Induction of osteogenic differentiation
Both the MM14 and C2C12 myoblasts are well characterized for their potential for myogenic differentiation and capability for forming mature myotubes in culture. These two cell lines were originally derived from adult muscle satellite cells. They both express muscle-specific markers and are competent for differentiating into myotubes and incorporate into functional muscle fibers after transplantation in vivo. In addition, the MM14 cells have been reported to exhibit different migratory phenotypes on Ln verses fibronectin substrates while C2C12 cells have been widely used for studies of myoblast differentiation and transplantation fate [12
Here we have analyzed the capacity of C2C12 and MM14 cells for their multipotent potential along osteoblastic lineages. Myoblasts were grown in the presence or absence of BMP2, an inducer of the osteogenic pathway. Both cells were able to form myotubes (). When either MM14 or C2C12 cells were treated with BMP2, they failed to differentiate into myotubes and instead differentiated along the osteogenic pathway as revealed by induction of alkaline phosphatase (). The cells remained as nonfused mononuclear polygonal cells. A majority of BMP2 treated cells strongly expressed the alkaline phosphatase, whereas control cells did not. These results confirm that both MM14 and C2C12 myoblasts when treated with BMP2 underwent osteogenic differentiation.
BMP2 induces osteogenic differentiation in C2C12 and MM14 myoblasts
To further define the potential of mouse MM14 and C2C12 myoblasts to differentiate along the osteogenic pathway, BMP2 treated myoblasts were analyzed by RT-PCR for expression of osteocalcin and Runx2, both markers for osteoblasts. Compared to the GAPDH control, osteocalcin and Runx2 were strongly detected in BMP2-treated myoblasts while expression was absent in untreated cells (). The expression of common myogenic markers such as desmin and MyoD following osteogenic differentiation was also examined. Desmin and MyoD were detected in control cells while loss of expression was observed in BMP2 treated myoblasts (). These results further support that BMP2 can induce mouse C2C12 and MM14 myoblasts along osteogenic differentiation pathway while inhibiting myogenesis.
BMP2-induced changes in adhesion and motility
ECM proteins are important for tissue-specific functions [24
]. These structural proteins contribute to the unique properties that define the niche for each tissue type, and help maintain stem cell function and specification [1
]. For the most part, the integrin expression profile reflects the type of differentiated cell type found in tissue subtypes. Consequently, we tested myoblasts for their ability to adhere to different ECM molecules and examined if this adhesion was altered during osteogenic differentiation. Adhesion to Ln-1 or Col-I substrates was assessed after BMP2 treatment and in the presence of function-perturbing mAbs to integrin receptors,. For C2C12 cells, anti-α7 mAb substantially inhibited cell adhesion, while anti-β1 mAb and the combination of anti-α6 and anti-α7 mAb completely blocked Ln adhesion (). Following induction of differentiation with BMP2 treatment, C2C12 adhesion to Ln substrates dramatically declined (). Adherence was sensitive to anti-α2 mAb but also to anti-β1. This is consistent with α2 integrin mediating the relatively poor adhesion to Ln in BMP2 treated cells. In contrast, anti-α7 had no effect on adhesion to Ln.
Adhesion and migration of mouse myoblasts is altered by BMP2
On Col-I substrates, C2C12 cells displayed poor adhesion and only a minor fraction of the cells adhered to this substrate (). However, following BMP2 induction, there was a strong increase in adhesion to Col-I () that was blocked by anti-α2 and by anti-α1 mAb to a lesser extent; other anti-α chain mAbs had no effect. Complete inhibition of adhesion was achieved with anti-β1 mAb. Similar results were also obtained for the MM14 myoblasts (not shown).
BMP2 affects on cell motility with Ln-1 or Col-I substrates was also examined. For Ln, the C2C12 myoblasts showed a strong locomotive response (). Consistent with the high expression of the α7 integrin, migration on Ln-1 was blocked with the anti-α7 mAb, and as expected, anti-β1 mAb was effective in inhibiting motility. Following treatment with BMP2, however, cell migration on Ln-1 was reduced as compared to controls (). The migration response was mediated by the α2 receptor, which is well known to function as a Ln receptor, a collagen receptor, or both [26
]. On Col-I substrates, C2C12 cells typically showed poor migration, but with BMP2, migration on Col-I dramatically increased (). This increase was dependent on the α2 integrin receptor, since antibodies to either α2 or β1 subunits completely abolished motility. For the MM14 cells, nearly identical results were observed (data not shown).
Modulation of integrin expression following BMP2 treatment revealed the reason for the inversed behavior in adhesion and migration on the two substrates. As such, changes in integrin expression following induction were assessed by FACS. For C2C12 cells, we found a diverse set of integrin α chains with low levels of α1, α2 and α5, significant levels of α3 and α6, and abundant α7 integrin expression (). A similar repertoire of integrins was expressed on MM14 cells (not shown). Following BMP2 treatment, there was a dramatic loss of α7 integrin (). Also, there was a reciprocal increase in the α2 integrin, the dominant Col-I receptor used by osteoblasts. Analysis of the dot plot data for α2 and α7 integrins shows that the majority of the cell population had inverted their relative integrin expression levels following BMP2 treatment. A minor subset of cells did not show complete induction of the α2 and loss of α7 receptor expression, suggesting that this small fraction of cells remained committed to muscle lineage and did not switch their specification. Similar results were obtained by immunofluoresent staining showing that in most cells, positive staining of the α7 integrin was lost following BMP2 treatment whereas α2 showed a strong increase after the treatment ().
BMP2 modulates adhesion receptor expression
Thus, following BMP2 induced conversion to the osteogenic lineage, cells expressed the α2 integrin, which is needed to bind to interstitial collagen matrix of bone, and thereby maintaining a fully differentiated phenotype. We confirmed that BMP2-induced alterations in the integrin profile seem to correlate with the changes in the adhesive and motile. Not only does BMP2 dramatically downregulate α7 integrin levels in C2C12 (and MM14) myoblasts, but induced a reciprocal increase in the α2 integrin, the dominant Col-I receptor for osteogenic cells.
BMP2 downregulated α7 integrin expression at the level of the promoter
Next, we tested gene expression by RT-PCR of Ln-I-binding α7 and Col-I-binding α2 integrins following BMP2-mediated differentiation into the osteogenic lineage (). The results indicate that compared to GAPDH controls, BMP2 downregulated the α7 integrin gene in myoblasts, and induced a reciprocal increase in the α2 integrin expression (Fig.4A).
α7 gene expression and promoter activity following osteogenic differentiation
Therefore, both FACS and RT-PCR analysis showed that BMP2 induced myoblasts to develop into the osteogenic pathway with a nearly complete loss of expression of the α7 integrin. To confirm decreased transcription was responsible for this change in mRNA levels, we checked promoter activity in myoblasts using the CAT reporter gene driven by the full-length 2.8-kb mouse α7 promoter. C2C12 cells were transiently transfected with deletion constructs of mouse α7 integrin promoter and cell lysates were tested for CAT promoter activity expressed as activity relative to that of empty vector pCAT (). In cells treated with BMP2, α7 promoter activity was nearly completely suppressed indicating that BMP2 regulates α7 integrin expression through a transcriptional mechanism.
The current study has identified that α7 integrin may be a specific marker that defines myoblasts with pluripotent stem cell potential. Also, modulation of integrin receptor expression occurs in parallel with the conversion of myoblasts cells to osteogenic lineage. These alterations in adhesion are potentially important for the optimization of cell functions such as adhesion, motility, remodeling, and repair of interstitial matrix molecules. Similar changes in adhesion receptors must occur in embryonic and other adult stem cells as they develop into highly differentiated cell types. More work is needed to define these changes. α7 integrin is rapidly lost following BMP2-induction. We have established that the mechanism for this suppression is at the transcriptional level. Since α7 levels appear to be an important marker of myoblasts with ‘stem cell’ characteristics and since α7 levels seem to be modulated by BMP2 treatment, we are interested in defining the mechanism of this regulation. In addition, it will be interesting to artificially downregulate α7 and determine if receptor loss facilitates osteogenic differentiation. Myoblasts bind to Ln-1 through the α7 receptor, but lack the α2 receptor and bind poorly to interstitial Col-I. In contrast, osteoblasts tend to favor binding to Col-I scaffolding through the α2 receptor and fail to express the α7 receptor. Regulation of α7 levels by BMP2 at the level of the promoter may be through direct or indirect mechanisms. BMP2 could affect the myogenic MRF family of factors including MyoD, myf5, myogenin, MRF4 or have an effect on the MEF2 family of proteins. It is also possible that Smads bind directly or through a protein complex to modulate expression of the α7 promoter. Future studies will investigate the mechanism by which BMP2 downregulates α7 integrin.