Alterations in VASP phosphorylation and profilin1 and cofilin1 expression in hyperoxic lung injury and BPD.

BACKGROUND: Hyperoxia is a frequently employed therapy for prematurely born infants, induces lung injury and contributes to development of bronchopulmonary dysplasia (BPD). BPD is characterized by decreased cellular proliferation, cellular migration, and failure of injury repair systems. Actin binding proteins (ABPs) such as VASP, cofilin1, and profilin1 regulate cell proliferation and migration via modulation of actin dynamics. Lung mesenchymal stem cells (L-MSCs) initiate repair processes by proliferating, migrating, and localizing to sites of injury. These processes have not been extensively explored in hyperoxia induced lung injury and repair. METHODS: ABPs and CD146+ L-MSCs were analyzed by immunofluorescence in human lung autopsy tissues from infants with and without BPD and by western blot in lung tissue homogenates obtained from our murine model of newborn hyperoxic lung injury. RESULTS: Decreased F-actin content, ratio of VASPpS157/VASPpS239, and profilin 1 expression were observed in human lung tissues but this same pattern was not observed in lungs from hyperoxia-exposed newborn mice. Increases in cofilin1 expression were observed in both human and mouse tissues at 7d indicating a dysregulation in actin dynamics which may be related to altered growth. CD146 levels were elevated in human and newborn mice tissues (7d). CONCLUSION: Altered phosphorylation of VASP and expression of profilin 1 and cofilin 1 in human tissues indicate that the pathophysiology of BPD involves dysregulation of actin binding proteins. Lack of similar changes in a mouse model of hyperoxia exposure imply that disruption in actin binding protein expression may be linked to interventions or morbidities other than hyperoxia alone.

Dendritic spine loss caused by AlCl3 is associated with inhibition of the Rac 1/cofilin signaling pathway.

Aluminum (Al) has neurotoxicity that can result in cognitive dysfunction. Hippocampal dendritic spine loss is a pathological characteristic of cognitive dysfunction. Our previous study reported that Al exposure caused dendritic spine loss in the hippocampus, but the underlying mechanism remains unclear. In this study, rats were orally administered 50, 150 or 450 mg/kg of AlCl3 for 90 days. The dendritic spine density of the CA1 and DG regions was detected by Golgi-Cox staining. The F-actin/G-actin ratio, the expression of drebrin A and the components of the Rac 1/cofilin pathway were measured in the hippocampus. The results obtained showed that AlCl3 caused dendritic spine loss and decreased the F-actin/G-actin ratio. In addition, it was found that AlCl3 downregulated the expression of Rac 1, p-PAK, p-LIMK, p-cofilin and drebrin A and upregulated cofilin expression. Altogether, these results demonstrated that Al inactivated the Rac 1/cofilin pathway by inhibiting the phosphorylation of cofilin and the polymerization of F-actin, resulting in dendritic spine loss in the hippocampus.

Study on the significance of Cofilin 1 overexpression in human bladder cancer.

PURPOSE: Cofilin 1 is a type of cytoskeletal protein. The overexpression of this gene has been regarded to hold a special relationship with the development and progress of some cancers. However, the detailed position of Cofilin 1 in human bladder cancer has not been investigated intensively. METHODS: In this study, we mainly explored the relationship between human bladder cancer and the expression of Cofilin 1. The expression of Cofilin 1 in bladder cancer tissues and paracancerous tissues of patients was evaluated with quantitative polymerase chain reaction, Western blot, and immunohistochemical staining. Downregulation of Cofilin 1 expression model was established with siRNA in human RT4 bladder cancer cell line, and the changing cell viability was analyzed to determine the role of Cofilin 1 in human bladder cancer. RESULTS: Our results showed that the expression of Cofilin 1 was much higher in both RNA level and protein level in human bladder cancer tissues than paracancerous tissues for 3 patients. Downregulation of Cofilin 1 expression could inhibit cell proliferation, cell migration, cell adhesion, and colony formation ability, and increase the percentage of cell apoptosis in RT4 cells. CONCLUSIONS: Our study indicates that Cofilin 1 holds an important position in the development and progression of human bladder cancer, and this gene might become a novel target in the diagnosis and treatment of human bladder cancer.

MicroRNA-138 inhibits migration and invasion of non-small cell lung cancer cells by targeting LIMK1.

MicroRNA (miR)-138 has previously been demonstrated to have a suppressive role in numerous types of human cancer, including non-small cell lung cancer (NSCLC). LIM domain kinase 1 (LIMK1) is a serine/threonine kinase that regulates actin polymerization via phosphorylation and inactivation of cofilin. Previous studies have reported that LIMK1 is associated with NSCLC; however, the underlying regulatory mechanism of LIMK1, and the association between LIMK1 and miR­138 in NSCLC cells, remains largely unknown. The present study aimed to reveal the regulatory roles of miR­138 and LIMK1 in NSCLC cell migration and invasion. Reverse transcription­quantitative polymerase chain reaction and western blot analysis were used to examine the mRNA and protein expression levels. Transwell and wound healing assays were conducted to determine cell invasion and migration. A luciferase reporter assay was used to determine the target association between miR­138 and LIMK1. The present study demonstrated that miR­138 was markedly downregulated in NSCLC tissues and cell lines, whereas the expression levels of LIMK1 were significantly upregulated. LIMK1 was further identified as a direct target of miR­138 in NSCLC H460 cells. Furthermore, overexpression of miR­138 significantly inhibited the protein expression of LIMK1, whereas knockdown of miR­138 upregulated the protein expression of LIMK1 in H460 cells. In addition, overexpression of miR­138 significantly inhibited the migration and invasion of NSCLC cells; however, overexpression of LIMK1 significantly promoted NSCLC cell migration and invasion. An investigation into the underlying molecular mechanism revealed that overexpression of miR­138 significantly decreased cofilin signaling activity, whereas knockdown of miR­138 notably enhanced cofilin signaling activity. In conclusion, the present study suggests that miR­138 may inhibit the migration and invasion of NSCLC cells by targeting the LIMK1/cofilin signaling pathway. Therefore, miR-138/LIMK1/cofilin may be considered a potential therapeutic target for the treatment of NSCLC.

Inhibition of the Rac1-WAVE2-Arp2/3 signaling pathway promotes radiosensitivity via downregulation of cofilin-1 in U251 human glioma cells.

The Ras-related C3 botulinum toxin substrate 1 (Rac1)-WASP-family verprolin-homologous protein-2 (WAVE2)-actin-related protein 2/3 (Arp2/3) signaling pathway has been identified to be involved in cell migration and invasion in various types of cancer cell. Cofilin­1 (CFL­1), which is regulated by the Rac1­WAVE2­Arp2/3 signaling pathway, may promote radioresistance in glioma. Therefore, the present study aimed to investigate the potential role of the Rac1­WAVE2­Arp2/3 signaling pathway in radioresistance in U251 human glioma cells and elucidate its affect on CFL­1 expression. Western blot analysis was performed to evaluate the protein expression of CFL­1. In the present study, Rac1 was inhibited by NSC 23766, WAVE2 was inhibited by transfection with short hairpin (sh)RNA­WAVE2 using Lipofectamine™ 2000 and Arp2/3 was inhibited by CK­666. Cell viability was measured using the 3­(4,5­dimethylthiazol­2­yl)-2,5­diphenyltetrazolium bromide assay, the cell migration ability was examined by a wound­healing assay, and the cell invasion ability was assessed using a Transwell culture chamber system. The results showed that inhibition of the Rac1­WAVE2­Arp2/3 signaling pathway using NSC 23766, shRNA­WAVE2 or CK­666 reduced the cell viability, migration and invasion abilities in U251 human glioma cells, concordant with a reduced expression of CFL­1. Furthermore, the expression of CFL­1 was significantly increased in radioresistant U251 glioma cells when compared with normal U251 human glioma cells. These findings indicate that inhibition of the Rac1­WAVE2­Arp2/3 signaling pathway may promote radiosensitivity, which may partially result from the downregulation of CFL­1 in U251 human glioma cells.

CFLAP1 and CFLAP2 Are Two bHLH Transcription Factors Participating in Synergistic Regulation of AtCFL1-Mediated Cuticle Development in Arabidopsis.

The cuticle is a hydrophobic lipid layer covering the epidermal cells of terrestrial plants. Although many genes involved in Arabidopsis cuticle development have been identified, the transcriptional regulation of these genes is largely unknown. Previously, we demonstrated that AtCFL1 negatively regulates cuticle development by interacting with the HD-ZIP IV transcription factor HDG1. Here, we report that two bHLH transcription factors, AtCFL1 associated protein 1 (CFLAP1) and CFLAP2, are also involved in AtCFL1-mediated regulation of cuticle development. CFLAP1 and CFLAP2 interact with AtCFL1 both in vitro and in vivo. Overexpression of either CFLAP1 or CFLAP2 led to expressional changes of genes involved in fatty acids, cutin and wax biosynthesis pathways and caused multiple cuticle defective phenotypes such as organ fusion, breakage of the cuticle layer and decreased epicuticular wax crystal loading. Functional inactivation of CFLAP1 and CFLAP2 by chimeric repression technology caused opposite phenotypes to the CFLAP1 overexpressor plants. Interestingly, we find that, similar to the transcription factor HDG1, the function of CFLAP1 in cuticle development is dependent on the presence of AtCFL1. Furthermore, both HDG1 and CFLAP1/2 interact with the same C-terminal C4 zinc finger domain of AtCFL1, a domain that is essential for AtCFL1 function. These results suggest that AtCFL1 may serve as a master regulator in the transcriptional regulation of cuticle development, and that CFLAP1 and CFLAP2 are involved in the AtCFL1-mediated regulation pathway, probably through competing with HDG1 to bind to AtCFL1.

MicroRNA-429 inhibits the migration and invasion of colon cancer cells by targeting PAK6/cofilin signaling.

MicroRNAs (miRs), a class of non-coding RNAs 18-25 nucleotides in length, can lead to mRNA degradation or inhibit protein translation by directly binding to the 3'-untranslational region (UTR) of their target mRNAs. The deregulation of miR-429 has been suggested to be involved in the development and progression of colon cancer. However, the detailed molecular mechanism involved remains to be determined. The aim of the present study was to investigate the role of miR-429 in the regulation of migration and invasion of colon cancer cells using RT-qPCR and western blotting. The results showed that the expression of miR-429 was reduced in colon cancer cell lines, when compared to a normal colon epithelial cell line. Treatment with DNA demethylation agent 5-aza-2'-deoxycytidine and histone deacetylase inhibitor phenylbutyrate (PBA), or transfection with the pre-miR-429 lentivirus plasmid led to the upregulation of miR-429 expression, as well as inhibition of migration and invasion in colon cancer cells. Investigation of the molecular mechanism showed that PAK6 was a novel target of miR-429, and the expression of PAK6 was upregulated in colon cancer tissues and cell lines, and was negatively regulated by miR-429 in colon cancer cells. Moreover, the cofilin signaling acted as a downstream effector of miR-429 in colon cancer cells. In conclusion, the results of the present study suggested that miR-429 inhibits the migration and invasion of colon cancer cells, partly at least, by mediating the expression of PAK6, as well as the activity of cofilin signaling. Therefore, miR-429 is as a potential molecular target for the treatment of colon cancer.

Morphotype transition and sexual reproduction are genetically associated in a ubiquitous environmental pathogen.

Sexual reproduction in an environmental pathogen helps maximize its lineage fitness to changing environment and the host. For the fungal pathogen Cryptococcus neoformans, sexual reproduction is proposed to have yielded hyper virulent and drug resistant variants. The life cycle of this pathogen commences with mating, followed by the yeast-hypha transition and hyphal growth, and it concludes with fruiting body differentiation and sporulation. How these sequential differentiation events are orchestrated to ensure developmental continuality is enigmatic. Here we revealed the genetic network of the yeast-to-hypha transition in Cryptococcus by analyzing transcriptomes of populations with a homogeneous morphotype generated by an engineered strain. Among this network, we found that a Pumilio-family protein Pum1 and the matricellular signal Cfl1 represent two major parallel circuits directing the yeast-hypha transition. Interestingly, only Pum1 coordinates the sequential morphogenesis events during a-α bisexual and α unisexual reproduction. Pum1 initiates the yeast-to-hypha transition, partially through a novel filament-specific secretory protein Fas1; Pum1 is also required to sustain hyphal growth after the morphological switch. Furthermore, Pum1 directs subsequent differentiation of aerial hyphae into fruiting bodies in both laboratory and clinical isolates. Pum1 exerts its control on sexual reproduction partly through regulating the temporal expression of Dmc1, the meiosis-specific recombinase. Therefore, Pum1 serves a pivotal role in bridging post-mating morphological differentiation events with sexual reproduction in Cryptococcus. Our findings in Cryptococcus illustrate how an environmental pathogen can ensure the completion of its life cycle to safeguard its long-term lineage success.

Differential expression of the proteome of myeloid dendritic cells in severe aplastic anemia.

Severe aplastic anemia (SAA) is a syndrome of severe bone marrow failure with high mortality. Our previous studies have demonstrated that both immature and activated DC1 increased in the bone marrow of SAA patients, and the balance of DC1 subsets shifted the stable form to active one, which might promote Th0 cells to polarize to Th1 cells and cause the over-function of T lymphocytes and hematopoiesis failure in SAA. So we assumed myeloid dendritic cells (mDCs) may be the key immune cells that cause destruction of hematopoietic cells in SAA, but the mechanism of activation of mDCs is unclear. Here, we investigated the proteome of mDCs in SAA patients to further explore the pathogenesis of SAA and the possible antigen that leads to immune activation in SAA. mDCs from 12 SAA patients, 12 remission patients and 12 controls were sorted by flow cytometry and examined by two-dimensional gel electrophoresis and mass spectrometry. Intensity changes of 41 spots were detected with statistical significance. Nine of the 41 spots were identified by MALDI-TOF/TOF tandem mass spectrometry. Changes in protein expression levels were found in the SAA group. These changes reveal that abnormal expression of cofilin, glucose-6-phosphate dehydrogenase and pyruvate kinase enzyme M2 in mDCs from SAA patients may be the reason for mDC hyperfunction.

Both NMDA and non-NMDA receptors mediate glutamate stimulation induced cofilin rod formation in cultured hippocampal neurons.

Cofilin is the major actin-depolymerizing factor in the CNS for the regulation of actin dynamics. Neurodegenerative stimuli can induce the formation of cofilin rod, a pathological structure composed of cofilin and actin. The formation of cofilin rod was found to disrupt synapse function and cause neurite loss. The aim of the present study is to study the whole process of cofilin rod formation pattern in cultured hippocampal neurons under excitotoxic stimulation and to explore its underlying pharmacological mechanism. By using live cell imaging of neurons overexpressing EGFP-tagged wild type cofilin, we found a two-phase pattern of rod formation induced by glutamate stimulation. The early phase of rod formation occurred shortly after stimulation (∼0.5h) but quickly dissolved within 2h. The second phase happened within a much longer time window, 8h after stimulation. Immunostaining of endogenous cofilin in neurons also confirmed this glutamate stimulation induced two-phase rod formation pattern. The first phase was co-related with intracellular calcium concentration and pH increase while the second phase was not. These two phases of cofilin rod formation induced by glutamate stimulation was antagonized by both non-NMDA and NMDA receptor antagonist DNQX and AP5, respectively. Our results for the first time demonstrate the dynamic cofilin rod formation pattern under stress stimulation in detail by time lapse imaging. These findings reveal a novel time course of excitotoxicity induced neuronal damage and indicate a potential target of neuropathy treatment of neurodegenerative diseases.

Identification of endometriosis-related genes by representational difference analysis of cDNA.

BACKGROUND: Accumulated evidence reveals that abnormally expressed genes in eutopic endometrium of endometriosis play a critical role in the pathogenesis of endometriosis. AIMS: Identification of endometriosis-related genes for further revealing the pathogenesis of endometriosis and offering the basis for developing the molecular-targeted diagnosis and therapy of endometriosis. METHODS: Forty women with endometriosis and forty control women without endometriosis during their secretory phase were selected for this study. cDNA representational difference analysis (cDNA-RDA) was performed to screen the up-regulated genes in eutopic endometrium samples of endometriosis (n = 10) compared with the controls (n = 10). To validate the results, MAT2A, the most abundantly expressed gene, was selected to detect mRNA and protein levels between eutopic endometrium of endometriosis (n = 40) and controls (n = 40) using immunohistochemistry, real-time fluorescent quantitative PCR and Western blotting. RESULTS: Ten up-regulated genes were identified in eutopic endometria of endometriosis compared with controls. Among these genes, COX-2, BRAF, NRAS and CFL1 have already been reported to be associated with the endometriosis in previous studies. MAT2A, SEPT9, ATAD3A and CADM2 have been reported to be involved in other diseases but not in endometriosis. NAA15 and CCDC21 have not reported in any diseases. Further study showed that MAT2A protein was localised in both endometrial glandular and stromal cells. Compared with controls, the mRNA and protein levels of MAT2A were significantly higher in eutopic endometrium of endometriosis (P < 0.05). CONCLUSIONS: cDNA-RDA can be used to effectively identify the endometriosis-related genes, which can provide novel experimental data and further understand the pathogenesis of endometriosis.

Nuclear transport of the serum response factor coactivator MRTF-A is downregulated at tensional homeostasis.

The serum response factor (SRF) coactivator myocardin-related transcription factor A (MAL/MKL1/MRTF-A), the nuclear transport and activity of which is regulated by monomeric actin, has been implicated in tension-based regulation of SRF-mediated transcriptional activity. However, the mechanisms involved remain unclear. We used fibroblasts grown within collagen matrices to explore whether MRTF-A transport is regulated by tissue tension. We show that MRTF-A nuclear accumulation following stimulation with serum, actin drugs or acute mechanical stress is prevented within mechanically loaded, anchored matrices at tensional homeostasis. This is accompanied by a higher G/F actin ratio, defective nuclear import and increased cofilin expression. We propose that tension regulates MRTF-A/SRF activity through cofilin-mediated modulation of actin dynamics.

Overexpression of cofilin 1 can predict progression-free survival in patients with epithelial ovarian cancer receiving standard therapy.

The aim of this study was to examine the relation between cofilin 1 expression and progression-free survival in advanced epithelial ovarian cancer. We performed quantitative reverse transcriptase polymerase chain reaction and immunohistochemical analysis in 78 patients with advanced epithelial ovarian cancer (excluding those with mucinous and clear-cell types). All patients received the standard therapy, including staging laparotomy and adjuvant chemotherapy consisting of carboplatin and paclitaxel. Of 78 samples, RNA from 62 samples was available for reverse transcriptase polymerase chain reaction analysis. We defined cofilin 1 high expression as relative gene expression equal to or higher than the median and low expression as gene expression lower than median. The progression-free survival was longer in cofilin 1 low-expression cases than in high-expression cases (P = .039). Multivariate analysis demonstrated that stage and cofilin 1 expression were significant predictors of progression-free survival. Of the 78 samples, 54 were appropriate for immunohistochemical study. In 35 of those 54 cases, cofilin 1 protein expression was detected. The progression-free survival was longer in cofilin 1 protein-negative cases than in protein-positive cases (P = .042). Expression of cofilin 1 may predict the progression-free survival of patients with advanced epithelial ovarian cancer receiving standard therapy.

Pentagalloylglucose downregulates cofilin1 and inhibits HSV-1 infection.

To investigate the anti-herpesvirus mechanism of pentagalloylglucose (PGG), we compared the proteomic changes between herpes simplex virus type 1 (HSV-1) infected MRC-5 cells with or without PGG-treatment, and between non-infected MRC-5 cells with or without PGG-treatment by 2-DE and MS-based analysis. Differentially expressed cellular proteins were mainly involved with actin cytoskeleton regulation. Significantly, PGG can down-regulate cofilin1, a key regulator of actin cytoskeleton dynamics. PGG can inhibit HSV-1-induced rearrangements of actin cytoskeleton which is important for infectivity. Furthermore, cofilin1 knockdown by siRNA also inhibited the HSV-1-induced actin-skeleton rearrangements. Both PGG-treatment and cofilin1 knockdown can reduce HSV-1 DNA, mRNA, protein synthesis and virus yields. Altogether, the results suggested that down-regulating cofilin1 plays a role in PGG inhibiting HSV-1 infection. PGG may be a promising anti-herpesvirus agent for drug development.

Decreased cofilin1 expression is important for compaction during early mouse embryo development.

Compaction, occurring at the eight-cell stage of mouse development, is the process of cell flattening and polarization by which cellular asymmetry is first established. During this process many molecules and organelles undergo polarized distribution, but the cytoskeletal basis for these distribution specifications remains to be explored. The present study focused on cofilin1, an actin-binding protein that depolymerizes actin filaments. We showed that cofilin1 expression decreased at the compaction stage, and that down-regulation of cofilin1 expression by siRNA microinjection accelerated compaction. Continuous observation using time-lapse video miscroscopy confirmed these findings. That is, the embryonic cells microinjected with anti-cofilin1 antibody exhibit earlier adherence properties compared to uninjected cells. Pronuclear microinjection of a site-directed mutated cofilin1 plasmid, in which cofilin1 is sustained in its active form produced embryos with blastomeres that did not adhere, suggesting that inactivation of cofilin1 is critical for cell flattening and adherence. Fluorescein-phalloidin staining indicated that decreased cofilin1 expression promoted the formation of the apical pole, which is a marker for polarity. Scanning electron microscopy results demonstrated the appearance of microvilli on the outer face of blastomeres in cofilin1 knockdown embryos. Our results suggest that cofilin1 plays an important role in cortical cytoplasmic organization during embryo compaction.

A tropomyosin 1 induced defect in cytokinesis can be rescued by elevated expression of cofilin.

Cytokinesis in eukaryotic cells is mediated by the contractile ring, an actomyosin-based structure which provides the force required to separate daughter cells. Isoforms of the actin-binding protein tropomyosin are also localised to the contractile ring in both fission yeast and human astrocytes. Although tropomyosin is required for cytokinesis in yeast, its precise role in the contractile ring is unknown. In this study we find that increased expression of a single tropomyosin isoform, tropomyosin 1, in U373MG astrocytoma cells leads to multinucleated cells and mitotic spindle defects. Furthermore, cells expressing increased levels of tropomyosin 1 usually fail to complete cytokinesis and this is accompanied by reduced accumulation of actin depolymerising factor/cofilin in the contractile ring. Adenovirus mediated expression of cofilin is able to relieve the tropomyosin 1 induced effects on cytokinesis. We conclude that tropomyosin 1 and cofilin play antagonistic roles within the contractile ring and that the balance between tropomyosin 1 and cofilin expression is important for cytokinesis.

Coordinated regulation of pathways for enhanced cell motility and chemotaxis is conserved in rat and mouse mammary tumors.

Correlating tumor cell behavior in vivo with patterns of gene expression has led to new insights into the microenvironment of tumor cells in the primary tumor. Until now, these studies have been done with cell line-derived tumors. In the current study, we have analyzed, in polyoma middle T oncogene (PyMT)-derived mammary tumors, tumor cell behavior and gene expression patterns of the invasive subpopulation of tumor cells by multiphoton-based intravital imaging and microarray-based expression profiling, respectively. Our results indicate that the patterns of cell behavior that contribute to invasion and metastasis in the PyMT tumor are similar to those seen previously in rat MTLn3 cell line-derived mammary tumors. The invasive tumor cells collected from PyMT mouse mammary tumors, like their counterparts from rat xenograft mammary tumors, are a population that is relatively nondividing and nonapoptotic but chemotherapy resistant and chemotactic. Changes in the expression of genes that occur uniquely in the invasive subpopulation of tumor cells in the PyMT mammary tumors that fall on the Arp2/3 complex, capping protein and cofilin pathways show a pattern like that seen previously in invasive tumor cells from the MTLn3 cell line-derived tumors. These changes predict an enhanced activity of the cofilin pathway, and this was confirmed in isolated invasive PyMT tumor cells. We conclude that changes in gene expression and their related changes in cell behavior, which were identified in the invasive tumor cells of cell line-derived tumors, are conserved in the invasive tumor cells of PyMT-derived mouse mammary tumors, although these tumor types have different genetic origins.

Expression of nonmuscle cofilin-1 and steroid responsiveness in severe asthma.

BACKGROUND: Glucocorticoids are the mainstay of asthma therapy; however, a proportion of patients with asthma has a severe form of the disease that fails to respond to therapy. Understanding the molecular mechanisms behind glucocorticoid-insensitive asthma is therefore of clinical importance. Evidence in glucocorticoid-unresponsive Henrietta Lack (HeLa) cells indicated that cofilin-1 could act as an inhibitor of glucocorticoid function. OBJECTIVE: To determine whether cofilin-1 expression is abnormally expressed in cells from patients with severe glucocorticoid-insensitive asthma and examine the effect of cofilin-1 overexpression on glucocorticoid function. METHODS: Peripheral blood CD4(+) T cells were purified from 16 subjects with severe glucocorticoid-insensitive asthma and 16 subjects with mild glucocorticoid-sensitive asthma, and cofilin-1 expression was determined by quantitative real-time RT-PCR and Western blotting. The effect of dexamethasone on cofilin-1 expression was determined in Jurkat T cells, and the effect of cofilin-1 overexpression on anti-CD3/CD28-stimulated IL-2 release was measured. RESULTS: Peripheral blood CD4(+) T cells from subjects with severe glucocorticoid-insensitive asthma are less responsive to dexamethasone than cells from subjects with mild glucocorticoid-sensitive asthma. Cells from these patients express significantly (P < .05) higher levels of cofilin-1 than cells from subjects with mild asthma. Dexamethasone did not affect cofilin-1 expression in Jurkat T cells. Functionally, dexamethasone suppression of anti-CD3/CD28-stimulated IL-2 was attenuated in Jurkat cells overexpressing cofilin-1. CONCLUSION: These results suggest that increased cofilin-1 expression may be important in the regulation of glucocorticoid sensitivity in peripheral blood lymphocytes of patients with severe treatment-insensitive asthma. CLINICAL IMPLICATIONS: Understanding the mechanisms of enhanced cofilin-1 expression may lead to the development of new therapies for severe treatment-insensitive asthma.

Inhibition of invasiveness of human lung cancer cell line H1299 by over-expression of cofilin.

The Rho-LIM-kinase (LIMK) signaling pathway, believed to be involved in the regulation of tumor invasion, specifically regulates the activity of cofilin. However, it is unclear whether cofilin plays a pivotal role in tumor invasiveness. In this paper we show using a tet-on gene expression system that over-expression of cofilin inhibits the invasiveness of human lung cancer H1299 cells. Over-expressed cofilin disrupts the actin cytoskeleton at the leading edge of the cell and up-regulates p27(kip1), which is known to be involved in regulating cell motility. Removal of cofilin over-expression normalizes the p27(kip1) level and concomitantly restores the invasiveness of the cultured cells. These findings suggest that excessive cofilin production might prevent cancer cell invasion.

Ligation of cell surface-associated glucose-regulated protein 78 by receptor-recognized forms of alpha 2-macroglobulin: activation of p21-activated protein kinase-2-dependent signaling in murine peritoneal macrophages.

Previous studies of the plasma proteinase inhibitor alpha2-macroglobulin (alpha2M) demonstrated that alpha2M-proteinase complexes (alpha2M*) modulate immune responses and promotes macrophage locomotion and chemotaxis. Alpha2M* binds to cell surface-associated glucose-regulated protein 78 (GRP78), which activates downstream signaling events. The role of p21-activated protein kinase-1 and -2 (PAK-1 and -2) in promoting cellular motility is well documented. In the current study, we examined the ability of alpha2M* to activate PAK-1 and PAK-2. Upon macrophage stimulation with alpha2M*, PAK-2 is autophosphorylated, resulting in increased kinase activity; however, PAK-1 is negligibly affected. Alpha2M*-stimulated macrophages showed a marked elevation in the levels of Rac x GTP. Receptor tyrosine phosphorylation upon binding of alpha2M* to GRP78, recruits PAK-2 to the plasma membrane via the adaptor protein NCK. Consistent with this hypothesis, silencing of GRP78 gene expression greatly attenuated the levels of membrane-associated PAK-2 and NCK. PAK-2 activity was markedly decreased by inhibition of tyrosine kinases and PI3K before alpha2M* stimulation. We further demonstrate that phosphorylation of Lin-11, Isl-1, Mec-3 (LIM) kinase and cofilin is promoted by treating macrophages with alpha2M*. Thus, alpha2M* regulates activation of the PAK-2-dependent motility mechanism in these cells.