[Molecular dynamics simulation of force-regulated interaction between glycoprotein Ib α and filamin].

In resting platelets, the 17 th domain of filamin a (FLNa17) constitutively binds to the platelet membrane glycoprotein Ibα (GPIbα) at its cytoplasmic tail (GPIbα-CT) and inhibits the downstream signal activation, while the binding of ligand and blood shear force can activate platelets. To imitate the pull force transmitted from the extracellular ligand of GPIbα and the lateral tension from platelet cytoskeleton deformation, two pulling modes were applied on the GPIbα-CT/FLNa17 complex, and the molecular dynamics simulation method was used to explore the mechanical regulation on the affinity and mechanical stability of the complex. In this study, at first, nine pairs of key hydrogen bonds on the interface between GPIbα-CT and FLNa17 were identified, which was the basis for maintaining the complex structural stability. Secondly, it was found that these hydrogen bonding networks would be broken down and lead to the dissociation of FLNa17 from GPIbα-CT only under the axial pull force; but, under the lateral tension, the secondary structures at both terminals of FLNa17 would unfold to protect the interface of the GPIbα-CT/FLNa17 complex from mechanical damage. In the range of 0~40 pN, the increase of pull force promoted outward-rotation of the nitrogen atom of the 563 rd phenylalanine (PHE 563-N) at GPIbα-CT and the dissociation of the complex. This study for the first time revealed that the extracellular ligand-transmitted axial force could more effectively relieve the inhibition of FLNa17 on the downstream signal of GPIbα than pure mechanical tension at the atomic level, and would be useful for further understanding the platelet intracellular force-regulated signal pathway.

Filamin-A-interacting protein 1 (FILIP1) is a dual compartment protein linking myofibrils and microtubules during myogenic differentiation and upon mechanical stress.

Variations in the gene encoding filamin-A-interacting protein 1 (FILIP1) were identified to be associated with a combination of neurological and muscular symptoms. While FILIP1 was shown to regulate motility of brain ventricular zone cells, a process important for corticogenesis, the function of the protein in muscle cells has been less well characterized. The expression of FILIP1 in regenerating muscle fibres predicted a role in early muscle differentiation. Here we analysed expression and localization of FILIP1 and its binding partners filamin-C (FLNc) and microtubule plus-end-binding protein EB3 in differentiating cultured myotubes and adult skeletal muscle. Prior to the development of cross-striated myofibrils, FILIP1 is associated with microtubules and colocalizes with EB3. During further myofibril maturation its localization changes, and FILIP1 localizes to myofibrillar Z-discs together with the actin-binding protein FLNc. Forced contractions of myotubes by electrical pulse stimulation (EPS) induce focal disruptions in myofibrils and translocation of both proteins from Z-discs to these lesions, suggesting a role in induction and/or repair of these structures. The immediate proximity of tyrosylated, dynamic microtubules and EB3 to lesions implies that also these play a role in these processes. This implication is supported by the fact that in nocodazole-treated myotubes that lack functional microtubules, the number of lesions induced by EPS is significantly reduced. In summary, we here show that FILIP1 is a cytolinker protein that is associated with both microtubules and actin filaments, and might play a role in the assembly of myofibrils and their stabilization upon mechanical stress to protect them from damage.

Peptide location fingerprinting identifies species- and tissue-conserved structural remodelling of proteins as a consequence of ageing and disease.

Extracellular matrices (ECMs) in the intervertebral disc (IVD), lung and artery are thought to undergo age-dependant accumulation of damage by chronic exposure to mechanisms such as reactive oxygen species, proteases and glycation. It is unknown whether this damage accumulation is species-dependant (via differing lifespans and hence cumulative exposures) or whether it can influence the progression of age-related diseases such as atherosclerosis. Peptide location fingerprinting (PLF) is a new proteomic analysis method, capable of the non-targeted identification of structure-associated changes within proteins. Here we applied PLF to publicly available ageing human IVD (outer annulus fibrosus), ageing mouse lung and human arterial atherosclerosis datasets and bioinformatically identified novel target proteins alongside common age-associated differences within protein structures which were conserved between three ECM-rich organs, two species, three IVD tissue regions, sexes and in an age-related disease. We identify peptide yield differences across protein structures which coincide with biological regions, potentially reflecting the functional consequences of ageing or atherosclerosis for macromolecular assemblies (collagen VI), enzyme/inhibitor activity (alpha-2 macroglobulin), activation states (complement C3) and interaction states (laminins, perlecan, fibronectin, filamin-A, collagen XIV and apolipoprotein-B). Furthermore, we show that alpha-2 macroglobulin and collagen XIV exhibit possible shared structural consequences in IVD ageing and arterial atherosclerosis, providing novel links between an age-related disease and intrinsic ageing. Crucially, we also demonstrate that fibronectin, laminin beta chains and filamin-A all exhibit conserved age-associated structural differences between mouse lung and human IVD, providing evidence that ECM, and their associating proteins, may be subjected to potentially similar mechanisms or consequences of ageing across both species, irrespective of differences in lifespan and tissue function.

Filamin A and parafibromin expression in parathyroid carcinoma.

OBJECTIVE: Parathyroid carcinoma (PC), atypical parathyroid tumours (APT) and parathyroid adenoma (PA), all present with hypercalcemia. Diminished calcium-sensing receptor (CaSR) expression is reported in PC but is rare in benign tumours. Filamin A (FLNA) binds to the CaSR and activates the mitogen-activated protein kinase (MAPK) signalling pathway. FLNA is related to tumour aggressiveness in several cancers, but its role in parathyroid neoplasia is unknown. DESIGN: We examined FLNA, CaSR and parafibromin expression in PCs (n = 32), APTs (n = 44) and PAs (n = 77) and investigated their potential as diagnostic and/or prognostic markers. METHODS: Tissue microarray slides were immunohistochemically stained with antibodies for FLNA, CaSR and parafibromin. Staining results were correlated with detailed clinical data. RESULTS: All tumours stained positively for CaSR, with two tumours (one PC and one APT) showing diminished expression. Carcinomas were characterized by increased cytoplasmic FLNA expression compared to APTs and PAs (P = 0.004). FLNA expression was not correlated with Ki-67 proliferation index or loss of parafibromin expression. Cytoplasmic FLNA expression was also associated with higher serum calcium, PTH concentrations and male sex (P = 0.014, P = 0.017 and P = 0.049 respectively). Using a combined marker score, we found that parathyroid tumours with low FLNA expression and positive parafibromin staining were extremely likely to be benign (P < 0.001). CONCLUSION: Cytoplasmic and membranous FLNA expression is increased in parathyroid carcinomas compared to benign tumours. A combined FLNA and parafibromin expression score shows potential as a prognostic predictor of indolent behaviour in parathyroid neoplasms.

Weighted gene coexpression network analysis reveals ESR1, FLNA and Furin as hub genes for DEHP-induced prepubertal testicular injury.

Di-(2-ethylhexyl) phthalate (DEHP) is an environmental endocrine disruptor that accumulates in organisms in various ways and induces male reproductive system disorders. In this study, we established a testicular injury model by gavage with different concentrations of DEHP. The testes were then collected for RNA sequencing (RNA-seq), and the results were analyzed by bioinformatics and verified by experiments. Our research results show that different concentrations of DEHP interfere with testicular development differently. Weighted gene coexpression network analysis (WGCNA) generated sixteen modules and identified the turquoise module as key. Then, estrogen receptor 1 (ESR1), filamin A (Flna) and Furin were identified as hub genes. qPCR and immunohistochemistry results revealed that all three hub genes were upregulated. We detected the locations of these genes by immunohistochemistry. ESR1 was mainly located in Leydig cells; Flna immunostaining is observed in the Leydig and some germ cells and Furin staining was seen in almost all types of testicular cells. Furthermore, Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analyses showed enrichment mainly in MAPK signaling pathways, p53 signaling pathways, HIF-1 signaling pathways, protein processing in the endoplasmic reticulum, apoptosis, the cell cycle, RNA degradation, etc. This is the first study using WGCNA to investigate the mechanism of DEHP-induced injury in the prepubertal testis, providing new research angles to further understand the mechanism of DEHP-induced injury in the prepubertal testis.

Role and mechanism of FLNa and UCP2 in the development of cervical cancer.

Recent studies have reported that filamin A (FLNa) and uncoupling protein 2 (UCP2) are highly expressed in various types of cancer, but little is currently known about their roles in cervical cancer (CC). In the present study, immunohistochemical staining of paraffin sections of cervical tissues was performed in order to compare the differential expression of FLNa, UCP2, p16 and Ki67 between CC and high­grade intraepithelial neoplasia (HSIL). UCP2 and FLNa were knocked down in CC cell lines to investigate the effects on cell proliferation, cell cycle arrest, apoptosis, migration and invasion. In addition, the present study investigated the expression of cell­associated proteins [extracellular signal­regulated kinase (ERK), phosphorylated (p) ERK, protein kinase B (AKT), p­AKT and B­cell lymphoma­2 (Bcl­2)] and the mRNA levels of cellular proteins such as Ras, matrix metalloproteinase (MMP)­2 and MMP­9. FLNa and UCP2 expression levels were significantly higher in CC tissues than in HSIL tissues, with no significant differential expression of p16 or Ki67. UCP2 expression was significantly different in patients with clinical stage II or higher or lymph node metastasis compared with in other patients with cervical cancer. FLNa or UCP2 knockdown slowed or decreased SiHa and HeLa cell proliferation, migration and invasion, with no significant change in apoptosis, and downregulated the protein levels of p­ERK1/2, and the mRNA levels of Ras, MMP­2 and MMP­9. UCP2 knockdown arrested the cell cycle at the G2 phase in SiHa and HeLa cells, while FLNa knockdown arrested the cell cycle at the G2 phase in HeLa cells. The results of the present study revealed that FLNa and UCP2 play roles in the development and progression of CC via the Ras/MAPK/ERK signalling pathway. FLNa and UCP2 are superior to p16 and Ki67 for early prediction of CC, indicating that FLNa and UCP2 may be used for the early diagnosis of CC. UCP2 may be used to predict the prognosis of CC.

Biological roles of filamin a in prostate cancer cells

ABSTRACT Objective This study aims to investigate the association of filamin A with the function and morphology of prostate cancer (PCa) cells, and explore the role of filamin A in the development of PCa, in order to analyze its significance in the evolvement of PCa. Materials and Methods A stably transfected cell line, in which filamin A expression was suppressed by RNA interference, was first established. Then, the effects of the suppression of filamin A gene expression on the biological characteristics of human PCa LNCaP cells were observed through cell morphology, in vitro cell growth curve, soft agar cloning assay, and scratch test. Results A cell line model with a low expression of filamin A was successfully constructed on the basis of LNCaP cells. The morphology of cells transfected with plasmid pSilencer-filamin A was the following: Cells were loosely arranged, had less connection with each other, had fewer tentacles, and presented a fibrous look. The growth rate of LNCap cells was faster than cells transfected with plasmid pSilencer-filamin A (P <0.05). The clones of LNCap cells in the soft agar cloning assay was significantly fewer than that of cells stably transfected with plasmid pSilencer-filamin A (P <0.05). Cells stably transfected with plasmid pSilencer-filamin A presented with a stronger healing and migration ability compared to LNCap cells (healing rate was 32.2% and 12.1%, respectively; P <0.05). Conclusion The expression of the filamin A gene inhibited the malignant development of LNCap cells. Therefore, the filamin A gene may be a tumor suppressor gene.

Biological roles of filamin a in prostate cancer cells.

OBJECTIVE: This study aims to investigate the association of filamin A with the function and morphology of prostate cancer (PCa) cells, and explore the role of filamin A in the development of PCa, in order to analyze its significance in the evolvement of PCa. MATERIALS AND METHODS: A stably transfected cell line, in which filamin A expression was suppressed by RNA interference, was first established. Then, the effects of the suppression of filamin A gene expression on the biological characteristics of human PCa LNCaP cells were observed through cell morphology, in vitro cell growth curve, soft agar cloning assay, and scratch test. RESULTS: A cell line model with a low expression of filamin A was successfully constructed on the basis of LNCaP cells. The morphology of cells transfected with plasmid pSilencer-filamin A was the following: Cells were loosely arranged, had less connection with each other, had fewer tentacles, and presented a fibrous look. The growth rate of LNCap cells was faster than cells transfected with plasmid pSilencer-filamin A (P<0.05). The clones of LNCap cells in the soft agar cloning assay was significantly fewer than that of cells stably transfected with plasmid pSilencer-filamin A (P<0.05). Cells stably transfected with plasmid pSilencer-filamin A presented with a stronger healing and migration ability compared to LNCap cells (healing rate was 32.2% and 12.1%, respectively; P<0.05). CONCLUSION: The expression of the filamin A gene inhibited the malignant development of LNCap cells. Therefore, the filamin A gene may be a tumor suppressor gene.

Decreased Filamin b expression regulates trophoblastic cells invasion through ERK/MMP-9 pathway in pre-eclampsia.

OBJECTIVES: The purpose of this study was to investigate the expression of Filamin b in the placental placenta of patients with early or late onset pre-eclampsia (PE) and its potential effects on the pathophysiology of the disease. METHODS AND METHODS: Immunohistochemistry staining, western blot assays and real time PCR were used to detect the expression level of FLN-b. The expression levels of MMP-2, MMP-9 and ERK1/2 proteins from control and FLN-b-silenced JEG-3 cells were also detected by western blot and JEG-3 cell invasion. RESULTS: Compared with normal term pregnancies placentas, the FLN-b expression was significantly lower than that of women with PE, its level in late-onset PE is lower than in early-onset PE. In FLN-b-silenced JEG-3 cells, the protein levels of MMP-2, MMP-9 and phosphorylated ERK1/2 decreased markedly and the number of cells penetrating through the transwell chamber membrane is also greatly reduced. CONCLUSIONS: Down-regulation of FLN-b inhibits the ERK/MMP-2 and MMP-9 pathways, leading to trophoblastic invasion disorders in the PE placenta.

Filamin A expression predicts early recurrence of hepatocellular carcinoma after hepatectomy.

BACKGROUND & AIMS: Recurrence of hepatocellular carcinoma (HCC) after hepatectomy is very high. A predictive marker of early recurrence (ER) capable of personalizing follow-up and developing a new target therapy would be beneficial. The overexpression of Filamin-A (FLNA), a cytoskeleton protein with scaffolding properties, has recently been associated with progression in tumours. The aim of this study was to test the expression of FLNA in a cohort of patients operated for HCC. METHODS: A retrospective cohort of patients who underwent hepatic resection at Humanitas Clinical and Research Center between January 2004 and December 2014 was analysed. FLNA was tested, using a tissue microarray, in the HCC and in the surrounding tissues. The endpoint was the role of FLNA expression in predicting ER of HCC after hepatectomy. Analyses were performed following the REMARK guidelines. RESULTS: A total of 113 patients were considered. FLNA was expressed only in the tumoral tissue. Several variables, including T stage, tumour number, tumour size, type of viral hepatitis, type of hepatectomy and intra and peritumoral immune-reactivity to FLNA were significantly associated with ER by univariate analysis. With multivariate analysis, only T stage (HR=2.108; P=.002), tumour number (HR=1.586; P=.023), intra-tumoral (HR=2.672; P<.001) and peritumoral immune-reactivity to FLNA (HR=2.569; P<.001), significantly correlated with ER. The logistic regression analysis revealed that advanced T stage (OR=2.985; P=.001), HCV-infection (OR=1.219; P=.008) and advanced tumour grading (OR=2.781; P=.002) were associated with intratumoral FLNA immune-reactivity. CONCLUSIONS: FLNA expression predicts recurrence of HCC after hepatectomy. This finding provides important insights that would help physicians to personalize follow-up strategies.

Disruption of TACE-filamin interaction can inhibit TACE-mediated ectodomain shedding.

Ectodomain shedding regulates functions of many membrane proteins through the cleavage of their juxtamembrane region mainly by a disintegrin and metalloproteinase family proteinases. Tumor necrosis factor-alpha converting enzyme (TACE) is known to be responsible for phorbol myristate acetate (PMA)-induced shedding of various membrane proteins. How PMA regulates TACE-dependent shedding and how TACE exhibits substrate specificity without proteolysis of other membrane proteins are questionable. Here, we show that TACE can interact with an actin-binding protein, filamin, through 20th filamin repeat. We found that the interaction between TACE and filamin was increased by PMA treatment. In addition, loss of filamin or specific disruption of TACE-filamin interaction inhibited ectodomain shedding of representative TACE substrates, CD44 and amyloid protein precursor. From these data, we suggest that filamin may work as a scaffold that can recruit TACE and its substrates in a PMA-dependent manner to achieve substrate specificity for TACE.

New cancer suppressor gene for colorectal adenocarcinoma: filamin A.

AIM: To determine the expression and significance of filamin A (FLNa) in colorectal adenocarcinoma tissue. METHODS: The expression of FLNa in 46 colorectal cancer tissues and normal tissues was detected by immunohistochemistry, reverse transcription polymerase chain reaction (RT-PCR) and Western blotting, and its relationship with clinical parameters and prognosis was analyzed. RESULTS: The positive expression of FLNa in cancer tissues was lower than that in normal mucosa, and the difference was statistically significant. The expression of FLNa correlated with liver metastasis, lymph node metastasis and rectal invasion depth, regardless of sex, age, tumor location, tumor size, gross shape and histological type of colorectal carcinoma. Multivariate analysis showed that FLNa was an independent risk factor for postoperative survival of patients with colorectal adenocarcinoma. Moreover, survival analysis showed that the expression level of FLNa was closely related with survival of patients with colorectal adenocarcinoma. The results of RT-PCR and Western blotting were consistent with those of immunohistochemistry. CONCLUSION: FLNa showed low expression in colorectal adenocarcinoma, high correlation with the incidence and development of colorectal cancer, and was considered an indicator of prognosis.

Analysis of the androgen receptor/filamin a complex in stromal cells.

The androgen receptor (AR), a ligand-regulated nuclear transcription factor, mediates differentiation and proliferation of target tissues. Its action is frequently associated with human proliferative diseases, mainly the prostate cancer. We have recently analyzed in mouse embryo NIH3T3 fibroblasts and human fibrosarcoma HT1080 cells the molecular basis and the biological role of AR interaction with the full-length filamin A (FLNa), an actin-cross-linking protein. Here, we describe a procedure revealing the AR/FLNa complex in stromal cells. Upon physiological (10 nM) androgen stimulation of quiescent NIH3T3 cells, FLNa co-immunoprecipitates with AR and co-localizes with the receptor at intermediate actin filaments. The AR/FLNa complex specifically regulates AR extranuclear functions leading to Rac1 activation and consequent cell motility. This complex adds a new and unexpected piece to the growing evidence of the role of signalling effectors, scaffolds, and cytoskeletal proteins in the rapid androgen action and in progression of hormone-dependent cancers.

Filamin A regulates MMP-9 expression and suppresses prostate cancer cell migration and invasion.

This study aims to analyze the expression and clinical significance of Filamin A (FLNA) in prostate carcinoma and the biological effect in its cell line by FLNA overexpression. Immunohistochemistry and Western blot were used to analyze FLNA protein expression in 68 cases of prostate cancer and 37 cases of normal tissues to study the influence of the upregulated expression of FLNA that might be found on PC-3 cell biological effect. In the immunohistochemical analysis, the level of FLNA protein expression was found to be significantly lower in prostate cancer tissue than in normal tissues (P < 0.05). In the Western blot analysis, the relative amount of FLNA protein in prostate cancer tissue was found to be significantly lower than in normal tissues (P < 0.05). The level of FLNA protein expression was not correlated with age and PSA concentration (P > 0.05), but it was correlated with T stages, lymph node metastasis, clinic stage, and Gleason score (P < 0.05). The result of biological function showed that PC-3 cell transfected FLNA had a lower survival fraction, a significant decrease in migration and invasion, and a lower matrix metallopeptidase 9 (MMP-9) protein expression compared with PC-3 cell untransfected FLNA (P < 0.05). FLNA expression decreased in prostate cancer and correlated significantly with T stages, lymph node metastasis, clinic stage, and Gleason score, suggesting that FLNA may play important roles as a negative regulator to prostate cancer PC-3 cell by promoting the degradation of MMP-9.

Absent expression of FLNA is correlated with poor prognosis of nasopharyngeal cancer.

This study aimed to analyze the expression, clinical significance of filamin A (FLNA) in nasopharyngeal carcinoma, and the biological effect in its cell line by FLNA overexpression. Immunohistochemistry and western blot were used to analyze FLNA protein expression in 63 cases of nasopharyngeal cancer and 21 cases of normal tissues to study the relationship between FLNA expression and clinical factors. FLNA lentiviral vector and empty vector were respectively transfected into nasopharyngeal cancer CNE2 cell line. Reverse transcription-polymerase chain reaction (RT-PCR) and western blot were used to detect the mRNA level and protein of FLNA. 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide, migration, and invasion assays were also conducted as to the influence of the upregulated expression of FLNA that might be found on CNE2 cell biological effect. Immunohistochemistry: the level of FLNA protein expression was found to be significantly lower in nasopharyngeal cancer tissue than normal tissues (P < 0.05). Western blot: the relative amount of FLNA protein in nasopharyngeal cancer tissue was found to be significantly lower than in normal tissues (P < 0.05). The level of FLNA protein expression was correlated with T stages, lymph node metastasis, clinic stage, and histological grade (P < 0.05). Loss of FLNA expression correlated significantly with poor overall survival time by Kaplan-Meier analysis (P < 0.05). The result of biological function has shown that CNE2 cell-transfected FLNA had a lower survival fraction, significant decrease in migration and invasion, and lower matrix metallopeptidase 9 (MMP-9) protein expression compared with CNE2 cell-untransfected FLNA (P < 0.05). FLNA expression decreased in nasopharyngeal cancer and correlated significantly lymph node metastasis, clinic stage, histological grade, and poor overall survival, suggesting that FLNA may play important roles as a negative regulator to nasopharyngeal cancer CNE2 cell by promoting degradation of MMP-9.