Identification of key genes involved in collagen hydrogel-induced chondrogenic differentiation of mesenchymal stem cells through transcriptome analysis: the role of m6A modification.

Collagen hydrogel has been shown promise as an inducer for chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs), contributing to the repair of cartilage defects. However, the precise molecular mechanism underlying this phenomenon remains poorly elucidated. Here, we induced chondrogenic differentiation of BMSCs using collagen hydrogel and identified 4451 differentially expressed genes (DEGs) through transcriptomic sequencing. Our analysis revealed that DEGs were enriched in the focal adhesion pathway, with a notable decrease in expression levels in the collagen hydrogel group compared to the control group. Protein-protein interaction network analysis suggested that actinin alpha 1 (ACTN1) and actinin alpha 4 (ACTN4), two proteins also involved in cytoskeletal recombination, may be crucial in collagen hydrogel-induced chondrogenic differentiation of BMSCs. Additionally, we found that N6-methyladenosine RNA methylation (m6A) modification was involved in collagen hydrogel-mediated chondrogenic differentiation, with fat mass and obesity-associated protein (FTO) implicated in regulating the expression of ACTN1 and ACTN4. These findings suggest that collagen hydrogel might regulate focal adhesion and actin cytoskeletal signaling pathways through down-regulation of ACTN1 and ACTN4 mRNA via FTO-mediated m6A modification, ultimately driving chondrogenic differentiation of BMSCs. In conclusion, our study provides valuable insights into the molecular mechanisms of collagen hydrogel-induced chondrogenic differentiation of BMSCs, which may aid in developing more effective strategies for cartilage regeneration.

Actin Cytoskeleton Remodeling Accompanied by Redistribution of Adhesion Proteins Drives Migration of Cells in Different EMT States.

Epithelial-mesenchymal transition (EMT) is a process during which epithelial cells lose epithelial characteristics and gain mesenchymal features. Here, we used several cell models to study migratory activity and redistribution of cell-cell adhesion proteins in cells in different EMT states: EGF-induced EMT of epithelial IAR-20 cells; IAR-6-1 cells with a hybrid epithelial-mesenchymal phenotype; and their more mesenchymal derivatives, IAR-6-1-DNE cells lacking adherens junctions. In migrating cells, the cell-cell adhesion protein α-catenin accumulated at the leading edges along with ArpC2/p34 and α-actinin. Suppression of α-catenin shifted cell morphology from fibroblast-like to discoid and attenuated cell migration. Expression of exogenous α-catenin in MDA-MB-468 cells devoid of α-catenin drastically increased their migratory capabilities. The Y654 phosphorylated form of ß-catenin was detected at integrin adhesion complexes (IACs). Co-immunoprecipitation studies indicated that α-catenin and pY654-ß-catenin were associated with IAC proteins: vinculin, zyxin, and α-actinin. Taken together, these data suggest that in cells undergoing EMT, catenins not participating in assembly of adherens junctions may affect cell migration.

AGR2-mediated unconventional secretion of 14-3-3ε and α-actinin-4, responsive to ER stress and autophagy, drives chemotaxis in canine mammary tumor cells.

BACKGROUND: Canine mammary tumors (CMTs) in intact female dogs provide a natural model for investigating metastatic human cancers. Our prior research identified elevated expression of Anterior Gradient 2 (AGR2), a protein disulfide isomerase (PDI) primarily found in the endoplasmic reticulum (ER), in CMT tissues, highly associated with CMT progression. We further demonstrated that increased AGR2 expression actively influences the extracellular microenvironment, promoting chemotaxis in CMT cells. Unraveling the underlying mechanisms is crucial for assessing the potential of therapeutically targeting AGR2 as a strategy to inhibit a pro-metastatic microenvironment and impede tumor metastasis. METHODS: To identify the AGR2-modulated secretome, we employed proteomics analysis of the conditioned media (CM) from two CMT cell lines ectopically expressing AGR2, compared with corresponding vector-expressing controls. AGR2-regulated release of 14-3-3ε (gene: YWHAE) and α-actinin 4 (gene: ACTN4) was validated through ectopic expression, knockdown, and knockout of the AGR2 gene in CMT cells. Extracellular vesicles derived from CMT cells were isolated using either differential ultracentrifugation or size exclusion chromatography. The roles of 14-3-3ε and α-actinin 4 in the chemotaxis driven by the AGR2-modulated CM were investigated through gene knockdown, antibody-mediated interference, and recombinant protein supplement. Furthermore, the clinical relevance of the release of 14-3-3ε and α-actinin 4 was assessed using CMT tissue-immersed saline and sera from CMT-afflicted dogs. RESULTS: Proteomics analysis of the AGR2-modulated secretome revealed increased abundance in 14-3-3ε and α-actinin 4. Ectopic expression of AGR2 significantly increased the release of 14-3-3ε and α-actinin 4 in the CM. Conversely, knockdown or knockout of AGR2 expression remarkably reduced their release. Silencing 14-3-3ε or α-actinin 4 expression diminished the chemotaxis driven by AGR2-modulated CM. Furthermore, AGR2 controls the release of 14-3-3ε and α-actinin 4 primarily via non-vesicular routes, responding to the endoplasmic reticulum (ER) stress and autophagy activation. Knockout of AGR2 resulted in increased α-actinin 4 accumulation and impaired 14-3-3ε translocation in autophagosomes. Depletion of extracellular 14-3-3ε or α-actinin 4 reduced the chemotaxis driven by AGR2-modulated CM, whereas supplement with recombinant 14-3-3ε in the CM enhanced the CM-driven chemotaxis. Notably, elevated levels of 14-3-3ε or α-actinin 4 were observed in CMT tissue-immersed saline compared with paired non-tumor samples and in the sera of CMT dogs compared with healthy dogs. CONCLUSION: This study elucidates AGR2's pivotal role in orchestrating unconventional secretion of 14-3-3ε and α-actinin 4 from CMT cells, thereby contributing to paracrine-mediated chemotaxis. The insight into the intricate interplay between AGR2-involved ER stress, autophagy, and unconventional secretion provides a foundation for refining strategies aimed at impeding metastasis in both canine mammary tumors and potentially human cancers.

KLF5 regulates actin remodeling to enhance the metastasis of nasopharyngeal carcinoma.

Transcription factors (TFs) engage in various cellular essential processes including differentiation, growth and migration. However, the master TF involved in distant metastasis of nasopharyngeal carcinoma (NPC) remains largely unclear. Here we show that KLF5 regulates actin remodeling to enhance NPC metastasis. We analyzed the msVIPER algorithm-generated transcriptional regulatory networks and identified KLF5 as a master TF of metastatic NPC linked to poor clinical outcomes. KLF5 regulates actin remodeling and lamellipodia formation to promote the metastasis of NPC cells in vitro and in vivo. Mechanistically, KLF5 preferentially occupies distal enhancer regions of ACTN4 to activate its transcription, whereby decoding the informative DNA sequences. ACTN4, extensively localized within actin cytoskeleton, facilitates dense and branched actin networks and lamellipodia formation at the cell leading edge, empowering cells to migrate faster. Collectively, our findings reveal that KLF5 controls robust transcription program of ACTN4 to modulate actin remodeling and augment cell motility which enhances NPC metastasis, and provide new potential biomarkers and therapeutic interventions for NPC.

Actinin-4 controls survival signaling in B cells by limiting the lateral mobility of B-cell antigen receptors.

The structure and dynamics of F-actin networks in the cortical area of B cells control the signal efficiency of B-cell antigen receptors (BCRs). Although antigen-induced signaling has been studied extensively, the role of cortical F-actin in antigen-independent tonic BCR signaling is less well understood. Because these signals are essential for the survival of B cells and are consequently exploited by several B-cell lymphomas, we assessed how the cortical F-actin structure influences tonic BCR signal transduction. We employed genetic variants of a primary cell-like B-cell line that can be rendered quiescent to show that cross-linking of actin filaments by α-actinin-4 (ACTN4), but not ACTN1, is required to preserve the dense architecture of F-actin in the cortical area of B cells. The reduced cortical F-actin density in the absence of ACTN4 resulted in increased lateral BCR diffusion. Surprisingly, this was associated with reduced tonic activation of BCR-proximal effector proteins, extracellular signal-regulated kinase, and pro-survival pathways. Accordingly, ACTN4-deficient B-cell lines and primary human B cells exhibit augmented apoptosis. Hence, our findings reveal that cortical F-actin architecture regulates antigen-independent tonic BCR survival signals in human B cells.

Cytosolic actin isoforms form networks with different rheological properties that indicate specific biological function.

The implications of the existence of different actins expressed in epithelial cells for network mechanics and dynamics is investigated by microrheology and confocal imaging. γ-actin predominately found in the apical cortex forms stiffer networks compared to ß-actin, which is preferentially organized in stress fibers. We attribute this to selective interactions with Mg2+-ions interconnecting the filaments' N-termini. Bundling propensity of the isoforms is different in the presence of Mg2+-ions, while crosslinkers such as α-actinin, fascin, and heavy meromyosin alter the mechanical response independent of the isoform. In the presence of myosin, ß-actin networks show a large number of small contraction foci, while γ-actin displays larger but fewer foci indicative of a stronger interaction with myosin motors. We infer that subtle changes in the amino acid sequence of actin isoforms lead to alterations of the mechanical properties on the network level with potential implications for specific biological functions.

ACTN1 promotes HNSCC tumorigenesis and cisplatin resistance by enhancing MYH9-dependent degradation of GSK-3ß and integrin ß1-mediated phosphorylation of FAK.

BACKGROUND: Head and neck squamous cell carcinoma (HNSCC) is one of the most common malignant tumors globally. Understanding the molecular basis of tumor progression and drug resistance can offer innovative strategies to enhance clinical outcomes for HNSCC patients. METHODS: The cytoskeletal remodeling genes associated with cisplatin resistance were screened using a PCR array. The role of alpha-actinin 1 (ACTN1) in modulating cisplatin resistance and tumorigenesis in HNSCC was evaluated both in vitro and in vivo. Co-immunoprecipitation (Co-IP), IP-mass spectrometry (MS), western blotting, dual-luciferase assay, and bioinformatics analysis were performed to elucidate the underlying mechanisms involved. RESULTS: Our study identifies ACTN1 as a crucial contributor to cisplatin resistance and tumorigenesis in HNSCC, as evidenced across cellular, animal, and patient-derived xenograft models. From a clinical perspective, overexpression of ACTN1 significantly correlates with a suboptimal response to neoadjuvant chemotherapy and reduced overall survival in HNSCC patients. Mechanistically, ACTN1 predominantly activates ß-catenin-mediated signaling by promoting the interaction between myosin heavy chain 9 (MYH9) and GSK-3ß, leading to the ubiquitin-dependent degradation of GSK-3ß. ACTN1 also interacts with integrin ß1, subsequently activating the FAK/PI3K/AKT pathway, providing an additional avenue for the activation of ß-catenin signaling. Our study also unveils that the ß-catenin/c-Myc axis transcriptionally regulates ACTN1, thereby creating a positive feedback loop promoting HNSCC tumorigenesis and drug resistance. CONCLUSIONS: These insights underscore the novel mechanisms that highlight ACTN1's pivotal role in driving HNSCC progression and resistance to chemotherapy, suggesting ACTN1 as a promising therapeutic target in HNSCC management.

Cardiomyocyte external mechanical unloading activates modifications of α-actinin differently from sarcomere-originated unloading.

Loss of myocardial mass in a neonatal rat cardiomyocyte culture is studied to determine whether there is a distinguishable cellular response based on the origin of mechano-signals. The approach herein compares the sarcomeric assembly and disassembly processes in heart cells by imposing mechano-signals at the interface with the extracellular matrix (extrinsic) and at the level of the myofilaments (intrinsic). Experiments compared the effects of imposed internal (inside/out) and external (outside/in) loading and unloading on modifications in neonatal rat cardiomyocytes. Unloading of the cellular substrate by myosin inhibition (1 µm mavacamten), or cessation of cyclic strain (1 Hz, 10% strain) after preconditioning, led to significant disassembly of sarcomeric α-actinin by 6 h. In myosin inhibition, this was accompanied by redistribution of intracellular poly-ubiquitin K48 to the cellular periphery relative to the poly-ubiquitin K48 reservoir at the I-band. Moreover, loading and unloading of the cellular substrate led to a three-fold increase in post-translational modifications (PTMs) when compared to the myosin-specific activation or inhibition. Specifically, phosphorylation increased with loading while ubiquitination increased with unloading, which may involve extracellular signal-regulated kinase 1/2 and focal adhesion kinase activation. The identified PTMs, including ubiquitination, acetylation, and phosphorylation, are proposed to modify internal domains in α-actinin to increase its propensity to bind F-actin. These results demonstrate a link between mechanical feedback and sarcomere protein homeostasis via PTMs of α-actinin that exemplify how cardiomyocytes exhibit differential responses to the origin of force. The implications of sarcomere regulation governed by PTMs of α-actinin are discussed with respect to cardiac atrophy and heart failure.

Induction of filopodia formation by α-Actinin-2 via RelA with a feedforward activation loop promoting overt bone marrow metastasis of gastric cancer.

BACKGROUND: Bone marrow metastasis (BMM) is underestimated in gastric cancer (GC). GC with BMM frequently complicate critical hematological abnormalities like diffused intravascular coagulation and microangiopathic hemolytic anemia, which constitute a highly aggressive GC (HAGC) subtype. HAGC present a very poor prognosis with peculiar clinical and pathological features when compared with not otherwise specified advanced GC (NAGC). But the molecular mechanisms underlying BMM from GC remain rudimentary. METHODS: The transcriptomic difference between HAGC and NAGC were analyzed. Genes that were specifically upregulated in HAGC were identified, and their effect on cell migration and invasion was studied. The function of ACTN2 gene were confirmed by GC cell lines, bone-metastatic animal model and patients' tissues. Furthermore, the molecular mechanism of ACTN2 derived-BMM was explored by multiple immunofluorescence staining, western blot, chromatin immunoprecipitation, and luciferase reporter assays. RESULTS: We elucidated the key mechanisms of BMM depending on the transcriptomic difference between HAGC and NAGC. Five genes specifically upregulated in HAGC were assessed their effect on cell migration and invasion. The ACTN2 gene encoding protein α-Actinin-2 was detected enhanced the metastatic capability and induced BMM of GC cells in mouse models. Mechanically, α-Actinin-2 was involved in filopodia formation where it promoted the Actin filament cross-linking by replacing α-Actinin-1 to form α-Actinin-2:α-Actinin-4 complexes in GC cells. Moreover, NF-κB subunit RelA and α-Actinin-2 formed heterotrimers in the nuclei of GC cells. As a direct target of RelA:α-Actinin-2 heterotrimers, the ACTN2 gene was a positive auto-regulatory loop for α-Actinin-2 expression. CONCLUSIONS: We demonstrated a link between filopodia, BMM and ACTN2 activation, where a feedforward activation loop between ACTN2 and RelA is established via actin in response to distant metastasis. Given the novel filopodia formation function and the new mechanism of BMM in GC, we propose ACTN2 as a druggable molecular vulnerability that may provide potential therapeutic benefit against BMM of GC.

Myosin and [Formula: see text]-actinin regulation of stress fiber contractility under tensile stress.

Stress fibers are actomyosin bundles that regulate cellular mechanosensation and force transduction. Interacting with the extracellular matrix through focal adhesion complexes, stress fibers are highly dynamic structures regulated by myosin motors and crosslinking proteins. Under external mechanical stimuli such as tensile forces, the stress fiber remodels its architecture to adapt to external cues, displaying properties of viscoelastic materials. How the structural remodeling of stress fibers is related to the generation of contractile force is not well understood. In this work, we simulate mechanochemical dynamics and force generation of stress fibers using the molecular simulation platform MEDYAN. We model stress fiber as two connecting bipolar bundles attached at the ends to focal adhesion complexes. The simulated stress fibers generate contractile force that is regulated by myosin motors and [Formula: see text]-actinin crosslinkers. We find that stress fibers enhance contractility by reducing the distance between actin filaments to increase crosslinker binding, and this structural remodeling ability depends on the crosslinker turnover rate. Under tensile pulling force, the stress fiber shows an instantaneous increase of the contractile forces followed by a slow relaxation into a new steady state. While the new steady state contractility after pulling depends only on the overlap between actin bundles, the short-term contractility enhancement is sensitive to the tensile pulling distance. We further show that this mechanical response is also sensitive to the crosslinker turnover rate. Our results provide new insights into the stress fiber mechanics that have significant implications for understanding cellular adaptation to mechanical signaling.

Proteins Secreted by Lung Cancer Cells Induce the Onset of Proteinuria via Focal Adhesion Kinase Signaling in Mice.

Paraneoplastic nephrotic syndrome (PNS) is a complication seen in cancer patients. Ultrastructural examination shows the accumulation of proteins and the presence of foot process (FP) effacement in the glomeruli of PNS patients. Previously, we reported that orthotopic xenografts of Lewis lung carcinoma 1 in C57BL/6 mice caused them to develop lung cancer with albuminuria. This implies that these mice can be used as a model of human disease and suggests that Lewis lung carcinoma 1 cell-secreted proteins (LCSePs) contain nephrotoxic molecules and cause inflammation in renal cells. As podocyte effacement was present in glomeruli in this model, such podocyte injury may be attributable to either soluble LCSeP or LCSeP deposits triggering pathological progression. LCSePs in conditioned media was concentrated for nephrotoxicity testing. Integrin-focal adhesion kinase (FAK) signaling and inflammatory responses were evaluated in podocytes either exposed to soluble LCSePs or seeded onto substrates with immobilized LCSePs. FAK phosphorylation and interleukin-6 expression were higher in podocytes attached to LCSePs substrates than in those exposed to soluble LCSePs. Notably, LCSeP-based haptotaxis gave rise to altered signaling in podocytes. When podocytes were stimulated by immobilized LCSePs, FAK accumulated at focal adhesions, synaptopodin dissociated from F-actin, and disrupting the interactions between synaptopodin and α-actinin was observed. When FAK was inhibited by PF-573228 in immobilized LCSePs, the association between synaptopodin and α-actinin was observed in the podocytes. The association of synaptopodin and α-actinin with F-actin allowed FP stretching, establishing a functional glomerular filtration barrier. Therefore, in this mouse model of lung cancer, FAK signaling prompts podocyte FP effacement and proteinuria, indicative of PNS.

Shenqi granule upregulates CD2AP and α-actinin4 and activates autophagy through regulation of mTOR/ULK1 pathway in MPC5 cells.

ETHNOPHARMACOLOGICAL RELEVANCE: The incidence of membranous nephropathy (MN) continues to rise globally. Shenqi granule (SQ), composed of thirteen Chinese medicinal herbs, has clinical efficacy in the treatment of MN and has been used in China for decades. However, the mechanism behind this effect remains unclear. AIM OF THE STUDY: In this study, we documented the effects of SQ on cultured mouse podocytes (MPC5) cytoskeletal proteins (CD2AP, α-actinin4) and autophagic activity, and identified the mechanism underlying the ameliorating effects of SQ on MN. MATERIALS AND METHODS: The main components of SQ was analysed using High-performance liquid chromatography (HPLC). We induced MPC5 cells with puromycin aminonucleoside (PAN) as a model of MN-like disease. Cyclosporine A (CsA) was used as a positive control drug. MPC5 cells viability was analysed using CCK-8 assays to select the PAN dose and SQ dose. CD2AP and α-actinin4 mRNA expression was examined by RT-PCR, CD2AP and α-actinin4 protein expression as well as autophagic activity (LC3, Beclin1) was examined by Western blot in MPC5 cells, and the mechanism of action of SQ granule was assessed by Western blot to detect the protein expression at the phosphorylation level of PI3K/AKT/mTOR pathway. RESULTS: In PAN-induced MPC5 cells, mRNA and protein expression of α-actinin-4 and CD2AP were significantly reduced, and SQ granule was able to alleviate this manifestation. In contrast to the inhibition of LC3 and Beclin1 expression in the PAN model, SQ granule was able to activate cellular autophagic activity. In addition to this, our study revealed that PAN could activate the mTOR/ULK1 pathway, resulting in a significant increase in p-mTOR and p-ULK1 protein expression, while the SQ group was able to significantly inhibit the phosphorylation level of this pathway. CONCLUSIONS: SQ granule attenuated PAN-induced MPC5 cell damage similar to MN. The mechanism may be to upregulate the expression of α-actinin-4 and CD2AP and activate autophagy activity, which may be achieved by inhibiting the phosphorylation level of mTOR/ULK1.

Chitinase-3 like-protein-1 promotes glioma progression via the NF-κB signaling pathway and tumor microenvironment reprogramming.

Background: Chitinase-3-like protein 1 (CHI3L1) is overexpressed in various types of tumors, especially in glioma, and contributes to tumor progression. However, the definite role of CHI3L1 and involved pathway in glioma progression are not completely understood. Methods: CHI3L1 expression in human gliomas and its association with patient survival was determined using enzyme-linked immunosorbent assay, western blot, immunohistochemistry, and public databases. Single-cell RNA-seq was used to characterize the landscape of tumor and myeloid cells. Human proteome microarray assay was applied to identify the binding partners of CHI3L1. Protein-protein interactions were analyzed by co-immunoprecipitation and cellular co-localization. The roles of CHI3L1 in glioma proliferation and invasion were investigated in tumor cell lines by gain- and loss- of function, as well as in vivo animal experiments. Results: CHI3L1 was up-regulated in all disease stages of glioma, which was closely related with tumor survival, growth, and invasion. CHI3L1 was primarily expressed in glioma cells, followed by neutrophils. Moreover, glioma cells with high expression of CHI3L1 were significantly enriched in NF-κB pathway. Pseudo-time trajectory analysis revealed a gradual transition from CHI3L1low to CHI3L1high glioma cells, along with the NF-κB pathway gradually reversed from inhibition to activation. Intriguingly, CHI3L1 binds to actinin alpha 4 (ACTN4) and NFKB1, and enhances the NF-κB signaling pathway by promoting the NF-κB subunit nuclear translocation in glioma cells. Further, CHI3L1 were released into the tumor microenvironment (TME) and interacted with CD44 expressed on tumor-associated macrophages to activate AKT pathway, thereby contributing to M2 macrophage polarization. In addition, CHI3L1 positively correlated to the expression of immune checkpoints, such as CD274 (PD-L1) and HAVCR2 (LAG3), which then remodeled the TME to an immunosuppressive phenotype. Conclusion: Our research revealed that CHI3L1 facilitated NF-κB pathway activation within glioma cells and reprogramed the TME, thereby serving as a promising therapeutic target for glioma.

The role of Actopaxin in tumor metastasis.

Actopaxin is a newly discovered focal adhesions (FAs) protein, actin-binding protein and pseudopodia-enriched molecule. It can not only bind to a variety of FAs proteins (such as Paxillin, ILK and PINCH) and non-FAs proteins (such as TESK1, CdGAP, ß2-adaptin, G3BP2, ADAR1 and CD29), but also participates in multiple signaling pathways. Thus, it plays a crucial role in regulating important processes of tumor metastasis, including matrix degradation, migration, and invasion, etc. This review covers the latest progress in the structure and function of Actopaxin, its interaction with other proteins as well as its involvement in regulating tumor development and metastasis. Additionally, the current limitations for Actopaxin related studies and the possible research directions on it in the future are also discussed. It is hoped that this review can assist relevant researchers to obtain a deep understanding of the role that Actopaxin plays in tumor progression, and also enlighten further research and development of therapeutic approaches for the treatment of tumor metastasis.

Parvin: A hub of intracellular signalling pathways regulating cellular behaviour and disease progression.

α-actinin superfamily houses the family of parvins, comprising α, ß and γ isoforms in the vertebrates and a single orthologue in the invertebrates. Parvin as an adaptor protein is a member of the ternary IPP-complex including Integrin Linked Kinase (ILK) and particularly-interesting-Cys-His-rich protein (PINCH). Each of the complex proteins showed a conserved lineage and was principally used by the evolutionarily primitive integrin-adhesome machinery to regulate cellular behaviour and signalling pathways. Parvin facilitated integrin mediated integration of the extracellular matrix with cytoskeletal framework culminating in regulation of cellular adhesion and spreading, cytoskeleton reorganisation and cell survival. Studies have established role of parvin in pregnancy, lactation, matrix degradation, blood vessel formation and in several diseases such as cancer, NAFLD and cardiac diseases etc. This review narrates the history of parvin discovery, its elaborate gene structure and conservation across phyla including cellular expression, localisation and interacting partners in vertebrates as well as invertebrates. The review further discusses how parvin acts as an epicentre of signalling pathways, its associated mutants and diseased conditions.

Complementary Nck1/2 Signaling in Podocytes Controls α Actinin-4-Mediated Actin Organization, Adhesion, and Basement Membrane Composition.

BACKGROUND: Maintenance of the kidney filtration barrier requires coordinated interactions between podocytes and the underlying glomerular basement membrane (GBM). GBM ligands bind podocyte integrins, which triggers actin-based signaling events critical for adhesion. Nck1/2 adaptors have emerged as essential regulators of podocyte cytoskeletal dynamics. However, the precise signaling mechanisms mediated by Nck1/2 adaptors in podocytes remain to be fully elucidated. METHODS: We generated podocytes deficient in Nck1 and Nck2 and used transcriptomic approaches to profile expression differences. Proteomic techniques identified specific binding partners for Nck1 and Nck2 in podocytes. We used cultured podocytes and mice deficient in Nck1 and/or Nck2, along with podocyte injury models, to comprehensively verify our findings. RESULTS: Compound loss of Nck1/2 altered expression of genes involved in actin binding, cell adhesion, and extracellular matrix composition. Accordingly, Nck1/2-deficient podocytes showed defects in actin organization and cell adhesion in vitro, with podocyte detachment and altered GBM morphology present in vivo. We identified distinct interactomes for Nck1 and Nck2 and uncovered a mechanism by which Nck1 and Nck2 cooperate to regulate actin bundling at focal adhesions via α actinin-4. Furthermore, loss of Nck1 or Nck2 resulted in increased matrix deposition in vivo, with more prominent defects in Nck2-deficient mice, consistent with enhanced susceptibility to podocyte injury. CONCLUSION: These findings reveal distinct, yet complementary, roles for Nck proteins in regulating podocyte adhesion, controlling GBM composition, and sustaining filtration barrier integrity.

Hsa_circ_0097922 promotes tamoxifen resistance and cell malignant behaviour of breast cancer cells by regulating ACTN4 expression via miR-876-3p.

An increasing number of findings have verified the critical roles of circular RNAs (circRNAs) in human cancers, and chemotherapy resistance is a poor prognostic factor for breast cancer (BC). This study is designed to explore the function of hsa_circ_0097922 in the tamoxifen resistance of breast cancer. Hsa_circ_0097922, microRNA-876-3p (miR-876-3p), and alpha-actinin 4 (ACTN4) level were detected by real-time quantitative polymerase chain reaction (RT-qPCR). Cell survival, proliferation, apoptosis, migration and invasion were detected by Cell Counting Kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and flow cytometry, wound healing and Transwell assays. Protein levels of proliferating cell nuclear antigen (PCNA), B-cell lymphoma-2 (Bcl-2), cleaved caspase 3, matrix metalloproteinase 9 (MMP9), and ACTN4 were determined using western blot assay. Using bioinformatics software, the binding between miR-876-3p and hsa_circ_0097922 or ACTN4 was predicted, followed by confirmation by RNA immunoprecipitation (RIP) and RNA pull-down assays. A xenograft tumour model in vivo analysed the biological role of hsa_circ_0097922 on BC tumour growth and drug resistance. Hsa_circ_0097922 and ACTN4 were increased, and miR-876-3p was decreased in tamoxifen resistance BC cells. Moreover, hsa_circ_0097922 knockdown can block BC cell malignant behaviour and tamoxifen resistance in vitro. Mechanically, hsa_circ_0097922 acted as a sponge of miR-876-3p to regulate ACTN4 expression. Hsa_circ_0097922 silencing increased the drug sensitivity of BC in vivo. Hsa_circ_0097922 might regulate BC cell malignant behaviour and tamoxifen resistance partly by regulating the miR-876-3p/ACTN4 axis, hinting at a promising therapeutic target for the BC treatment.

RNF38 suppress growth and metastasis via ubiquitination of ACTN4 in nasopharyngeal carcinoma.

BACKGROUND: Accumulated evidence suggests that RING finger proteins (RNFs) are involved in the carcinogenesis of cancers. However, RNF38, a member of the RNF protein family, has not been studied in nasopharyngeal carcinoma (NPC). METHODS: RNF38 expression was analyzed by RT-PCR, Western blotting and Immunohistochemistry. Biological functions of RNF38 were evaluated by cell growth, colony formation, apoptosis, migration and invasion assays in vitro. Xenograft growth and lung metastasis models were conducted to investigate the effect of RNF38 in vivo. Liquid chromatography coupled with tandem mass spectrometry, co-immunoprecipitation, and CHX assay were implemented to detect the interaction among RNF38 and ACTN4. RESULTS: RNF38 was significantly downregulated in NPC cells and tissues. Immunohistochemistry implied that loss of RNF38 was an independent prognostic factor for poor outcomes of NPC patients. Gain- and loss-of-function experiments showed that RNF38 inhibited proliferation and metastasis in NPC in vitro and in vivo. Upregulation of RNF38 promoted apoptosis of NPC cells to etoposide but not cisplatin. ACTN4 was upregulated in NPC and negatively correlated with RNF38. Mechanistic investigations suggested that RNF38 inactivates the NF-𝛋B and ERK1/2 signaling pathways by inducing ubiquitination and degradation of ACTN4. RNF38 suppress the development of NPC by interacting with ACTN4. CONCLUSIONS: RNF38 plays a potential cancer suppressor gene role in NPC tumorigenesis and is a prognostic biomarker in NPC.

H3K27ac-activated EGFR-AS1 promotes cell growth in cervical cancer through ACTN4-mediated WNT pathway.

BACKGROUND: Recently, extensive studies unveiled that lncRNAs exert critical function in the development and progression of cervical cancer (CC). EGFR-AS1 is a novel lncRNA which has not been well-explored in CC. AIMS: Our study aimed to research the function and molecular mechanism of EGFR-AS1 in CC cells. qRT-PCR analysis was performed to detect gene expression. Colony formation, EdU, flow cytometry, TUNEL, western blot and transwell assays were performed to assess the effect of EGFR-AS1 on CC cell growth. The regulatory mechanism of EGFR-AS1 was dug out through mechanism experiments. RESULTS: EGFR-AS1 was notably overexpressed in CC cell lines. Loss-of-functional experiments revealed that EGFR-AS1 promoted CC cell proliferation, migration and invasion, and suppressed cell apoptosis. Mechanistically, up-regulation of EGFR-AS1 was attributed to the activation of H3K27 acetylation (H3K27ac). Further, EGFR-AS1 was revealed to function as miR-2355-5p sponge. Additionally, miR-2355-5p was down-regulated in CC cells and ACTN4 was identified as a target gene of miR-2355-5p. Ultimately, overexpressed ACTN4 could reserve the suppressive role of EGFR-AS1 silencing in CC cell growth. Last but not least, EGFR-AS1 facilitated CC cell growth via ACTN4-mediated WNT pathway. CONCLUSIONS: H3K27ac-activated EGFR-AS1 sponged miR-2355-5p and promoted CC cell growth through ACTN4-mediated WNT pathway.