The Actin-binding Protein, Actinin Alpha 4 (ACTN4), Is a Nuclear Receptor Coactivator that Promotes Proliferation of MCF-7 Breast Cancer Cells

Abstract

Alpha actinins (ACTNs) are known for their ability to modulate cytoskeletal organization and cell motility by cross-linking actin filaments. We show here that ACTN4 harbors a functional LXXLL receptor interaction motif, interacts with nuclear receptors in vitro and in mammalian cells, and potently activates transcription mediated by nuclear receptors. Whereas overexpression of ACTN4 potentiates estrogen receptor ␣ (ER␣)-mediated transcription in transient transfection reporter assays, knockdown of ACTN4 decreases it. In contrast, histone deacetylase 7 (HDAC7) inhibits estrogen receptor ␣ (ER␣)-mediated transcription. Moreover, the ACTN4 mutant lacking the CaM (calmodulin)-like domain that is required for its interaction with HDAC7 fails to activate transcription by ER␣. Chromatin immunoprecipitation (ChIP) assays demonstrate that maximal associations of ACTN4 and HDAC7 with the pS2 promoter are mutually exclusive. Knockdown of ACTN4 significantly decreases the expression of ER␣ target genes including pS2 and PR and also affects cell proliferation of MCF-7 breast cancer cells with or without hormone, whereas knockdown of HDAC7 exhibits opposite effects. Interestingly, overexpression of wild-type ACTN4, but not the mutants defective in interacting with ER␣ or HDAC7, results in an increase in pS2 and PR mRNA accumulation in a hormone-dependent manner. In summary, we have identified ACTN4 as a novel, atypical coactivator that regulates transcription networks to control cell growth.The alpha actinins (ACTNs) 2 belong to a family of cytoskeletal proteins that bind actin filaments to maintain cytoskeletal structure and cell morphology (1). Among the four members of the family, ACTN2 and ACTN3 are expressed primarily in muscle, while ACTN1 and ACTN4 are ubiquitously expressed (2). All four members of the actinin family share high sequence homology with conserved functional domains including an N-terminal actin-binding domain containing two highly conserved calponin homology (CH1 and CH2) domains, a central domain consisting of four spectrin repeats (SR), two EF hand calcium-binding domains and a C-terminal calmodulin (CaM)-like domain (3). Although predominantly localized in the cytoskeleton, ACTN4 is also found in the nucleus of certain cell types and is capable of translocating to the nucleus in response to extracellular stimuli (4). In addition to its role in the cytosol, we have recently identified a novel function for ACTN4 in transcriptional regulation by myocyte enhancer factor 2 (MEF2) (5). Another study has also demonstrated an association between ACTN4 and nuclear factor B (NF-B) (6). These findings suggest that ACTN4 may play an unexpected role in transcriptional regulation.Nuclear hormone receptors, including vitamin D receptor (VDR) and steroid hormone receptors such as estrogen receptors (ER) are ligand-activated transcription factors that control aspects of homeostasis, cell differentiation, proliferation, and development (7-9). Transcriptional regulation by nuclear receptors is thought to occur through t...

RETRACTED ARTICLE: Regulation of estrogen receptor signaling in breast carcinogenesis and breast cancer therapy

“…Recently, actinin α4, a cytoskeletal modulator, has been identified as a novel, atypical ERα coactivator that regulates transcription networks to control cell growth. Actinin α4 interacts with ERα through its functional LXXLL receptor interaction motif and potentiates ERα gene expression in MCF7 cells [183]. The DEAD-box RNA helicases p68 (DDX5) and p72 (DDX17), which are primarily involved in RNA splicing, also act as ERα coactivators in breast cancer cells.…”

Identification of a Novel LXXLL Motif in α-Actinin 4-spliced Isoform That Is Critical for Its Interaction with Estrogen Receptor α and Co-activators

“…48 h after transfection, the cells were trypsinized, and equal numbers of cells were seeded and grown in DMEM supplemented with 10% charcoal-stripped fetal bovine serum, 50 units/ml penicillin G, and 50 g/ml streptomycin sulfate. Cell numbers were determined by a Cyquant cell proliferation assay kit (C7026) according to our published protocol (24). An aliquot of cells was also used to prepare whole cell lysates to examine expression levels of transfected HA-ACTN4 (Iso).…”

“…Antibodies and Chemicals-Anti-ACTN4 antibody has been previously described (23,24). Anti-HA-conjugated ␣-horseradish peroxidase was purchased from Roche Applied Science.…”

“…We have recently shown that ACTN4 (full length) is also capable of potentiating transcriptional activation by VDR and ER␣ and that knockdown of ACTN4 significantly compromised the ability of estrogen to induce ER␣ target genes (24). In addition to missing part of SR2, SR3, and SR4 because of exon exclusion, ACTN4 (Iso) harbors a putative LXXLL nuclear receptor interaction motif with distinct amino acid sequences C-terminal to the LXXLL motif when compared with ACTN4 (full length) (Fig.…”

“…Notably, although originally identified as an actin-binding protein, ACTN4 is localized in both the cytoplasm and the nucleus (22,23). Indeed, we have previously shown that ACTN4 interacts with and potentiates MEF2 transcription factors as well as estrogen receptor (ER) ␣ and VDR (23,24). The ability of ACTN4 to interact with and potentiate NR activity depends on an intact LXXLL motif present in ACTN4.…”