Heterozygous CAPZA2 mutations cause global developmental delay, hypotonia with epilepsy: a case report and the literature review.

CAPZA2 encodes the α2 subunit of CAPZA, which is vital for actin polymerization and depolymerization in humans. However, understanding of diseases associated with CAPZA2 remains limited. To date, only three cases have been documented with neurodevelopmental abnormalities such as delayed motor development, speech delay, intellectual disability, hypotonia, and a history of seizures. In this study, we document a patient who exhibited seizures, mild intellectual disability, and impaired motor development yet did not demonstrate speech delay or hypotonia. The patient also suffered from recurrent instances of respiratory infections, gastrointestinal and allergic diseases. A novel de novo splicing variant c.219+1 G > A was detected in the CAPZA2 gene through whole-exome sequencing. This variant led to exon 4 skipping in mRNA splicing, confirmed by RT-PCR and Sanger sequencing. To our knowledge, this is the third study on human CAPZA2 defects, documenting the fourth unambiguously diagnosed case. Furthermore, this splicing mutation type is reported here for the first time. Our research offers additional support for the existence of a CAPZA2-related non-syndromic neurodevelopmental disorder. Our findings augment our understanding of the phenotypic range associated with CAPZA2 deficiency and enrich the knowledge of the mutational spectrum of the CAPZA2 gene.

Exosome-Delivered circSTAU2 Inhibits the Progression of Gastric Cancer by Targeting the miR-589/CAPZA1 Axis.

Background: Circular RNAs (circRNAs) are endogenous noncoding RNAs that play vital roles in many biological processes, particularly in human cancer. Recent studies indicate that circRNAs play an important role in tumor progression through exosomes. However, the specific functions of gastric cancer-derived exosomes and the role of circSTAU2 in gastric cancer (GC) remain largely unknown. Methods: Differentially expressed circRNAs in GC were identified by circRNA microarrays analysis and quantitative real-time polymerase chain reaction (qRT-PCR). The role of circSTAU2 in GC was verified by circSTAU2 knockdown and overexpression with functional assays both in vitro and in vivo. Fluorescence in situ hybridization (FISH), immunofluorescence, RNA immunoprecipitation (RIP), dual-luciferase reporter assay, qRT-PCR and Western blot were adopted to evaluate the expression and regulatory mechanism of MBNL1, circSTAU2, miR-589 and CAPZA1. Furthermore, the role of exosomes was demonstrated by transmission electron microscopy and nano-sight particle tracking analysis. Results: CircSTAU2, mainly localized in the cytoplasm, was significantly downregulated in GC. CircSTAU2 overexpression inhibited GC cell proliferation, invasion and migration both in vitro and in vivo, while circSTAU2 knockdown had the inverse effect. CircSTAU2 could be wrapped in exosomes and delivered to recipient cells, and functioned as a sponge for miR-589 to relieve its inhibitory effect on CAPZA1, thus inhibiting GC progression. Furthermore, MBNL1 acted as the upstream RNA-binding protein of circSTAU2 and significantly influenced the circularization and expression of circSTAU2. Conclusion: Exosome-delivered circSTAU2 may act as a tumor suppressor that restrains GC progression via miR-589/CAPZA1 axis, which demonstrates a potential therapeutic target for GC.

Capping protein regulates endosomal trafficking by controlling F-actin density around endocytic vesicles and recruiting RAB5 effectors.

Actin filaments (F-actin) have been implicated in various steps of endosomal trafficking, and the length of F-actin is controlled by actin capping proteins, such as CapZ, which is a stable heterodimeric protein complex consisting of α and ß subunits. However, the role of these capping proteins in endosomal trafficking remains elusive. Here, we found that CapZ docks to endocytic vesicles via its C-terminal actin-binding motif. CapZ knockout significantly increases the F-actin density around immature early endosomes, and this impedes fusion between these vesicles, manifested by the accumulation of small endocytic vesicles in CapZ-knockout cells. CapZ also recruits several RAB5 effectors, such as Rabaptin-5 and Rabex-5, to RAB5-positive early endosomes via its N-terminal domain, and this further activates RAB5. Collectively, our results indicate that CapZ regulates endosomal trafficking by controlling actin density around early endosomes and recruiting RAB5 effectors.

Vacuolin-1 inhibits endosomal trafficking and metastasis via CapZß.

Metastasis is the fundamental cause of cancer mortality, but there are still very few anti-metastatic drugs available. Endosomal trafficking has been implicated in tumor metastasis, and we have previously found that small chemical vacuolin-1 (V1) potently inhibits autophagosome-lysosome fusion and general endosomal-lysosomal degradation. Here, we assessed the anti-metastatic activity of V1 both in vitro and in vivo. V1 significantly inhibits colony formation, migration, and invasion of various cancer cells in vitro. It also compromises the assembly-disassembly dynamics of focal adhesions (FAs) by inhibiting the recycling and degradation of integrins. In various experimental or transgenic mouse models, V1 significantly suppresses the metastasis and/or tumor growth of breast cancer or melanoma. We further identified capping protein Zß (CapZß) as a V1 binding protein and showed that it is required for the V1-mediated inhibition of migration and metastasis of cancer cells. Collectively, our results indicate that V1 targets CapZß to inhibit endosomal trafficking and metastasis.

Actin-binding protein profilin1 promotes aggressiveness of clear-cell renal cell carcinoma cells.

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.

Cytoskeleton regulators CAPZA2 and INF2 associate with CFTR to control its plasma membrane levels under EPAC1 activation.

Cystic Fibrosis (CF), the most common lethal autosomic recessive disorder among Caucasians, is caused by mutations in the gene encoding the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) protein, a cAMP-regulated chloride channel expressed at the apical surface of epithelial cells. Cyclic AMP regulates both CFTR channel gating through a protein kinase A (PKA)-dependent process and plasma membane (PM) stability through activation of the exchange protein directly activated by cAMP1 (EPAC1). This cAMP effector, when activated promotes the NHERF1:CFTR interaction leading to an increase in CFTR at the PM by decreasing its endocytosis. Here, we used protein interaction profiling and bioinformatic analysis to identify proteins that interact with CFTR under EPAC1 activation as possible regulators of this CFTR PM anchoring. We identified an enrichment in cytoskeleton related proteins among which we characterized CAPZA2 and INF2 as regulators of CFTR trafficking to the PM. We found that CAPZA2 promotes wt-CFTR trafficking under EPAC1 activation at the PM whereas reduction of INF2 levels leads to a similar trafficking promotion effect. These results suggest that CAPZA2 is a positive regulator and INF2 a negative one for the increase of CFTR at the PM after an increase of cAMP and concomitant EPAC1 activation. Identifying the specific interactions involving CFTR and elicited by EPAC1 activation provides novel insights into late CFTR trafficking, insertion and/or stabilization at the PM and highlighs new potential therapeutic targets to tackle CF disease.

Variants in CAPZA2, a member of an F-actin capping complex, cause intellectual disability and developmental delay.

The actin cytoskeleton is regulated by many proteins including capping proteins that stabilize actin filaments (F-actin) by inhibiting actin polymerization and depolymerization. Here, we report two pediatric probands who carry damaging heterozygous de novo mutations in CAPZA2 (HGNC: 1490) and exhibit neurological symptoms with shared phenotypes including global motor development delay, speech delay, intellectual disability, hypotonia and a history of seizures. CAPZA2 encodes a subunit of an F-actin-capping protein complex (CapZ). CapZ is an obligate heterodimer consisting of α and ß heterodimer conserved from yeast to human. Vertebrate genomes contain three α subunits encoded by three different genes and CAPZA2 encodes the α2 subunit. The single orthologue of CAPZA genes in Drosophila is cpa. Loss of cpa leads to lethality in early development and expression of the human reference; CAPZA2 rescues this lethality. However, the two CAPZA2 variants identified in the probands rescue this lethality at lower efficiency than the reference. Moreover, expression of the CAPZA2 variants affects bristle morphogenesis, a process that requires extensive actin polymerization and bundling during development. Taken together, our findings suggest that variants in CAPZA2 lead to a non-syndromic neurodevelopmental disorder in children.

A new 1p36.13-1p36.12 microdeletion syndrome characterized by learning disability, behavioral abnormalities, and ptosis.

Two 1p36 contiguous gene deletion syndromes are known so far: the terminal 1p36 deletion syndrome and a 1p36 deletion syndrome with a critical region located more proximal at 1p36.23-1p36.22. We present even more proximally located overlapping deletions from seven individuals, with the smallest region of overlap comprising 1 Mb at 1p36.13-1p36.12 (chr1:19077793-20081292 (GRCh37/hg19)) defining a new contiguous gene deletion syndrome. The characteristic features of this new syndrome are learning disability or mild intellectual disability, speech delay, behavioral abnormalities, and ptosis. The genes UBR4 and CAPZB are considered the most likely candidate genes for the features of this new syndrome.

Comparative Analysis of CPI-Motif Regulation of Biochemical Functions of Actin Capping Protein.

The heterodimeric actin capping protein (CP) is regulated by a set of proteins that contain CP-interacting (CPI) motifs. Outside of the CPI motif, the sequences of these proteins are unrelated and distinct. The CPI motif and surrounding sequences are conserved within a given protein family, when compared to those of other CPI-motif protein families. Using biochemical assays with purified proteins, we compared the ability of CPI-motif-containing peptides from different protein families (a) to bind to CP, (b) to allosterically inhibit barbed-end capping by CP, and (c) to allosterically inhibit interaction of CP with V-1, another regulator of CP. We found large differences in potency among the different CPI-motif-containing peptides, and the different functional assays showed different orders of potency. These biochemical differences among the CPI-motif peptides presumably reflect interactions between CP and CPI-motif peptides involving amino acid residues that are conserved but are not part of the strictly defined consensus, as it was originally identified in comparisons of sequences of CPI motifs across all protein families [Hernandez-Valladares, M., et al. (2010) Structural characterization of a capping protein interaction motif defines a family of actin filament regulators. Nat. Struct. Mol. Biol. 17, 497-503; Bruck, S., et al. (2006) Identification of a Novel Inhibitory Actin-capping Protein Binding Motif in CD2-associated Protein. J. Biol. Chem. 281, 19196-19203]. These biochemical differences may be important for conserved distinct functions of CPI-motif protein families in cells with respect to the regulation of CP activity and actin assembly near membranes.

Cancer Stem-Cell Marker CD44v9-Positive Cells Arise From Helicobacter pylori-Infected CAPZA1-Overexpressing Cells.

BACKGROUND & AIMS: CD44 variant 9 (CD44v9)-positive cancer stem-like cells strongly contribute to the development and recurrence of gastric cancer. However, the origin of CD44v9-positive cells is uncertain. METHODS: CD44v9, ß-catenin, and epithelial splicing regulatory protein 1 signals were assessed by real-time reverse-transcription polymerase chain reaction, immunoblot analysis, or immunofluorescence microscopy. Capping actin protein of muscle Z-line α subunit 1 (CAPZA1) expression was assessed by immunoblot analysis or immunohistochemical analysis of Mongolian gerbils' gastric mucosa or human biopsy specimens. Levels of oxidative stress were assessed by measuring malondialdehyde and protein carbonylation. Histone H3 acetylation levels in the CAPZA1 proximal promoter region were measured by using chromatin immunoprecipitation analysis with an antibody against the acetylated histone H3 in human gastric carcinoma cell line (AGS) cells. RESULTS: CD44v9 is expressed in CAPZA1-overexpressing cells in human gastric cancer tissues. CAPZA1 overexpression enhanced expression of ß-catenin, which is a transcription factor for CD44, and epithelial splicing regulatory protein 1, which increases alternative splicing of CD44 to generate CD44v9. CAPZA1-overexpressing cells after cytotoxin-associated gene A accumulation showed CD44v9 expression by inducing nuclear accumulation of ß-catenin, concomitant with the enhancement of expression of Sal-like protein 4 and Krüppel-like factor 5, which encode reprogramming factors. Oxidative stress increased the CAPZA1 expression in AGS cells through the enhancement of histone H3 acetylation of CAPZA1 promoter. CAPZA1 expression was increased depending on oxidative stress in H pylori-infected gastric mucosa. CONCLUSIONS: CD44v9 expression is evoked from CAPZA1-overexpressing cells after accumulation of cytotoxin-associated gene A. Our findings provide important insights into the mechanisms underlying the development of CD44v9-positive cells.

Force-history dependence and cyclic mechanical reinforcement of actin filaments at the single molecular level.

The actin cytoskeleton is subjected to dynamic mechanical forces over time and the history of force loading may serve as mechanical preconditioning. While the actin cytoskeleton is known to be mechanosensitive, the mechanisms underlying force regulation of actin dynamics still need to be elucidated. Here, we investigated actin depolymerization under a range of dynamic tensile forces using atomic force microscopy. Mechanical loading by cyclic tensile forces induced significantly enhanced bond lifetimes and different force-loading histories resulted in different dissociation kinetics in G-actin-G-actin and G-actin-F-actin interactions. Actin subunits at the two ends of filaments formed bonds with distinct kinetics under dynamic force, with cyclic mechanical reinforcement more effective at the pointed end compared to that at the barbed end. Our data demonstrate force-history dependent reinforcement in actin-actin bonds and polarity of the actin depolymerization kinetics under cyclic tensile forces. These properties of actin may be important clues to understanding regulatory mechanisms underlying actin-dependent mechanotransduction and mechanosensitive cytoskeletal dynamics.This article has an associated First Person interview with the first author of the paper.

CAPZA1 determines the risk of gastric carcinogenesis by inhibiting Helicobacter pylori CagA-degraded autophagy.

Helicobacter pylori-derived CagA, a type IV secretion system effector, plays a role as an oncogenic driver in gastric epithelial cells. However, upon delivery into gastric epithelial cells, CagA is usually degraded by macroautophagy/autophagy. Hence, the induction of autophagy in H. pylori-infected epithelial cells is an important host-protective ability against gastric carcinogenesis. However, the mechanisms by which autophagosome-lysosome fusion is regulated, are unknown. Here, we report that enhancement of LAMP1 (lysosomal associated membrane protein 1) expression is necessary for autolysosome formation. LAMP1 expression is induced by nuclear translocated LRP1 (LDL receptor related protein 1) intracellular domain (LRP1-ICD) binding to the proximal LAMP1 promoter region. Nuclear translocation of LRP1-ICD is enhanced by H. pylori infection. In contrast, CAPZA1 (capping actin protein of muscle Z-line alpha subunit 1) inhibits LAMP1 expression via binding to LRP1-ICD in the nuclei. The binding of CAPZA1 to LRP1-ICD prevents LRP1-ICD binding to the LAMP1 proximal promoter. Thus, in CAPZA1-overexpressing gastric epithelial cells infected with H. pylori, autolysosome formation is inhibited and CagA escapes autophagic degradation. These findings identify CAPZA1 as a novel negative regulator of autolysosome formation and suggest that deregulation of CAPZA1 expression leads to increased risk of gastric carcinogenesis. Abbreviations: CagA: cytotoxin-associated gene A; CAPZA1: capping actin protein of muscle Z-line alpha subunit 1; ChIP: chromatin immunoprecipitation; GTF2I: general transcription factor IIi; HDAC: histone deacetylase; LAMP1: lysosomal associated membrane protein 1; LRP1: LDL receptor related protein 1; LRP1-ICD: CagA intracellular domain; qPCR: quantitative polymerase chain reaction; VacA: vacuolating cytotoxin.

Heterodimeric capping protein is required for stereocilia length and width regulation.

Control of the dimensions of actin-rich processes like filopodia, lamellipodia, microvilli, and stereocilia requires the coordinated activity of many proteins. Each of these actin structures relies on heterodimeric capping protein (CAPZ), which blocks actin polymerization at barbed ends. Because dimension control of the inner ear's stereocilia is particularly precise, we studied the CAPZB subunit in hair cells. CAPZB, present at ∼100 copies per stereocilium, concentrated at stereocilia tips as hair cell development progressed, similar to the CAPZB-interacting protein TWF2. We deleted Capzb specifically in hair cells using Atoh1-Cre, which eliminated auditory and vestibular function. Capzb-null stereocilia initially developed normally but later shortened and disappeared; surprisingly, stereocilia width decreased concomitantly with length. CAPZB2 expressed by in utero electroporation prevented normal elongation of vestibular stereocilia and irregularly widened them. Together, these results suggest that capping protein participates in stereocilia widening by preventing newly elongating actin filaments from depolymerizing.

An RNAi Screen in a Novel Model of Oriented Divisions Identifies the Actin-Capping Protein Z ß as an Essential Regulator of Spindle Orientation.

Oriented cell divisions are controlled by a conserved molecular cascade involving Gαi, LGN, and NuMA. We developed a new cellular model of oriented cell divisions combining micropatterning and localized recruitment of Gαi and performed an RNAi screen for regulators acting downstream of Gαi. Remarkably, this screen revealed a unique subset of dynein regulators as being essential for spindle orientation, shedding light on a core regulatory aspect of oriented divisions. We further analyze the involvement of one novel regulator, the actin-capping protein CAPZB. Mechanistically, we show that CAPZB controls spindle orientation independently of its classical role in the actin cytoskeleton by regulating the assembly, stability, and motor activity of the dynein/dynactin complex at the cell cortex, as well as the dynamics of mitotic microtubules. Finally, we show that CAPZB controls planar divisions in vivo in the developing neuroepithelium. This demonstrates the power of this in cellulo model of oriented cell divisions to uncover new genes required in spindle orientation in vertebrates.

Replication confirms the association of loci in FOXE1, PDE8B, CAPZB and PDE10A with thyroid traits: a Genetics of Diabetes Audit and Research Tayside study (GoDARTS).

OBJECTIVE: Replication of associations in genome-wide association studies is desirable to ensure that such signals are potentially clinically meaningful. This study aimed to replicate associations of selected single-nucleotide polymorphisms (SNPs) with hypothyroidism and serum thyroid-stimulating hormone (TSH) using electronic medical records (EMRs). PATIENTS AND METHODS: A cross-sectional study was carried out among patients of European Caucasian ethnicity from the Genetics of Diabetes Audit and Research Tayside recruited in Tayside (Scotland, UK). EMRs (biochemistry, prescribing, hospital admissions and demographics) were used to ascertain patients with hypothyroidism and their controls as well as average serum TSH concentration, and linked to genetic biobank data. Genetic tests of association were performed using logistic and linear regression models. RESULTS: We analysed 1703 cases of hypothyroidism and 9457 controls. All four SNPs located on chromosome 9 at FOXE1 were associated with hypothyroidism with similar effect estimates (odds ratio=0.75-0.76, P<5e-08). Also, loci on chromosomes 1 (PTPN22), six (HLA-E/HLA-C) and 12 (SH2B3) were replicated. For serum TSH, we confirmed 12 SNPs previously reported at PDE8B, CAPZB, PDE10A, LOC105371356, NR3C2, VEGFA, IGFBP5, INSR, PRDM11, NFIA, ITPK1 and ABO. Overall, these SNPs accounted for 6.8% of the serum TSH variation (P<1e-04). CONCLUSION: EMRs linked to genomic data in large populations enable validation of genome-wide association studies discoveries without additional genotyping costs. Our replication confirmed at genome-wide significance the association of loci at FOXE1 with hypothyroidism, and PDE8B, CAPZB and PDE10A with serum TSH. A total of 12 SNPs seemed to explain nearly 7% of the serum TSH variation.

Actin cortex architecture regulates cell surface tension.

Animal cell shape is largely determined by the cortex, a thin actin network underlying the plasma membrane in which myosin-driven stresses generate contractile tension. Tension gradients result in local contractions and drive cell deformations. Previous cortical tension regulation studies have focused on myosin motors. Here, we show that cortical actin network architecture is equally important. First, we observe that actin cortex thickness and tension are inversely correlated during cell-cycle progression. We then show that the actin filament length regulators CFL1, CAPZB and DIAPH1 regulate mitotic cortex thickness and find that both increasing and decreasing thickness decreases tension in mitosis. This suggests that the mitotic cortex is poised close to a tension maximum. Finally, using a computational model, we identify a physical mechanism by which maximum tension is achieved at intermediate actin filament lengths. Our results indicate that actin network architecture, alongside myosin activity, is key to cell surface tension regulation.

Genetic analysis of VCP and WASH complex genes in a German cohort of sporadic ALS-FTD patients.

Mutations of the human valosin-containing protein, p97 (VCP) and Wiskott-Aldrich syndrome protein and SCAR homolog (WASH) complex genes cause motor neuron and cognitive impairment disorders. Here, we analyzed a cohort of German patients with sporadic amyotrophic lateral sclerosis and frontotemporal lobar degeneration comorbidity (ALS/FTD) for VCP and WASH complex gene mutations. Next-generation panel sequencing of VCP, WASH1, FAM21C, CCDC53, SWIP, strumpellin, F-actin capping protein of muscle Z-line alfa 1 (CAPZA1), and CAPZB genes was performed in 43 sporadic ALS/FTD patients. Subsequent analyses included Sanger sequencing, in silico analyses, real-time PCR, and CCDC53 immunoblotting. We identified 1 patient with the heterozygous variant c.26C>T in CAPZA1, predicted to result in p.Ser9Leu, and a second with the heterozygous start codon variant c.2T>C in CCDC53. In silico analysis predicted structural changes in the N-terminus of CAPZα1, which may interfere with CAPZα:CAPZß dimerization. Though the translation initiation codon of CCDC53 is mutated, real-time PCR and immunoblotting did neither reveal any evidence for a CCDC53 haploinsufficiency nor for aberrant CCDC53 protein species. Moreover, a disease-causing C9orf72 repeat expansion mutation was later on identified in this patient. Thus, with the exception of a putatively pathogenic heterozygous c.26C>T CAPZA1 variant, our genetic analysis did not reveal mutations in VCP and the remaining WASH complex subunits.

Tankyrase-Binding Protein TNKS1BP1 Regulates Actin Cytoskeleton Rearrangement and Cancer Cell Invasion.

Tankyrase, a PARP that promotes telomere elongation and Wnt/ß-catenin signaling, has various binding partners, suggesting that it has as-yet unidentified functions. Here, we report that the tankyrase-binding protein TNKS1BP1 regulates actin cytoskeleton and cancer cell invasion, which is closely associated with cancer progression. TNKS1BP1 colocalized with actin filaments and negatively regulated cell invasion. In TNKS1BP1-depleted cells, actin filament dynamics, focal adhesion, and lamellipodia ruffling were increased with activation of the ROCK/LIMK/cofilin pathway. TNKS1BP1 bound the actin-capping protein CapZA2. TNKS1BP1 depletion dissociated CapZA2 from the cytoskeleton, leading to cofilin phosphorylation and enhanced cell invasion. Tankyrase overexpression increased cofilin phosphorylation, dissociated CapZA2 from cytoskeleton, and enhanced cell invasion in a PARP activity-dependent manner. In clinical samples of pancreatic cancer, TNKS1BP1 expression was reduced in invasive regions. We propose that the tankyrase-TNKS1BP1 axis constitutes a posttranslational modulator of cell invasion whose aberration promotes cancer malignancy. Cancer Res; 77(9); 2328-38. ©2017 AACR.

CAPZA1 modulates EMT by regulating actin cytoskeleton remodelling in hepatocellular carcinoma.

BACKGROUND: Epithelial-mesenchymal transition (EMT) elicits dramatic changes, including cytoskeleton remodelling as well as changes in gene expression and cellular phenotypes. During this process, actin filament assembly plays an important role in maintaining the morphology and movement of tumour cells. Capping protein, a protein complex referred to as CapZ, is an actin-binding complex that can regulate actin cytoskeleton remodelling. CAPZA1 is the α1 subunit of this complex, and we hypothesized that CAPZA1 regulates EMT through the regulation of actin filaments assembly, thus reducing the metastatic ability of hepatocellular carcinoma (HCC) cells. METHODS: Immunohistochemistry was used to detect CAPZA1 expression in 129 HCC tissues. Western blotting and qPCR were used to detect CAPZA1, EMT markers and EMT transcription factors in HCC cells. Transwell migration and invasion assays were performed to observe the migration and invasion of HCC cells. Cell Counting Kit-8 (CCK-8) was used to detect the proliferation of HCC cells. Immunoprecipitation was used to detect the interaction between CAPZA1 and actin filaments. Finally, a small animal magnetic resonance imager (MRI) was used to observe metastases in HCC cell xenografts in the liver. RESULTS: CAPZA1 expression levels were negatively correlated with the biological characteristics of primary HCC and patient prognosis. CAPZA1 expression was negatively correlated with the migration and invasion of HCC cells. CAPZA1 down regulation promoted the migration and invasion of HCC cells. Conversely, CAPZA1 overexpression significantly inhibited the migration and invasion of HCC cells. Moreover, CAPZA1 expression levels were correlated with the expression of the EMT markers E-cadherin, N-cadherin and Vimentin. Furthermore, the expression of Snail1 and ZEB1 were negatively correlated with CAPZA1 expression levels. Similarly, CAPZA1 significantly inhibited intrahepatic metastases of HCC cells in an orthotopic transplantation tumour model. CONCLUSIONS: CAPZA1 inhibits EMT in HCC cells by regulating actin cytoskeleton remodelling, thereby reducing the metastatic ability of the cells. Together, our data suggest that CAPZA1 could be a useful biomarker for clinical determination of the prognosis of HCC patients.

Morphological change of skin fibroblasts induced by UV Irradiation is involved in photoaging.

Human dermal fibroblasts (HDFs) are typically flattened or extensible shaped and play a critical role in the metabolism of extracellular matrix components. As the properties of fibroblasts in the dermis are considered to be influenced by their morphology, we investigated the morphological changes induced in fibroblasts by ultraviolet (UV) irradiation as well as the relationship between these changes and collagen metabolism. In this study, we showed that UVA exposure induced morphological changes and reduced collagen contents in HDFs. These morphological changes were accompanied a reduction in actin filaments and upregulation of the actin filament polymerization inhibitor, capping protein muscle Z-line ɑ1 (CAPZA1). External actin filament growth inhibitors also affected the shape of HDFs and reduced collagen levels. These results suggest that UVA exposure may inhibit the polymerization of actin filaments and induce morphological changes in skin fibroblasts. These morphological changes in fibroblasts may accelerate reductions in collagen synthesis. This mechanism may be one of the processes responsible for collagen reductions observed in photoaged skin. When natural materials that suppress these morphological changes in HDFs were evaluated, we found that an extract of Lilium 'Casa Blanca' (LCB) suppressed UVA-induced alterations in the shape of HDFs, which are typically followed by inhibition of collagen reduction. An analysis of the active compounds in LCB extract led to the identification of regaloside I, which had a structure of phenylpropanoid glycerol glucoside, as the active compound inhibiting the upregulation of CAPZA1. Therefore, inhibition of UVA-induced morphological changes in HDFs is considered to be promising way for the suppression of collagen reduction in photoaging.