Maintenance of muscle stem-cell quiescence by microRNA-489.

Among the key properties that distinguish adult mammalian stem cells from their more differentiated progeny is the ability of stem cells to remain in a quiescent state for prolonged periods of time. However, the molecular pathways for the maintenance of stem-cell quiescence remain elusive. Here we use adult mouse muscle stem cells (satellite cells) as a model system and show that the microRNA (miRNA) pathway is essential for the maintenance of the quiescent state. Satellite cells that lack a functional miRNA pathway spontaneously exit quiescence and enter the cell cycle. We identified quiescence-specific miRNAs in the satellite-cell lineage by microarray analysis. Among these, miRNA-489 (miR-489) is highly expressed in quiescent satellite cells and is quickly downregulated during satellite-cell activation. Further analysis revealed that miR-489 functions as a regulator of satellite-cell quiescence, as it post-transcriptionally suppresses the oncogene Dek, the protein product of which localizes to the more differentiated daughter cell during asymmetric division of satellite cells and promotes the transient proliferative expansion of myogenic progenitors. Our results provide evidence of the miRNA pathway in general, and of a specific miRNA, miR-489, in actively maintaining the quiescent state of an adult stem-cell population.

Alternative polyadenylation mediates microRNA regulation of muscle stem cell function.

Pax3, a key myogenic regulator, is transiently expressed during activation of adult muscle stem cells, or satellite cells (SCs), and is also expressed in a subset of quiescent SCs (QSCs), but only in specific muscles. The mechanisms regulating these variations in expression are not well understood. Here we show that Pax3 levels are regulated by miR-206, a miRNA with a previously demonstrated role in myogenic differentiation. In most QSCs and activated SCs, miR-206 expression suppresses Pax3 expression. Paradoxically, QSCs that express high levels of Pax3 also express high levels of miR-206. In these QSCs, Pax3 transcripts are subject to alternative polyadenylation, resulting in transcripts with shorter 3' untranslated regions (3'UTRs) that render them resistant to regulation by miR-206. Similar alternate polyadenylation of the Pax3 transcript also occurs in myogenic progenitors during development. Our findings may reflect a general role of alternative polyadenylation in circumventing miRNA-mediated regulation of stem cell function.

Focal adhesion kinase signaling regulates the expression of caveolin 3 and beta1 integrin, genes essential for normal myoblast fusion.

An essential phase of skeletal myogenesis is the fusion of mononucleated myoblasts to form multinucleated myotubes. Many cell adhesion proteins, including integrins, have been shown to be important for myoblast fusion in vertebrates, but the mechanisms by which these proteins regulate cell fusion remain mostly unknown. Here, we focused on the role of focal adhesion kinase (FAK), an important nonreceptor protein tyrosine kinase involved in integrin signaling, as a potential mediator by which integrins may regulate myoblast fusion. To test this hypothesis in vivo, we generated mice in which the Fak gene was disrupted specifically in muscle stem cells ("satellite cells") and we found that this resulted in impaired myotube formation during muscle regeneration after injury. To examine the role of FAK in the fusion of myogenic cells, we examined the expression of FAK and the effects of FAK deletion on the differentiation of myoblasts in vitro. Differentiation of mouse primary myoblasts was accompanied by a rapid and transient increase of phosphorylated FAK. To investigate the requirement of FAK in myoblast fusion, we used two loss-of-function approaches (a dominant-negative inhibitor of FAK and FAK small interfering RNA [siRNA]). Inhibition of FAK resulted in markedly impaired fusion but did not inhibit other biochemical measures of myogenic differentiation, suggesting a specific role of FAK in the morphological changes of cell fusion as part of the differentiation program. To examine the mechanisms by which FAK may be regulating fusion, we used microarray analysis to identify the genes that failed to be normally regulated in cells that were fusion defective due to FAK inhibition. Several genes that have been implicated in myoblast fusion were aberrantly regulated during differentiation when FAK was inhibited. Intriguingly, the normal increases in the transcript of caveolin 3 as well as an integrin subunit, the beta1D isoform, were suppressed by FAK inhibition. We confirmed this also at the protein level and show that direct inhibition of beta1D subunit expression by siRNA inhibited myotube formation with a prominent effect on secondary fusion. These data suggest that FAK regulation of profusion genes, including caveolin 3 and the beta1D integrin subunit, is essential for morphological muscle differentiation.

Focal adhesion kinase is essential for costamerogenesis in cultured skeletal muscle cells.

A central question in muscle biology is how costameres are formed and become aligned with underlying myofibrils in mature tissues. Costameres are composed of focal adhesion proteins, including vinculin and paxillin, and anchor myofibril Z-bands to the sarcolemma. In the present study, we investigated the process of costamere formation ("costamerogenesis") in differentiating primary mouse myoblasts. Using vinculin and paxillin as costameric markers, we found that two additional focal adhesion components, alpha5beta1 integrin and focal adhesion kinase (FAK), are associated with costameres. We have characterized costamerogenesis as occurring in three distinct stages based on the organizational pattern of these costameric proteins. We show that both costamerogenesis and myofibrillogenesis are initiated at sites of membrane contacts with the extracellular matrix and that their maturation is tightly coupled. To test the importance of FAK signaling in these processes, we analyzed cells expressing a dominant negative form of FAK (dnFAK). When cells expressing dnFAK were induced to differentiate, both costamerogenesis and myofibrillogenesis were disrupted although the expression of constituent proteins was not inhibited. Likewise, inhibiting FAK activity by reducing FAK levels using an siRNA approach also resulted in an inhibition of costamerogenesis and myofibrillogenesis. The relationship between costamere and myofibril formation was tested further by treating myotube cultures with potassium or tetrodotoxin to block contraction and disrupt myofibril organization. This also resulted in inhibition of costamere maturation. We present a model of costamerogenesis whereby signaling through FAK is essential for both normal costamerogenesis and normal myofibrillogenesis which are tightly coupled during skeletal myogenesis.