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.

E-cadherin mediated lateral interactions between neighbor cells necessary for collective migration.

Collective cell movement is critical in pathological processes such as wound healing and cancer invasion. It entails complex interactions between adjacent cells and between cells-extracellular matrices. Most studies measure the migration patterns and force propagation by placing cells on flat, patterned substrates. The cooperative behavior resulting from cell-cell interactions is not well understood. We have developed a multi-channel microfluidic device that has junctional protein E-cadherin coated onto the sidewalls of the channels that enables the cells' lateral interactions with their neighbors to be studied. Our study reveals that epithelial cells rely on lateral E-cadherin-based adhesions to maintain the cohesion of the group. Cells move faster in narrower channels, but the average velocity along the channels is reduced in E-cadherin coated channels versus non-adhesive channels. We have directly measured the forces in the cross-linking protein, alpha-actinin, using FRET sensors during cell migration, and found that higher tension exists at the cell edges adjacent to the walls coated with E-cadherin, the implication being E-cadherin transmits the shear forces but does not provide a driving force for this migration.

α-Actinin-4 confers radioresistance coupled invasiveness in breast cancer cells through AKT pathway.

Acquired radioresistance accompanied with increased metastatic potential is a major hurdle in effective radiotherapy of breast cancers. However, the nature of their inter-dependence and the underlying mechanism remains largely intangible. By employing radioresistant (RR) cell lines, we herein demonstrate that MCF-7 RR cells display phenotypic and molecular alterations evocative of epithelial to mesenchymal transition (EMT) with increased traction forces and membrane ruffling culminating in boosted invasiveness. We then show that these changes can be attributed to overexpression of alpha-actinin-4 (ACTN4), with ACTN4 knockdown near-completely abrogating both radioresistance and EMT-associated changes. We further found that in MCF-7 RR cells, ACTN4 mediates the observed effects by activating AKT, and downstream AKT/GSK3ß signalling. Though ACTN4 plays a similar role in mediating radioresistance and invasiveness in MDA-MB-231 RR cells, co-immunoprecipitation studies reveal that these changes are effected through increased association with AKT and not by overexpression of AKT. Taken together, our study identifies ACTN4/AKT/GSK3ß as a novel pathway regulating radioresistance coupled invasion which can be further explored to improve the radiotherapeutic gain.

Role of α-Actinin 2 in Cytoadherence and Cytotoxicity of Trichomonas vaginalis.

Trichomonas vaginalis is a pathogen that triggers severe immune responses in hosts. T. vaginalis α-actinin 2 (Tvα-actinin 2) has been used to diagnose trichomoniasis. Tvα-actinin 2 was dissected into three parts; the N-terminal, central, and C-terminal portions of the protein (#1, #2, and #3, respectively). Western blot of these Tvα-actinin 2 proteins with pooled patients' sera indicated that #2 and #3, but not #1, reacted with those sera. Immunofluorescence assays of two different forms of T. vaginalis (trophozoites and amoeboid forms), using anti-Tvα-actinin 2 antibodies, showed localization of Tvα-actinin 2 close to the plasma membranes of the amoeboid form. Fractionation experiments indicated the presence of Tvα-actinin 2 in cytoplasmic, membrane, and secreted proteins of T. vaginalis. Binding of fluorescence-labeled Trichomonas to vaginal epithelial cells and prostate cells was decreased in the antibody blocking experiment using anti-Tvα-actinin 2 antibodies. Pretreatment of T. vaginalis with anti-rTvα-actinin 2 antibodies also resulted in reduction in its cytotoxicity. Flow cytometry, ligand-binding immunoblotting assay, and observation by fluorescence microscopy were used to detect the binding of recombinant Tvα-actinin 2 to human epithelial cell lines. Specifically, the truncated N-terminal portion of Tvα-actinin 2, Tvα-actinin 2 #1, was shown to bind directly to vaginal epithelial cells. These data suggest that α-actinin 2 is one of the virulence factors responsible for the pathogenesis of T. vaginalis by serving as an adhesin to the host cells.

Evidence for ACTN3 as a Speed Gene in Isolated Human Muscle Fibers.

PURPOSE: To examine the effect of α-actinin-3 deficiency due to homozygosity for the ACTN3 577X-allele on contractile and morphological properties of fast muscle fibers in non-athletic young men. METHODS: A biopsy was taken from the vastus lateralis of 4 RR and 4 XX individuals to test for differences in morphologic and contractile properties of single muscle fibers. The cross-sectional area of the fiber and muscle fiber composition was determined using standard immunohistochemistry analyses. Skinned single muscle fibers were subjected to active tests to determine peak normalized force (P0), maximal unloading velocity (V0) and peak power. A passive stretch test was performed to calculate Young's Modulus and hysteresis to assess fiber visco-elasticity. RESULTS: No differences were found in muscle fiber composition. The cross-sectional area of type IIa and IIx fibers was larger in RR compared to XX individuals (P<0.001). P0 was similar in both groups over all fiber types. A higher V0 was observed in type IIa fibers of RR genotypes (P<0.001) but not in type I fibers. The visco-elasticity as determined by Young's Modulus and hysteresis was unaffected by fiber type or genotype. CONCLUSION: The greater V0 and the larger fast fiber CSA in RR compared to XX genotypes likely contribute to enhanced whole muscle performance during high velocity contractions.

The non-muscle functions of actinins: an update.

α-Actinins are a major class of actin filament cross-linking proteins expressed in virtually all cells. In muscle, actinins cross-link thin filaments from adjacent sarcomeres. In non-muscle cells, different actinin isoforms play analogous roles in cross-linking actin filaments and anchoring them to structures such as cell-cell and cell-matrix junctions. Although actinins have long been known to play roles in cytokinesis, cell adhesion and cell migration, recent studies have provided further mechanistic insights into these functions. Roles for actinins in synaptic plasticity and membrane trafficking events have emerged more recently, as has a 'non-canonical' function for actinins in transcriptional regulation in the nucleus. In the present paper we review recent advances in our understanding of these diverse cell biological functions of actinins in non-muscle cells, as well as their roles in cancer and in genetic disorders affecting platelet and kidney physiology. We also make two proposals with regard to the actinin nomenclature. First, we argue that naming actinin isoforms according to their expression patterns is problematic and we suggest a more precise nomenclature system. Secondly, we suggest that the α in α-actinin is superfluous and can be omitted.

α-actinin1 and 4 tyrosine phosphorylation is critical for stress fiber establishment, maintenance and focal adhesion maturation.

In polarized, migrating cells, stress fibers are a highly dynamic network of contractile acto-myosin structures composed of bundles of actin filaments held together by actin cross-linking proteins such as α-actinins. As such, α-actinins influence actin cytoskeleton organization and dynamics and focal adhesion maturation. In response to environmental signals, α-actinins are tyrosine phosphorylated and this affects their binding to actin stress fibers; however, the cellular role of α-actinin tyrosine phosphorylation remains largely unknown. We found that non-muscle α-actinin1/4 are critical for the establishment of dorsal stress fibers and maintenance of transverse arc stress fibers. Analysis of cells genetically depleted of α-actinin1 and 4 reveals two distinct modes for focal adhesion maturation. An α-actinin1 or 4 dependent mode that uses dorsal stress fiber precursors as a template for establishing focal adhesions and their maturation, and an α-actinin-independent manner that uses transverse arc precursors to establish focal adhesions at both ends. Focal adhesions formed in the absence of α-actinins are delayed in their maturation, exhibit altered morphology, have decreased amounts of Zyxin and VASP, and reduced adhesiveness to extracellular matrix. Further rescue experiments demonstrate that the tyrosine phosphorylation of α-actinin1 at Y12 and α-actinin4 at Y265 is critical for dorsal stress fiber establishment, transverse arc maintenance and focal adhesion maturation.

Modulation of stretch-induced myocyte remodeling and gene expression by nitric oxide: a novel role for lipoma preferred partner in myofibrillogenesis.

Prolonged hemodynamic load as a result of hypertension eventually leads to maladaptive cardiac adaptation and heart failure. The signaling pathways that underlie these changes are still poorly understood. The adaptive response to mechanical load is mediated by mechanosensors that convert the mechanical stimuli into a biological response. We examined the effect of cyclic mechanical stretch on myocyte adaptation using neonatal rat ventricular myocytes with 10% (adaptive) or 20% (maladaptive) maximum strain at 1 Hz for 48 h to mimic in vivo mechanical stress. Cells were also treated with and without nitro-L-arginine methyl ester (L-NAME), a general nitric oxide synthase (NOS) inhibitor to suppress NO production. Maladaptive 20% mechanical stretch led to a significant loss of intact sarcomeres that were rescued by L-NAME (P < 0.05; n ≥ 5 cultures). We hypothesized that the mechanism was through NO-induced alteration of myocyte gene expression. L-NAME upregulated the mechanosensing proteins muscle LIM protein (MLP; by 100%; P < 0.05; n = 5 cultures) and lipoma preferred partner (LPP), a novel cardiac protein (by 80%; P < 0.05; n = 4 cultures). L-NAME also significantly altered the subcellular localization of LPP and MLP in a manner that favored growth and adaptation. These findings suggest that NO participates in stretch-mediated adaptation. The use of isoform selective NOS inhibitors indicated a complex interaction between inducible NOS and neuronal NOS isoforms regulate gene expression. LPP knockdown by small intefering RNA led to formation of α-actinin aggregates and Z bodies showing that myofibrillogenesis was impaired. There was an upregulation of E3 ubiquitin ligase (MUL1) by 75% (P < 0.05; n = 5 cultures). This indicates that NO contributes to stretch-mediated adaptation via the upregulation of proteins associated with mechansensing and myofibrillogenesis, thereby presenting potential therapeutic targets during the progression of heart failure.

Actinin-4 in keratinocytes regulates motility via an effect on lamellipodia stability and matrix adhesions.

During wound repair, epidermal cells at the edge of an injury establish front-rear polarity through orchestrated changes in their cytoskeleton and adhesion structures. The polarity and directed migration of such cells is determined by the assembly, extension, and stabilization of a lamellipodium. Actinin-4 associates with lamellipodia and has been implicated in regulating lamellipodial structure, function and assembly. To study the functions of actinin-4 in human keratinocytes, we used shRNA to generate knockdown cells and compared their motility behavior and matrix adhesion assembly to scrambled shRNA treated control keratinocytes. Actinin-4 knockdown keratinocytes lack polarity, assemble multiple lamellipodia with a 2× increased area over controls, display reduced activity of the actin remodeling protein cofilin, and fail to migrate in a directional manner. This motility defect is rescued by plating knockdown cells on preformed laminin-332 matrix. In actinin-4-knockdown keratinocytes, focal contact area is increased by 25%, and hemidesmosome proteins are mislocalized. Specifically, α6ß4 integrin localizes to large lamellipodial extensions, displays reduced dynamics, and fails to recruit its bullous pemphigoid antigen binding partners. Together, our data indicate a role for actinin-4 in regulating the steering mechanism of keratinocytes via profound effects on their matrix adhesion sites.

Role of alpha-actinin-3 in contractile properties of human single muscle fibers: a case series study in paraplegics.

A common nonsense polymorphism in the ACTN3 gene results in the absence of α-actinin-3 in XX individuals. The wild type allele has been associated with power athlete status and an increased force output in numeral studies, though the mechanisms by which these effects occur are unclear. Recent findings in the Actn3(-/-) (KO) mouse suggest a shift towards 'slow' metabolic and contractile characteristics of fast muscle fibers lacking α-actinin-3. Skinned single fibers from the quadriceps muscle of three men with spinal cord injury (SCI) were tested regarding peak force, unloaded shortening velocity, force-velocity relationship, passive tension and calcium sensitivity. The SCI condition induces an 'equal environment condition' what makes these subjects ideal to study the role of α-actinin-3 on fiber type expression and single muscle fiber contractile properties. Genotyping for ACTN3 revealed that the three subjects were XX, RX and RR carriers, respectively. The XX carrier's biopsy was the only one that presented type I fibers with a complete lack of type II(x) fibers. Properties of hybrid type II(a)/II(x) fibers were compared between the three subjects. Absence of α-actinin-3 resulted in less stiff type II(a)/II(x) fibers. The heterozygote (RX) exhibited the highest fiber diameter (0.121±0.005 mm) and CSA (0.012±0.001 mm(2)) and, as a consequence, the highest peak force (2.11±0.14 mN). Normalized peak force was similar in all three subjects (P = 0.75). Unloaded shortening velocity was highest in R-allele carriers (P<0.001). No difference was found in calcium sensitivity. The preservation of type I fibers and the absence of type II(x) fibers in the XX individual indicate a restricted transformation of the muscle fiber composition to type II fibers in response to long-term muscle disuse. Lack of α-actinin-3 may decrease unloaded shortening velocity and increase fiber elasticity.

MDM2 binding protein, a novel metastasis suppressor.

MDM2 binding protein (MTBP) is a protein that interacts with oncoprotein murine double minute (MDM2), a major inhibitor of the tumor suppressor p53. Overexpression of MTBP leads to p53-independent cell proliferation arrest, which is in turn blocked by simultaneous overexpression of MDM2. Importantly, reduced expression of MTBP in mice increases tumor metastasis and enhances migratory potential of mouse embryonic fibroblasts regardless of the presence of p53. Clinically, loss of MTBP expression in head and neck squamous cell carcinoma is associated with reduced patient survival, and is shown to serve as an independent prognostic factor when p53 is mutated in tumors. These results indicate the involvement of MTBP in suppressing tumor progression. Our recent findings demonstrate that overexpression of MTBP in human osteosarcoma cells lacking wild-type p53 inhibits metastasis, but not primary tumor growth, when cells are transplanted in femurs of immunocompromised mice. These data indicate that MTBP functions as a metastasis suppressor independent of p53 status. Furthermore, overexpression of MTBP suppresses cell migration and filopodia formation, in part, by inhibiting function of an actin crosslinking protein α-actinin-4. Thus, increasing evidence indicates the significance of MTBP in tumor progression. We summarize published results related to MTBP function and discuss caveats and future directions in this review article.

EWI-2 association with α-actinin regulates T cell immune synapses and HIV viral infection.

EWI motif-containing protein 2 (EWI-2) is a member of the Ig superfamily that links tetraspanin-enriched microdomains to the actin cytoskeleton. We found that EWI-2 colocalizes with CD3 and CD81 at the central supramolecular activation cluster of the T cell immune synapse. Silencing of the endogenous expression or overexpression of a cytoplasmic truncated mutant of EWI-2 in T cells increases IL-2 secretion upon Ag stimulation. Mass spectrometry experiments of pull-downs with the C-term intracellular domain of EWI-2 revealed the specific association of EWI-2 with the actin-binding protein α-actinin; this association was regulated by PIP2. α-Actinin regulates the immune synapse formation and is required for efficient T cell activation. We extended these observations to virological synapses induced by HIV and found that silencing of either EWI-2 or α-actinin-4 increased cell infectivity. Our data suggest that the EWI-2-α-actinin complex is involved in the regulation of the actin cytoskeleton at T cell immune and virological synapses, providing a link between membrane microdomains and the formation of polarized membrane structures involved in T cell recognition.

Dynamic and structural signatures of lamellar actomyosin force generation.

The regulation of cellular traction forces on the extracellular matrix is critical to cell adhesion, migration, proliferation, and differentiation. Diverse lamellar actin organizations ranging from contractile lamellar networks to stress fibers are observed in adherent cells. Although lamellar organization is thought to reflect the extent of cellular force generation, understanding of the physical behaviors of the lamellar actin cytoskeleton is lacking. To elucidate these properties, we visualized the actomyosin dynamics and organization in U2OS cells over a broad range of forces. At low forces, contractile lamellar networks predominate and force generation is strongly correlated to actomyosin retrograde flow dynamics with nominal change in organization. Lamellar networks build ∼60% of cellular tension over rapid time scales. At high forces, reorganization of the lamellar network into stress fibers results in moderate changes in cellular tension over slower time scales. As stress fibers build and tension increases, myosin band spacing decreases and α-actinin bands form. On soft matrices, force generation by lamellar networks is unaffected, whereas tension-dependent stress fiber assembly is abrogated. These data elucidate the dynamic and structural signatures of the actomyosin cytoskeleton at different levels of tension and set a foundation for quantitative models of cell and tissue mechanics.

Alpha-actinin 1 and alpha-actinin 4: contrasting roles in the survival, motility, and RhoA signaling of astrocytoma cells.

Alpha-actinin is a prominent actin filament associated protein for which different isoforms exist. Here, we have examined whether the two highly homologous non-muscle alpha-actinin isoforms 1 and 4 exhibit functional differences in astrocytoma cells. The protein levels of these isoforms were differentially regulated during the development and progression of astrocytomas, as alpha-actinin 1 was higher in astrocytomas compared to normal brains whereas alpha-actinin 4 was elevated in high-grade astrocytomas compared to normal brains and low grade astrocytomas. RNAi demonstrated contrasted contributions of alpha-actinin 1 and 4 to the malignant behavior of U-373, U-87 and A172 astrocytoma cells. While alpha-actinin 1 appeared to favor the expansion of U-373, U-87 and A172 astrocytoma cell populations, alpha-actinin 4 played this role only for U-373 cells. On the other hand, downregulation of alpha-actinin 4, but not 1, reduced cell motility, adhesion, cortical actin, and RhoA levels. Finally, in the three astrocytoma cell lines examined, alpha-actinin 1 and 4 had contrasted biochemical properties as alpha-actinin 4 was significantly more abundant in the actin cytoskeleton than alpha-actinin 1. Collectively, these findings suggest that alpha-actinin 1 and 4 are differentially regulated during the development and progression of astrocytomas because each of these isoforms uniquely contributes to distinct malignant properties of astrocytoma cells.

The role of actinin-4 in bladder cancer invasion.

OBJECTIVES: To examine actinin-4 expression levels in bladder cancer, in particular its levels during cellular growth and invasion. Actinin-4 is an actin-binding protein that is associated with cell motility and cancer metastasis. METHODS: Relative messenger ribonucleic acid (mRNA) and protein expression of actinin-4 in normal bladder and bladder cancer cell lines was determined by quantitative real-time polymerase chain reaction and Western blot analysis. Actinin-4 expression was also localized in bladder cancer cells and tissues using immunohistochemistry. The growth and invasion activity of bladder cancer cells was evaluated using cell growth and in vitro cell invasion assays, and compared with that of bladder cancer cells treated with actinin-4 small interfering ribonucleic acids. RESULTS: Actinin-4 mRNA and protein levels were elevated in bladder cancer cells that are known to exhibit increased growth and invasion activity. Protein expression was predominantly observed in the cytoplasm of the invasive bladder cancer cells and tissues. Treatment of bladder cancer cell lines with actinin-4 small interfering ribonucleic acids suppressed the invasive potential of the cells, but did not alter their growth. CONCLUSIONS: The current study demonstrates that actinin-4 mRNA and protein levels are elevated in bladder cancer cells lines that exhibit increased growth and invasion activity. In addition, actinin-4 knockdown inhibited invasion of bladder cancer cells, but did not alter their growth. In conclusion, we hypothesize that the accumulation of actinin-4 in the cell cytoplasm is related to an increased susceptibility of tumor invasion and metastasis.

Intermediate filament protein synemin contributes to the migratory properties of astrocytoma cells by influencing the dynamics of the actin cytoskeleton.

We have shown previously that, in astrocytoma cells, synemin is present at the leading edge, an unusual localization for an intermediate filament (IF) protein. Here, we report that synemin down-regulation with specific small hairpin RNAs (shRNAs) sharply decreased the migration of astrocytoma cells. The presence of synemin at the leading edge also correlated with a high migratory potential, as shown by comparing astrocytoma cells to carcinoma cells without synemin at the leading edge. Synemin-silenced astrocytoma cells were smaller and spread more slowly than controls. In addition, synemin silencing reduced proliferation without increasing apoptosis. The adhesion to substratum and distribution of vinculin in focal contacts of synemin-silenced astrocytoma cells were similar to those of controls. Synemin-silenced cells, however, exhibited a reduction in the amount of filamentous (F) -actin and of alpha-actinin, but not of vinculin, associated with F-actin. Altogether, these results demonstrate that synemin is important for the malignant behavior of astrocytoma cells and that it contributes to the high motility of these cells by modulating the dynamics of alpha-actinin and actin.

HER2/Neu (ErbB2) signaling to Rac1-Pak1 is temporally and spatially modulated by transforming growth factor beta.

In HER2 (ErbB2)-overexpressing cells, transforming growth factor beta (TGF-beta), via activation of phosphoinositide-3 kinase (PI3K), recruits actin and actinin to HER2, which then colocalizes with Vav2, activated Rac1, and Pak1 at cell protrusions. This results in prolonged Rac1 activation, enhanced motility and invasiveness, Bad phosphorylation, uncoupling of Bad/Bcl-2, and enhanced cell survival. The recruitment of the HER2/Vav2/Rac1/Pak1/actin/actinin complex to lamellipodia was abrogated by actinin siRNAs, dominant-negative (dn) p85, gefitinib, and dn-Rac1 or dn-Pak1, suggesting that the reciprocal interplay of PI3K, HER2 kinase, and Rac GTPases with the actin cytoskeleton is necessary for TGF-beta action in oncogene-overexpressing cells. Thus, by recruiting the actin skeleton, TGF-beta "cross-links" this signaling complex at cell lamellipodia; this prolongs Rac1 activation and increases metastatic properties and survival of HER2-overexpressing cells.

Myomaxin is a novel transcriptional target of MEF2A that encodes a Xin-related alpha-actinin-interacting protein.

The physiological targets regulated by MEF2 in striated muscle are not completely known. Several recent studies have identified novel downstream target genes and shed light on the global transcriptional network regulated by MEF2 in muscle. In our continuing effort to identify novel, downstream pathways controlled by MEF2, we have used mef2a knock-out mice to find those genes dependent on MEF2A transcriptional activity. Here, we describe the characterization of a direct, downstream target gene for the MEF2A transcription factor encoding a large, muscle-specific protein that localizes to the Z-disc/costameric region in striated muscle. This gene, called myomaxin, was identified as a gene markedly down-regulated in MEF2A knock-out hearts. Myomaxin is the mouse ortholog of a partial human cDNA of unknown function named cardiomyopathy associated gene 3 (CMYA3). Myomaxin is expressed as a single, large transcript of approximately 11 kilobases in adult heart and skeletal muscle with an open reading frame of 3,283 amino acids. The protein encoded by the myomaxin gene is related to the actin-binding protein Xin and interacts with the sarcomeric Z-disc protein, alpha-actinin-2. Our findings demonstrate that Myomaxin functions directly downstream of MEF2A at the peripheral Z-disc complex in striated muscle potentially playing a role in regulating cytoarchitectural integrity.