Actin-associated protein palladin is required for migration behavior and differentiation potential of C2C12 myoblast cells

https://doi.org/10.1016/j.bbrc.2014.08.143Get rights and content

Highlights

  • Palladin is involved in myogenesis in vitro.

  • Palladin knockdown by siRNA increases myoblast proliferation, viability and differentiation.

  • Palladin knockdown decreases C2C12 myoblast migration ability.

Abstract

The actin-associated protein palladin has been shown to be involved in differentiation processes in non-muscle tissues. However, but its function in skeletal muscle has rarely been studied. Palladin plays important roles in the regulation of diverse actin-related signaling in a number of cell types. Since intact actin-cytoskeletal remodeling is necessary for myogenesis, in the present study, we pursue to investigate the role of actin-associated palladin in skeletal muscle differentiation. Palladin in C2C12 myoblasts is knocked-down using specific small interfering RNA (siRNA). The results show that down-regulation of palladin decreased migratory activity of mouse skeletal muscle C2C12 myoblasts. Furthermore, the depletion of palladin enhances C2C12 vitality and proliferation. Of note, the loss of palladin promotes C2C12 to express the myosin heavy chain, suggesting that palladin has a role in the modulation of C2C12 differentiation. It is thus proposed that palladin is required for normal C2C12 myogenesis in vitro.

Introduction

Skeletal muscle is a heterogeneous tissue that is required for almost all vertebrate conscious action. The differentiation of muscle progenitor cells into mature myotubes, called myogenesis, is a highly ordered process [1], [2], [3]. Initially, mononucleoted myoblasts proliferate, irreversibly withdraw from the cell-cycle, migrate and align with one another. After alignment, myoblasts fuse to form multinucleated myotubes and terminal differentiated myofibers [4], [5]. Skeletal muscle differentiation necessitates intensive actin cytoskeleton rearrangement to undergo differentiation with complex changes, including maintenance of cell shape, coordinated cell locomotion, adhesion, fusion, cell–extracellular matrix interaction, and muscle contraction [6]. Growing evidence suggests that the sub-cellular coordination of the cytoskeleton and its regulatory, scaffolding and cytoskeletal cross-linking proteins are responsible for these dynamic reorganizations and maintaining the normal actin-cytoskeleton during myogenesis [7], [8], [9].

Palladin was characterized independently by two research groups, namely those of Dr. Otey and Dr. Carpén, a decade ago [10], [11]. This phosphoprotein belongs to the cytoskeleton-associated protein family named palladin–myotilin–myopalladin [12]. Palladin functions as a scaffold protein that interacts with actin and numerous actin-associated proteins that are required for organizing the actin-cytoskeleton [13], [14], [15], [16]. Palladin is widely expressed in both muscle and non-muscle cells and tissues. In vertebrates, several palladin isoforms are transcribed from a single gene through alternative splicing [12], [17], [18]. Three canonical isoforms of palladin have been exhaustively characterized, with molecular weights of 200, 140, and 90 kDa, respectively [10], [19]. Over the past few years, many studies have suggested the contribution of palladin in organizing actin arrays, which is necessary for controlling cell shape, migration, invasion, and development in a variety of cell types and tissues [10], [16], [19], [20]. Indeed, palladin presents in actin-based structures such as stress fibers, focal adhesions, membrane ruffles, and podosomes [10], [21]. Palladin knockout mouse had the embryonic lethal phenotype due to neural tube closure defects [18]. In addition, suppression of palladin expression in neurons resulted in decreasing of neurite outgrowth [22]. Moreover, fibroblasts derived from palladin-deficient mice showed defects in cell motility, adhesion, and actin organization [23]. Nevertheless, the overexpression of palladin in Cos-7 cells and astrocytes increased the number and size of actin bundles [24]. Importantly, 140 kDa-palladin was up-regulated by TGF-β1 during myofibroblast differentiation [25]. Likewise, palladin also played an important role in smooth muscle cell differentiation [26]. However, fewer studies have reported the role of palladin in skeletal muscle differentiation.

Given that palladin is involved in the regulation of diverse actin-related signaling processes, it is speculated that palladin plays potential roles in skeletal muscle differentiation, at least through actin-cytoskeleton remodeling activities. The present study examines whether palladin is related to the differentiation of murine skeletal muscle C2C12 myoblasts. Our data demonstrat for the first time that a depletion of palladin by siRNA inhibit the migration of skeletal muscle cells, but promot their proliferation and differentiation.

Section snippets

Mouse skeletal muscle cell line C2C12 culture

C2C12 (from Bioresource Collection and Research Center, Taiwan) was propagated as myoblasts in growth medium, GM (Dulbecco’s modified Eagle’s medium, DMEM, Hyclone, USA) supplemented with 10% fetal bovine serum (Hyclone) at 37 °C in a humidified atmosphere of air and 5% CO2. For differentiation into myotubes, the myoblasts were switched to differentiation medium, DM (DMEM with 2% horse serum (GIBCO, USA)). The myotubes began to form in 2–4 days post-differentiation.

For 3-(4, 5-dimethyl

Palladin expression was significantly depleted by siRNA in C2C12 myoblasts

The expression of the palladin isoforms during the transition from proliferation to early myotube differentiation of C2C12 cells induced by serum starvation was initially examined. As shown in the control cells, the amount of the 90 kDa isoform rapidly increased whereas the amount of the 140 kDa isoform was slightly decreased upon myogenesis in C2C12 myoblasts (Fig. 1B and C). These results are consistent with previously published reports on C2C12 myogenesis [18]. To explore the possible

Discussion

It is commonly accepted that skeletal muscle differentiation is associated with dramatic cytoskeletal changes. Cytoskeletons and their related proteins are well known to be involved in the intracellular signal transduction and cytoskeleton re-organization that could be necessary for myoblast proliferation, fusion and migration to form myotubes. Palladin is a cytoskeleton-associated protein that interacts with many proteins that are required for cell mobility [20], [21], [31], intracellular

Acknowledgments

This work was supported by Grant NSC-100-2311-B-006-007 from the National Science Council of Taiwan, and MOST102-2320-B-006-036 from the Ministry of Science and Technology of Taiwan, and National Cheng Kung University’s Aim for the Top University Project.

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