ABSTRACT
Ubiquitin-mediated protein degradation is the main mechanism for controlled proteolysis, which is crucial for muscle development and maintenance. The ankyrin repeat-containing protein with a suppressor of cytokine signaling box 2 gene (ASB2) encodes the specificity subunit of an E3 ubiquitin ligase complex involved in differentiation of hematopoietic cells. Here, we provide the first evidence that a novel ASB2 isoform, ASB2beta, is important for muscle differentiation. ASB2beta is expressed in muscle cells during embryogenesis and in adult tissues. ASB2beta is part of an active E3 ubiquitin ligase complex and targets the actin-binding protein filamin B (FLNb) for proteasomal degradation. Thus, ASB2beta regulates FLNb functions by controlling its degradation. Knockdown of endogenous ASB2beta by shRNAs during induced differentiation of C2C12 cells delayed FLNb degradation as well as myoblast fusion and expression of muscle contractile proteins. Finally, knockdown of FLNb in ASB2beta knockdown cells restores myogenic differentiation. Altogether, our results suggest that ASB2beta is involved in muscle differentiation through the targeting of FLNb to destruction by the proteasome.
Subject(s)
Carrier Proteins/metabolism , Cell Differentiation , Contractile Proteins/metabolism , Microfilament Proteins/metabolism , Myoblasts/metabolism , Proteasome Endopeptidase Complex/metabolism , Suppressor of Cytokine Signaling Proteins/metabolism , Ubiquitin-Protein Ligases/metabolism , Adaptor Proteins, Signal Transducing , Animals , Cell Line , Chickens , Contractile Proteins/deficiency , Filamins , Gene Knockdown Techniques , Humans , Mice , Microfilament Proteins/deficiency , RNA Interference , RNA, Messenger/metabolism , Suppressor of Cytokine Signaling Proteins/deficiency , Suppressor of Cytokine Signaling Proteins/geneticsABSTRACT
Although transcription factors are still the main focus to understanding leukemogenesis, recent results strongly suggest that alteration of a receptor and/or subsequent signaling plays a critical and co-operative role in the pathogenesis of acute myeloid leukemia (AML). The t(15;17) translocation, found in 95% of APL, encodes a PML-RARalpha fusion protein. A main model proposed for acute promyelocytic leukemia (APL) is that PML-RARalpha exerts its oncogenic effects by repressing retinoic acid-inducible genes critical to myeloid differentiation. Dysregulation of these genes may result in abnormal signaling, thereby freeing pre-leukemic cells from controls which normally induce the onset of differentiation. It is also likely that treatment of APL cells by retinoic acid induces de novo up-regulation of the same genes which are dominantly repressed by PML-RARalpha and whose expression is required for reactivation of the differentiation program. Identification of such genes together with the signaling pathways interrupted at the early stages of leukemia transformation and reactivated during retinoic acid-induced differentiation in APL cells will contribute to the development of new molecular targets for treatment of leukemia.
Subject(s)
Leukemia, Promyelocytic, Acute/metabolism , Signal Transduction , Chromosomes, Human, Pair 15 , Chromosomes, Human, Pair 17 , Humans , Leukemia, Promyelocytic, Acute/genetics , Leukemia, Promyelocytic, Acute/pathology , Translocation, GeneticABSTRACT
A pivotal role has been assigned to Myb in the control of myeloid cell growth. Although Myb is a target of retinoic acid, little is known about the mechanisms by which it may contribute to induced growth arrest in leukemia cells. Indeed, few Myb target genes are known to be linked to proliferation. Myeloblastin is involved in the control of proliferation in myeloid leukemia cells. It is expressed early during hematopoiesis and is a granulocyte colony-stimulating factor-responsive gene. Myeloblastin can confer factor-independent growth to hematopoietic cells, an early step in leukemia transformation. The myeloblastin promoter contains PU.1, C/EBP, and Myb binding sites, each of which are critical for constitutive expression in myeloid cells. Inhibition of myeloblastin expression in leukemia cells growth-arrested by retinoic acid is demonstrated to depend on Myb down-regulation. Myb is shown to induce myeloblastin expression and abolish its down-regulation by retinoic acid. Altogether, the data offer a clue as to how a myeloid-specific transcriptional machinery can be accessible to regulation by retinoic acid and point to myeloblastin as a novel target of Myb. This link between Myb and myeloblastin suggests a previously nonidentified Myb pathway through which growth arrest is induced by retinoic acid in myeloid leukemia cells.
Subject(s)
Antineoplastic Agents/pharmacology , Gene Expression Regulation, Leukemic/drug effects , Neoplasm Proteins/genetics , Proto-Oncogene Proteins c-myb/physiology , Serine Endopeptidases/genetics , Transcription Factors , Transcription, Genetic , Tretinoin/pharmacology , Animals , Base Sequence , Binding Sites , CCAAT-Enhancer-Binding Protein-alpha/metabolism , CCAAT-Enhancer-Binding Protein-beta/metabolism , CCAAT-Enhancer-Binding Protein-delta , CCAAT-Enhancer-Binding Proteins/metabolism , COS Cells , Cell Division/drug effects , Chlorocebus aethiops , Genes, myb , Molecular Sequence Data , Myeloblastin , Neoplasm Proteins/biosynthesis , Promoter Regions, Genetic , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-myb/biosynthesis , Recombinant Fusion Proteins/metabolism , Sequence Deletion , Trans-Activators/metabolism , TransfectionABSTRACT
The t(15;17) translocation, found in 95% of acute promyelocytic leukemia, encodes a promyelocytic leukemia (PML)-retinoic acid receptor alpha (RARalpha) fusion protein. Complete remission of acute promyelocytic leukemia can be obtained by treating patients with all-trans retinoic acid, and PML-RARalpha plays a major role in mediating retinoic acid effects in leukemia cells. A main model proposed for acute promyelocytic leukemia is that PML-RARalpha exerts its oncogenic effects by repressing the expression of retinoic acid-inducible genes critical to myeloid differentiation. By applying subtraction cloning to acute promyelocytic leukemia cells, we identified a retinoic acid-induced gene, PRAM-1 (PML-RARalpha target gene encoding an Adaptor Molecule-1), which encodes a novel adaptor protein sharing structural homologies with the SLAP-130/fyb adaptor. PRAM-1 is expressed and regulated during normal human myelopoiesis. In U937 myeloid precursor cells, PRAM-1 expression is inhibited by expression of PML-RARalpha in the absence of ligand and de novo superinduced by retinoic acid. PRAM-1 associates with other adaptors, SLP-76 and SKAP-55HOM, in myeloid cell lines and with protein tyrosine kinase lyn. By providing the first evidence that PML-RARalpha dysregulates expression of an adaptor protein, our data open new insights into signaling events that are disrupted during transformation by PML-RARalpha and induced by retinoic acid during de novo differentiation of acute promyelocytic leukemia cells.
Subject(s)
Leukemia, Promyelocytic, Acute/metabolism , Neoplasm Proteins/physiology , Oncogene Proteins, Fusion/physiology , Proteins/metabolism , Tretinoin/pharmacology , Adaptor Proteins, Signal Transducing , Amino Acid Sequence , Base Sequence , Cell Differentiation , Cloning, Molecular , DNA, Complementary , Gene Expression Regulation, Neoplastic/drug effects , Humans , Leukemia, Promyelocytic, Acute/pathology , Molecular Sequence Data , Proteins/chemistry , Proteins/genetics , RNA, Messenger/genetics , Tumor Cells, Cultured , U937 CellsABSTRACT
Hematopoiesis depends on a pool of quiescent hematopoietic stem/progenitor cells. When exposed to specific cytokines, a portion of these cells enters the cell cycle to generate an amplified progeny. Myeloblastin (MBN) initially was described as involved in proliferation of human leukemia cells. The granulocyte colony-stimulating factor (G-CSF), which stimulates the proliferation of granulocytic precursors, up-regulates MBN expression. Here we show that constitutive overexpression of MBN confers factor-independent growth to murine bone marrow-derived Ba/F3/G-CSFR cells. Our results point to MBN as a G-CSF responsive gene critical to factor-independent growth and indicate that expression of the G-CSF receptor is a prerequisite to this process. A 91-bp MBN promoter region containing PU.1, C/EBP, and c-Myb binding sites is responsive to G-CSF treatment. Although PU.1, C/EBP, and c-Myb transcription factors all were critical for expression of MBN, its up-regulation by G-CSF was associated mainly with PU.1. These findings suggest that MBN is an important target of PU.1 and a key protease for factor-independent growth of hematopoietic cells.
