Filamin A in Cardiovascular Remodeling

Filamin A (FLNA) is a large actin-binding cytoskeletal protein that stabilizes actin networks and integrates them with cell membranes. FLNA is therefore important for cell motility and organ development. We recently discovered that a C-terminal fragment of FLNA (FLNACT) can be cleaved off by calpain and stimulate angiogenesis by transporting transcription factors into the nucleus. However, little is known about the role of FLNA in cell types that participate in the pathogenesis of vascular diseases where angiogenesis typically plays an important role. In this thesis, we defined the impact of inactivating Flna in mouse vascular endothelial cells and macrophages on the pathogenesis of myocardial infarction (MI) and atherosclerosis, respectively—and made several exciting discoveries. In Study I, we induced MI by ligating the left descending coronary artery in wt control mice and mice lacking FLNA in endothelial cells. The Flna-knockout mice developed larger MI lesions than controls, and exhibited larger and thinner left ventricles, impaired cardiac function, elevated plasma levels of the cardiac damage biomarker NT-proBNP, and reduced plasma levels of the angiogenesis-promoting factor VEGF-A. Hearts from the Flna-knockout mice exhibited reduced capillary structures within infarcted regions; and cultured Flna-deficient endothelial cells showed impaired migration and tubular formation, along with reduced levels of the signaling molecules p-ERK and p-AKT and the small GTPase RAC1. In Study II, we first discovered that FLNA expression was higher in human carotid arteries with advanced atherosclerotic plaques than with intermediate plaques. We generated mice lacking FLNA in macrophages and found that their macrophages proliferated and migrated less compared with littermate controls. Moreover, Flna-deficient macrophages exhibited reduced levels of p-ERK and p-AKT, and reduced lipid uptake and increased cholesterol efflux. In two different mouse atherosclerosis models, the knockout of FLNA in macrophages markedly reduced lesion size and number of CD68-positive lesional macrophages. Interestingly, the calpain-cleaved FLNACT fragment interacted strongly with STAT3 in wt macrophages. Inhibiting FLNA cleavage with the calpain inhibitor calpeptin reduced nuclear p-STAT3 levels and subsequent IL-6 secretion in vitro; and reduced atherosclerotic lesions in vivo. We conclude that FLNA interacts with transcription factors and thereby regulates angiogenesis and inflammatory responses which are important events in the progression of MI and atherosclerosis. These findings identify FLNA as an important new mediator of cardiovascular remodeling and as a potential target for therapy

Blocking the Cleavage of Filamin A by Calpain Inhibitor Decreases Tumor Cell Growth

Background/Aim: Filamin A (FLNA) is the most abundant and widely expressed isoform of filamin in human tissues. It is cleaved by calpain at the hinge 1 and 2 domains, producing a 90-kDa carboxyl-terminal fragment (FLNA(CT)). Recently, it has been shown that FLNA(CT) mediates cell signaling and transports transcription factors into the cell nucleus. However, the significance of cleavage of FLNA by calpain has not been studied in cancer cell growth. Calpeptin is a chemical inhibitor of both calpain 1 and 2 that cleaves FLNA. In this study, we questioned if inhibiting calpain using calpeptin would decrease tumor cell proliferation, migration, invasion, and colony formation. Materials and Methods: Human melanoma (A7), prostate cancer (PC3), mouse fibrosarcoma (T241) and endothelial (MS1) cells were assayed for proliferation, migration, invasion and colony formation after treatment with calpeptin. Cell lysates were immunoblotted for FLNA and FLNA(CT). Results: Calpeptin treatment of these cells resulted in a decreased production of FLNA(CT). Calpeptin-treated human and mouse tumor cells displayed impaired proliferation, migration, and colony formation. Conclusion: These data suggest that the cleavage of FLNA by calpain is an important cellular event in the regulation of tumor cell growth

The Risk-Associated Long Noncoding RNA NBAT-1 Controls Neuroblastoma Progression by Regulating Cell Proliferation and Neuronal Differentiation

Neuroblastoma is an embryonal tumor of the sympathetic nervous system and the most common extracranial tumor of childhood. By sequencing transcriptonnes of low- and high-risk neuroblastomas, we detected differentially expressed annotated and nonannotated long noncoding RNAs (lncRNAs). We identified a lncRNA neuroblastoma associated transcript-1 (NBAT-1) as a biomarker significantly predicting clinical outcome of neuroblastoma. CpG methylation and a high-risk neuroblastoma associated SNP on chromosome 6p22 functionally contribute to NBAT-1 differential expression. Loss of NBAT-1 increases cellular proliferation and invasion. It controls these processes via epigenetic silencing of target genes. NBAT-1 loss affects neuronal differentiation through activation of the neuronal-specific transcription factor NRSF/REST. Thus, loss of NBAT-1 contributes to aggressive neuroblastoma by increasing proliferation and impairing differentiation of neuronal precursors

Sense-Antisense lncRNA Pair Encoded by Locus 6p22.3 Determines Neuroblastoma Susceptibility via the USP36-CHD7-SOX9 Regulatory Axis

Trait-associated loci often map to genomic regions encoding long noncoding RNAs (lncRNAs), but the role of these lncRNAs in disease etiology is largely unexplored. We show that a pair of sense/antisense lncRNA (6p22lncRNAs) encoded by CASC15 and NBAT1 located at the neuroblastoma (NB) risk-associated 6p22.3 locus are tumor suppressors and show reduced expression in high-risk NBs. Loss of functional synergy between 6p22lncRNAs results in an undifferentiated state that is maintained by a gene-regulatory network, including SOX9 located on 17q, a region frequently gained in NB. 6p22lncRNAs regulate SOX9 expression by controlling CHD7 stability via modulating the cellular localization of USP36, encoded by another 17q gene. This regulatory nexus between 6p22.3 and 17q regions may lead to potential NB treatment strategies