Cell-to-cell contacts are crucial for cell differentiation. The promyogenic cell surface protein, Cdo, functions as a component of multiprotein clusters to mediate cell adhesion signaling. Connexin43, the main connexin forming gap junctions, also plays a key role in myogenesis. At least part of its effects are independent of the intercellular channel function, but the mechanisms underlying are unknown. Here, using multiple optical approaches, we provided the first evidence that Cx43 physically interacts with Cdo to form dynamic complexes during myoblast differentiation, offering clues for considering this interaction a structural basis of the channel-independent function of Cx43.
Sphingosine 1-phosphate (S1P) is a bioactive lipid that is abundantly present in the serum and mediates multiple biological responses. With the aim of extending our knowledge on the role played by S1P in the regulation of cytoskeletal reorganization, native as well as C2C12 myoblasts stably transfected with green fluorescent protein (GFP)-tagged alpha- and beta-actin constructs were stimulated with S1P (1 microM) and observed under confocal and multiphoton microscopes. The addition of S1P induced the appearance of actin stress fibres and focal adhesions through Rho- and phospholipase D (PLD)-mediated pathways. The cytoskeletal response was dependent on the extracellular action of S1P through its specific surface receptors, since the intracellular delivery of the sphingolipid by microinjection was unable to modify the actin cytoskeletal assembly. Interestingly, it was revealed by whole-cell patch-clamp that S1P-induced stress fibre formation was associated with increased ion currents and conductance through stretch-activated channels (SACs), thereby suggesting a possible regulatory role for organized actin in channel sensitivity. Experiments aimed at stretching the plasma membrane of C2C12 cells, using the cantilever of an atomic force microscope, indicated that there was a Ca2+ influx through putative SACs. In conclusion, the present data suggest novel mechanisms of S1P signalling involving actin cytoskeletal reorganization and Ca2+ elevation through SACs that might influence myoblastic functions.
Considerable attention has been paid to the high cytotoxic potential of small, prefibrillar aggregates of proteins/peptides, either associated or not associated with amyloid diseases. Recently, we reported that different cell types are variously affected by early aggregates of the N‐terminal domain of the prokaryotic hydrogenase maturation factor HypF (HypF‐N), a protein not involved in any disease. In this study, we provide detailed information on a chain of events triggered in Hend murine endothelial cells and IMR90 fibroblasts, which have previously been shown to be highly vulnerable or very resistant, respectively, to HypF‐N aggregates. Initially, both cell lines displayed impaired viability upon exposure to HypF‐N toxic aggregates; however, at longer exposure times, IMR90 cells recovered completely, whereas Hend cells did not. In particular, significant initial mitochondrial permeability transition (MPT) pore opening was found in IMR90 cells followed by a sudden repair of membrane integrity with rapid and efficient inhibition of cytochrome c and AIF release, and upregulation of Bcl‐2. The greater resistance of IMR90 fibroblasts may also be due to a higher cholesterol content in the plasma membrane, which disfavours interaction with the aggregates. In contrast, Hend cells, which have less membrane cholesterol, showed delayed MPT opening with prolonged translocation of cytochrome c into the cytosol. Finally, the caspase 9 active fragment was increased significantly in both Hend and IMR90 cells; however, only Hend cells showed caspase 8 and caspase 3 activation with DNA fragmentation. From our data, the different responses of the two cell types to the same aggregates appear to be associated with two key events: (a) aggregate interaction with the plasma membrane, disfavoured by a high level of membrane cholesterol; and (b) alterations in mitochondrial functionality, leading to the release of pro‐apoptotic stimuli, which are counteracted by upregulation of Bcl‐2.
Injured skeletal muscle can undergo repair spontaneously via satellite cells activation. However, increasing evidence suggest that the mere presence of these cells is not sufficient to ensure a rapid functional regeneration of the injured muscle, and that the release of appropriate factors and the establishment of suitable microenvironment are even more important in determining the effectiveness of the myogenic response. In particular, extracellular matrix remodelling is essential for myoblast migration, differentiation and fusion at the sites of muscle injury. On these basis, in the present study we evaluated the ability of C2C12 myoblasts and satellite cells to express and release metalloproteases in the course of differentiation and whether this activity could be regulated by factors released by MSCs. This with the aim of expanding our knowledge on the paracrine effects of MSCs on skeletal muscle repair/regeneration. The results showed that the administration of conditioned medium from MSCs (MSC-CM) up-regulated the expression and promoted MMP-2 and MMP-9 activation in the cultured myoblasts, as judged by Western blotting, confocal immunofluorescence and gelatinase/collagenase assay. Similar results were obtained when MSCs were separated from the myoblasts by polycarbonate membranes, enabling diffusion of soluble factors while preventing the physical contact between the two cell types. Interestingly, in the single fiber experiments, it was possible to reveal that MSC-CM administration promoted satellite cell mobilization and fusion into multinucleated myotubes. These findings add new information on the effects of MSC on the skeletal muscle healing, and suggest that growth factors and cytokines released by these cells may play a pivotal role not only in the modulation of tissue fibrosis but also in the differentiation of satellite cells.
