Metaplasia, the conversion of one cell type into another, has been suggested to be, at the molecular level, the consequence of a change in the expression of key developmental genes. In normal development, particular combinations of these master genes are activated in each embryo region following the expression of local inducing signals. Postnatally, numerous examples of pathologic metaplasia are known, but the underlying mechanisms are poorly understood [40, 41]. Metaplasias are nearly always associated with situations of tissue regeneration, in the absence of many of the signals that are present during development. In this study we have demonstrated that when myoblasts are cultured under conditions that affect or modify ECM synthesis and assembly, an important and significative increment in the expression of osteogenic markers is observed. This occurs through a mechanism that did not involve either the turn-off or turn-on of master genes such as Myf-5, MyoD, myogenin or Cbfa-1. The expression of these osteogenic markers was fully reverted by the addition of an exogenous ECM. Furthermore, we show that the ECM produced by myoblasts induced to transdifferentiate into osteoblasts by BMP-2 treatment was able to induce ALP activity in C2C12 myoblasts under normal skeletal muscle differentiation conditions. Both phenomena seem to be BMP-2 independent because the induction of osteogenic markers was not inhibited by competence with a soluble form of the BMP-2 receptor ectodomain. These results suggest that signals arising from the ECM induce the expression of osteogenic markers in myoblasts and emphasizes that a proper ECM is required for correct skeletal muscle differentiation.
We have previously shown that skeletal muscle differentiation was strongly inhibited under conditions where the assembly of the ECM was affected by inhibitors of proteoglycans synthesis as sodium chlorate or β-D-xyloside [11, 12]. As a consequence of these treatments, a decrease in focal adhesion kinase (FAK) phosphorylation was observed . This enzyme is activated upon interaction of integrins with ECM constituents [42, 43], but interference of this interactions by the addition of RGDS peptides affects normal skeletal muscle differentiation [10, 11]. These observations indicate that a proper interaction and signaling between cells and their environment is crucial for adequate terminal differentiation.
Myoblasts are considered fully committed to muscle differentiation due to the expression of the MRFs Myf-5 and MyoD. We have previously shown that, under differentiation conditions, proper expression and the nuclear localization of the MRF myogenin is not sufficient to drive a successful myogenesis and that myoblasts-ECM interactions are also required . Here we show that when myoblasts are grown in the absence of an appropriate ECM, they express ALP and osteocalcin concomitant to the expression of skeletal muscle determination-associated MRFs in the same cells. Therefore down-regulation of MyoD or Myf-5 is not necessary for the appearance of an osteoblastic-like phenotype. Furthermore we show that the induction of Cbfa-1, a transcription factor essential for osteogenesis [34, 35], is not required for the initial expression of osteogenic markers in skeletal muscle cells. Our observation is coincident with a previous report showing that ALP induction after BMP-2 treatment of mouse myogenic cells preceded the down-regulation of MyoD expression, and that Cbfa-1 was normally expressed in committed myogenic cells . All these evidences support the proposed stock options model of differentiation in skeletal muscle cells, where multiple determination genes can be expressed and depending on the differentiation-inducing signals the cells follow a terminal differentiation pathway .
Several examples of transdifferentation, the conversion of one differentiated cell type to another, are known in the literature. Myoblasts can be made to transdifferentiate into adipocytes after introduction of the transcription factors C/EBPα and peroxisome-proliferator-activated receptor (PPAR)-γ . It is also known that C2C12 myoblasts can be converted to adipocytes after transfection with a dominant-negative version of the transcription factor TCF4  and, as mentioned before, myoblasts can differentiate into osteoblasts by BMP-2 treatment . Although these observations suggest that muscle cells preserve multi-potentiality, the exact requirements to achieve the different phenotypes are still not clear. It is well known that the MRF MyoD will convert several cell lines into muscle , but, as discussed above, the forced expression of a particular gene might not be sufficient to drive metaplasia without the proper environmental signals.
It has been shown that BMP-2/4 associated to the ECM are essential for differentiation of osteoblastic cells . The fact that a soluble BMP-2 receptor ectodomain was unable to inhibit the induction of osteogenic markers in myoblasts cultured in a deficient ECM indicates that we are in presence of a different mechanism of osteogenic induction. Likely, changes in or the absence of some type of signals between the ECM and the cells, presumably through integrins, are sufficient to trigger an osteoblastic phenotype. This concept is reinforced by the fact that the ECM obtained from BMP-2 treated myoblasts was able to induce ALP activity in normal myoblasts. If this is the result of a new type of interaction between ECM and its cell surface receptors or of the absence of some other interactions requires further investigation. We have experimental data that indicate that the proteoglycan population synthesized by BMP-2 treated myoblasts is different in composition , but changes in other ECM constituents may also have a role. It has been shown that fibronectin supports the induction of ALP by ascorbic acid in fibroblasts through its interaction with integrin α5β1, whereas type I collagen fibrils cause the suppression of ALP expression . However, the possibility that BMP-2 may be inducing the expression of another osteogenic factor, such as BMP-7, which could be retained at the ECM can not be excluded from our experiments.
Heterotopic bone formation within skeletal muscle is a widely observed pathologic phenomenon, specially after repeated trauma, but it is exacerbated in rare genetic diseases such as Fibrodysplasia Ossificans Progressiva . Osteoprogenitor cells are thought to reside in skeletal muscle, although their identity in the tissue has not been clearly determined . On the other hand, it is known that diseased muscles show increased ALP activity, as in DMD, facioscapulohumeral dystrophy, polymyositis, etc. [20, 21, 50]. As mdx skeletal muscle is under constant rounds of degeneration-regeneration and shows enhanced ECM remodeling and deposition , we thought it would be an appropriate model to evaluate muscle ALP expression in an in vivo situation. We found not only an increase in ALP activity but a relocalization of the activity in dystrophic muscle, in contrast to normal mice where the ALP was localized in scattered cells and blood vessels around individual fibers. In mdx muscle ALP activity was localized within muscle fibers that formed small groups in the tissue. Expression of ALP activity by skeletal muscle fibers had been previously reported in DMD and congenital muscular dystrophy (CMD) muscle biopsies, and its expression was suggested to be localized in immature fibers . We evaluated if there was a correlation between the relocalization of ALP activity and the formation of new muscle fibers. We found that although some new muscle fibers, determined by the expression of EM, were positive for ALP this was not always the case and EM-positive fibers containing no ALP activity were also found. When ALP expression during skeletal muscle regeneration was evaluated, we also found ALP activity within a small number of newly formed myotubes. ALP expression in muscle fibers lasted for longer than the expression of embryonic myosin, but one month after the induction of damage ALP activity was found again localized in the endomysium. It is known that during skeletal muscle regeneration, fusing myoblasts are in contact with a scaffold of remnant ECM from degenerated fibers before they begin to synthesize their own ECM [38, 39]. Considering that muscle tissue possess cells with osteogenic potential that may also act as muscle precursor cells [31, 36] and our findings of ALP expression by endomysial cells in normal muscle and ALP induction in groups of fibers during muscle regeneration, it is interesting to speculate whether this phenomenon, if exacerbated by repetition in time or by impairment of its regulatory mechanisms, may be related to heterotopic ossification.
The induction and relocalization of ALP activity found under these two in vivo experimental conditions of skeletal muscle differentiation and ECM remodeling, extend our observations of co-expression of differentiation markers for distinct lineages and suggest that they are perhaps a consequence of a different interaction between the muscle fibers and the surrounding ECM. This concept is supported by observations of ALP increase in the dystrophic muscle of dy/dy mouse  and the presence of positive myofibers for ALP in CMD , phenotypes that lack of laminin α2 expression, so the normal interaction between the basement membrane and the dystrophin-glycoprotein complex or integrins is missing .
It is known from cell transplantation studies that cells of different origins, such as bone marrow  and neural tissue , can be incorporated into skeletal muscle. Furthermore, muscle-derived cells different from satellite cells, like the side population of dissociated muscle cells or cells present in the interstitial space can differentiate into muscle and hematopoietic cells or muscle and endothelial cells, respectively [53–55]. In this work we have shown that C2C12 myoblasts can be induced to express osteogenic markers by signals from the ECM. The mechanisms of metaplasia of these different cell populations are not clear, but what it might be critical in vivo is the fact that these cells can become reprogrammed or prepared to acquire different fates when surrounded by a particular environment.