Search

An emerging instigator of endothelial dysfunction in type 2 diabetes (T2D) is the stiffening of the cell. Previous reports suggest that polymerization of filamentous actin (F-actin) is a potential mediator of endothelial stiffening. Actin polymerization is limited by active cofilin, an F-actin-severing protein that can be oxidized, leading to its inactivation and loss of severing capability. Yet, whether these mechanisms are implicated in endothelial stiffening in T2D remains unknown. Herein, we report that endothelial cells exposed to plasma from male and female subjects with T2D, and the aortic endothelium of diabetic male mice ( db/db ), exhibit evidence of increased oxidative stress, F-actin, and stiffness. Furthermore, we show reactive oxygen species, including H 2 O 2 , are increased in the endothelium of mesenteric arteries isolated from db/db male mice, and that exposure of endothelial cells to H 2 O 2 induces F-actin formation. We also demonstrate, in vitro, that cofilin-1 can be oxidized by H 2 O 2 , leading to reduced F-actin severing activity. Finally, we provide evidence that genetic silencing or pharmacological inhibition of LIM kinase 1, an enzyme that phosphorylates and thus inactivates cofilin, reduces F-actin and cell stiffness. In aggregate, this work supports the inactivation of cofilin as a potential novel mechanism underlying endothelial stiffening in T2D. NEW & NOTEWORTHY Cell stiffening is an emerging contributor to endothelial dysfunction, a classic feature of type 2 diabetes (T2D). However, the mechanisms underlying endothelial stiffening remain largely unknown. This work provides evidence that oxidative stress-induced inactivation of cofilin, a key F-actin severing protein, may be implicated in increasing endothelial F-actin and cell stiffness in T2D.

Chronic systemic inflammation (CSI) results in neuroinflammation and neurodegeneration. Cofilin1 is a stress protein that activates microglia and induces neuroinflammation, but its role in CSI at different aging stages remains unidentified. Therefore, the study aims to identify cofilin1 and its upstream regulators LIMK1 and SSH1 after CSI in young-, middle-, and advanced-aged mice. CSI was induced by injecting the male and female mice with a sub-lethal dose of Lipopolysaccharide weekly for six weeks. The results showed that normal male mice did not show cofilin pathway dysregulation, but a significant dysregulation was observed in CSI advanced-aged mice. In females, cofilin1 dysregulation was observed in healthy and CSI advanced-aged mice, while significant cofilin1 dysregulation was observed in middle-aged mice during CSI. Furthermore, cofilin1 pathway dysregulations correlated with Alzheimer's disease (AD) biomarkers in the brain and saliva, astrocyte activation, synaptic degeneration, neurobehavioral impairments, gut-microbiota abnormalities, and circulatory inflammation. These results provide new insights into cofilin1 sex and age-dependent mechanistic differences that might help identify targets for modulating neuroinflammation and early onset of AD., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Objective: This study investigated the potential anticancer properties of Myo-inositol on the DU-145 prostate cancer cell line., Methods: The DU-145 cells have been treated to different doses of Myo-inositol in order to ascertain the half-maximal inhibitory concentration (IC50) using the trypan blue exclusion assay. The impact of Myo-inositol on proteomic profiles was evaluated using 2D gel electrophoresis and liquid chromatography-mass spectrometry (LC-MS)., Results: Myo-inositol significantly reduced DU-145 cell viability with an IC50 of 0.06 mg/ml (p<0.05). Proteomic analysis highlighted marked differences in protein expression between treated and untreated cells, particularly in proteins related to cytoskeletal regulation, apoptosis, and stress response. LC-MS further identified significant alterations in protein profiles, with suppression of proteins like Annexin A2 and Cofilin-1-A in controls, and upregulation of proteins such as Rho GTPase-activating protein, Apoptotic protease-activating factor 1 (APAF1), and TNF receptor-associated factor 2 (TRAF2) in treated samples (p<0.001), indicating modulation of key signaling pathways involved in tumor suppression and oncogenesis., Conclusion: Myo-inositol exhibits anticancer properties in prostate cancer cells by impacting cell viability and altering protein expression. While promising as an adjunctive treatment, further studies are needed to understand its mechanisms and potential in combination therapies for managing CRPC.

Purpose: Despite its recognized aggressive clinical manifestations, invasive micropapillary carcinoma has a controversial prognosis in comparison to invasive ductal carcinoma of the breast. This retrospective study aimed to explore the prognosis and underlying molecular mechanisms of invasive micropapillary carcinoma., Methods: Through the SEER database, we compared patients survival outcomes with invasive micropapillary carcinoma versus invasive ductal carcinoma, and developed a nomogram to predict the overall survival of the former group. We explored gene profiles of invasive micropapillary carcinoma in the GEO database. Hub genes were identified as the top ten genes in the PPI network with the highest degrees of connectivity, and three of them were selected for validation by immunohistochemistry., Results: Invasive micropapillary carcinoma patients had better overall survival and breast cancer-specific survival than invasive ductal carcinoma patients did. Multivariate analysis revealed age, marital status, TN stage, ER status, and chemotherapy as independent prognostic factors for invasive micropapillary carcinoma patients, which were used to construct a nomogram with good performance. A total of 294 DEGs were identified, with ten hub genes, including MMP2, FLNA and CFL1, which were expressed at lower levels in invasive micropapillary carcinoma patients than in invasive ductal carcinoma patients, indicating favorable outcomes., Conclusions: Patients with invasive micropapillary carcinoma generally have a better prognosis than those with invasive ductal carcinoma does, which could be attributed to the lower expression of pro-oncogenic genes in the former group; however, the underlying mechanism needs further investigation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Spermatogenesis is a biological process within the testis that produces haploid spermatozoa for the continuity of species. Sertoli cells are somatic cells in the seminiferous epithelium that orchestrate spermatogenesis. Cyclic reorganization of the Sertoli cell actin cytoskeleton is vital for spermatogenesis, but the underlying mechanism remains largely unclear. Here, we report that the RNA-binding protein PTBP1 controls Sertoli cell actin cytoskeleton reorganization by programming alternative splicing of actin cytoskeleton regulators. This splicing control enables ectoplasmic specializations, the actin-based adhesion junctions, to maintain the blood-testis barrier and support spermatid transport and transformation. Particularly, we show that PTBP1 promotes actin bundle formation by repressing the inclusion of exon 14 of Tnik, a kinase present at the ectoplasmic specialization. Our results thus reveal a novel mechanism wherein Sertoli cell actin cytoskeleton dynamics are controlled post-transcriptionally by utilizing functionally distinct isoforms of actin regulatory proteins, and PTBP1 is a critical regulatory factor in generating such isoforms., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Oxidative stress on cysteine (Cys)-containing proteins has been associated with physiological disorders, as suggested for the human cofilin-1 (CFL-1) protein, in which the oxidized residues are likely implicated in the aggregation process of α-synuclein, leading to severe neuronal injuries. Considering the relevance of the oxidation state of cysteine, quantification of thiols may offer a guide for the development of effective therapies. This work presents, for the very first time, thiol quantification within CFL-1 in solution and on the surface following classic and adapted versions of Ellman's assay. The 1:1 stoichiometric Ellman's reaction occurs between 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB), and the free thiol of the cysteine residue, producing two 2-nitro-5-thiobenzoate (TNB 2- ) ions, one of which is released into the medium. While in solution, the thiol concentration was determined by the absorbance of the released TNB 2- , on the surface, the mass of the attached TNB 2- ion to the protein allowed the quantification by means of the multiparametric surface plasmon resonance (MP-SPR) technique. The SPR angle change after the interaction of DTNB with immobilized CFL-1 gave a surface coverage of 26.5 pmol cm -2 for the TNB 2- ions (Γ TNB2- ). The ratio of this value to the surface coverage of CFL-1, Γ CFL-1 = 6.5 ± 0.6 pmol cm -2 (also determined by MP-SPR), gave 4.1 as expected for this protein, i.e., CFL-1 contains four Cys residues in its native form (reduced state). A control experiment with adsorbed oxidized protein showed no SPR angle change, thus proving the reliability of adapting Ellman's assay to the surface using the MP-SPR technique. The results presented in this work provide evidence of the heterogenization of Ellman's assay, offering a novel perspective for studying thiol-containing species within proteins. This may be particularly useful to ensure further studies on drug-like molecules that can be carried out with validated oxidized or reduced CFL-1 or other analogous systems.

Peroxiredoxin 1 (PRDX1) is an important member of the peroxiredoxin family (PRDX) and is upregulated in a variety of tumors. Previous studies have found that high PRDX1 expression is closely related to the metastasis of oral squamous cell carcinoma (OSCC), but the specific molecular mechanism is elusive. To elucidate the role of PRDX1 in the metastasis process of OSCC, we evaluated the expression of PRDX1 in OSCC clinical specimens and its impact on the prognosis of OSCC patients. Then, the effect of PRDX1 on OSCC metastasis and cytoskeletal reconstruction was explored in vitro and in nude mouse tongue cancer models, and the molecular mechanisms were also investigated. PRDX1 can directly interact with the actin-binding protein Cofilin, inhibiting the phosphorylation of its Ser3 site, accelerating the depolymerization and turnover of actin, promoting OSCC cell movement, and aggravating the invasion and metastasis of OSCC. In clinical samples and mouse tongue cancer models, PRDX1 also increased lymph node metastasis of OSCC and was negatively correlated with the phosphorylation of Cofilin; PRDX1 also reduced the overall survival rate of OSCC patients. In summary, our study identified that PRDX1 may be a potential therapeutic target to inhibit OSCC metastasis., (© 2024 UICC.)

This article focuses on screening the major secreted proteins by the ischemia-challenged cardiac stromal fibroblasts (CF), the assessment of their expression status and functional role in the post-ischemic left ventricle (LV) and in the ischemia-challenged CF culture and to phenotype CF at single cell resolution based on the positivity of the identified mediators. The expression level of CRSP2, HSP27, IL-8, Cofilin-1, and HSP90 in the LV tissues following coronary artery bypass graft (CABG) and myocardial infarction (MI) and CF cells followed the screening profile derived from the MS/MS findings. The histology data unveiled ECM disorganization, inflammation and fibrosis reflecting the ischemic pathology. CRSP2, HSP27, and HSP90 were significantly upregulated in the LV-CABG tissues with a concomitant reduction ion LV-MI whereas Cofilin-1, IL8, Nrf2, and Troponin I were downregulated in LV-CABG and increased in LV-MI. Similar trends were exhibited by ischemic CF. Single cell transcriptomics revealed multiple sub-phenotypes of CF based on their respective upregulation of CRSP2, HSP27, IL-8, Cofilin-1, HSP90, Troponin I and Nrf2 unveiling pathological and pro-healing phenotypes. Further investigations regarding the underlying signaling mechanisms and validation of sub-populations would offer novel translational avenues for the management of cardiac diseases., (© 2024. The Author(s).)

Objective: In this study, we investigated the mechanism of action of LIMK1 in cervical cancer progression., Methods: The biological role of LIMK1 in regulating the growth, invasion, and metastasis of cervical cancer was studied in SiHa, CaSki cells and nude mice tumor models. The role of LIMK1 in the growth of cervical cancer was evaluated by HE staining. The role of LIMK1 in the invasion, metastasis, and proliferation of cervical cancer was evaluated by cell scratch, Transwell, and monoclonal experiments. The interaction among LIMK1, ROS, and Src was evaluated by Western blotting. The effects of regulating ROS and p-Src expression on LIMK1 in the migration/invasion and proliferation of cervical cancer cells were evaluated through cellular functional assays., Results: Overexpression of LIMK1 promoted tumor growth in nude mice. Cell scratch, Transwell, and monoclonal experiments suggested that LIMK1 promoted the invasion, metastasis, and proliferation of cervical cancer cells. Western blotting suggested that LIMK1 can promote the expression of ROS-related proteins NOX2, NOX4, p-Src, and downstream proteins p-FAK, p-ROCK1/2, p-Cofilin-1, F-actin and inhibit the expression of p-SHP2 protein. Correction experiments showed that LIMK1 regulated the expression of p-FAK and p-Cofilin-1 proteins by regulating ROS and p-Src. Through the detection of cervical cancer cell functions, it was found that the activation of ROS and p-Src induced by LIMK1 is an early event that promotes the migration, proliferation, and invasion of cervical cancer cells., Conclusions: LIMK1 promotes the expression of F-actin and promotes the development of cervical cancer by regulating the oxidative stress/Src-mediated p-FAK/p-ROCK1/2/p-Cofilin-1 pathway.

The anti-tumor capacity of natural killer (NK) cells heavily relies on their ability to migrate towards their target cells. This process is based on dynamic actinrearrangement, so-called actin treadmilling, andis tightly regulated by proteins such as cofilin-1. The aim of the present study was to identify the role of cofilin-1 (CFL-1) in the migratory behavior of NK cells and to investigate a possible impact of an obesity-associated micromilieu on these cells, as it is known that obesity correlates with various impaired NK cell functions. CFL-1 was knocked-down via transfection of NK-92 cells with respective siRNAs. Obesity associated micromilieu was mimicked by incubation of NK-92 cells with adipocyte-conditioned medium from human preadipocyte SGBS cells or leptin. Effects on CFL-1 levels, the degree of phosphorylation to the inactive pCFL-1 as well as NK-92 cell motility were analyzed. Surprisingly, siRNA-mediated CFL-1 knockdown led to a significant increase of migration, as determined by enhanced velocity and accumulated distance of migration. No effect on CFL-1 nor pCFL-1 expression levels, proportion of phosphorylation and cell migratory behavior could be demonstrated under the influence of an obesity-associated microenvironment. In conclusion, the results indicate a significant effect of a CFL-1 knockdown on NK cell motility.

Intracellular actin networks assemble through the addition of ATP-actin subunits at the growing barbed ends of actin filaments. This is followed by "aging" of the filament via ATP hydrolysis and subsequent phosphate release. Aged ADP-actin subunits thus "treadmill" through the filament before being released back into the cytoplasmic monomer pool as a result of depolymerization at filament pointed ends. The necessity for aging before filament disassembly is reinforced by preferential binding of cofilin to aged ADP-actin subunits over newly-assembled ADP-P i actin subunits in the filament. Consequently, investigations into how cofilin influences pointed-end depolymerization have, thus far, focused exclusively on aged ADP-actin filaments. Using microfluidics-assisted Total Internal Reflection Fluorescence (mf-TIRF) microscopy, we reveal that, similar to their effects on ADP filaments, cofilin and cyclase-associated protein (CAP) also promote pointed-end depolymerization of ADP-P i filaments. Interestingly, the maximal rates of ADP-P i filament depolymerization by CAP and cofilin together remain approximately 20-40 times lower than for ADP filaments. Further, we find that the promotion of ADP-P i pointed-end depolymerization is conserved for all three mammalian cofilin isoforms. Taken together, the mechanisms presented here open the possibility of newly-assembled actin filaments being directly disassembled from their pointed-ends, thus bypassing the slow step of P i release in the aging process., Competing Interests: Declaration of Competing Interest None, (Copyright © 2024 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Prevalence of metabolic dysfunction-associated steatotic liver disease (MASLD), formerly known as non-alcoholic fatty liver disease, increases worldwide and associates with type 2 diabetes and other cardiometabolic diseases. Here we demonstrate that Sema3a is elevated in liver sinusoidal endothelial cells of animal models for obesity, type 2 diabetes and MASLD. In primary human liver sinusoidal endothelial cells, saturated fatty acids induce expression of SEMA3A, and loss of a single allele is sufficient to reduce hepatic fat content in diet-induced obese mice. We show that semaphorin-3A regulates the number of fenestrae through a signaling cascade that involves neuropilin-1 and phosphorylation of cofilin-1 by LIM domain kinase 1. Finally, inducible vascular deletion of Sema3a in adult diet-induced obese mice reduces hepatic fat content and elevates very low-density lipoprotein secretion. Thus, we identified a molecular pathway linking hyperlipidemia to microvascular defenestration and early development of MASLD., (© 2024. The Author(s).)

Cofilin, a key actin-binding protein, orchestrates the dynamics of the actomyosin network through its actin-severing activity and by promoting the recycling of actin monomers. Recent experiments suggest that cofilin forms functionally distinct oligomers via thiol post-translational modifications (PTMs) that promote actin nucleation and assembly. Despite these advances, the structural conformations of cofilin oligomers that modulate actin activity remain elusive because there are combinatorial ways to oxidize thiols in cysteines to form disulfide bonds rapidly. This study employs molecular dynamics simulations to investigate human cofilin 1 as a case study for exploring cofilin dimers via disulfide bond formation. Utilizing a biasing scheme in simulations, we focus on analyzing dimer conformations conducive to disulfide bond formation. Additionally, we explore potential PTMs arising from the examined conformational ensemble. Using the free energy profiling, our simulations unveil a range of probable cofilin dimer structures not represented in current Protein Data Bank entries. These candidate dimers are characterized by their distinct population distributions and relative free energies. Of particular note is a dimer featuring an interface between cysteines 139 and 147 residues, which demonstrates stable free energy characteristics and intriguingly symmetrical geometry. In contrast, the experimentally proposed dimer structure exhibits a less stable free energy profile. We also evaluate frustration quantification based on the energy landscape theory in the protein-protein interactions at the dimer interfaces. Notably, the 39-39 dimer configuration emerges as a promising candidate for forming cofilin tetramers, as substantiated by frustration analysis. Additionally, docking simulations with actin filaments further evaluate the stability of these cofilin dimer-actin complexes. Our findings thus offer a computational framework for understanding the role of thiol PTM of cofilin proteins in regulating oligomerization, and the subsequent cofilin-mediated actin dynamics in the actomyosin network.

Within the bloodstream, monocytes must traverse the microvasculature to prevent leukostasis, which is the entrapment of monocytes within the confines of the microvasculature. Using the model cell line, THP-1, and VCAM-1 coated channels to simulate the microvasculature surface, we demonstrate that monocytes predominantly adopt an amoeboid phenotype, which is characterized by the formation of blebs. As opposed to cortical actin flow in leader blebs, cell movement is correlated with myosin contraction at the cell rear. It was previously documented that cofilin-1 promotes cortical actin turnover at leader bleb necks in melanoma cells. In monocytes, our data suggest that cofilin-1 promotes the local upregulation of myosin contractility through actin cytoskeleton remodeling. In support of this concept, cofilin-1 is found to localize to a single cell edge. Moreover, the widespread upregulation of myosin contractility was found to inhibit migration. Thus, monocytes within the microvasculature may avoid entrapment by adopting an amoeboid mode of migration., (© 2024. The Author(s).)

Purpose: The purpose of this study was to explore the expression, prognostic value and immune correlation of Cofilin 1 protein(CFL-1) in head and neck squamous cell carcinoma(HNSCC)., Methods: The expression and prognostic value of CFL-1 in head and neck squamous cell carcinoma(HNSCC) was explored in the cancer genome map database (TCGA) and gene expression total databases (GEO), and the potential immune pathway of CFL-1 in HNSCC was revealed by GESA and cibersoft analysis. The data were statistically analyzed using SPSS 26.0 software package., Results: CFL-1 was significantly up-regulated in HNSCC tissue. The expression level of CFL-1 was significantly correlated with the overall survival status of HNSCC. High expression of CFL-1 was significantly associated with a lower overall survival rate. In addition, multivariate Cox survival analysis showed that CFL-1 expression was independent predictors of poor prognosis of HNSCC. GESA and cibersoft analysis showed that the imbalance of CFL-1 expression affected multiple signal pathways and infiltration of immune cells., Conclusions: CFL-1 is highly expressed in HNSCC and is significantly associated with poor prognosis of NHSCC. It is a potential prognostic marker of HNSCC.

To mount an adaptive immune response, dendritic cells must migrate to lymph nodes to present antigens to T cells. Critical to 3D migration is the nucleus, which is the size-limiting barrier for migration through the extracellular matrix. Here, we show that inflammatory activation of dendritic cells leads to the nucleus becoming spherically deformed and enables dendritic cells to overcome the typical 2- to 3-μm diameter limit for 3D migration through gaps in the extracellular matrix. We show that the nuclear shape change is partially attained through reduced cell adhesion, whereas improved 3D migration is achieved through reprogramming of the actin cytoskeleton. Specifically, our data point to a model whereby the phosphorylation of cofilin-1 at serine 41 drives the assembly of a cofilin-actomyosin ring proximal to the nucleus and enhances migration through 3D collagen gels. In summary, these data describe signaling events through which dendritic cells deform their nucleus and enhance their migratory capacity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cofilin family proteins have essential roles in remodeling the cytoskeleton through filamentous actin depolymerization and severing. The short, unstructured N-terminal region of cofilin is critical for actin binding and harbors the major site of inhibitory phosphorylation. Atypically for a disordered sequence, the N-terminal region is highly conserved, but specific aspects driving this conservation are unclear. Here, we screen a library of 16,000 human cofilin N-terminal sequence variants for their capacity to support growth in S. cerevisiae in the presence or absence of the upstream regulator LIM kinase. Results from the screen and biochemical analysis of individual variants reveal distinct sequence requirements for actin binding and regulation by LIM kinase. LIM kinase recognition only partly explains sequence constraints on phosphoregulation, which are instead driven to a large extent by the capacity for phosphorylation to inactivate cofilin. We find loose sequence requirements for actin binding and phosphoinhibition, but collectively they restrict the N-terminus to sequences found in natural cofilins. Our results illustrate how a phosphorylation site can balance potentially competing sequence requirements for function and regulation., (© 2024. The Author(s).)

Background: Primary Sjögren's syndrome (pSS) is a chronic systemic autoimmune disease. There is no relevant research on whether the migratory ability of bone marrow mesenchymal stem cells (BM-MSC) is impaired in patients with pSS (pSS-BMMSC)., Methods: Trajectories and velocities of BM-MSC were analyzed. Transwell migration assay and wound healing assay were used to investigate the migratory capacity of BM-MSC. The proliferative capacity of BM-MSC was evaluated by EDU and CCK8 assay. RNA-seq analysis was then performed to identify the underlying mechanism of lentivirus-mediated cofilin-1 overexpression BM-MSC (BMMSC CFL1 ). The therapeutic efficacy of BMMSC CFL1 was evaluated in NOD mice., Results: The migratory capacity of pSS-BMMSC was significantly reduced compared to normal volunteers (HC-BMMSC). The expression of the motility-related gene CFL1 was decreased in pSS-BMMSC. Lentivirus-mediated CFL1 overexpression of pSS-BMMSC promoted the migration capacity of pSS-BMMSC. Furthermore, RNA-seq revealed that CCR1 was the downstream target gene of CFL1. To further elucidate the mechanism of CFL1 in regulating BM-MSC migration and proliferation via the CCL5/CCR1 axis, we performed a rescue experiment using BX431 (a CCR1-specific inhibitor) to inhibit CCR1. The results showed that CCR1 inhibitors suppressed the migration and proliferation capacity of MSC induced by CFL1., Conclusion: The pSS-BMMSC leads to impaired migration and proliferation, and overexpression of CFL1 can rescue the functional deficiency and alleviate disease symptoms in NOD mice. Mechanically, CFL1 can regulate the expression level of the downstream CCL5/CCR1 axis to enhance the migration and proliferation of BM-MSC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

The molecular mechanisms mediating the aggregation and transmission of tau in AD remain unclear. Here, we show that the actin-binding protein cofilin is cleaved by a cysteine protease asparagine endopeptidase (AEP) at N138 in the brains of patients with AD. The AEP-generated cofilin 1-138 fragment interacts with tau and promotes its aggregation. The mixed fibrils consisting of cofilin 1-138 and tau are more pathogenic to cells than pure tau fibrils. Furthermore, overexpression of cofilin 1-138 in the brain facilitates the propagation of pathological tau aggregates and promotes AD-like cognitive impairments in tau P301S mice. However, mice infected with adeno-associated viruses (AAVs) encoding an AEP-uncleavable cofilin mutant show attenuated tau pathology and cognitive impairments compared with mice injected with AAVs encoding wild-type cofilin. Together, these observations support the role of the cofilin 1-138 fragment in the aggregation and transmission of tau pathology during the onset and progression of AD., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Inc. All rights reserved.)

Background: Air pollutants exacerbate chronic airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD). However, the underlying mechanisms are yet to be determined. While a number of studies have reported adverse effects of nanoparticles on humans, little is known about their effects on the respiratory system., Objectives: To examine the protein expression in human lung microvascular endothelial cells (HMVEC-L) exposed to titanium dioxide (TiO2) nanoparticles, a common air pollutant., Material and Methods: A proteomics approach using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and matrix-assisted laser desorption/ionization time-of-flight/time-of-flight mass spectrometry (MALDI-TOF/TOF MS) was used to determine the differences in protein expression at 8 h and 24 h, following the treatment of HMVEC-L with 20-μM or 40-μM TiO2 nanoparticles., Results: Human lung microvascular endothelial cells treated with 20-μM TiO2 nanoparticles showed alterations of 7 protein spots, including molecules related to calcium regulation, transport, cytoskeleton, and muscle contraction. The treatment of HMVEC-L with 40-μM TiO2 nanoparticles resulted in alterations of 4 protein spots, with molecular functions related to the cytoskeleton, myosin regulation, actin modulation, as well as guanosine diphosphate (GDP) and guanosine triphosphate (GTP) regulation. To validate these results, immunohistochemical staining and western blotting analyses were performed on lung tissues collected from mice exposed to TiO2 nanoparticles. Cofilin-1 and profilin-1 were expressed in the endothelium, epithelium and inflammatory cells, and decreased in lung tissues of TiO2 nanoparticle-exposed mice compared to sham-treated controls., Conclusions: These results suggest that some of the differentially expressed proteins may play important roles in airway diseases caused by TiO2 nanoparticle exposure.

Background and Aims: Intrahepatic cholangiocarcinoma (ICC) is not fully investigated, and how stromal cells contribute to ICC formation is poorly understood. We aimed to uncover ICC origin, cellular heterogeneity, and critical modulators during ICC initiation/progression, and to decipher how fibroblast and endothelial cells in the stromal compartment favor ICC progression., Approach and Results: We performed single-cell RNA sequencing (scRNA-seq) using AKT/Notch intracellular domain-induced mouse ICC tissues at early, middle, and late stages. We analyzed the transcriptomic landscape, cellular classification and evolution, and intercellular communication during ICC initiation/progression. We confirmed the findings using quantitative real-time PCR, western blotting, immunohistochemistry or immunofluorescence, and gene knockout/knockdown analysis. We identified stress-responding and proliferating subpopulations in late-stage mouse ICC tissues and validated them using human scRNA-seq data sets. By integrating weighted correlation network analysis and protein-protein interaction through least absolute shrinkage and selection operator regression, we identified zinc finger, MIZ-type containing 1 (Zmiz1) and Y box protein 1 (Ybx1) as core transcription factors required by stress-responding and proliferating ICC cells, respectively. Knockout of either one led to the blockade of ICC initiation/progression. Using two other ICC mouse models (YAP/AKT, KRAS/p19) and human ICC scRNA-seq data sets, we confirmed the orchestrating roles of Zmiz1 and Ybx1 in ICC occurrence and development. In addition, hes family bHLH transcription factor 1, cofilin 1, and inhibitor of DNA binding 1 were identified as driver genes for ICC. Moreover, periportal liver sinusoidal endothelial cells could differentiate into tip endothelial cells to promote ICC development, and this was Dll4-Notch4-Efnb2 signaling-dependent., Conclusions: Stress-responding and ICC proliferating subtypes were identified, and Zmiz1 and Ybx1 were revealed as core transcription factors in these subtypes. Fibroblast-endothelial cell interaction promotes ICC development., (© 2022 The Authors. Hepatology published by Wiley Periodicals LLC on behalf of American Association for the Study of Liver Diseases.)

Background: Long non-coding RNAs (lncRNAs) are closely associated with the pathogenesis of numerous diseases including cancers. LncRNA AGAP2 Antisense RNA 1 (AGAP2-AS1) has been found to participate in the tumorigenesis of several kinds of human cancers. Nonetheless, its potential function in colorectal cancer (CRC) was still poorly investigated., Methods: The expression level of RNAs or proteins was assessed by RT-qPCR or western blot analysis. Functional experiments were performed to analyze the role of AGAP2-AS1 in CRC in vitro and in vivo. Mechanism investigations were fulfilled to determine the potential mechanism of the molecules., Results: AGAP2-AS1 expression was significantly elevated in CRC cells and could be transcriptionally activated by E2F Transcription Factor 4 (E2F4). Down-regulated AGAP2-AS1 could weaken CRC cell growth, migration, invasion, and epithelial-mesenchymal transition (EMT). MicroRNA-182-5p (miR-182-5p) was the target downstream molecule of AGAP2-AS1. Furthermore, Cofilin 1 (CFL1) was proved as the target of miR-182-5p. Mechanically, AGAP2-AS1 could boost the CFL1 expression via competitively binding to miR-182-5p in CRC. Importantly, CFL1 restoration could counteract the in vitro and in vivo suppression of depleted AGAP2-AS1 on CRC progression., Conclusion: E2F4-stimulated AGAP2-AS1 aggravated CRC development through regulating miR-182-5p/CFL1 axis, implying that AGAP2-AS1 might become a potent new target for future therapies for CRC., Competing Interests: Conflict of Interest None., (Copyright © 2021 Editrice Gastroenterologica Italiana S.r.l. Published by Elsevier Ltd. All rights reserved.)

The actin cytoskeleton reorganization during sperm capacitation is essential for the occurrence of acrosomal exocytosis (AR) in several mammalian species. Here, we demonstrate that in mouse sperm, within the first minutes of exposure upon capacitating conditions, the activity of RHOA/C and RAC1 is essential for LIMK1 and COFILIN phosphorylation. However, we observed that the signaling pathway involving RAC1 and PAK4 is the main player in controlling actin polymerization in the sperm head necessary for the occurrence of AR. Moreover, we show that the transient phosphorylation of COFILIN is also influenced by the Slingshot family of protein phosphatases (SSH1). The activity of SSH1 is regulated by the dual action of two pathways. On one hand, RHOA/C and RAC1 activity promotes SSH1 phosphorylation (inactivation). On the other hand, the activating dephosphorylation is driven by okadaic acid-sensitive phosphatases. This regulatory mechanism is independent of the commonly observed activating mechanisms involving PP2B and emerges as a new finely tuned modulation that is, so far, exclusively observed in mouse sperm. However, persistent phosphorylation of COFILIN by SSH1 inhibition or okadaic acid did not altered actin polymerization and the AR. Altogether, our results highlight the role of small GTPases in modulating actin dynamics required for AR., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

The cofilin-1 protein, encoded by CFL1, is an actin-binding protein that regulates F-actin depolymerization and nucleation activity through phosphorylation and dephosphorylation. CFL1 has been implicated in the development of neurodegenerative diseases (Alzheimer's disease and Huntington's disease), neuronal migration disorders (lissencephaly, epilepsy, and schizophrenia), and neural tube closure defects. Mutations in CFL1 have been associated with impaired neural crest cell migration and neural tube closure defects. In our study, various computational approaches were utilized to explore single-nucleotide polymorphisms (SNPs) in CFL1. The Variation Viewer and gnomAD databases were used to retrieve CFL1 SNPs, including 46 nonsynonymous SNPs (nsSNPs). The functional and structural annotation of SNPs was performed using 12 sequence-based web applications, which identified 20 nsSNPs as being the most likely to be deleterious or disease-causing. The conservation of cofilin-1 protein structures was illustrated using the ConSurf and PROSITE web servers, which projected the 12 most deleterious nsSNPs onto conserved domains, with the potential to disrupt the protein's functionality. These 12 nsSNPs were selected for protein structure construction, and the DynaMut/DUET servers predicted that the protein variants V7G, L84P, and L99A were the most likely to be damaging to the cofilin-1 protein structure or function. The evaluation of molecular docking studies demonstrated that the L99A and L84P cofilin-1 variants reduce the binding affinity for actin compared with the native cofilin-1 structure, and molecular dynamic simulation studies confirmed that these variants might destabilize the protein structure. The consequences of putative mutations on protein-protein interactions and post-translational modification sites in the cofilin-1 protein structure were analyzed. This study represents the first complete approach to understanding the effects of nsSNPs within the actin-depolymerizing factor/cofilin family, which suggested that SNPs resulting in L84P (rs199716082) and L99A (rs267603119) variants represent significant CFL1 mutations associated with disease development., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

Actin polymerization dynamics regulated by actin-binding proteins are essential for various cellular functions. The cofilin family of proteins are potent regulators of actin severing and filament disassembly. The structural basis for cofilin-isoform-specific severing activity is poorly understood as their high-resolution structures in complex with filamentous actin (F-actin) are lacking. Here, we present the atomic-resolution structure of the muscle-tissue-specific isoform, cofilin-2 (CFL2), assembled on ADP-F-actin, determined by magic-angle-spinning (MAS) NMR spectroscopy and data-guided molecular dynamics (MD) simulations. We observe an isoform-specific conformation for CFL2. This conformation is the result of a unique network of hydrogen bonding interactions within the α2 helix containing the non-conserved residue, Q26. Our results indicate F-site interactions that are specific between CFL2 and ADP-F-actin, revealing mechanistic insights into isoform-dependent F-actin disassembly., (© 2022. The Author(s).)

An inhibitory effect of estradiol (E2) on HIV-1 infection was suggested by several reports. We previously identified increased gene expression of actin-binding protein cofilin 1 (CFL1) in endocervix in the E2-dominated proliferative phase of the menstrual cycle. Actin cytoskeleton has an integral role in establishing and spreading HIV-1 infection. Herein, we studied in vitro effects of E2 on HIV-1 infection and on CFL1 expression to gain insight into the mechanism of HIV-1 inhibition by E2. E2 dose-dependently inhibited HIV-1 BaL infection in peripheral blood mononuclear cells (PBMCs) and endocervix. In PBMCs and endocervix, E2 increased protein expression of total CFL1 and phosphorylated CFL1 (pCFL1) and pCFL1/CFL1 ratios. LIMKi3, a LIM kinase 1 and 2 inhibitor, abrogated the phenotype and restored infection in both PBMCs and endocervix; inhibited E2-induced expression of total CFL1, pCFL1; and decreased pCFL1/CFL1 ratios. Knockdown of CFL1 in PBMCs also abrogated the phenotype and partially restored infection. Additional analysis of soluble mediators revealed decreased concentrations of pro-inflammatory chemokines CXCL10 and CCL5 in infected tissues incubated with E2. Our results suggest a link between E2-mediated anti-HIV-1 activity and expression of CFL1 in PBMCs and endocervical mucosa. The data support exploration of cytoskeletal signaling pathway targets for the development of prevention strategies against HIV-1., (© 2022. The Author(s).)

Cofilin-1 (Cfl1), a member of the ADF/cofilin family, has been identified as one of differentially expressed proteins in human dendritic cells challenged with lipopolysaccharide (LPS), suggesting that it may be involved in immune response. Here we showed that zebrafish cfl1 was markedly up-regulated by LPS and LTA treatment. We also showed that zebrafish recombinant Cfl1 (rCfl1) not only bound to the Gram-negative and positive bacteria A. hydrophila and S. aureus as well as their signature molecules LPS and LTA but also inhibited the growth of the bacteria. Moreover, we found that the heparin-binding motif-containing regions of Cfl1, i.e., Cfl1 9-25 , Cfl1 34-51 and Cfl1 108-125 , like rCfl1, were also able to bind to LPS and LTA and to inhibit the bacterial growth. rCfl1, Cfl1 9-25 , Cfl1 34-51 , and Cfl1 108-125 were all able to cause bacterial cell destruction, to induce membrane depolarization, and to stimulate intracellular ROS production. Finally, we showed that zebrafish Cfl1 could protect developing embryos/larvae against attack by the potential pathogen A. hydrophila. These data together indicate that zebrafish Cfl1 plays an immune-relevant role as a newly-characterized antimicrobial protein., (Copyright © 2021. Published by Elsevier Ltd.)

Taxol is commonly used chemotherapy regimen for esophageal squamous cell carcinoma (ESCC). Study of the underlying mechanisms of Taxol chemoresistance provides better understanding of esophageal cancer treatment and may provide a rational molecular target for diagnosis and intervention. Here we showed FBXO31, which was reported to be highly expressed in ESCC and significantly associated with poor prognosis, could regulate ESCC chemosensitivity to Taxol. Silencing of FBXO31 in ESCC cells sensitized cells to Taxol treatment, evidenced by FACS analysis and TUNEL assay, showing as an increased apoptotic population in FBXO31-knockdown cells compared to the control cells. The mass spectrometry data and coimmunoprecipitation results showed FBXO31 could bind with cofilin-1. Cofilin-1 knockdown in FBXO31-overexpression cells reversed FBXO31-induced suppression of cell apoptosis, suggesting FBXO31-mediated Taxol chemoresistance is associated with cofilin-1. Furthermore, in vivo experiments confirmed that knockdown of FBXO31 sensitized ESCC to Taxol treatment. This finding substantiated a pivotal role of FBOX31 in ESCC chemoresistance, indicating that FBXO31 may be a potential indicator or target for drug resistance in ESCC., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Previous studies showed that neural stem cells (NSCs) have an ability to differentiate into neurons, astrocytes and oligodendrocytes. However, the mechanisms that govern the fate of neural stem cell determination have not yet been fully clarified. In this study, we demonstrated that expression and activation of cofilin 1, a F-actin depolymerizing factor, are significantly changed during the development of brain, cortex or NSCs. Using Neuro-2a cells as a model, we found that overexpression of cofilin 1 significantly inhibit the cell differentiation and neurite outgrowth, while inhibition of intracellular cofilin 1 phosphorylation was significantly promoted. In cultured NSCs, we observed that cofilin 1 reduced the proportion of neurons derived from NSC due to inhibition of the phosphorylation, while the morphological maturation of neurons was promoted. Together, our findings revealed that cofilin 1 plays dynamic regulatory role on NSC cell fate determination and enhance neuronal maturation through regulating its activity and expression., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Many cell death pathways, including apoptosis, regulated necrosis, and ferroptosis, are relevant for neuronal cell death and share common mechanisms such as the formation of reactive oxygen species (ROS) and mitochondrial damage. Here, we present the role of the actin-regulating protein cofilin1 in regulating mitochondrial pathways in oxidative neuronal death. Cofilin1 deletion in neuronal HT22 cells exerted increased mitochondrial resilience, assessed by quantification of mitochondrial ROS production, mitochondrial membrane potential, and ATP levels. Further, cofilin1-deficient cells met their energy demand through enhanced glycolysis, whereas control cells were metabolically impaired when challenged by ferroptosis. Further, cofilin1 was confirmed as a key player in glutamate-mediated excitotoxicity and associated mitochondrial damage in primary cortical neurons. Using isolated mitochondria and recombinant cofilin1, we provide a further link to toxicity-related mitochondrial impairment mediated by oxidized cofilin1. Our data revealed that the detrimental impact of cofilin1 on mitochondria depends on the oxidation of cysteine residues at positions 139 and 147. Overall, our findings show that cofilin1 acts as a redox sensor in oxidative cell death pathways of ferroptosis, and also promotes glutamate excitotoxicity. Protective effects by cofilin1 inhibition are particularly attributed to preserved mitochondrial integrity and function. Thus, interfering with the oxidation and pathological activation of cofilin1 may offer an effective therapeutic strategy in neurodegenerative diseases., (© 2021. The Author(s).)

The fusion gene of ABL1 is closely related to tumor proliferation, invasion, and migration. It has been reported recently that ABL1 itself is required for T-cell acute lymphoblastic leukemia (T-ALL) cell migration induced by CXCL12. Further experiments revealed that ABL1 inhibitor Nilotinib inhibited leukemia cell migration induced by CXCL12, indicating the possible application of Nilotinib in T-ALL leukemia treatment. However, the interacting proteins of ABL1 and the specific mechanisms of their involvement in this process need further investigation. In the present study, ABL1 interacting proteins were characterized and their roles in the process of leukemia cell migration induced by CXCL12 were investigated. Co-immunoprecipitation in combination with mass spectrometry analysis identified 333 proteins that interact with ABL1, including Cofilin1. Gene ontology analysis revealed that many of them were enriched in the intracellular organelle or cytoplasm, including nucleic acid binding components, transfectors, or co-transfectors. Kyoto Encyclopedia of Genes and Genomes analysis showed that the top three enriched pathways were translation, glycan biosynthesis, and metabolism, together with human diseases. ABL1 and Cofilin1 were in the same complex. Cofilin1 binds the SH3 domain of ABL1 directly; however, ABL1 is not required for the phosphorylation of Cofilin1. Molecular docking analysis shows that ABL1 interacts with Cofilin1 mainly through hydrogen bonds and ionic interaction between amino acid residues. The mobility of leukemic cells was significantly decreased by Cofilin1 siRNA. These results demonstrate that Cofilin1 is a novel ABL1 binding partner. Furthermore, Cofilin1 participates in the migration of leukemia cells induced by CXCL12. These data indicate that ABL1 and Cofilin1 are possible targets for T-ALL treatment., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Center for Excellence in Molecular Cell Science, Chinese Academy of Sciences. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Phagosome-lysosome fusion in innate immune cells like macrophages and neutrophils marshal an essential role in eliminating intracellular microorganisms. In microbe-challenged macrophages, phagosome-lysosome fusion occurs 4 to 6 h after the phagocytic uptake of the microbe. However, live pathogenic mycobacteria hinder the transfer of phagosomes to lysosomes, up to 20 h post-phagocytic uptake. This period is required to evade pro-inflammatory response and upregulate the acid-stress tolerant proteins. The exact sequence of events through which mycobacteria retards phagolysosome formation remains an enigma. The macrophage coat protein Coronin1(Cor1) is recruited and retained by mycobacteria on the phagosome membrane to retard its maturation by hindering the access of phagosome maturation factors. Mycobacteria-infected macrophages exhibit an increased cAMP level, and based on receptor stimulus, Cor1 expressing cells show a higher level of cAMP than non-Cor1 expressing cells. Here we have shown that infection of bone marrow-derived macrophages with H37Rv causes a Cor1 dependent rise of intracellular cAMP levels at the vicinity of the phagosomes. This increased cAMP fuels cytoskeletal protein Cofilin1 to depolymerize F-actin around the mycobacteria-containing phagosome. Owing to reduced F-actin levels, the movement of the phagosome toward the lysosomes is hindered, thus contributing to the retarded phagosome maturation process. Additionally, Cor1 mediated upregulation of Cofilin1 also contributes to the prevention of phagosomal acidification, which further aids in the retardation of phagosome maturation. Overall, our study provides first-hand information on Cor1 mediated retardation of phagosome maturation, which can be utilized in developing novel peptidomimetics as part of host-directed therapeutics against tuberculosis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Saha, Hazra, Ghatak, Singh, Roy and BoseDasgupta.)

Cofilin-1, an actin dynamizing protein, forms actin-cofilin rods, which is one of the major events that exacerbates the pathophysiology of amyloidogenic diseases. Cysteine oxidation in cofilin-1 under oxidative stress plays a crucial role in the formation of these rods. Others and we have reported that cofilin-1 possesses a self-oligomerization property in vitro and in vivo under physiological conditions. However, it remains elusive if cofilin-1 itself forms amyloid-like structures. We, therefore, hypothesized that cofilin-1 might form amyloid-like assemblies, with a potential to intensify the pathophysiology of amyloid-linked diseases. We used various in silico and in vitro techniques and examined the amyloid-forming propensity of cofilin-1. The study confirms that cofilin-1 possesses an intrinsic tendency of aggregation and forms amyloid-like structures in vitro. Further, we studied the effect of cysteine oxidation on the stability and structural features of cofilin-1. Our data show that oxidation at Cys-80 renders cofilin-1 unstable, leading to a partial loss of protein structure. The results substantiate our hypothesis and establish a strong possibility that cofilin-1 aggregation might play a role in cofilin-mediated pathology and the progression of several amyloid-linked diseases., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cofilins are important for the regulation of the actin cytoskeleton, sarcomere organization, and force production. The role of cofilin-1, the non-muscle-specific isoform, in muscle function remains unclear. Mutations in LMNA encoding A-type lamins, intermediate filament proteins of the nuclear envelope, cause autosomal Emery-Dreifuss muscular dystrophy (EDMD). Here, we report increased cofilin-1 expression in LMNA mutant muscle cells caused by the inability of proteasome degradation, suggesting a protective role by ERK1/2. It is known that phosphorylated ERK1/2 directly binds to and catalyzes phosphorylation of the actin-depolymerizing factor cofilin-1 on Thr25. In vivo ectopic expression of cofilin-1, as well as its phosphorylated form on Thr25, impairs sarcomere structure and force generation. These findings present a mechanism that provides insight into the molecular pathogenesis of muscular dystrophies caused by LMNA mutations., Competing Interests: Declaration of interests H.J.W. is on the scientific advisory board and owns equity in AlloMek Therapeutics. The remaining authors declare no competing interests., (Copyright © 2021 The Author(s). Published by Elsevier Inc. All rights reserved.)

Calpain-2 (CAPN2) is a processing enzyme ubiquitously expressed in mammalian tissues whose pleiotropic functions depend on the role played by its cleaved-products. Nuclear interaction networks, crucial for a number of molecular processes, could be modified by CAPN2 activity. However, CAPN2 functions in cell nucleus are poorly understood. To unveil CAPN2 functions in this compartment, the result of CAPN2-mediated interactions in cell nuclei was studied in breast cancer cell (BCC) lines. CAPN2 abundance was found to be determinant for its nucleolar localization during interphase. Those CAPN2-dependent components of nucleolar proteome, including the actin-severing protein cofilin-1 (CFL1), were identified by proteomic approaches. CAPN2 binding, cleavage and activation of LIM Kinase-1 (LIMK1), followed by CFL1 phosphorylation was studied. Upon CAPN2-depletion, full-length LIMK1 levels increased and CFL1/LIMK1 binding was inhibited. In addition, LIMK1 accumulated at the cell periphery and perinucleolar region and, the mitosis-specific increase of CFL1 phosphorylation and localization was altered, leading to aberrant mitosis and cell multinucleation. These findings uncover a mechanism for the role of CAPN2 during mitosis, unveil the critical role of CAPN2 in the interactions among nuclear components and, identifying LIMK1 as a new CAPN2-target, provide a novel mechanism for LIMK1 activation. CFL1 is crucial for cytoskeleton remodeling and mitosis, but also for the maintenance of nuclear structure, the movement of chromosomes and the modulation of transcription frequently altered in cancer cells. Consequently, the role of CAPN2 in the nuclear compartment might be extended to other actin-associated biological and pathological processes., (© 2021. The Author(s).)

Fibrocytes originate from the bone marrow monocyte lineage and participate in the pathogenesis of pulmonary fibrosis. Research providing a comprehensive picture of fibrocytes is still limited. Cofilin-1 (CFL-1) is an important protein that regulates cell proliferation, migration and differentiation. Whether CFL-1 can induce monocyte differentiation into fibrocytes and promote the process of pulmonary fibrosis is unknown. Compared with that of healthy controls, the expression of CFL-1 was significantly increased in the plasma and peripheral blood mononuclear cells (PBMCs) from idiopathic pulmonary fibrosis (IPF) and connective tissue disease-associated interstitial lung disease (CTD-ILD) patients (P < 0.05). The percentages of peripheral blood fibrocytes in the IPF group (4.2550 ± 0.3483%) and CTD-ILD group (4.7100 ± 0.4811%) were higher than that in the control group (1.6340 ± 0.2549%) (both P < 0.05). In vitro, PBMCs transfected with siRNA-CFL-1 showed lower expression of CFL-1, and the percentage of fibrocytes was lower than that of the control (P < 0.05). PBMCs transfected with Lv-CFL-1 to increase the expression of CFL-1 showed a higher percentage of fibrocytes than the control (P < 0.05). In mice with bleomycin-induced pulmonary fibrosis, the relative expression of CFL-1 was increased, and the percentage of fibrocytes was higher than that in the saline group (P < 0.05). In bleomycin-induced mice, interference with Lv-CFL-1 decreased the expression of CFL-1, the percentage of fibrocytes was lower, and the lung tissue showed less fibrosis (P < 0.05). The overexpression of CFL-1 is associated with pulmonary fibrogenesis. CFL-1 could promote the differentiation of fibrocytes from monocyte peripheral blood mononuclear cells and promote pulmonary fibrosis., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Melanoma cells have been shown to undergo fast amoeboid (leader bleb-based) migration, requiring a single large bleb for migration. In leader blebs, is a rapid flow of cortical actin that drives the cell forward. Using RNAi, we find that co-depleting cofilin-1 and actin depolymerizing factor (ADF) led to a large increase in cortical actin, suggesting that both proteins regulate cortical actin. Furthermore, severing factors can promote contractility through the regulation of actin architecture. However, RNAi of cofilin-1 but not ADF led to a significant decrease in cell stiffness. We found cofilin-1 to be enriched at leader bleb necks, whereas RNAi of cofilin-1 and ADF reduced bleb sizes and the frequency of motile cells. Strikingly, cells without cofilin-1 and ADF had blebs with abnormally long necks. Many of these blebs failed to retract and displayed slow actin turnover. Collectively, our data identifies cofilin-1 and ADF as actin remodeling factors required for fast amoeboid migration., Competing Interests: MU, JL No competing interests declared, (© 2021, Ullo and Logue.)

Objective: To investigate the molecular mechanism of high phosphorylation levels of cofilin-1 (p-CFL-1) associated with paclitaxel resistance in epithelial ovarian cancer (EOC) cells., Methods: Cells displaying varying levels of p-CFL-1 and CFL-1 were created by plasmid transfection and shRNA interference. Cell inhibition rate indicating paclitaxel efficacy was assessed by Cell Counting Kit-8 (CCK-8) assay. Apoptosis was assessed by flow cytometry and protein levels were detected by western blotting. Quantitative real-time polymerase chain reaction (qRT-PCR) was used to measure the expression levels of phosphokinases and phosphatases of CFL-1. Survival analysis evaluated the correlation between the prognosis of EOC patients and the levels of p-CFL-1 and slingshot-1 (SSH-1)., Results: High levels of p-CFL-1 were observed in EOC cells that survived treatment with high doses of paclitaxel. SKOV3 cell mutants with upregulated p-CFL-1 showed impaired paclitaxel efficacy, as well as decreased apoptosis rates and pro-survival patterns of apoptosis-specific protein expression. Cytoplasmic accumulation of p-CFL-1 inhibited paclitaxel-induced mitochondrial apoptosis. SSH-1 silencing mediated CFL-1 phosphorylation in paclitaxel-resistant SKOV3 cells. Clinically, the high level of p-CFL-1 and the low level of SSH-1 in EOC tissues were closely related to chemotherapy resistance and poor prognosis in EOC patients., Conclusion: The SSH-1/p-CFL-1 signaling pathway mediates paclitaxel resistance by apoptosis inhibition in EOC and is expected to be a potential prognostic predictor., (Copyright © 2021 The Editorial Board of Biomedical and Environmental Sciences. Published by China CDC. All rights reserved.)

Background: Hepatocellular carcinoma (HCC) is the fourth fatal malignant tumour type worldwide. However, the exact molecular mechanism involved in HCC progression remains unclear., Methods: Three pairs of HCC and matched portal vein tumour thrombus (PVTT) tissue samples were analysed by isobaric tags for relative and absolute quantification (iTRAQ) assay to investigate the differentially expressed proteins. Real-time quantitative PCR, immunostaining, and immunoblotting were performed to detect cofilin 1 (CFL1) in HCC and non-tumour tissues. CCK8 and EdU, and Transwell assays, respectively, determined cell proliferation, migration, and invasion of HCC cells. Further, subcutaneous and tail vein injection were performed in nude mice for investigating HCC growth and lung metastasis in vivo. Regulatory effect of hypoxia-inducible factor-1α (HIF-1α) on CFL1 was confirmed by chromatin immunoprecipitation (ChIP) assay. Finally, interaction between CFL1 and phospholipase D1 (PLD1) was studied using immunoprecipitation (IP) assay., Results: The iTRAQ analysis identified expression of CFL1 to be significantly upregulated in PVTT than in HCC tissues. Increased expression of CFL1 was closely associated with unfavourable clinical features, and was an independent risk predictor of overall survival in HCC patients. The knockdown of CFL1 inhibited cell growth viability, invasiveness, and epithelial-mesenchymal transformation (EMT) in HCC cells. Furthermore, CFL1 silencing significantly suppressed the growth and lung metastasis of HCC cells in nude mice. Next, HIF-1α directly regulated CFL1 transcription by binding to the hypoxia-responsive element (HRE) in the promoter. Moreover, we disclosed the interaction between CFL1 and PLD1 in HCC cells using IP assay. Mechanistically, CFL1 maintained PLD1 expression by repressing ubiquitin-mediated protein degradation, thereby activating AKT signalling in HCC cells. Notably, the CFL1/PLD1 axis was found mediating the hypoxia-induced activation of the AKT pathway and EMT., Conclusion: The analysis suggests that hypoxia-induced CFL1 increases the proliferation, migration, invasion, and EMT in HCC by activating the PLD1/AKT pathway., (© 2021 The Authors. Clinical and Translational Medicine published by John Wiley & Sons Australia, Ltd on behalf of Shanghai Institute of Clinical Bioinformatics.)

Depression afflicts more than 300 million people worldwide, but there is currently no universally effective drug in clinical practice. In this study, chronic restraint stress (CRS)-induced mice depression model was used to study the antidepressant effects of resveratrol and its mechanism. Our results showed that resveratrol significantly attenuated depression-like behavior in mice. Consistent with behavioral changes, resveratrol significantly attenuated CRS-induced reduction in the density of dendrites and dendritic spines in both hippocampus and medial prefrontal cortex (mPFC). Meanwhile, in hippocampus and mPFC, resveratrol consistently alleviated CRS-induced cofilin1 activation by increasing its ser3 phosphorylation. In addition, cofilin1 immunofluorescence distribution in neuronal inner peri-membrane in controls, and cofilin1 diffusely distribution in the cytoplasm in CRS group were common in hippocampus. However, the distribution of cofilin1 in mPFC was reversed. Pearson's correlation analysis revealed that there was a significant positive correlation found between the sucrose consumption in sucrose preference test and the dendrite density in multiple sub-regions of hippocampus and mPFC, and a significant negative correlation between the immobility time in tail suspension test and the dendrite/dendritic spine density in several different areas of hippocampus and mPFC. P-cofilin1 was significantly positively correlated with the overall dendritic spine density in mPFC as well as with the overall dendrite density or BDNF in the hippocampus. Our results suggest that the BDNF/cofilin1 pathway, in which cofilin1 may be activated in a brain-specific manner, was involved in resveratrol's attenuating the dendrite and dendritic spine loss and behavioral abnormality.

Background: Chemoresistance remains the main obstacle in hepatocellular carcinoma (HCC) therapy. Despite significant advances in HCC therapy, HCC still has a poor prognosis. Thus, there is an urgent need to identify a treatment target to reverse HCC chemotherapy resistance. Platycodon grandiflorus (PG) is a perennial herb that has been used as food and traditional Chinese medicine for thousands of years in Northeast Asia. Platycodin D (PD), a main active triterpenoid saponin found in the root of PG, has been reported to possess anticancer properties in several cancer cell lines, including HCC; however, the reversal effect of this molecule on HCC chemoresistance remains largely unknown., Purpose: This study aimed to investigate the role and the mechanism of PD-mediated reversal of the histone deacetylase inhibitor (HDACi) resistance in HCC cells., Methods: Human HCC cells (HA22T) and HDACi-resistant (HDACi-R) cells were used. Cell viability was measured using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Combination index was used to calculate the synergism potential. Expression of ERK1/2 (total/phospho), cofilin-1 (total/phospho) and apoptosis-related protein was determined using western blotting. Mitochondrial membrane potential was assessed using the JC-1 (5,5',6,6'-tetrachloro-1,1',3,3'-tetraethylbenzimidazolocarbocyanine iodide) probe. Apoptosis was detected using the terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Mitochondrial reactive oxygen species generation was measured using the MitoSOX Red fluorescent probe., Results: We found that PD treatment inhibited cell viability both in HA22T HCC and HDACi-R cells. Inhibition of ERK1/2 by PD98059 could reverse drug resistance in HDACi-R cells treated with PD98059 and PD. Nevertheless, pre-treatment with U46619, an ERK1/2 activator, rescued PD-induced apoptosis by decreasing levels of apoptosis-related proteins in HCC cells. The combined treatment of PD with apicidin a powerful HDACi, dramatically enhanced the apoptotic effect in HDACi-R cells., Conclusion: For the first time, we showed that PD reversed HDACi resistance in HCC by repressing ERK1/2-mediated cofilin-1 phosphorylation. Thus, PD can potentially be a treatment target to reverse HCC chemotherapy resistance in future therapeutic trials., (Copyright © 2020. Published by Elsevier GmbH.)

Background: Rheumatoid arthritis (RA) is an autoimmune rheumatic disease that carries a substantial burden for both patients and society. Early diagnosis of RA is essential to prevent disease progression and select an optimal therapeutic strategy. However, RA diagnosis is challenging, partly due to a lack of reliable biomarkers. Here, we aimed to explore the diagnostic signature and establish a predictive model of RA., Methods: The mRNA expression profiling data of GSE17755, containing blood samples of 112 RA patients and 53 healthy control patients, were obtained from the Gene Expression Omnibus (GEO) database, followed by differential expression, GO (Gene Ontology), and KEGG (Kyoto Encyclopedia of Genes and Genomes) enrichment analysis. A PPI network was constructed to select candidate hub genes, then logistic regression and random forest models were established based on the identified genes., Results: Significantly, we identified 52 differentially expressed genes (DEGs), including 16 upregulated genes and 36 downregulated genes in RA samples compared with control samples. GO and KEGG analysis showed that several immune-related cellular processes were particularly enriched. We identified nine hub genes in the PPI network, including CFL1, COTL1, ACTG1, PFN1, LCP1, LCK, HLA-E, FYN, and HLA-DRA. The logistic regression and random forest models based on the nine identified genes reliably distinguished the RA samples from the healthy samples with substantially high AUC., Conclusion: The diagnostic logistic regression and random forest models based on nine hub genes reliably predicted the occurrence of RA. Our findings could provide new insights into RA diagnostics.

An accumulation of evidence indicates that long noncoding RNAs are involved in the tumorigenesis and progression of pancreatic cancer (PC). In this study, we investigated the functions and molecular mechanism of action of LINC00941 in PC. Quantitative PCR was used to examine the expression of LINC00941 and miR-335-5p in PC tissues and cell lines, and to investigate the correlation between LINC00941 expression and clinicopathological features. Plasmid vectors or lentiviruses were used to manipulate the expression of LINC00941, miR-335-5p, and ROCK1 in PC cell lines. Gain or loss-of-function assays and mechanistic assays were employed to verify the roles of LINC00941, miR-335-5p, and ROCK1 in PC cell growth and metastasis, both in vivo and in vitro. LINC00941 and ROCK1 were found to be highly expressed in PC, while miR-335-5p exhibited low expression. High LINC00941 expression was strongly associated with larger tumor size, lymph node metastasis, and poor prognosis. Functional experiments revealed that LINC00941 silencing significantly suppressed PC cell growth, metastasis and epithelial-mesenchymal transition. LINC00941 functioned as a molecular sponge for miR-335-5p, and a competitive endogenous RNA (ceRNA) for ROCK1, promoting ROCK1 upregulation, and LIMK1/Cofilin-1 pathway activation. Our observations lead us to conclude that LINC00941 functions as an oncogene in PC progression, behaving as a ceRNA for miR-335-5p binding. LINC00941 may therefore have potential utility as a diagnostic and treatment target in this disease.

The main cause of death in non-small-cell lung cancer (NSCLC) is tumor progression, in which metastasis and invasion play an important role. The metastatic cascade is marked by a change in morphological, biological, biochemical and functional characteristics, including the acquisition of cellular mobility. The migration activity of tumor cells determines the work of actin-binding proteins that cause their functional partners CAP1 and cofilin. Of interest is the study of the regulation of working tandem CAP1/cofilin in NSCLC. The mechanism that regulates the level of proteins in cells is proteolysis, carried out by proteasomes and calpains. Therefore, the aim of this study was to estimate the expression of CAP1/CFL1 mRNA and their protein level in NSCLC tissues, and to analyze the possible mechanisms of their regulation by the proteasome and calpain systems. Samples of NSCLC and histological unchanged lung tissue were used (n = 42). The CAP1 and CFL1 mRNA expressions were determined by real-time PCR, the contents of proteins encoded by them were determined by Western blotting, and the activity of proteasomes and calpains by the fluorimetric method. There was an increase in the expression of mRNA and protein levels of CAP1 and cofilin in the tumor tissue compared with the unchanged lung tissue. The expression of mRNA and the level of CAP1 in tumor tissue increased during growth of the primary tumor. The cofilin level in the tumor tissue decreases against the background of increased expression of its mRNA. At the same time, during tumor growth, the activity of proteasomes and calpains increased. A negative regression relationships between the activity of proteasomes and the levels of CAP1 and cofilin, as well as the activity of calpains and the level of cofilin, were found. It can be assumed that proteasomes and calpains are involved in the degradation of CAP1 and cofilin. The data obtained suggest the importance of CAP1, cofilin and proteolytic systems in the tumor transformation and lymphogenous metastasis.

Morphological change is an explicit characteristic of cell senescence, but the underlying mechanisms remains to be addressed. Here, we demonstrated, after a survey of various actin-binding proteins, that the post-translational up-regulation of cofilin-1 was essential for the reduced rate of actin depolymerization morphological enlargement in senescent cells. Additionally, up-regulated cofilin-1 mainly existed in the serine-3 phosphorylated form, according to the 2D gel immunoblotting assay. The up-regulation of cofilin-1 was also detected in aged mammalian tissues. The over-expression of wild-type cofilin-1 and constitutively phosphorylated cofilin-1 promoted cell senescence with an increased cell size. Additionally, senescent phenotypes were also reduced by knockdown of total cofilin-1, which led to a decrease in phosphorylated cofilin-1. The senescence induced by the over-expression of cofilin-1 was dependent on p27 Kip1 , but not on the p53 and p16 INK4 expressions. The knockdown of p27 Kip1 alleviated cell senescence induced by oxidative stress or replicative stress. We also found that the over-expression of cofilin-1 induced the expression of p27 Kip1 through transcriptional suppression of the transcriptional enhancer factors domain 1 (TEAD1) transcription factor. The TEAD1 transcription factor played a transrepressive role in the p27 Kip1  gene promoter, as determined by the promoter deletion reporter gene assay. Interestingly, the down-regulation of TEAD1 was accompanied by the up-regulation of cofilin-1 in senescence. The knockdown and restoration of TEAD1 in young cells and old cells could induce and inhibit p27 Kip1 and senescent phenotypes, respectively. Taken together, the current data suggest that cofilin-1/TEAD1/p27 Kip1 signaling is involved in senescence-related morphological change and growth arrest., (© 2020 The Authors. Aging Cell published by the Anatomical Society and John Wiley & Sons Ltd.)

Chemoresistance is one of the main factors of treatment failure of cervical cancer (CC). Here, we intended to discover the role and mechanism of miR-509-5p in the paclitaxel chemoresistance of CC cells. RT-PCR was conducted to verify miR-509-3p expression. HCC94 and C-33A paclitaxel-resistant CC cell models were constructed. Additionally, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and flow cytometry were performed to verify the viability and apoptosis of HCC94 and C-33A cells after upregulating miR-509-3p. Besides, the downstream target of miR-509-3p was analyzed by bioinformatics, and the targeted relationship between miR-509-3p and RAC1 was identified by the dual-luciferase reporter assay and RNA immunoprecipitation (RIP) assay. Further, the expression of apoptotic proteins (Bcl2, Bax, and Caspase3) and the RAC1/PAK1/LIMK1/Cofilin pathway was monitored by Western blot. The result showed that upregulating miR-509-3p markedly inhibited the viability and promoted the apoptosis of CC cells. On the other hand, miR-509-3p was distinctly downregulated in paclitaxel-resistant HCC94 and C-33A cells (vs. normal cells). The transfection of miR-509-3p mimics notably increased their sensitivity to paclitaxel. Meanwhile, RAC1 was found as the potential target of miR-509-3p in bioinformatics analysis. Moreover, the RAC1/p21 (RAC1) activated kinase 1 (PAK1)/LIM kinase 1 (LIMK1)/Cofilin pathway was significantly activated in paclitaxel-resistant HCC94 and C-33A cells, while miR-509-3p overexpression significantly inactivated this pathway. Additionally, downregulation of RAC1 also partly reversed the paclitaxel-resistance of CC cells and inhibited PAK1/LIMK1/Cofilin. All in all, miR-509-3p enhances the apoptosis and chemosensitivity of CC cells by regulating the RAC1/PAK1/LIMK1/Cofilin pathway.

All‑trans retinoic acid (ATRA) and arsenic trioxide (As2O3) are currently first‑line treatments for acute promyelocytic leukemia (APL). However, a number of patients with APL are resistant to ATRA but still sensitive to As2O3, and the underlying mechanisms of this remain unclear. In the present study, two‑dimensional gel electrophoresis, mass spectrometry and other proteomic methods were applied to screen and identify the differentially expressed proteins between the retinoic acid‑sensitive cell lines and drug‑resistant cell lines. The results demonstrated that in retinoic acid‑resistant NB4‑R1 cells, the protein expression of cofilin‑1 was markedly increased compared with that in the drug‑sensitive NB4 cells. Subsequently, the effects of cofilin‑1 on As2O3‑induced apoptosis in NB4‑R1 cells were further investigated. The results revealed that cell viability was markedly suppressed and apoptosis was increased in the As2O3‑treated NB4‑R1 cells, with increased expression levels of cleaved‑poly (ADP‑ribose) polymerase and cleaved‑caspase 12. Cofilin‑1 expression was significantly decreased at both the mRNA and protein levels in the As2O3‑treated group compared with the control. Western blotting further revealed that As2O3 treatment decreased the cytoplasmic cofilin‑1 level but increased its expression in the mitochondrion. However, the opposite effects of As2O3 on the cytochrome C distribution were found in NB4‑R1 cells. This suggested that As2O3 can induce the transfer of cofilin‑1 from the cytoplasm to mitochondria and trigger the release of mitochondrial cytochrome C in NB4‑R1 cells. Moreover, cofilin‑1 knockdown by its specific short hairpin RNA significantly suppressed As2O3‑induced NB4‑R1 cell apoptosis and inhibited the release of mitochondrial cytochrome C. Whereas, overexpression of cofilin‑1 using a plasmid vector carrying cofilin‑1 increased the release of cytochrome C into the cytoplasm from the mitochondria in As2O3‑treated NB4‑R1 cells. In conclusion, cofilin‑1 played a role in As2O3‑induced NB4‑R1 cell apoptosis and it might be a novel target for APL treatment.

Clear-cell renal cell carcinoma (ccRCC), the most common subtype of renal cancer, has a poor clinical outcome. A hallmark of ccRCC is genetic loss-of-function of VHL (von Hippel-Lindau) that leads to a highly vascularized tumor microenvironment. Although many ccRCC patients initially respond to antiangiogenic therapies, virtually all develop progressive, drug-refractory disease. Given the role of dysregulated expressions of cytoskeletal and cytoskeleton-regulatory proteins in tumor progression, we performed analyses of The Cancer Genome Atlas (TCGA) transcriptome data for different classes of actin-binding proteins to demonstrate that increased mRNA expression of profilin1 (Pfn1), Arp3, cofilin1, Ena/VASP, and CapZ, is an indicator of poor prognosis in ccRCC. Focusing further on Pfn1, we performed immunohistochemistry-based classification of Pfn1 staining in tissue microarrays, which indicated Pfn1 positivity in both tumor and stromal cells; however, the vast majority of ccRCC tumors tend to be Pfn1-positive selectively in stromal cells only. This finding is further supported by evidence for dramatic transcriptional up-regulation of Pfn1 in tumor-associated vascular endothelial cells in the clinical specimens of ccRCC. In vitro studies support the importance of Pfn1 in proliferation and migration of RCC cells and in soluble Pfn1's involvement in vascular endothelial cell tumor cell cross-talk. Furthermore, proof-of-concept studies demonstrate that treatment with a novel computationally designed Pfn1-actin interaction inhibitor identified herein reduces proliferation and migration of RCC cells in vitro and RCC tumor growth in vivo Based on these findings, we propose a potentiating role for Pfn1 in promoting tumor cell aggressiveness in the setting of ccRCC., Competing Interests: Conflict of interest—The authors declare that they have no conflicts of interest with the contents of this article., (© 2020 Allen et al.)