Actin-associated proteins regulate multiple cellular processes, including proliferation and differentiation, but the molecular mechanisms underlying these processes are unclear. Here, we report that the actin-binding protein filamin A (FlnA) physically interacts with the actin-nucleating protein formin 2 (Fmn2). Loss of FlnA and Fmn2 impairs proliferation, thereby generating multiple embryonic phenotypes, including microcephaly. FlnA interacts with the Wnt co-receptor Lrp6. Loss of FlnA and Fmn2 impairs Lrp6 endocytosis, downstream Gsk3β activity, and β-catenin accumulation in the nucleus. The proliferative defect in Flna and Fmn2 null neural progenitors is rescued by inhibiting Gsk3β activity. Our findings thus reveal a novel mechanism whereby actin-associated proteins regulate proliferation by mediating the endocytosis and transportation of components in the canonical Wnt pathway. Moreover, the Fmn2-dependent signaling in this pathway parallels that seen in the non-canonical Wnt-dependent regulation of planar cell polarity through the Formin homology protein Daam. These studies provide evidence for integration of actin-associated processes in directing neuroepithelial proliferation.

• MeSH
• beta Catenin metabolism   MeSH
• Cell Differentiation MeSH
• Cell Membrane physiology   MeSH
• Cell Line MeSH
• Endocytosis physiology   MeSH
• Filamins genetics   metabolism   MeSH
• Glycogen Synthase Kinase 3 beta antagonists & inhibitors   metabolism   MeSH
• HEK293 Cells MeSH
• Nuclear Proteins genetics   metabolism   MeSH
• Low Density Lipoprotein Receptor-Related Protein-6 metabolism   MeSH
• Humans MeSH
• Microcephaly genetics   MeSH
• Microfilament Proteins genetics   metabolism   MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Cell Proliferation genetics   physiology   MeSH
• Wnt Proteins metabolism   MeSH
• Wnt Signaling Pathway physiology   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH

Filamin B (FlnB) is an actin-binding protein thought to transduce signals from various membrane receptors and intracellular proteins onto the actin cytoskeleton. Formin1 (Fmn1) is an actin-nucleating protein, implicated in actin assembly and intracellular signaling. Human mutations in FLNB cause several skeletal disorders associated with dwarfism and early bone fusion. Mouse mutations in Fmn1 cause aberrant fusion of carpal digits. We report here that FlnB and Fmn1 physically interact, are co-expressed in chondrocytes in the growth plate and share overlapping expression in the cell cytoplasm and nucleus. Loss of FlnB leads to a dramatic decrease in Fmn1 expression at the hypertrophic-to-ossification border. Loss of Fmn1-FlnB in mice leads to a more severe reduction in body size, weight and growth plate length, than observed in mice following knockout of either gene alone. Shortening of the long bone is associated with a decrease in chondrocyte proliferation and an overall delay in ossification in the double-knockout mice. In contrast to FlnB null, Fmn1 loss results in a decrease in the width of the prehypertrophic zone. Loss of both proteins, however, causes an overall decrease in the width of the proliferation zone and an increase in the differentiated hypertrophic zone. The current findings suggest that Fmn1 and FlnB have shared and independent functions. FlnB loss promotes prehypertrophic differentiation whereas Fmn1 leads to a delay. Both proteins, however, regulate chondrocyte proliferation, and FlnB may regulate Fmn1 function at the hypertrophic-to-ossification border, thereby explaining the overall delay in ossification.

• MeSH
• Cell Differentiation * MeSH
• Chondrocytes metabolism   pathology   MeSH
• Fetal Proteins deficiency   metabolism   MeSH
• Filamins deficiency   metabolism   MeSH
• Calcification, Physiologic MeSH
• Hypertrophy MeSH
• Nuclear Proteins deficiency   metabolism   MeSH
• Humans MeSH
• Microfilament Proteins deficiency   metabolism   MeSH
• Mice, Knockout MeSH
• Cell Proliferation MeSH
• Receptor, Parathyroid Hormone, Type 1 metabolism   MeSH
• Growth Plate metabolism   pathology   MeSH
• Protein Transport MeSH
• Protein Binding MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Research Support, N.I.H., Extramural MeSH

• MeSH
• Drug Resistance, Neoplasm MeSH
• Cisplatin therapeutic use   MeSH
• Cytoskeleton MeSH
• Gene Expression MeSH
• Filamins * MeSH
• Immunohistochemistry MeSH
• Clinical Studies as Topic MeSH
• Combined Modality Therapy MeSH
• Humans MeSH
• Neoplasm Metastasis MeSH
• Survival Rate MeSH
• Biomarkers, Tumor * MeSH
• Carcinoma, Non-Small-Cell Lung * drug therapy   surgery   MeSH
• DNA Repair MeSH
• Disease-Free Survival MeSH
• Prognosis MeSH
• Disease Progression MeSH
• Proportional Hazards Models MeSH
• Antineoplastic Combined Chemotherapy Protocols MeSH
• Neoplasm Grading MeSH
• Check Tag
• Humans MeSH
• Male MeSH
• Female MeSH
• Publication type
• Clinical Trial MeSH
• Research Support, Non-U.S. Gov't MeSH

BACKGROUND: FLNC is one of the few genes associated with all types of cardiomyopathies, but it also underlies neuromuscular phenotype. The combination of concomitant neuromuscular and cardiac involvement is not often observed in filaminopathies and the impact of this on the disease prognosis has hitherto not been analyzed. RESULTS: Here we provide a detailed clinical, genetic, and structural prediction analysis of distinct FLNC-associated phenotypes based on twelve pediatric cases. They include early-onset restrictive cardiomyopathy (RCM) in association with congenital myopathy. In all patients the initial diagnosis was established during the first year of life and in five out of twelve (41.7%) patients the first symptoms were observed at birth. RCM was present in all patients, often in combination with septal defects. No ventricular arrhythmias were noted in any of the patients presented here. Myopathy was confirmed by neurological examination, electromyography, and morphological studies. Arthrogryposes was diagnosed in six patients and remained clinically meaningful with increasing age in three of them. One patient underwent successful heart transplantation at the age of 18 years and two patients are currently included in the waiting list for heart transplantation. Two died due to congestive heart failure. One patient had ICD instally as primary prevention of SCD. In ten out of twelve patients the disease was associated with missense variants and only in two cases loss of function variants were detected. In half of the described cases, an amino acid substitution A1186V, altering the structure of IgFLNc10, was found. CONCLUSIONS: The present description of twelve cases of early-onset restrictive cardiomyopathy with congenital myopathy and FLNC mutation, underlines a distinct unique phenotype that can be suggested as a separate clinical form of filaminopathies. Amino acid substitution A1186V, which was observed in half of the cases, defines a mutational hotspot for the reported combination of myopathy and cardiomyopathy. Several independent molecular mechanisms of FLNC mutations linked to filamin structure and function can explain the broad spectrum of FLNC-associated phenotypes. Early disease presentation and unfavorable prognosis of heart failure demanding heart transplantation make awareness of this clinical form of filaminopathy of great clinical importance.

• MeSH
• Phenotype MeSH
• Filamins chemistry   genetics   metabolism   MeSH
• Cardiomyopathies * genetics   metabolism   MeSH
• Humans MeSH
• Muscular Diseases * MeSH
• Cardiomyopathy, Restrictive * genetics   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH

Spondylocarpotarsal syndrome (SCT) is a rare musculoskeletal disorder characterized by short stature and vertebral, carpal, and tarsal fusions resulting from biallelic nonsense mutations in the gene encoding filamin B (FLNB). Utilizing a FLNB knockout mouse, we showed that the vertebral fusions in SCT evolved from intervertebral disc (IVD) degeneration and ossification of the annulus fibrosus (AF), eventually leading to full trabecular bone formation. This resulted from alterations in the TGFβ/BMP signaling pathway that included increased canonical TGFβ and noncanonical BMP signaling. In this study, the role of FLNB in the TGFβ/BMP pathway was elucidated using in vitro, in vivo, and ex vivo treatment methodologies. The data demonstrated that FLNB interacts with inhibitory Smads 6 and 7 (i-Smads) to regulate TGFβ/BMP signaling and that loss of FLNB produces increased TGFβ receptor activity and decreased Smad 1 ubiquitination. Through the use of small molecule inhibitors in an ex vivo spine model, TGFβ/BMP signaling was modulated to design a targeted treatment for SCT and disc degeneration. Inhibition of canonical and noncanonical TGFβ/BMP pathway activity restored Flnb-/- IVD morphology. These most effective improvements resulted from specific inhibition of TGFβ and p38 signaling activation. FLNB acts as a bridge for TGFβ/BMP signaling crosstalk through i-Smads and is key for the critical balance in TGFβ/BMP signaling that maintains the IVD. These findings further our understanding of IVD biology and reveal new molecular targets for disc degeneration as well as congenital vertebral fusion disorders.

• Publication type
• Journal Article MeSH

Medulloblastoma comprises four main subgroups (WNT, SHH, Group 3 and Group 4) originally defined by transcriptional profiling. In primary medulloblastoma tissues, these groups are thought to be distinguishable using the immunohistochemical detection of β-catenin, filamin A, GAB1 and YAP1 protein markers. To investigate the utility of these markers for in vitro studies using medulloblastoma cell lines, immunoblotting and indirect immunofluorescence were employed for the detection of β-catenin, filamin A, GAB1 and YAP1 in both DAOY and D283 Med reference cell lines and the panel of six medulloblastoma cell lines derived in our laboratory from the primary tumor tissues of known molecular subgroups. Immunohistochemical detection of these markers was performed on formalin-fixed paraffin-embedded tissue of the matching primary tumors. The results revealed substantial divergences between the primary tumor tissues and matching cell lines in the immunoreactivity pattern of medulloblastoma-subgroup-specific protein markers. Regardless of the molecular subgroup of the primary tumor, all six patient-derived medulloblastoma cell lines exhibited a uniform phenotype: immunofluorescence showed the nuclear localization of YAP1, accompanied by strong cytoplasmic positivity for β-catenin and filamin A, as well as weak positivity for GAB1. The same immunoreactivity pattern was also found in both DAOY and D283 Med reference medulloblastoma cell lines. Therefore, we can conclude that various medulloblastoma cell lines tend to exhibit the same characteristics of protein marker expression under standard in vitro conditions. Such a finding emphasizes the importance of the analyses of primary tumors in clinically oriented medulloblastoma research and the urgent need to develop in vitro models of improved clinical relevance, such as 3D cultures and organotypic slice cultures.

• MeSH
• Child MeSH
• Adult MeSH
• Fluorescent Antibody Technique MeSH
• Immunoblotting MeSH
• Immunohistochemistry MeSH
• Infant MeSH
• Humans MeSH
• Medulloblastoma pathology   MeSH
• Adolescent MeSH
• Young Adult MeSH
• Cerebellum pathology   MeSH
• Biomarkers, Tumor analysis   MeSH
• Tumor Cells, Cultured MeSH
• Cerebellar Neoplasms pathology   MeSH
• Child, Preschool MeSH
• Check Tag
• Child MeSH
• Adult MeSH
• Infant MeSH
• Humans MeSH
• Adolescent MeSH
• Young Adult MeSH
• Male MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH

Nuclear actin plays an important role in such processes as chromatin remodeling, transcriptional regulation, RNA processing, and nuclear export. Recent research has demonstrated that actin in the nucleus probably exists in dynamic equilibrium between monomeric and polymeric forms, and some of the actin-binding proteins, known to regulate actin dynamics in cytoplasm, have been also shown to be present in the nucleus. In this paper, we present ultrastructural data on distribution of actin and various actin-binding proteins (alpha-actinin, filamin, p190RhoGAP, paxillin, spectrin, and tropomyosin) in nuclei of HeLa cells and resting human lymphocytes. Probing extracts of HeLa cells for the presence of actin-binding proteins also confirmed their presence in nuclei. We report for the first time the presence of tropomyosin and p190RhoGAP in the cell nucleus, and the spatial colocalization of actin with spectrin, paxillin, and alpha-actinin in the nucleolus.

• MeSH
• Actinin MeSH
• Actins analysis   MeSH
• Cell Nucleus chemistry   ultrastructure   MeSH
• Financing, Organized MeSH
• HeLa Cells MeSH
• Nuclear Proteins analysis   MeSH
• Humans MeSH
• Lymphocytes chemistry   ultrastructure   MeSH
• Microfilament Proteins analysis   MeSH
• Paxillin MeSH
• GTPase-Activating Proteins MeSH
• Spectrin MeSH
• Tropomyosin MeSH
• Check Tag
• Humans MeSH

Naltrexón sa používa v liečbe závislosti od alkoholu v dávkach 50 mg naltrexóniumchloridu denne (v USA aj v liečbe závislosti od opioidov, orálne aj injekčne), v kombinácii s bupropiónom pri liečbe obezity. V nízkych dávkach (1-5 mg denne) sa špecificky viaže na TLR-4 receptory, kde pôsobí antagonisticky. Pôsobí protizápalovo prostredníctvom buniek mikroglie. Jeho nízke dávky redukujú syntézu faktoru nekrózy nádoru (TNF)-α a interferónu-β. Nízkodávkový naltrexón (LDN) je modulačným nástrojom neuroimunitnej osi, tá interferuje s neuroendokrinnou osou. Prechodne sa zvyšuje signalizácia opioidového rastového faktoru (OGFr). LDN pôsobí stereoselektívne. Podaním (+)-naltrexónu nie je zasiahnutá opioidná signalizácia, ale iba TLR-4 signalizácia. Je prínosom pri liečbe fibromyalgie, Crohnovej choroby, sklerózy multiplex, tumorov, pri chronickom benígnom pemphigu (Hailey-Hailey disease) aj Gulf War Illness. Ultranízke dávky naltrexónu (ULDN) sú nižšie ako 1 μg. Analgetický efekt sprostredkováva druhý posol filamín A Využíva sa hlavne v postoperačnej kontrole analgézie. Dávky medzi 1 μg a 1 mg sú veľmi nízke dávky naltrexónu (VLDN). Sú efektívne ako „add on“ pri metadónovej udržiavacej liečbe.

• MeSH
• Hormesis drug effects   MeSH
• Humans MeSH
• Naltrexone * administration & dosage   MeSH
• Check Tag
• Humans MeSH