Débora M. Portilho

Cholesterol depletion by methyl-β-cyclodextrin enhances myoblast fusion and induces the formation of myotubes with disorganized nuclei

The formation of a skeletal muscle fiber begins with the withdrawal of committed mononucleated precursors from the cell cycle. These myoblasts elongate while aligning with each other, guided by recognition between their membranes. This step is followed by cell fusion and the formation of long striated multinucleated myotubes. We used methyl-β-cyclodextrin (MCD) in primary cultured chick skeletal muscle cells to deplete membrane cholesterol and investigate its role during myogenesis. MCD promoted a significant increase in the expression of troponin T, enhanced myoblast fusion, and induced the formation of large multinucleated myotubes with nuclei being clustered centrally and not aligned at the cell periphery. MCD myotubes were striated, as indicated by sarcomeric α-actinin staining, and microtubule and desmin filament distribution was not altered. Pre-fusion MCD-treated myoblasts formed large aggregates, with cadherin and β-catenin being accumulated in cell adhesion contacts. We also found that the membrane microdomain marker GM1 was not present as clusters in the membrane of MCD-treated myoblasts. Our data demonstrate that cholesterol is involved in the early steps of skeletal muscle differentiation.

A soluble and active form of Wnt-3a protein is involved in myogenic differentiation after cholesterol depletion

Cholesterol is one of the major lipids of plasma membranes. Recently, we have shown that cholesterol depletion by methyl‐β‐cyclodextrin (MβCD) induces the activation of the Wnt/β‐catenin pathway and enhances myogenic differentiation. Here, we show that MβCD‐conditioned media accelerates myogenesis in a similar way as MβCD does, suggesting that the effects induced by MβCD could be caused by soluble factors present in the culture medium. Soluble Wnt‐3 protein is significantly enhanced in MβCD‐conditioned medium. Wnt‐3a‐enriched media induces myogenesis as much as MβCD does, whereas Wnt‐5a‐enriched media inhibits. We suggest that Wnt‐3a is involved in the myogenic induction observed after cholesterol depletion.

Membrane Cholesterol Depletion by Methyl-β-Cyclodextrin Enhances the Expression of Cardiac Differentiation Markers

Cholesterol is a sterol lipid that plays pleiotropic roles in the plasma membrane; it is involved in maintaining membrane fluidity and permeability and the structure of lipid microdomains. Despite its importance, the consequences of membrane cholesterol depletion during cardiac differentiation have not been described. Therefore, we investigated the cellular and molecular mechanisms associated with cholesterol depletion in cultures of chick cardiac cells. We used methyl-β-cyclodextrin (MCD) to deplete membrane cholesterol and investigate its role in cardiac differentiation by following the expression of several markers including the transcriptional factor Nkx2.5, the myofibrillar protein tropomyosin, the cytoskeletal intermediate filament protein desmin, the caveolar protein caveolin-3, the cadherin/β-catenin adhesion complex, and the junctional protein connexin 43. Confocal microscopy showed that desmin-positive cells were located more externally in the aggregates in relation to the more internally located caveolin-3-positive cells. Desmin and caveolin-3 were co-localized in filamentous structures in the subsarcolemmal region of well-spread cells outside the aggregates. β-Catenin was concentrated in regions of cell-cell contact, and tropomyosin in sarcomeric structures. Western blot tests showed that immediately following cholesterol depletion, there was a slight decrease in the expression of caveolin-3 and desmin, and at the same time there was a sharp increase in the expression of cadherin, tropomyosin, Nkx2.5 and connexin 43. Further, we found an increase in the expression of cardiac β-myosin heavy chain 7, a marker of the cardiac hypertrophic phenotype. These observations suggest that membrane cholesterol plays a significant role in regulating cardiomyocyte differentiation.

The Wnt signaling pathway regulates Nalm-16 b-cell precursor acute lymphoblastic leukemic cell line survival and etoposide resistance.

B-cell precursor acute lymphoblastic leukemia (BCP-ALL) is the most common malignancy in children. The Wnt signaling pathway has been found to be extensively involved in cancer onset and progression but its role in BCP-ALL remains controversial. We evaluate the role of the Wnt pathway in maintenance of BCP-ALL cells and resistance to chemotherapy. Gene expression profile revealed that BCP-ALL cells are potentially sensitive to modulation of Wnt pathway. Nalm-16 and Nalm-6 cell lines displayed low levels of canonical activation, as reflected by the virtually complete absence of total beta-catenin in Nalm-6 and the beta-catenin cell membrane distribution in Nalm-16 cell line. Canonical activation with Wnt3a induced nuclear beta-catenin translocation and led to BCP-ALL cell death. Lithium chloride (LiCl) also induced a cytotoxic effect on leukemic cells. In contrast, both Wnt5a and Dkk-1 increased Nalm-16 cell survival. Also, Wnt3a enhanced the in vitro sensitivity of Nalm-16 to etoposide (VP-16) while treatment with canonical antagonists protected leukemic cells from chemotherapy-induced cell death. Overall, our results suggest that canonical activation of the Wnt pathway may exerts a tumor suppressive effect, thus its inhibition may support BCP-ALL cell survival.

Association between the muscle-specific proteins desmin and caveolin-3 in muscle cells

The muscle-specific intermediate filament protein desmin is expressed in mononucleated myoblasts and in differentiated myotubes. Desmin has been shown to associate with the sarcolemma in specific structures, such as neuromuscular junctions and the dystrophin-associated protein complex. Since these are specialized membrane regions, the study of a possible association between desmin and liquid-ordered membrane microdomains is of particular interest. We have carried out an analysis of the association between desmin and the muscle-specific protein caveolin-3, a major component of caveolar microdomains. Our results demonstrate that (1) desmin precisely co-localizes with caveolin-3 in myoblasts and multinucleated myotubes, (2) caveolin-3 is up-regulated during in vitro chick muscle development, (3) desmin is detectable in caveolae-enriched membrane fractions prepared from skeletal muscle, and (4) caveolin-3 co-immunoprecipitates with desmin. We have thus shown, for the first time, an association between the intermediate filament protein desmin and caveolin-3 in myogenic cells.

Desmin filaments are stably associated with the outer nuclear surface in chick myoblasts

Eukaryotic cells have highly organized, interconnected intracellular compartments. The nuclear surface and cytoplasmic cytoskeletal filaments represent compartments involved in such an association. Intermediate filaments are the major cytoskeletal elements in this association. Desmin is a muscle-specific structural protein and one of the earliest known muscle-specific genes to be expressed during cardiac and skeletal muscle development. Desmin filaments have been shown to be associated with the nuclear surface in the myogenic cell line C2C12. Previous studies have revealed that mice lacking desmin develop imperfect muscle, exhibiting the loss of nuclear shape and positioning. In the present work, we have analyzed the association between desmin filaments and the outer nuclear surface in nuclei isolated from pectoral skeletal muscle of chick embryos and in primary chick myogenic cell cultures by using immunofluorescence microscopy, negative staining, immunogold, and transmission electron microscopy. We show that desmin filaments remain firmly attached to the outer nuclear surface after the isolation of nuclei. Furthermore, positive localization of desmin persists after gentle washing of the nuclei with high ionic strength solutions. These data suggest that desmin intermediate filaments are stably and firmly connected to the outer nuclear surface in skeletal muscles cells in vivo and in vitro.

α-Cyclodextrin enhances myoblast fusion and muscle differentiation by the release of IL-4.

Muscle fibers are formed during embryonic development by the fusion of mononucleated myoblasts. The spatial structure and molecular composition of the sarcolemma are crucial for the myoblast recognition and fusion steps. Cyclodextrins are a group of substances that have the ability to solubilize lipids through the formation of molecular inclusion complexes. Previously, we have shown that methyl-β-cyclodextrin (MbCD) enhances muscle differentiation. Here, we analyzed the effects of α-cyclodextrin (aCD) during myogenesis. Myogenic cultures treated with aCD showed an increase in myoblast fusion and in the expression of myogenin, sarcomeric tropomyosin and desmin. aCD-conditioned media accelerates myogenesis in a similar way as aCD does, and increased levels of IL-4 were found in aCD-conditioned media. aCD-induced effects on myogenesis were inhibited by an anti-IL4 antibody. These results show that α-cyclodextrin induces myogenic differentiation by the release of IL-4.

13-P118 Wnt/β-catenin pathway activation and myogenic differentiation are induced by cholesterol depletion

In the embryonic telencephalon, Pax6 is expressed in the progenitors of the pallium in a caudal to rostral gradient and governs the anteroposterior and dorsoventral patterning of the telencephalon. The pallial–subpallial boundary (PSPB) is both a physical and gene expression boundary separating dorsal and ventral telencephalon. Previous studies have indicated that Pax6 is required for establishment and maintenance of this boundary. However, it is largely unknown how Pax6 exerts its effects at the molecular level at the PSPB. We have performed microarray analysis on tissue from the lateral telencephalon, including the PSPB, to compare gene expression at the onset of neurogenesis (E12) in Pax6 DTy54 and Pax6 DTy54 embryos. The novel DTy54 Pax6 reporter mouse in which cells capable of expressing Pax6 are tauGFP positive allows the PSPB to be visualized by the high expression of tauGFP. The GFP positive cells which express Pax6 were isolated by fluorescence activated cell sorting (FACS) and dissociated to carry out the microarray comparison of gene expression. This analysis identified many novel downstream targets of Pax6 as well as some novel biological processes with which Pax6 has not formerly been associated. Cell cycle processes are prominent themes in the up-regulated gene set of the microarray data. This is consistent with the important role of Pax6 in regulating cell cycle progression in the cortical ventricular zone. To address this issue, we are testing the changes in cell cycle and cell proliferation at the PSPB in Pax6 DTy54 and Pax6 DTy54 embryos using bromodeoxyuridine (BrdU) and iododeoxyuridine (IddU) double labeling.