A dynamic role for HDAC7 in MEF2-mediated muscle differentiation

The overlapping expression profile of MEF2 and the class-II histone deacetylase, HDAC7, led us to investigate the functional interaction and relationship between these regulatory proteins. HDAC7 expression inhibits the activity of MEF2 (-A, -C, and -D), and in contrast MyoD and Myogenin activities are not affected. Glutathione S-transferase pulldown and immunoprecipitation demonstrate that the repression mechanism involves direct interactions between MEF2 proteins and HDAC7 and is associated with the ability of MEF2 to interact with the N-terminal 121 amino acids of HDAC7 that encode repression domain 1. The MADS domain of MEF2 mediates the direct interaction of MEF2 with HDAC7, MEF2 inhibition by HDAC7 is dependent on the N-terminal repression domain and surprisingly does not involve the C-terminal deacetylase domain. HDAC7 interacts with CtBP and other class-I and -II HDACs suggesting that silencing of MEF2 activity involves corepressor recruitment. Furthermore, we show that induction of muscle differentiation by serum withdrawal leads to the translocation of HDAC7 from the nucleus into the cytoplasm. This work demonstrates that HDAC7 regulates the function of MEF2 proteins and suggests that this class-II HDAC regulates this important transcriptional (and pathophysiological) target in heart and muscle tissue. The nucleocytoplasmic trafficking of HDAC7 and other class-II HDACs during myogenesis provides an ideal mechanism for the regulation of HDAC targets during mammalian development and differentiation.

The coactivator-associated arginine methyltransferase is necessary for muscle differentiation - CARM1 coactivates myocyte enhancer factor-2

Studies with the myogenic basic helix-loop-helix and MADS box factors suggest that efficient transactivation is dependent on the recruitment of the steroid receptor coactivator (SRC) and the cofactors p300 and p300/CBP-associated factor. SRCs have been demonstrated to recruit CARM1 (coactivator-associated arginine methyltransferase-1), a member of the S-adenOSyl-L-methionine-dependent PRMTI-5 (protein-arginine N-methyltransferase-1-5) family, which catalyzes the methylation of arginine residues. This prompted us to investigate the functional role of CARM1/PRMT4 during skeletal myogenesis. We demonstrate that CARM1 and the SRC cofactor GRIP-1 cooperatively stimulate the activity of myocyte enhancer factor-2C (MEF2C). Moreover, there are direct interactions among MEF2C, GRIP-1, and CARM1. Chromatin immunoprecipitation demonstrated the in vivo recruitment of MEF2 and CARM1 to the endogenous muscle creatine kinase promoter in a differentiation-dependent manner. Furthermore, CARM1 is expressed in somites during embryogenesis and in the nuclei of muscle cells. Treatment of myogenic cells with the methylation inhibitor adenosine dialdehyde or tet-regulated CARM1 antisense expression did not affect expression of MyoD. However, inhibition of CARM1. inhibited differentiation and abrogated the expression of the key transcription factors (myogenin and MEF2) that initiate the differentiation cascade. This work clearly demonstrates that the arginine methyltransferase CARM1 potentiates myogenesis and supports the positive role of arginine methylation in mammalian differentiation.

Role of HuR in skeletal myogenesis through coordinate regulation of muscle differentiation genes

In this report, we investigate the role of the RNA-binding protein HuR during skeletal myogenesis. At the onset of myogenesis in differentiating C2C12 myocytes and in vivo in regenerating mouse muscle, HuR cytoplasmic abundance increased dramatically, returning to a predominantly nuclear presence upon completion of myogenesis. mRNAs encoding key regulators of myogenesis-specific transcription (myogenin and MyoD) and cell cycle withdrawal (p21), bearing AU-rich regions, were found to be targets of HuR in a differentiation-dependent manner. Accordingly, mRNA half-lives were highest during differentiation, declining when differentiation was completed. Importantly, HuR-overexpressing C2C12 cells displayed increased target mRNA expression and half-life and underwent precocious differentiation. Our findings underscore a critical function for HuR during skeletal myogenesis linked to HuR's coordinate regulation of muscle differentiation genes.

Strong smooth muscle differentiation is dependent on myocardin gene amplification in most human retroperitoneal leiomyosarcomas.

Myocardin (MYOCD), a serum response factor (SRF) transcriptional cofactor, is essential for cardiac and smooth muscle development and differentiation. We show here by array-based comparative genomic hybridization, fluorescence in situ hybridization, and expression analysis approaches that MYOCD gene is highly amplified and overexpressed in human retroperitoneal leiomyosarcomas (LMS), a very aggressive well-differentiated tumor. MYOCD inactivation by shRNA in a human LMS cell line with MYOCD locus amplification leads to a dramatic decrease of smooth muscle differentiation and strongly reduces cell migration. Moreover, forced MYOCD expression in three undifferentiated sarcoma cell lines and in one liposarcoma cell line confers a strong smooth muscle differentiation phenotype and increased migration abilities. Collectively, these results show that human retroperitoneal LMS differentiation is dependent on MYOCD amplification/overexpression, suggesting that in these well-differentiated LMS, differentiation could be a consequence of an acquired genomic alteration. In this hypothesis, these tumors would not necessarily derive from cells initially committed to smooth muscle differentiation. These data also provide new insights on the cellular origin of these sarcomas and on the complex connections between oncogenesis and differentiation in mesenchymal tumors.

Smooth muscle differentiation identifies two classes of poorly differentiated pleomorphic sarcomas with distinct outcome.

The clinical relevance of accurately diagnosing pleomorphic sarcomas has been shown, especially in cases of undifferentiated pleomorphic sarcomas with myogenic differentiation, which appear significantly more aggressive. To establish a new smooth muscle differentiation classification and to test its prognostic value, 412 sarcomas with complex genetics were examined by immunohistochemistry using four smooth muscle markers (calponin, h-caldesmon, transgelin and smooth muscle actin). Two tumor categories were first defined: tumors with positivity for all four markers and tumors with no or incomplete phenotypes. Multivariate analysis demonstrated that this classification method exhibited the strongest prognostic value compared with other prognostic factors, including histological classification. Secondly, incomplete or absent smooth muscle phenotype tumor group was then divided into subgroups by summing for each tumor the labeling intensities of all four markers for each tumors. A subgroup of tumors with an incomplete but strong smooth muscle differentiation phenotype presenting an intermediate metastatic risk was thus identified. Collectively, our results show that the smooth muscle differentiation classification method may be a useful diagnostic tool as well as a relevant prognostic tool for undifferentiated pleomorphic sarcomas.

Influence of the insecticide pyriproxyfen on the flight muscle differentiation of Apis mellifera (Hymenoptera, Apidae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Ultrastructure of haemocytes present during flight muscle differentiation of Melipona quadrifasciata anthidioides, Lepidoptera (Hymenoptera, Apidae)

The present work deals with the ultrastructure of the haemocytes present in the dorsal thoracic region of larvae and pre-pupae of Melipona quadrifasciata anthidioides. This is a region of intense muscular differentiation in this phase of life of the insect. Only plasmatocytes, granulocytes, and adiphoaemocytes were found in this area, all showing signs of being intermediate forms.

Does alveolar soft-part sarcoma exhibit skeletal muscle differentiation? An immunocytochemical and biochemical study of myogenic regulatory protein expression

There has been persistent controversy regarding the nature of cell differentiation in alveolar soft-part sarcoma (ASPS) since its first description in 1952. Some studies suggest that ASPS might represent an unusual variant of skeletal muscle tumor, Given the availability of new monoclonal antibodies to probe for skeletal muscle differentiation and the rapid advance in immunocytochemical techniques for deparaffinized, formalin-fixed tissue sections, we wished to test the proposed hypothesis that ASPS might represent a new type of rhabdomyosarcoma Twelve archival samples of ASPS were retrieved, and we investigated the expression of two myogenic regulatory proteins, MyoD1 and myogenin, as rvell as other muscle-associated proteins, using sensitive immunocytochemical techniques. Despite the presence of desmin immunostaining in six ASPSs, no tumors were positive for either muscle actin or myoglobin Most importantly, no specimen showed nuclear expression of MyoD1 or myogenin, In 11 tumors, however, there was considerable granular immunostaining in the tumor cell cytoplasm with the anti-MyoD1 monoclonal antibody 5.8A, a phenomenon observed in various nonmuscle normal and neoplastic tissues with this antibody, To analyze the exact nature of immunostaining of MyoD1 and desmin in ASPS, biochemical analyses using available fresh frozen tumor tissue were performed, Although a 53-kDa band was noted with antidesmin antibody on Western blot analysis, no specific protein band that corresponds to the 45-kDa MyoD1 was detected with antibody 5.8A. These results confirm the presence of desmin in ASPS but argue against authentic expression of MyoD1, They also suggest that the cytoplasmic immunostaining observed with anti-MyoD1 antibody 5.8A most likely represents a nonspecific cross-reaction with an unknown cytoplasmic antigen, Considering the master role that MyoD1 and myogenin play in skeletal muscle commitment and differentiation and the lack of expression of these two proteins in ASPS as determined immunocytochemically and biochemically, we think that the histogenesis of ASPS remains unknown.

Studies on regulation of skeletal muscle differentiation by growth factors

Skeletal muscle differentiation involves sequential events in which proliferating undifferentiated myoblasts withdraw from the cell cycle and fuse to form multinucleated myotubes. The process of fusion is accompanied by the disappearance of proteins associated with cell proliferation and the coordinate induction of a battery of muscle-specific gene products, which includes the muscle isoenzyme of creatine kinase, nicotinic acetylcholine receptor, and contractile proteins such as alpha-actin. The molecular events associated with myogenesis are particularly amenable to experimental analysis because the events which occur in vivo can be recapitulated in vitro using established muscle cell lines. Initiation of myogenic differentiation in vitro can be achieved by removing serum from the culture medium. Myogenesis, therefore, can be considered to be regulated through a repression-type of mechanism by components in serum. The objectives of this project were to identify the components involved in regulation of myogenesis and approach the mechanism(s) whereby these components achieve their regulatory function. Initially, the effects of a series of polypeptide growth factors on myogenesis were examined. Among them TGF$\beta$ and FGF were found to be potent inhibitors of myogenic differentiation which did not affect cell proliferation. The inhibitory effects of these growth factors on differentiation requires their persistent presence in the culture medium. After myoblasts have undergone fusion, they become refractory to the inhibitory effects of TGF$\beta$, FGF, and serum. When fusion is inhibited by the presence of EGTA, a Ca$\sp{2+}$ chelator, muscle-specific genes are expressed reversibly upon removal of inhibitory growth factors. Subsequent exposure of biochemically differentiated cells to serum or TGF$\beta$ leads to down-regulation of muscle-specific genes. Stimulation with serum also leads to reentry of myocytes into the cell cycle, whereas fused myotubes are irreversibly and terminally differentiated. Measurement of levels of TGF$\beta$ receptors reveals that under non-fusing conditions, TGF$\beta$ receptor levels in biochemically differentiated myocytes remained as high as in undifferentiated myoblasts, while during terminal differentiation, TGF$\beta$ receptors decreased at least five-fold. Thus, down-regulation of TGF$\beta$ receptors is coupled to irreversible differentiation, but not reversible differentiation in the absence of fusion. The possible involvement of second messenger systems, such as cAMP and protein kinase C, in the pathway(s) by which TGF$\beta$, FGF, or serum factors transduce their signals from the cell surface to the nucleus was also examined. The results showed that myogenic differentiation is subject to negative regulation through cAMP elevation-dependent and cAMP elevation-independent pathways and that serum mitogens, TGF$\beta$ and FGF inhibit differentiation through a mechanism independent of cAMP-elevation or protein kinase C activation. ^

Direct Involvement of N-Cadherin–mediated Signaling in Muscle Differentiation

Cell–cell interactions, mediated by members of the cadherin family of Ca2+-dependent adhesion molecules, play key roles in morphogenetic processes as well as in the transduction of long-range growth and differentiation signals. In muscle differentiation cell adhesion is involved in both early stages of myogenic induction and in later stages of myoblast interaction and fusion. In this study we have explored the involvement of a specific cadherin, namely N-cadherin, in myogenic differentiation. For that purpose we have treated different established lines of cultured myoblasts with beads coated with N-cadherin–specific ligands, including a recombinant N-cadherin extracellular domain, and anti-N-cadherin antibodies. Immunofluorescent labeling for cadherins and catenins indicated that treatment with the cadherin-reactive beads for several hours enhances the assembly of cell–cell adherens-type junctions. Moreover, immunofluorescence and immunoblotting analyses indicated that treatment with the beads for 12–24 h induces myogenin expression and growth arrest, which are largely independent of cell plating density. Upon longer incubation with the beads (2–3 d) a major facilitation in the expression of several muscle-specific sarcomeric proteins and in cell fusion into myotubes was observed. These results suggest that surface clustering or immobilization of N-cadherin can directly trigger signaling events, which promote the activation of a myogenic differentiation program.

Golgi Complex Reorganization during Muscle Differentiation: Visualization in Living Cells and Mechanism

During skeletal muscle differentiation, the Golgi complex (GC) undergoes a dramatic reorganization. We have now visualized the differentiation and fusion of living myoblasts of the mouse muscle cell line C2, permanently expressing a mannosidase-green fluorescent protein (GFP) construct. These experiments reveal that the reorganization of the GC is progressive (1–2 h) and is completed before the cells start fusing. Fluorescence recovery after photobleaching (FRAP), immunofluorescence, and immunogold electron microscopy demonstrate that the GC is fragmented into elements localized near the endoplasmic reticulum (ER) exit sites. FRAP analysis and the ER relocation of endogenous GC proteins by phospholipase A2 inhibitors demonstrate that Golgi-ER cycling of resident GC proteins takes place in both myoblasts and myotubes. All results support a model in which the GC reorganization in muscle reflects changes in the Golgi-ER cycling. The mechanism is similar to that leading to the dispersal of the GC caused, in all mammalian cells, by microtubule-disrupting drugs. We propose that the trigger for the dispersal results, in muscle, from combined changes in microtubule nucleation and ER exit site localization, which place the ER exit sites near microtubule minus ends. Thus, changes in GC organization that initially appear specific to muscle cells, in fact use pathways common to all mammalian cells.

Delta like ligand 4 is a critical regulator of bone marrow cell differentiation into pericytes/vascular smooth muscle cells and is essential for the vasculogenesis that supports the growth of Ewing’s sarcoma

We have previously shown that vasculogenesis, the process by which bone marrow-derived cells are recruited to the tumor and organized to form a blood vessel network de novo, is essential for the growth of Ewing’s sarcoma. We further demonstrated that these bone marrow cells differentiate into pericytes/vascular smooth muscle cells(vSMC) and contribute to the formation of the functional vascular network. The molecular mechanisms that control bone marrow cell differentiation into pericytes/vSMC in Ewing’s sarcoma are poorly understood. Here, we demonstrate that the Notch ligand Delta like ligand 4 (DLL4) plays a critical role in this process. DLL4 is essential for the formation of mature blood vessels during development and in several tumor models. Inhibition of DLL4 causes increased vascular sprouting, decreased pericyte coverage, and decreased vessel functionality. We demonstrate for the first time that DLL4 is expressed by bone marrow-derived pericytes/vascular smooth muscle cells in two Ewing’s sarcoma xenograft models and by perivascular cells in 12 out of 14 patient samples. Using dominant negative mastermind to inhibit Notch, we demonstrate that Notch signaling is essential for bone marrow cell participation in vasculogenesis. Further, inhibition of DLL4 using either shRNA or the monoclonal DLL4 neutralizing antibody YW152F led to dramatic changes in blood vessel morphology and function. Vessels in tumors where DLL4 was inhibited were smaller, lacked lumens, had significantly reduced numbers of bone marrow-derived pericyte/vascular smooth muscle cells, and were less functional. Importantly, growth of TC71 and A4573 tumors was significantly inhibited by treatment with YW152F. Additionally, we provide in vitro evidence that DLL4-Notch signaling is involved in bone marrow-derived pericyte/vascular smooth muscle cell formation outside of the Ewing’s sarcoma environment. Pericyte/vascular smooth muscle cell marker expression by whole bone marrow cells cultured with mouse embryonic stromal cells was reduced when DLL4 was inhibited by YW152F. For the first time, our findings demonstrate a role for DLL4 in bone marrow-derived pericyte/vascular smooth muscle differentiation as well as a critical role for DLL4 in Ewing’s sarcoma tumor growth.

Subcellular localization of the steroid receptor coactivators (SRCs) and MEF2 in muscle and rhabdomyosarcoma cells

Skeletal muscle differentiation and the activation of muscle-specific gene expression are dependent on the concerted action of the MyoD family and the MADS protein, MEF2, which function in a cooperative manner. The steroid receptor coactivator SRC-2/GRIP-1/TIF-2, is necessary for skeletal muscle differentiation, and functions as a cofactor for the transcription factor, MEF2. SRC-P belongs to the SRC family of transcriptional coactivators/cofactors that also includes SRC-1 and SRC-3/RAC-3/ACTR/ AIB-1. In this study we demonstrate that SRC-P is essentially localized in the nucleus of proliferating myoblasts; however, weak (but notable) expression is observed in the cytoplasm. Differentiation induces a predominant localization of SRC-P to the nucleus; furthermore, the nuclear staining is progressively more localized to dot-like structures or nuclear bodies. MEF2 is primarily expressed in the nucleus, although we observed a mosaic or variegated expression pattern in myoblasts; however, in myotubes all nuclei express MEF2. GRIP-1 and MEF2 are coexpressed in the nucleus during skeletal muscle differentiation, consistent with the direct interaction of these proteins. Rhabdomyosarcoma (RMS) cells derived from malignant skeletal muscle tumors have been proposed to be deficient in cofactors. Alveolar RMS cells very weakly express the steroid receptor coactivator, SRC-P, in a diffuse nucleocytoplasmic staining pattern. MEF2 and the cofactors, SRC-1 and SRC-3 are abundantly expressed in alveolar and embryonal RMS cells; however, the staining is not localized to the nucleus. Furthermore, the subcellular localization and transcriptional activity of MEF2C and a MEF2-dependent reporter are compromised in alveolar RMS cells. In contrast, embryonal RMS cells express SRC-2 in the nucleus, and MEF2 shuttles from the cytoplasm to the nucleus after serum withdrawal. In conclusion, this study suggests that the steroid receptor coactivator SRC-P and MEF2 are localized to the nucleus during the differentiation process. In contrast, RMS cells display aberrant transcription factor SRC localization and expression, which may underlie certain features of the RMS phenotype.

A morphological view of the relationship between indirect flight muscle maturation and the flying needs of two species of advanced eusocial bees

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Function of GCIP (CCNDBP1), a helix -loop -helix leucine -zip protein, in cell differentiation and tumorigenesis

Cell growth and differentiation are complex and well-organized processes in which cells respond to stimuli from the environment by carrying out genetic programs. Transcription factors with helix-loop-helix (HLH) motif play critical roles in controlling the expression of genes involved in lineage commitment, cell fate determination, proliferation and tumorigenesis. This study has examined the roles of GCIP (CCNDBP1) in cell differentiation and tumorigenesis. GCIP is a recently identified HLH-leucine zipper protein without a basic region like the Id family of proteins. However, GCIP shares little sequence homology with the Id proteins and has domains with high acidic amino acids and leucine-rich regions following the HLH domain like c-Myc. Here we firstly demonstrate that GCIP is a transcription regulator related to muscle differentiation program. Overexpression of GCIP in C2C12 cells not only promotes myotube formation but also upregulates myogenic differentiation biomarkers, including MHC and myogenein. On the other hand, our finding also suggests that GCIP is a potential tumor suppressor related to cell cycle control. Expression of GCIP was significantly down-regulated in colon tumors as compared to normal colon tissues. Overexpression of GCIP in SW480 colon cancer cell line resulted in a significant inhibition on tumor cell colony formation on soft agar assays while silencing of GCIP expression by siRNA can promote cell proliferation and colony formation. In addition, results from transgenic mice specifically expressing GCIP in liver also support the idea that GCIP is involved in the early stage of hepatocarcinogenesis and decreased susceptibility to chemical hepatocarcinogenesis. ^