplex virus type 1 (HSV-1) in humans and pseudorabies virus in pigs]. HSV-1 causes a range of clinical manifestations from cold sores to blindness and encephalitis. There are no vaccines or curative therapies available for HSV-1. A fundamental feature of these viruses is their establishment of lifelong infection of the nervous system in their respective hosts. This outcome is possible due to a potent neuroinvasive property that is coordinated by two proteins: pUL36 and pUL37. In this study, we explore the cellular protein network in proximity to pUL36 and pUL37 during infection and examine the impact of knocking down the expression of these proteins upon infection.

Force-activated zyxin assemblies coordinate actin nucleation and crosslinking to orchestrate stress fiber repair.

As the cytoskeleton sustains cell and tissue forces, it incurs physical damage that must be repaired to maintain mechanical homeostasis. The LIM-domain protein zyxin detects force-induced ruptures in actin-myosin stress fibers, coordinating downstream repair factors to restore stress fiber integrity through unclear mechanisms. Here, we reconstitute stress fiber repair with purified proteins, uncovering detailed links between zyxin's force-regulated binding interactions and cytoskeletal dynamics. In addition to binding individual tensed actin filaments (F-actin), zyxin's LIM domains form force-dependent assemblies that bridge broken filament fragments. Zyxin assemblies engage repair factors through multi-valent interactions, coordinating nucleation of new F-actin by VASP and its crosslinking into aligned bundles by ɑ-actinin. Through these combined activities, stress fiber repair initiates within the cores of micron-scale damage sites in cells, explaining how these F-actin depleted regions are rapidly restored. Thus, zyxin's force-dependent organization of actin repair machinery inherently operates at the network scale to maintain cytoskeletal integrity.

The role of zyxin in signal transduction and its relationship with diseases.

This review highlighted the pivotal role of zyxin, an essential cell focal adhesions protein, in cellular biology and various diseases. Zyxin can orchestrate the restructuring and dynamic alterations of the cellular cytoskeleton, which is involved in cell proliferation, adhesion, motility, and gene transcription. Aberrant zyxin expression is closely correlated with tumor cell activity and cardiac function in both tumorigenesis and cardiovascular diseases. Moreover, in fibrotic and inflammatory conditions, zyxin can modulate cellular functions and inflammatory responses. Therefore, a comprehensive understanding of zyxin is crucial for deciphering signal transduction networks and disease pathogenesis. Investigating its role in diseases holds promise for novel avenues in early diagnosis and therapeutic strategies. Nevertheless, targeting zyxin as a therapeutic focal point presents challenges in terms of specificity, safety, drug delivery, and resistance. Nonetheless, in-depth studies on zyxin and the application of precision medicine could offer new possibilities for personalized treatment modalities.

Endothelial knockdown of the tumor suppressor, WWOX, increases inflammation in ventilator-induced lung injury.

Chronic cigarette smoke exposure decreases lung expression of WWOX which is known to protect the endothelial barrier during infectious models of acute respiratory distress syndrome (ARDS). Proteomic analysis of WWOX-silenced endothelial cells (ECs) was done using tandem mass tag mass spectrometry (TMT-MS). WWOX-silenced ECs as well as those isolated from endothelial cell Wwox knockout (EC Wwox KO) mice were subjected to cyclic stretch (18% elongation, 0.5 Hz, 4 h). Cellular lysates and media supernatant were harvested for assays of cellular signaling, protein expression, and cytokine release. These were repeated with dual silencing of WWOX and zyxin. Control and EC Wwox KO mice were subjected to high tidal volume ventilation. Bronchoalveolar lavage fluid and mouse lung tissue were harvested for cellular signaling, cytokine secretion, and histological assays. TMT-MS revealed upregulation of zyxin expression during WWOX knockdown which predicted a heightened inflammatory response to mechanical stretch. WWOX-silenced ECs and ECs isolated from EC Wwox mice displayed significantly increased cyclic stretch-mediated secretion of various cytokines (IL-6, KC/IL-8, IL-1ß, and MCP-1) relative to controls. This was associated with increased ERK and JNK phosphorylation but decreased p38 mitogen-activated kinases (MAPK) phosphorylation. EC Wwox KO mice subjected to VILI sustained a greater degree of injury than corresponding controls. Silencing of zyxin during WWOX knockdown abrogated stretch-induced increases in IL-8 secretion but not in IL-6. Loss of WWOX function in ECs is associated with a heightened inflammatory response during mechanical stretch that is associated with increased MAPK phosphorylation and appears, in part, to be dependent on the upregulation of zyxin.NEW & NOTEWORTHY Prior tobacco smoke exposure is associated with an increased risk of acute respiratory distress syndrome (ARDS) during critical illness. Our laboratory is investigating one of the gene expression changes that occurs in the lung following smoke exposure: WWOX downregulation. Here we describe changes in protein expression associated with WWOX knockdown and its influence on ventilator-induced ARDS in a mouse model.

Machine learning interpretable models of cell mechanics from protein images.

Cellular form and function emerge from complex mechanochemical systems within the cytoplasm. Currently, no systematic strategy exists to infer large-scale physical properties of a cell from its molecular components. This is an obstacle to understanding processes such as cell adhesion and migration. Here, we develop a data-driven modeling pipeline to learn the mechanical behavior of adherent cells. We first train neural networks to predict cellular forces from images of cytoskeletal proteins. Strikingly, experimental images of a single focal adhesion (FA) protein, such as zyxin, are sufficient to predict forces and can generalize to unseen biological regimes. Using this observation, we develop two approaches-one constrained by physics and the other agnostic-to construct data-driven continuum models of cellular forces. Both reveal how cellular forces are encoded by two distinct length scales. Beyond adherent cell mechanics, our work serves as a case study for integrating neural networks into predictive models for cell biology.

Upregulated Cytoskeletal Proteins Promote Pathological Angiogenesis in Moyamoya Disease.

BACKGROUND: Moyamoya disease (MMD) is a rare progressive vascular disease that leads to intracranial internal carotid artery stenosis and eventual occlusion. However, its pathogenesis remains unclear. The purpose of this study is to explore the role of abnormally expressed proteins in the pathogenesis of MMD. METHODS: Data-independent acquisition mass spectrometry identifies the differentially expressed proteins in MMD serum by detecting the serum from 60 patients with MMD and 20 health controls. The differentially expressed proteins were validated using enzyme linked immunosorbent assays. Immunofluorescence for superficial temporal artery and middle cerebral artery specimens was used to explore the morphological changes of vascular wall in MMD. In vitro experiments were used to explore the changes and mechanisms of differentially expressed proteins on endothelial cells. RESULTS: Proteomic analysis showed that a total of 14 726 peptides and 1555 proteins were quantified by mass spectrometry data. FLNA (filamin A) and ZYX (zyxin) proteins were significantly higher in MMD serum compared with those in health controls (Log2FC >2.9 and >2.8, respectively). Immunofluorescence revealed an intimal hyperplasia in superficial temporal artery and middle cerebral artery specimens of MMD. FLNA and ZYX proteins increased the proportion of endothelial cells in S phase and promoted their proliferation, angiogenesis, and cytoskeleton enlargement. Mechanistic studies revealed that AKT (serine/threonine kinase)/GSK-3ß (glycogen synthase kinase 3ß)/ß-catenin signaling pathway plays a major role in these FLNA- and ZYX-induced changes in endothelial cells. CONCLUSIONS: This study provides proteomic data on a large sample size of MMD. The differential expression of FLNA and ZYX in patient with MMD and following in vitro experiments suggest that these upregulated proteins are related to the pathology of cerebrovascular intimal hyperplasia in MMD and are involved in MMD pathogenesis, with diagnostic and therapeutic ramifications.

Zyxin inhibits the epithelial-mesenchymal transition process in gastric cancer by upregulating SIRT1.

Tumor development relies on the stemness of cancer stem cells, which is regulated by environmental cues. Previous studies have shown that zyxin can inhibit the expression of genes for embryonic stem cell status. In the present study, the expression levels of zyxin protein in cancer tissues and adjacent noncancerous tissues from 73 gastric cancer patients with different clinical stages were analyzed by Western blot. We showed that the relative expression levels of zyxin in gastric cancer tissues (cancer tissues/adjacent tissues) were significantly downregulated in advanced clinical stages. Overexpression of zyxin inhibited the stemness and epithelial-mesenchymal transition (EMT) processes in gastric cancer cells. Zyxin also inhibited the proliferation, migration, and invasion but increased the sensitivity of cancer cells to drugs. Overexpression of zyxin in MKN45 cells inhibited tumor growth in nude mice. We show that the interactions between zyxin and SIRT1 led to the upregulation of SIRT1, reduced acetylation levels of histone H3 K9 and K23, decreased transcription levels of SNAI 1/2, and inhibition of the EMT process. This study demonstrated that zyxin negatively regulates the progression of gastric cancer by inhibiting the stemness of cancer stem cells and EMT. Our findings shed new light on the pathogenesis of gastric cancer.

Zyxin promotes hepatocellular carcinoma progression via the activation of AKT/mTOR signaling pathway.

Hepatocellular carcinoma (HCC) is a common malignancy that is driven by multiple genes and pathways. The aim of this study was to investigate the role and specific mechanism of the actin-interacting protein zyxin (ZYX) in HCC. We found that the expression of ZYX was significantly higher in HCC tissues compared to that in normal liver tissues. In addition, overexpression of ZYX in hepatoma cell lines (PLC/PRF/5, HCCLM3) enhanced their proliferation, migration and invasion, whereas ZYX knockdown had the opposite effects (SK HEP-1, Huh-7). Furthermore, the change in the expression levels of ZYX also altered that of proteins related to cell cycle, migration and invasion. Similar results were obtained with xenograft models. The AKT/mTOR signaling pathway is one of the key mediators of cancer development. While ZYX overexpression upregulated the levels of phosphorylated AKT/mTOR proteins, its knockdown had the opposite effect. In addition, the AKT inhibitor MK2206 neutralized the pro-oncogenic effects of ZYX on the HCC cells, whereas the AKT activator SC79 restored the proliferation, migration and invasion of HCC cells with ZYX knockdown. Taken together, ZYX promotes the malignant progression of HCC by activating AKT/mTOR signaling pathway, and is a potential therapeutic target in HCC.

Enhancement of Zyxin Promotes Skin Fibrosis by Regulating FAK/PI3K/AKT and TGF-ß Signaling Pathways via Integrins.

Skin fibrosis is a common pathological manifestation in systemic sclerosis (SSc), keloid, and localized scleroderma (LS) characterized by fibroblast activation and excessive extracellular matrix (ECM) deposition. However, few effective drugs are available to treat skin fibrosis due to its unclear mechanisms. In our study, we reanalyzed skin RNA-sequencing data of Caucasian, African, and Hispanic SSc patients from the Gene Expression Omnibus (GEO) database. We found that the focal adhesion pathway was up-regulated and Zyxin appeared to be the primary focal adhesion protein involved in skin fibrosis, and we further verified its expression in Chinese skin tissues of several fibrotic diseases, including SSc, keloid, and LS. Moreover, we found Zyxin inhibition could significantly alleviate skin fibrosis using Zyxin knock-down and knock-out mice, nude mouse model and skin explants of human keloid. Double immunofluorescence staining showed that Zyxin was highly expressed in fibroblasts. Further analysis revealed pro-fibrotic gene expression and collagen production increased in Zyxin over-expressed fibroblasts, and decreased in Zyxin interfered SSc fibroblasts. In addition, transcriptome and cell culture analyses revealed Zyxin inhibition could effectively attenuate skin fibrosis by regulating the FAK/PI3K/AKT and TGF-ß signaling pathways via integrins. These results suggest Zyxin appears a potential new therapeutic target for skin fibrosis.

Discoidin domain receptor-2 enhances secondary alveolar septation in mice by activating integrins and modifying focal adhesions.

The extracellular matrix (ECM) of the pulmonary parenchyma must maintain the structural relationships among resident cells during the constant distortion imposed by respiration. This dictates that both the ECM and cells adapt to changes in shape, while retaining their attachment. Membrane-associated integrins and discoidin domain receptors (DDR) bind collagen and transmit signals to the cellular cytoskeleton. Although the contributions of DDR2 to collagen deposition and remodeling during osseous development are evident, it is unclear how DDR2 contributes to lung development. Using mice (smallie, Slie/Slie, DDR2Δ) bearing a spontaneous inactivating deletion within the DDR2 coding region, we observed a decrease in gas-exchange surface area and enlargement of alveolar ducts. Compared with fibroblasts isolated from littermate controls, DDR2Δ fibroblasts, spread more slowly, developed fewer lamellipodia, and were less responsive to the rigidity of neighboring collagen fibers. Activated ß1-integrin (CD29) was reduced in focal adhesions (FA) of DDR2Δ fibroblasts, less phospho-zyxin localized to and fewer FA developed over ventral actin stress fibers, and the adhesions had a lower aspect ratio compared with controls. However, DDR2 deletion did not reduce cellular displacement of the ECM. Our findings indicate that DDR2, in concert with collagen-binding ß1-integrins, regulates the timing and location of focal adhesion formation and how lung fibroblasts respond to ECM rigidity. Reduced rigidity sensing and mechano-responsiveness may contribute to the distortion of alveolar ducts, where the fiber cable-network is enriched and tensile forces are concentrated. Strategies targeting DDR2 could help guide fibroblasts to locations where tensile forces organize parenchymal repair.

A Photopolymerized Hydrogel System with Dual Stiffness Gradients Reveals Distinct Actomyosin-Based Mechano-Responses in Fibroblast Durotaxis.

Durotaxis, migration of cells directed by a stiffness gradient, is critical in development and disease. To distinguish durotaxis-specific migration mechanisms from those on uniform substrate stiffnesses, we engineered an all-in-one photopolymerized hydrogel system containing areas of stiffness gradients with dual slopes (steep and shallow), adjacent to uniform stiffness (soft and stiff) regions. While fibroblasts rely on nonmuscle myosin II (NMII) activity and the LIM-domain protein Zyxin, ROCK and the Arp2/3 complex are surprisingly dispensable for durotaxis on either stiffness gradient. Additionally, loss of either actin-elongator Formin-like 3 (FMNL3) or actin-bundler fascin has little impact on durotactic response on stiffness gradients. However, lack of Arp2/3 activity results in a filopodia-based durotactic migration that is equally as efficient as that of lamellipodia-based durotactic migration. Importantly, we uncover essential and specific roles for FMNL3 and fascin in the formation and asymmetric distribution of filopodia during filopodia-based durotaxis response to the stiffness gradients. Together, our tunable all-in-one hydrogel system serves to identify both conserved as well as distinct molecular mechanisms that underlie mechano-responses of cells experiencing altered slopes of stiffness gradients.

Quantifying Strain-Sensing Protein Recruitment During Stress Fiber Repair.

A family of proteins have been identified that recognize damaged, strained actin filaments in stress fibers. These proteins are often referred to as strain- or force-sensing and trigger downstream signaling mechanisms that can facilitate repair at these strain sites. Here we describe a method using high-resolution microscopy to screen and quantify the mechanosensitive recruitment of proteins to these stress fiber strain sites. Strain sites are induced using spatially controlled illumination of UV light to locally damage actin stress fibers. Recruitment of potential strain-sensing proteins can then either be compared to (Blanchoin, Physiol Rev 94, 235-263, 2014) a known control (e.g., zyxin-GFP) or (Hoffman, Mol Biol Cell 23, 1846-1859, 2012) the pre-damaged stress fiber protein distribution. With this method, strain-sensing proteins and their dynamic association with stress fiber strain sites can be reproducibly measured and compared.

Cytoskeletal protein Zyxin in embryonic development: from controlling cell movements and pluripotency to regulating embryonic patterning.

The Lim-domain protein Zyxin was initially identified as a minor actin cytoskeleton protein that regulates the assembly and repair of actin filaments. At the same time, additional functions revealed for Zyxin in recent decades indicate that this protein can also play an important role in regulating gene expression and cell differentiation. In this review, we analysed the data in the literature pointing to Zyxin as one of the possible molecular hubs linking morphogenetic cell movements with gene expression, stem cell status regulation and pattern formation during the most complex processes in organism life, embryogenesis.

TES-1/Tes and ZYX-1/Zyxin protect junctional actin networks under tension during epidermal morphogenesis in the C. elegans embryo.

LIM-domain-containing repeat (LCR) proteins are recruited to strained actin filaments within stress fibers in cultured cells,1,2,3 but their roles at cell-cell junctions in living organisms have not been extensively studied. Here, we show that the Caenorhabditis elegans LCR proteins TES-1/Tes and ZYX-1/Zyxin are recruited to apical junctions during embryonic elongation when junctions are under tension. In genetic backgrounds in which embryonic elongation fails, junctional recruitment is severely compromised. The two proteins display complementary patterns of expression: TES-1 is expressed in lateral (seam) epidermal cells, whereas ZYX-1 is expressed in dorsal and ventral epidermal cells. tes-1 and zyx-1 mutant embryos display junctional F-actin defects. The loss of either protein strongly enhances morphogenetic defects in hypomorphic mutant backgrounds for cadherin/catenin complex (CCC) components. The LCR regions of TES-1 and ZYX-1 are recruited to stress fiber strain sites (SFSSs) in cultured vertebrate cells. Together, these data establish TES-1 and ZYX-1 as components of a multicellular, tension-sensitive system that stabilizes the junctional actin cytoskeleton during embryonic morphogenesis.

Expression of Zyxin in Non-Small Cell Lung Cancer-A Preliminary Study.

BACKGROUND: The potential involvement of zyxin (ZYX) in carcinogenesis has been investigated in many cancer types. However, there are a limited number of studies on the role of ZYX in the progression of non-small cell lung cancer (NSCLC). Since lung cancer is one of the most frequently diagnosed carcinomas, the aim of our study was to determine the localization and expression levels of ZYX in NSCLC and to correlate the results with the clinicopathological data. MATERIALS AND METHODS: The expression of ZYX was assessed in NSCLC cases and in cell lines representing this tumor type. Levels of ZYX were determined in the clinical material using immunohistochemistry (IHC) and Western Blot. Real-time PCR was used to assess ZYX mRNA levels. The expression of ZYX was also checked in NSCLC cell lines using real-time PCR, Western Blot, and immunofluorescence/immunocytochemistry. RESULTS: The results showed lower levels of ZYX in NSCLC cells compared with control tissues. This trend was observed at the protein and mRNA levels. The assays on the NSCLC model also demonstrated lower levels of ZYX in cancer cells compared with control cells. CONCLUSIONS: The decreased expression of ZYX in NSCLC may indicate a suppressor role of this protein in NSCLC.

Functional Remodeling of the Contractile Smooth Muscle Cell Cortex, a Provocative Concept, Supported by Direct Visualization of Cortical Remodeling.

Considerable controversy has surrounded the functional anatomy of the cytoskeleton of the contractile vascular smooth muscle cell. Recent studies have suggested a dynamic nature of the cortical cytoskeleton of these cells, but direct proof has been lacking. Here, we review past studies in this area suggesting a plasticity of smooth muscle cells. We also present images testing these suggestions by using the technique of immunoelectron microscopy of metal replicas to directly visualize the cortical actin cytoskeleton of the contractile smooth muscle cell along with interactions by representative cytoskeletal binding proteins. We find the cortical cytoskeletal matrix to be a branched, interconnected network of linear actin bundles. Here, the focal adhesion proteins talin and zyxin were localized with nanometer accuracy. Talin is reported in past studies to span the integrin-cytoplasm distance in fibroblasts and zyxin is known to be an adaptor protein between alpha-actinin and VASP. In response to activation of signal transduction with the alpha-agonist phenylephrine, we found that no movement of talin was detectable but that the zyxin-zyxin spacing was statistically significantly decreased in the smooth muscle cells examined. Contractile smooth muscle is often assumed to have a fixed cytoskeletal structure. Thus, the results included here are important in that they directly support the concept at the electron microscopic level that the focal adhesion of the contractile smooth muscle cell has a dynamic nature and that the protein-protein interfaces showing plasticity are protein-specific.

miR-16-5p Is a Novel Mediator of Venous Smooth Muscle Phenotypic Switching.

Vein graft failure after coronary artery bypass grafting (CABG) is primarily caused by intimal hyperplasia, which results from the phenotypic switching of venous smooth muscle cells (SMCs). This study investigates the role and underlying mechanism of miR-16-5p in the phenotypic switching of venous SMCs. In rats, neointimal thickness and area increased over time within 28 days after CABG, as did the time-dependent miR-16-5p downregulation and SMC phenotypic switching. Platelet-derived growth factor-BB-induced miR-16-5p downregulation in HSVSMCs was accompanied by and substantially linked with alterations in phenotypic switching indicators. Furthermore, miR-16-5p overexpression increased SMCs differentiation marker expression while suppressing HSVSMCs proliferation and migration and drastically inhibiting neointimal development in vein grafts. The miR-16-5p inhibited zyxin expression, which was necessary for HSVSMCs phenotypic switching. The miR-16-5p/zyxin axis is a novel, potentially therapeutic target for preventing and treating venous graft intimal hyperplasia.

The Cytoskeletal Protein Zyxin Inhibits Retinoic Acid Signaling by Destabilizing the Maternal mRNA of the RXRγ Nuclear Receptor.

Zyxin is an LIM-domain-containing protein that regulates the assembly of F-actin filaments in cell contacts. Additionally, as a result of mechanical stress, Zyxin can enter nuclei and regulate gene expression. Previously, we found that Zyxin could affect mRNA stability of the maternally derived stemness factors of Pou5f3 family in Xenopus laevis embryos through binding to Y-box factor1. In the present work, we demonstrate that Zyxin can also affect mRNA stability of the maternally derived retinoid receptor Rxrγ through the same mechanism. Moreover, we confirmed the functional link between Zyxin and Rxrγ-dependent gene expression. As a result, Zyxin appears to play an essential role in the regulation of the retinoic acid signal pathway during early embryonic development. Besides, our research indicates that the mechanism based on the mRNA destabilization by Zyxin may take part in the control of the expression of a fairly wide range of maternal genes.