Alpha-actinin 4 Research Articles

Characterization of ACTN4 as a novel antiviral target against SARS-CoV-2

The various mutations in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pose a substantial challenge in mitigating the viral infectivity. The identification of novel host factors influencing SARS-CoV-2 replication holds potential for discovering new targets for broad-spectrum antiviral drugs that can combat future viral mutations. In this study, potential host factors regulated by SARS-CoV-2 infection were screened through different high-throughput sequencing techniques and further identified in cells. Subsequent analysis and experiments showed that the reduction of m6A modification level on ACTN4 (Alpha-actinin-4) mRNA leads to a decrease in mRNA stability and translation efficiency, ultimately inhibiting ACTN4 expression. In addition, ACTN4 was demonstrated to target nsp12 for binding and characterized as a competitor for SARS-CoV-2 RNA and the RNA-dependent RNA polymerase complex, thereby impeding viral replication. Furthermore, two ACTN4 agonists, YS-49 and demethyl-coclaurine, were found to dose-dependently inhibit SARS-CoV-2 infection in both Huh7 cells and K18-hACE2 transgenic mice. Collectively, this study unveils the pivotal role of ACTN4 in SARS-CoV-2 infection, offering novel insights into the intricate interplay between the virus and host cells, and reveals two potential candidates for future anti-SARS-CoV-2 drug development.

Terpene extract from the stem of Celastrus orbiculatus inhibits actin cytoskeleton remodelling in gastric cancer cells by regulating the protein interaction between PTBP1 and ACTN4

Adjuvant chemoradiotherapy, molecular targeted therapy, and immunotherapy are frequently employed to extend the survival of patients with advanced gastric cancer (GC). However, most of these treatments have toxic side effects, drug resistance, and limited improvements in survival and quality of life. Therefore, it is crucial to discover and develop new medications targeting GC that are highly effective and have minimal toxicity. In previous studies, the total terpene extract from the stem of Celastrus orbiculatus demonstrated anti-GC activity; however, the specific mechanism was unclear. Our research utilising co-immunoprecipitation-mass spectrometry (Co-IP-MS), polypyrimidine tract binding protein 1 (ptbp1) clustered regularly interspaced short palindromic repeat-associated protein 9 (Cas9)-knockout (KO) mouse model, tissue microarray, and functional experiments suggests that alpha actinin-4 (ACTN4) could be a significant biomarker of GC. PTBP1 influences actin cytoskeleton restructuring in GC cells by interacting with ACTN4. Celastrus orbiculatus stem extract (COE) may directly target ACTN4 and affect the interaction between PTBP1 and ACTN4, thereby exerting anti-GC effects.

PHF23 promotes NSCLC proliferation, metastasis, and chemoresistance via stabilization of ACTN4 and activation of the ERK pathway.

At present, non-small cell lung cancer (NSCLC) is still one of the leading causes of cancer-related deaths. Chemotherapy remains the standard treatment for NSCLC. However, the emergence of chemoresistance is one of the major obstacles to lung cancer treatment. Plant homologous structural domain finger protein 23 (PHF23) plays crucial roles in multiple cell fates. However, the clinical significance and biological role of PHF23 in NSCLC remain elusive. The Cancer Genome Atlas data mining, NCBI/GEO data mining, and western blotting analysis were employed to characterize the expression of PHF23 in NSCLC cell lines and tissues. Statistical analysis of immunohistochemistry and the Kaplan-Meier Plotter database were used to investigate the clinical significance of PHF23. A series of in vivo and in vitro assays, including assays for colony formation, cell viability, 5-ethynyl-2'-deoxyuridine (EDU incorporation) and Transwell migration, flow cytometry, RT-PCR, gene set enrichment analysis, co-immunoprecipitation analysis, and a xenograft tumor model, were performed to demonstrate the effects of PHF23 on the chemosensitivity of NSCLC cells and to clarify the underlying molecular mechanisms. PHF23 is overexpressed in NSCLC cell lines and tissues. High PHF23 levels correlate with short survival times and a poor response to chemotherapy in NSCLC patients. PHF23 overexpression facilitates cell proliferation, migration and sensitizes NSCLC cells to Cisplatin and Docetaxel by promoting DNA damage repair. Alpha-actinin-4 (ACTN4), as a downstream regulator, interacts with PHD domain of PHF23. Moreover, PHF23 is involved in ACTN4 stabilization by inhibiting its ubiquitination level. These results show that PHF23 plays an important role in the development and progression of NSCLC and suggest that PHF23 may serve as a therapeutic target in NSCLC patients.

Single Nucleotide Polymorphism rs9277336 Controls the Nuclear Alpha Actinin 4-Human Leukocyte Antigen-DPA1 Axis and Pulmonary Endothelial Pathophenotypes in Pulmonary Arterial Hypertension.

Background Pulmonary arterial hypertension (PAH) is a complex, fatal disease where disease severity has been associated with the single nucleotide polymorphism (SNP) rs2856830, located near the human leukocyte antigen DPA1 (HLA-DPA1) gene. We aimed to define the genetic architecture of functional variants associated with PAH disease severity by identifying allele-specific binding transcription factors and downstream targets that control endothelial pathophenotypes and PAH. Methods and Results Electrophoretic mobility shift assays of oligonucleotides containing SNP rs2856830 and 8 SNPs in linkage disequilibrium revealed functional SNPs via allele-imbalanced binding to human pulmonary arterial endothelial cell nuclear proteins. DNA pulldown proteomics identified SNP-binding proteins. SNP genotyping and clinical correlation analysis were performed in 84 patients with PAH at University of Pittsburgh Medical Center and in 679 patients with PAH in the All of Us database. SNP rs9277336 was identified as a functional SNP in linkage disequilibrium (r2>0.8) defined by rs2856830, and the minor allele was associated with decreased hospitalizations and improved cardiac output in patients with PAH, an index of disease severity. SNP pulldown proteomics showed allele-specific binding of nuclear ACTN4 (alpha actinin 4) protein to rs9277336 minor allele. Both ACTN4 and HLA-DPA1 were downregulated in pulmonary endothelium in human patients and rodent models of PAH. Via transcriptomic and phenotypic analyses, knockdown of HLA-DPA1 phenocopied knockdown of ACTN4, both similarly controlling cell structure pathways, immune pathways, and endothelial dysfunction. Conclusions We defined the pathogenic activity of functional SNP rs9277336, entailing the allele-specific binding of ACTN4 and controlling expression of the neighboring HLA-DPA1 gene. Through inflammatory or genetic means, downregulation of this ACTN4-HLA-DPA1 regulatory axis promotes endothelial pathophenotypes, providing a mechanistic explanation for the association between this SNP and PAH outcomes.

Aortic Cellular Diversity and Quantitative Genome-Wide Association Study Trait Prioritization Through Single-Nuclear RNA Sequencing of the Aneurysmal Human Aorta.

Mural cells in ascending aortic aneurysms undergo phenotypic changes that promote extracellular matrix destruction and structural weakening. To explore this biology, we analyzed the transcriptional features of thoracic aortic tissue. Single-nuclear RNA sequencing was performed on 13 samples from human donors, 6 with thoracic aortic aneurysm, and 7 without aneurysm. Individual transcriptomes were then clustered based on transcriptional profiles. Clusters were used for between-disease differential gene expression analyses, subcluster analysis, and analyzed for intersection with genetic aortic trait data. We sequenced 71 689 nuclei from human thoracic aortas and identified 14 clusters, aligning with 11 cell types, predominantly vascular smooth muscle cells (VSMCs) consistent with aortic histology. With unbiased methodology, we found 7 vascular smooth muscle cell and 6 fibroblast subclusters. Differentially expressed genes analysis revealed a vascular smooth muscle cell group accounting for the majority of differential gene expression. Fibroblast populations in aneurysm exhibit distinct behavior with almost complete disappearance of quiescent fibroblasts. Differentially expressed genes were used to prioritize genes at aortic diameter and distensibility genome-wide association study loci highlighting the genes JUN, LTBP4 (latent transforming growth factor beta-binding protein 1), and IL34 (interleukin 34) in fibroblasts, ENTPD1, PDLIM5 (PDZ and LIM domain 5), ACTN4 (alpha-actinin-4), and GLRX in vascular smooth muscle cells, as well as LRP1 in macrophage populations. Using nuclear RNA sequencing, we describe the cellular diversity of healthy and aneurysmal human ascending aorta. Sporadic aortic aneurysm is characterized by differential gene expression within known cellular classes rather than by the appearance of novel cellular forms. Single-nuclear RNA sequencing of aortic tissue can be used to prioritize genes at aortic trait loci.

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.

Alpha-actinin-4 (ACTN4) gene amplification is a predictive biomarker for adjuvant chemotherapy with tegafur/uracil in stage I lung adenocarcinomas.

Although adjuvant tegafur/uracil (UFT) is recommended for patients with completely resected stage I non‐small‐cell lung cancer (NSCLC) in Japan, only one‐third of cases has received adjuvant chemotherapy (ADJ) according to real‐world data. Therefore, robust predictive biomarkers for selecting ADJ or observation (OBS) without ADJ are needed. Patients who underwent complete resection of stage I lung adenocarcinoma with or without adjuvant UFT were enrolled. The status of ACTN4 gene amplification was analyzed by FISH. Statistical analyses to determine whether the status of ACTN4 gene amplification affected recurrence‐free survival (RFS) were carried out. Formalin‐fixed, paraffin‐embedded samples from 1136 lung adenocarcinomas were submitted for analysis of ACTN4 gene amplification. Ninety‐nine (8.9%) of 1114 cases were positive for ACTN4 gene amplification. In the subgroup analysis of patients aged 65 years or older, the ADJ group had better RFS than the OBS group in the ACTN4‐positive cohort (hazard ratio [HR], 0.084, 95% confidence interval [CI], 0.009‐0.806; P = .032). The difference in RFS between the ADJ group and the OBS group was not significant in ACTN4‐negative cases (all ages: HR, 1.214; 95% CI, 0.848‐1.738; P = .289). Analyses of ACTN4 gene amplification contributed to the decision regarding postoperative ADJ for stage I lung adenocarcinomas, preventing recurrence, improving the quality of medical care, preventing the unnecessary side‐effects of ADJ, and saving medical costs.

Abstract 11643: ACTN4 Regulates HLA-DPA1-Related Pulmonary Endothelial Pathophenotypes in Pulmonary Arterial Hypertension Through Allele-Specific Binding to Functional SNP rs9277336

Introduction: Pulmonary arterial hypertension (PAH) is a progressive disease characterized by increased pulmonary arterial pressure and vascular resistance. This vascular injury promotes immune dysregulation, further worsening the vasculopathy. A genome-wide association study identified a single nucleotide polymorphism (SNP) associated with PAH risk (rs2856830) at Human Leukocyte Antigen DPA1 (HLA-DPA1) locus, a gene known to be involved in adaptive immunity. However, little is known about the mechanism underlying this clinical association. Hypothesis: Allele-specific binding of transcription factors to a disease-associated functional SNP (fSNP) controls HLA-DPA1 expression, driving pulmonary vascular cell dysfunction and PAH. Methods: Oligonucleotides containing alleles of rs2856830 and eight SNPs in linkage disequilibrium (LD) were subjected to electrophoretic mobility shift assays (EMSA) to identify fSNPs characterized by allele-imbalanced binding to human pulmonary arterial endothelial cells (PAEC) nuclear proteins. SNP genotyping was done using blood from PAH (n=134) and control patients (n=131) at UPMC. Transcriptomics, angiogenesis, and migration were assessed in siRNA mediated HLA-DPA1 and Alpha Actinin 4 (ACTN4) knockdown (KD) in PAECs. Results: Through EMSA, rs9277336 (LD r 2 =0.87) was identified as a fSNP in HLA-DPA1 locus. SNP pulldown and immunoblotting confirmed allele-specific binding of nuclear ACTN4 protein to rs9277336 risk allele A (3.26 ± 0.49, p=0.001). SNP genotyping revealed a 2.04 ± 0.67 fold enrichment of risk allele A in PAH patients (χ 2 test, p=0.025). ACTN4 KD in cultured human PAECs decreased HLA-DPA1 transcript (0.30 ± 0.10, p<0.001) . Transcriptomics revealed overlap of several cell structure and immune system-related pathways by HLA-DPA1 and ACTN4 KD. HLA-DPA1 and ACTN4 KD affected PAH associated pathophenotypes, including angiogenesis and migration. Conclusions: Our functional analysis identified ACTN4 as a novel regulator to disease-associated gene HLA-DPA1, affecting the endothelial pathophenotypes in PAH. The allele-specific binding to fSNP rs9277336 provides a mechanistic explanation for the clinical association between HLA-DPA1 locus and PAH outcomes.

The deubiquitinase OTUD3 stabilizes ACTN4 to drive growth and metastasis of hepatocellular carcinoma.

OTU domain-containing protein 3 (OTUD3), a deubiquitinating enzyme, has been shown to participate in progression of multiple malignancies. The accurate function of OTUD3 in hepatocellular carcinoma (HCC) progression remains elusive. We found that OTUD3 was significantly overexpressed in HCC, and higher OTUD3 expression was correlated with larger tumor size, more distant metastasis, and worse TNM stage. A series of gain- and loss-of-function assays were also performed to examine the oncogenic function of OTUD3 in promoting HCC cell growth and metastasis in vitro. Using a xenograft mouse model, we showed that OTUD3 accelerated HCC progression in vivo. Furthermore, alpha-actinin 4 (ACTN4) was identified as a downstream target of OTUD3 through mass spectrometry analysis, and the ACTN4 protein level was significantly related to OTUD3 expression. Additionally, OTUD3 directly bound with ACTN4 and deubiquitinated ACTN4 to stabilize it. Finally, ACTN4 was found to be essential for OTUD3-mediated HCC proliferation and metastasis in vitro and in vivo. Collectively, our findings identify the oncogenic role of OTUD3 in HCC and suggest that OTUD3 can be considered as a pivotal prognostic biomarker and a potential therapeutic target.

Alpha\u2010actinin\u20104 is essential for maintaining normal trophoblast proliferation and differentiation during early pregnancy

BackgroundProper differentiation of trophoblasts in the human placenta is essential for a successful pregnancy, whereas abnormal regulation of this process may lead to adverse pregnancy outcomes, especially preeclampsia (PE). However, the underlying mechanism of trophoblast differentiation remains unclear. Previous studies have reported the involvement of alpha-actinin-4 (ACTN4) in the actin cytoskeleton dynamics and motility. Hence, we hypothesized that ACTN4 may act as an important regulator in the normal proliferation and differentiation of trophoblasts during early pregnancy.Method:To test this hypothesis, we collected villous tissues from women undergoing a legal pregnancy termination during 6–10 weeks of gestation and explanted them for cell culture and siRNA transfection. We also obtained placental tissues from PE patients and healthy pregnant women and isolated the primary cytotrophoblast (CTB) cells. The expression of ACTN4 in the CTBs of placental villi and during the differentiation of CTBs into STBs was detected by immunofluorescence, immunohistochemistry (IHC), and EdU proliferation assays. Besides, villous explant, Matrigel invasion, transwell migration assay, and Wound-healing assay were performed to identify the possible role of ACTN4 in the outgrowth of explants and the invasion, migration, and proliferation of cell column trophoblasts (CCTs). Western blot analysis was carried out to compare the protein expression level of AKT, Snail activities, and epithelial-to-mesenchymal transition (EMT) in the villi or HTR8/SVneo cells with ACTN4 knockdown.ResultsACTN4 was highly expressed in CTB cells and interstitial extravillous trophoblast (iEVT) cells but not found in the syncytiotrophoblast (STB) cells in the first trimester villi. Downregulation of ACTN4 led to reduced trophoblast proliferation and explant outgrowth ex vivo, as well as iEVT invasion and migration in vitro due to disrupt of actin filaments organization. Such ACTN4 inhibition also decreased AKT and Snail activities and further impeded the EMT process. In addition, ACTN4 expression was found to be downregulated in the iEVTs from preeclamptic placentas.ConclusionsOur findings suggest that ACTN4 may act as an important regulator of trophoblast proliferation and differentiation during early pregnancy, and dysregulation of this protein may contribute to preeclampsia pathogenesis.

RETRACTED: circPHIP promotes oral squamous cell carcinoma progression by sponging miR-142-5p and regulating PHIP and ACTN4 expression

Circular RNA (circRNA) is a newly discovered class of noncoding RNAs that plays key regulatory role in pathological development, including the regulation of several solid tumors. However, the effects of circRNA expression on oral squamous cell carcinoma (OSCC) remain unclear. With the use of high-throughput RNA sequencing data on eight paired oral cancer and adjacent healthy tissues, we observed that circRNA derived from the gene encoding pleckstrin homology domain-interacting protein (circPHIP) was highly expressed in OSCC. Additionally, circPHIP was highly expressed in other OSCC-related cell lines and was associated with tumor metastasis, TNM stage, and human papilloma virus infection status. The inhibition of circPHIP expression reduced OSCC cell migration, invasion, and proliferation. We found that circPHIP could adsorb microRNA (miR)-142-5p and upregulate the expression of PHIP and alpha-actinin 4 (ACTN4), both of which are potential oncogenes closely related to OSCC prognosis. The inhibition of miR-142-5p or overexpressing PHIP or ACTN4 reversed the circPHIP depletion-induced attenuation of OSCC malignancy. In conclusion, circPHIP is overexpressed in OSCC and enhances its malignancy via an miR-142-5p/PHIP-ACTN4/AKT serine/threonine kinase 1 signaling axis. Therefore, circPHIP may represent a novel target for treating OSCC.

PDLIM1 Inhibits Tumor Metastasis Through Activating Hippo Signaling in Hepatocellular Carcinoma.

Tumor metastasis is a major factor of high recurrence and mortality in hepatocellular carcinoma (HCC), but its underlying mechanism remains elusive. We report that PDZ and LIM domain protein 1 (PDLIM1) is significantly down-regulated in metastatic human HCC tissues, which predicts unfavorable prognosis, suggesting that PDLIM1 may play an important inhibitory role during HCC metastasis. Functional studies indicate that PDLIM1 knockdown induces epithelial-to-mesenchymal transition (EMT) of HCC cells, elevates their invasive capacity, and promotes metastasis in vitro and in vivo, whereas overexpression of PDLIM1 exhibits opposite phenotypes. Mechanistically, PDLIM1 competitively binds to the cytoskeleton cross-linking protein alpha-actinin 4 (ACTN4), leading to the disassociation of ACTN4 from F-actin, thus preventing F-actin overgrowth. In contrast, loss of PDLIM1 induces excessive F-actin formation, resulting in dephosphorylation of large tumor suppressor kinase 1 and activation of Yes-associated protein, thereby promoting HCC metastasis. Moreover, Asn145 (N145) of PDLIM1 is critical for its interaction with ACTN4, and N145A mutation abolishes its regulatory function in Hippo signaling and HCC metastasis. Our findings indicate that PDLIM1 suppresses HCC metastasis by modulating Hippo signaling, suggesting that PDLIM1 may be a potential prognostic marker for metastatic HCC.

Focal segmental glomerulosclerosis ACTN4 mutants binding to actin: regulation by phosphomimetic mutations

Natural mutations such as lysine 255 to glutamic acid (K to E), threonine 259 to isoleucine (T to I) and serine 262 to proline (S to P) that occur within the actin binding domain of alpha-actinin-4 (ACTN4) cause an autosomal dominant form of focal segmental glomerulosclerosis (FSGS) in affected humans. This appears due to elevated actin binding propensity in podocytes resulting in a ‘frozen’ cytoskeleton. What is challenging is how this cellular behavior would be compatible with other cell functions that rely on cytoskeleton plasticity. Our previous finding revealed that wild type ACTN4 can be phosphorylated at tyrosine 4 and 31 upon stimulation by epidermal growth factor (EGF) to reduce the binding to actin cytoskeleton. We queried whether the elevated actin binding activity of FSGS mutants can be downregulated by EGF-mediated phosphorylation, to discern a mechanism by which the actin-cytoskeleton can be released in FSGS. In this manuscript, we first constructed variants with Y4/31E to mimic the phosphorylation at tyrosines 4 and 31 based on earlier modeling simulations that predicted that this would bury the actin binding domains and lead to a decrease in actin binding activity. We found that Y4/31E significantly reduced the actin binding activity of K255E, T259I and S262P, dramatically preventing them from aggregating in, and inhibiting motility of, podocytes, fibroblasts and melanoma cells. A putative kinase target site at Y265 in the actin binding domain was also generated as a phosphomimetic ACTN4 Y265E that demonstrated even greater binding to actin filaments than K255E and the other FSGS mutants. That the tyrosine kinase regulation of FSGS mutation binding to actin filaments can occur in cells was shown by phosphorylation on Y4 and Y31 of the K225E after extended exposure of cells to EGF, with a decrease in ACTN4 aggregates in fibroblasts. These findings will provide evidence for targeting the N-termini of FSGS ACTN4 mutants to downregulate their actin binding activities for ameliorating the glomerulosclerotic phenotype of patients.

Alpha-actinin 4 and tumorigenesis of hepatocellular carcinoma

Background: We examined the expression of alpha-actinin 4 (ACTN4) in hepatocellular carcinoma (HCC) tissues, to investigate the relationships between the expression of ACTN4 and the clinicopathological features of HCC patients. We have also analyzed the relevance of ACTN4 expression and survival time after HCC patients undergo surgery. Methods: The expression of ACTN4 was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry in 87 HCC patients. Statistical analyses were conducted with SPSS 22.0 software. Results: qRT-PCR revealed that the expression of ACTN4 mRNA was significantly higher in HCC tissues than in adjacent tissues. Immunohistochemistry revealed that the positivity rate of ACTN4 was increased in HCC tissues compared with adjacent tissues (P=0.029). A Mann-Whitney U analysis revealed that positive ACNT4 expression was statistically significantly associated with the lymph node statuses of the HCC patients. Kaplan-Meier curves revealed that positive ACTN4 expression was inversely correlated with the 5-year survival times of HCC patients after surgery, and this relationship was statistically significant. Conclusions: ACTN4 plays an important role in the carcinogenesis and progression of HCC.

Dysregulated expression of ACTN4 contributes to endothelial cell injury via the activation of the p38-MAPK/p53 apoptosis pathway in preeclampsia.

Preeclampsia (PE) is a hypertensive disease associated with increased endothelial cell dysfunction caused by systemic oxidative stress. Alpha-actinin-4 (ACTN4) is a member of the α-actinin family of actin crosslinking proteins that are upregulated in several types of cancer. However, its role in PE remains unclear. In this study, we found that ACTN4 was localized in placenta vascular endothelial cells (ECs), and its expression was downregulated in primary human umbilical vein endothelial cells (HUVECs) from severe preeclamptic patients compared to that in HUVECs from normotensive pregnant women. ACTN4 expression was also decreased in normotensive HUVECs treated with H2O2. Downregulation of ACTN4 by siRNA or H2O2 treatment promoted normotensive HUVEC apoptosis and increased p38-MAPK phosphorylation along with elevated levels of p53 phosphorylation, caspase cascade proteins, and bax and repressed expression of bcl-2. Conversely, upregulation of ACTN4 in PE HUVECs significantly inhibited apoptosis and decreased p38-MAPK phosphorylation compared to that of the PE HUVEC controls. In addition, overexpression of ACTN4 in normotensive HUVECs attenuated H2O2 treatment-induced apoptosis with decreased p53 phosphorylation, caspase cascade, and bax expression levels and increased expression of bcl-2 compared to that of only H2O2 treatment. Moreover, the suppression of ACTN4 induced apoptosis, which could be blocked by the p38-MAPK inhibitor SB202190. Collectively, these results demonstrate that dysregulated ACTN4 expression may be associated with PE due to its effects on endothelial cell apoptosis via the p38-MAPK/p53 apoptosis pathway.

Alpha-actinin 4 and tumorigenesis of hepatocellular carcinoma.

BackgroundWe examined the expression of alpha-actinin 4 (ACTN4) in hepatocellular carcinoma (HCC) tissues, to investigate the relationships between the expression of ACTN4 and the clinicopathological features of HCC patients. We have also analyzed the relevance of ACTN4 expression and survival time after HCC patients undergo surgery.MethodsThe expression of ACTN4 was analyzed by quantitative reverse transcription polymerase chain reaction (qRT-PCR) and immunohistochemistry in 87 HCC patients. Statistical analyses were conducted with SPSS 22.0 software.ResultsqRT-PCR revealed that the expression of ACTN4 mRNA was significantly higher in HCC tissues than in adjacent tissues. Immunohistochemistry revealed that the positivity rate of ACTN4 was increased in HCC tissues compared with adjacent tissues (P=0.029). A Mann-Whitney U analysis revealed that positive ACNT4 expression was statistically significantly associated with the lymph node statuses of the HCC patients. Kaplan-Meier curves revealed that positive ACTN4 expression was inversely correlated with the 5-year survival times of HCC patients after surgery, and this relationship was statistically significant.ConclusionsACTN4 plays an important role in the carcinogenesis and progression of HCC.

ACTN4 Promotes the Proliferation, Migration, Metastasis of Osteosarcoma and Enhances its Invasive Ability through the NF-\u03baB Pathway

Alpha-actinin-4 (ACTN4) is associated with different types of tumors, but its role in osteosarcoma (OS) is not known. We aimed to investigate the effect of ACTN4 on the growth, migration, invasion and metastasis of OS. We further explored the possible mechanism of how ACTN4 affects the development of OS. First, the expression of ACTN4 in OS tissues and OS cell lines was analyzed by PCR. Second, the role of ACTN4 in the development of OS was explored by the proliferation, scratch, and invasion assays. We further explored the effect of ACTN4 on OS growth in an orthotopic xenograft model of nude mice. In addition, we used hematoxylin and eosin (HE) staining of lung tissues in nude mice to observe the effect of ACTN4 on lung metastasis of OS. Finally, rescue experiments further investigated the role of NF-κB on ACTN4 in the development of OS. ACTN4 was highly expressed in OS tissues and OS cell lines. In vitro experiments demonstrated that reducing ACTN4 expression inhibited the proliferation, migration, and invasion of OS. In contrast, overexpression of ACTN4 promotes these effects. In vivo experiments further validated that ACTN4 promoted the growth of OS. The HE staining of lungs in nude mice revealed that ACTN4 promoted lung metastasis of OS. In addition, we found that ACTN4 enhanced the ability of OS to invade, through the NF-κB pathway. ACTN4 promotes the proliferation, migration, metastasis of OS and enhances its invasion ability through the NF-κB pathway.

WAVE2 is associated with poor prognosis in pancreatic cancers and promotes cell motility and invasiveness via binding to ACTN4.

WAVE2 is a member of the WASP/WAVE family of actin cytoskeletal regulatory proteins; unfortunately, little is known about its function in pancreatic cancers. In this study, we report the role of WAVE2 in the motility and invasiveness of pancreatic cancer cells. High WAVE2 expression in human pancreatic cancer tissues was correlated with overall survival. WAVE2 accumulated in the cell protrusions of pancreatic cancer cell lines. Downregulation of WAVE2 by small interfering RNA decreased the cell protrusions and inhibited the motility and invasiveness of pancreatic cancer cells. WAVE2 promoted pancreatic cancer cell motility and invasion by forming a complex with the actin cytoskeletal protein alpha‐actinin 4 (ACTN4). Downregulation of ACTN4 by small interfering RNA also inhibited the motility and invasiveness of the cells through a decrease in cell protrusions. Further investigation showed that WAVE2/ACTN4 signaling selectively stimulated p27 phosphorylation and thereby increased the motility and invasiveness of the cells. These results suggest that WAVE2 and ACTN4 stimulate p27 phosphorylation and provide evidence that WAVE2 promotes the motility and invasiveness of pancreatic cancer cells.

Erratum: Actin-binding protein alpha-actinin 4 (ACTN4) is a transcriptional co-activator of RelA/p65 sub-unit of NF-kB.

[This corrects the article DOI: 10.18632/oncotarget.901.].

Mechanical Relaxation of Alpha-Actinin in the Cellular Cytoplasm Probed with Magnetic Tweezers

The actin cytoskeleton is a key determinant of cell force production, mechanical integrity and structure, and mechanosensation. An essential yet often overlooked element of the actin cytoskeleton is the crosslinker, alpha-actinin 4 (ACTN4), which in vitro has been shown to exhibit catch-bond behavior and determine the mechanical relaxation of purified actin networks. ACTN4's role in determining the mechanical properties of the cytoplasm, however, is unclear. Here we use Magnetic Tweezers (MTs) to probe the moduli and creep behavior of the cytoplasm in living cells. We use biolistic injection of 200 nm fluorescent super-paramagnetic particles as probes, and then apply a step-stress magnetic field using the MTs, and quantify the time-dependent displacement of particles in the viscoelastic cytoplasm. To evaluate the role of ACTN4, we employ two cell lines: wild type ACTN4, and a mutated variant (K255E), which has previously been shown to exhibit stronger actin binding, and has been implicated in kidney pathology. These results present some of the first cytoplasmic creep measurements related to crosslinker binding kinetics, and provide insight into the detailed cytoskeleton relaxation responsible for cell mechanics.