The Nuclear Import of the Small GTPase Rac1 is Mediated by the Direct Interaction with Karyopherin α2

The small GTPase Rac1 is involved in multiple cytosolic functions but recent data point out that Rac1 also translocates to the nucleus to regulate signalling pathways that control gene expression and progression through the cell cycle. Here, we identify the nuclear import receptor karyopherin α2 (KPNA2) as a direct interaction partner of Rac1. The C‐terminal polybasic region of Rac1 contains a nuclear localization signal (NLS), whereas Rac2 and Rac3 lack a functional NLS and do not bind to KPNA2. The presence of the NLS in Rac1 determines the specificity of the interaction and is a prerequisite for the nuclear import. Although this interaction is independent of the Rac1 GDP/GTP loading, the induction of the translocation requires Rac1 activation. The activation of Rac1 via the cytotoxic necrotizing factor 1 and the concurrent inhibition of its proteasomal degradation are crucial for the nuclear accumulation of Rac1. Conversely, the reduction of KPNA2 expression inhibits the nuclear import of Rac1. For the first time, our results show a direct interaction between Rac1 and KPNA2 and argue for a KPNA2‐dependent nuclear import of Rac1. Liquid chromatography tandem mass spectrometry (LC‐MS/MS) analysis revealed that nuclear Rac1 coimmunoprecipitates with numerous proteins. In the nucleus, Rac1 may participate in a variety of so far uncharacterized processes.     

Stem cell antigen-1 regulates the tempo of muscle repair through effects on proliferation of alpha7 integrin-expressing myoblasts

Skeletal muscle repair occurs through a programmed series of events including myogenic precursor activation, myoblast proliferation, and differentiation into new myofibers. We previously identified a role for Stem cell antigen-1 (Sca-1) in myoblast proliferation and differentiation in vitro. We demonstrated that blocking Sca-1 expression resulted in sustained myoblast cell division. Others have since demonstrated that Sca-1-null myoblasts display a similar phenotype when cultured ex vivo. To test the importance of Sca-1 during myogenesis in vivo, we employed a myonecrotic injury model in Sca-1(-/-) and Sca-1(+/+) mice. Our results demonstrate that Sca-1(-/-) myoblasts exhibit a hyperproliferative response consisting of prolonged and accelerated cell division in response to injury. This leads to delayed myogenic differentiation and muscle repair. These data provide the first in vivo evidence for Sca-1 as a regulator of myoblast proliferation during muscle regeneration. These studies also suggest that the balance between myogenic precursor proliferation and differentiation is critical to normal muscle repair.     

Stac3 Inhibits Myoblast Differentiation into Myotubes

The functionally undefined Stac3 gene, predicted to encode a SH3 domain- and C1 domain-containing protein, was recently found to be specifically expressed in skeletal muscle and essential to normal skeletal muscle development and contraction. In this study we determined the potential role of Stac3 in myoblast proliferation and differentiation, two important steps of muscle development. Neither siRNA-mediated Stac3 knockdown nor plasmid-mediated Stac3 overexpression affected the proliferation of C2C12 myoblasts. Stac3 knockdown promoted the differentiation of C2C12 myoblasts into myotubes as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA and protein expression of myogenic markers including myogenin and myosin heavy chain. In contrast, Stac3 overexpression inhibited the differentiation of C2C12 myoblasts into myotubes as evidenced by decreased fusion index, decreased number of nuclei per myotube, and decreased mRNA and protein expression of myogenic markers. Compared to wild-type myoblasts, myoblasts from Stac3 knockout mouse embryos showed accelerated differentiation into myotubes in culture as evidenced by increased fusion index, increased number of nuclei per myotube, and increased mRNA expression of myogenic markers. Collectively, these data suggest an inhibitory role of endogenous Stac3 in myoblast differentiation. Myogenesis is a tightly controlled program; myofibers formed from prematurely differentiated myoblasts are dysfunctional. Thus, Stac3 may play a role in preventing precocious myoblast differentiation during skeletal muscle development.     

Nuclear movement during myotube formation is microtubule and dynein dependent and is regulated by Cdc42, Par6 and Par3

Cells actively position their nucleus within the cytoplasm. One striking example is observed during skeletal myogenesis. Differentiated myoblasts fuse to form a multinucleated myotube with nuclei positioned in the centre of the syncytium by an unknown mechanism. Here, we describe that the nucleus of a myoblast moves rapidly after fusion towards the central myotube nuclei. This movement is driven by microtubules and dynein/dynactin complex, and requires Cdc42, Par6 and Par3. We found that Par6β and dynactin accumulate at the nuclear envelope of differentiated myoblasts and myotubes, and this accumulation is dependent on Par6 and Par3 proteins but not on microtubules. These results suggest a mechanism where nuclear movement after fusion is driven by microtubules that emanate from one nucleus that are pulled by dynein/dynactin complex anchored to the nuclear envelope of another nucleus.     

TIEG1 negatively controls the myoblast pool indispensable for fusion during myogenic differentiation of C2C12 cells

The transforming growth factor (TGF)-β inducible early gene (TIEG)-1 is implicated in the control of cell proliferation, differentiation, and apoptosis in some cell types. Since TIEG1 functioning may be associated with TGF-β, a suppressor of myogenesis, TIEG1 is also likely to be involved in myogenesis. Therefore, we investigated the function of TIEG1 during myogenic differentiation in vitro using the murine myoblasts cell line, C2C12. TIEG1 expression increased during differentiation of C2C12 cells. Constitutive expression of TIEG1 reduced survival and decreased myotube formation. Conversely, knocking down TIEG1 expression increased the number of viable cells during differentiation, and accelerated myoblast fusion into multinucleated myotubes. However, expression of the myogenic differentiation marker, myogenin, remained unaffected by TIEG1 knockdown. The mechanism underlying these events was investigated by focusing on the regulation of myoblast numbers after induction of differentiation. The knockdown of TIEG1 led to changes in cell cycle status and inhibition of apoptosis during the initial stages of differentiation. Microarray and real-time PCR analyses showed that the regulators of cell cycle progression were highly expressed in TIEG1 knockdown cells. Therefore, TIEG1 is a negative regulator of the myoblast pool that causes inhibition of myotube formation during myogenic differentiation.     

Evidence on the participation of protein kinase C alpha in the proliferation of cultured myoblasts.

There is evidence involving protein kinase C (PKC) in the signal transduction pathways that regulate the differentiation of myoblasts into mature multinucleated muscle cells (myotubes). In order to obtain information on the possible role of individual PKC isozymes in myogenesis, in the present work we investigated the differential expression of PKC isoforms alpha, beta, delta, epsilon, and zeta during muscle cell development in vitro. Chick embryo myoblasts cultured from 1 to 6 days were used as experimental model. Morphological characterization and measurement of specific biochemical parameters in cultures, e.g., DNA synthesis, creatine kinase activity, and myosin levels, revealed a typical muscle cell developmental pattern consisting of an initial proliferation of myoblasts followed by their differentiation into myotubes. PKC activity was high at the proliferation stage, decreased as myoblasts elongated and fused, and increased again in differentiated myotubes. In proliferating myoblasts, the PKC inhibitors calphostin C and bisindolylmaleimide I decreased DNA synthesis whereas in myoblasts undergoing differentiation they exerted the opposite effect, suggesting that PKC plays a role at both stages of myogenesis. Western blot analysis of changes in the expression of PKC isoforms during muscle cell development showed high levels of PKC alpha in the proliferating phase which markedly decreased as myoblasts differentiated. Treatment with TPA of proliferative myoblasts inhibited DNA synthesis and selectively down-regulated PKC alpha, suggesting that this isozyme may have an important role in maintaining myoblast proliferation. On the other hand, an increase in the expression of PKC beta, delta, and epsilon was detected during myogenesis, suggesting that one or more of these isoforms may participate in the differentiation process of myoblasts.     

MURC, a muscle-restricted coiled-coil protein, is involved in the regulation of skeletal myogenesis

Skeletal myogenesis is a multistep process by which multinucleated mature muscle fibers are formed from undifferentiated, mononucleated myoblasts. However, the molecular mechanisms of skeletal myogenesis have not been fully elucidated. Here, we identified muscle-restricted coiled-coil (MURC) protein as a positive regulator of myogenesis. In skeletal muscle, MURC was localized to the cytoplasm with accumulation in the Z-disc of the sarcomere. In C2C12 myoblasts, MURC expression occurred coincidentally with myogenin expression and preceded sarcomeric myosin expression during differentiation into myotubes. RNA interference (RNAi)-mediated knockdown of MURC impaired differentiation in C2C12 myoblasts, which was accompanied by impaired myogenin expression and ERK activation. Overexpression of MURC in C2C12 myoblasts resulted in the promotion of differentiation with enhanced myogenin expression and ERK activation during differentiation. During injury-induced muscle regeneration, MURC expression increased, and a higher abundance of MURC was observed in immature myofibers compared with mature myofibers. In addition, ERK was activated in regenerating tissue, and ERK activation was detected in MURC-expressing immature myofibers. These findings suggest that MURC is involved in the skeletal myogenesis that results from modulation of myogenin expression and ERK activation. MURC may play pivotal roles in the molecular mechanisms of skeletal myogenic differentiation.     

Wip1 cooperates with KPNA2 to modulate the cell proliferation and migration of colorectal cancer via a p53-dependent manner

Due to the increasing incidence and mortality, the early diagnosis, specific targeted therapies, and prognosis for colorectal cancer (CRC) attract more and more attention. Wild-type p53-induced phosphatase 1 (Wip1) and karyopherin α2 (KPNA2) have been regarded as oncogenes in many cancers, including CRC. Wip1 dephosphorylates p53 to inactivate it. TP53 activator and Wip1 inhibitor downregulate KPNA2 expression. Therefore, we speculate that Wip1 may co-operate with KPNA2 to modulate CRC progression in a p53-dependent manner. Here, Wip1 and KPNA2 messenger RNA expression and protein levels are significantly increased in CRC tissues and cell lines and are positively correlated with each other. Wip1 silence increases p53 phosphorylation while decreases KPNA2 protein. Wip1 knockdown remarkably suppresses CRC cell proliferation and migration while KPNA2 overexpression exerts an opposing effect. KPNA2 overexpression could partially rescue Wip1 silence-inhibited CRC cell proliferation and migration. Finally, Wip1 interacts with KPNA2 to modulate the activation of AKT/GSK-3β signaling and metastasis-related factors. In summary, Wip1 could co-operate with KPNA2 to modulate CRC cell proliferation and migration, possibly via a p53-dependent manner, through downstream AKT/GSK-3β pathway. We provided a novel mechanism of Wip1 interacting with KPNA2, therefore modulating CRC cell proliferation and migration.     

Knockdown of hypoxia-inducible factor-1alpha by siRNA inhibits C2C12 myoblast differentiation

We analyzed the role of Hypoxia-inducible factor (HIF)-1alpha in myoblast differentiation by examining the expression and regulation of HIF-1alpha in proliferating and differentiating C2C12 myoblast, and by knocking down HIF-1alpha of C2C12 myoblasts with small interfering RNA (siRNA), given that HIF-1alpha has been shown to be involved in differentiative process in non-muscle tissues. Although HIF-1alpha mRNA was constantly expressed in C2C12 myoblasts both under growth and differentiating phase, HIF-1alpha protein was hardly detectable in the growth phase but became detectable only during myogenic differentiation even under normoxia. During early stage of C2C12 myogenesis, HIF-1alpha accumulated in the nuclei of myogenin-positive myoblasts. The inhibition of proteasome in the growth phase led to HIF-1alpha protein accumulation, whereas in the differentiation phase the inhibition of Hsp90, which stabilizes HIF-1alpha, suppressed HIF-1alpha accumulation. Therefore, we suggest that the level of HIF-1alpha protein expression is regulated by a proteasome-and chaperon-dependent pathway in C2C12 myoblast. Knockdown of HIF-1alpha effectively blocked myotube formation and myosin heavy chain (MHC) expression. Finally, HIF-1alpha expression in vivo was confirmed in the regenerative muscle tissue of mice after eccentric exercise. We conclude that HIF-1alpha is required for C2C12 myogenesis in vitro, and suggest that HIF-1alpha may have an essential role in regenerative muscle tissue in vivo.     

miR-152 regulates the proliferation and differentiation of C2C12 myoblasts by targeting <Emphasis Type="Italic">E2F3</Emphasis>

The development of skeletal muscle is a complex process involving the proliferation, differentiation, apoptosis, and changing of muscle fiber types in myoblasts. Many reports have described the involvement of microRNAs in the myogenesis of myoblasts. In this study, we found that the expression of miR-152 was gradually down-regulated during myoblast proliferation, but gradually up-regulated during the differentiation of myoblasts. Transfection with miR-152 mimics restrained cell proliferation and decreased the expression levels of cyclin E, CDK4, and cyclin D1, but promoted myotube formation and significantly increased the mRNA expression levels of MyHC, MyoD, MRF4, and MyoG in C2C12 myoblasts. However, treatment with miR-152 inhibitors promoted cell proliferation and restrained differentiation. Moreover, over-expression of miR-152 significantly decreased E2F3 production in C2C12 myoblasts. A luciferase assay confirmed that miR-152 could bind to the 3′ UTR of E2F3. In conclusion, this study showed that miR-152 inhibited proliferation and promoted myoblast differentiation by targeting E2F3.     

Differentiation-dependent regulation of skeletal myogenesis by neuregulin-1

Neuregulins comprise a group of growth factor proteins that regulate the differentiation of skeletal muscle. Here, we report that neuregulins are regulators of myogenic differentiation and stimulate mitogenesis in L6 skeletal myoblasts. The mitogenic response to neuregulin-1 was differentiation-dependent and observed only in aligned, differentiating cells. Treatment of these cells with neuregulin-1 increased [3H]thymidine incorporation and cell proliferation by 2- to 5-fold, while a minimal increase was seen in proliferating myoblasts. Neuregulin-1 did not induce DNA synthesis in fused, multinucleated myotubes. The increased DNA synthesis correlated with downregulation of myogenin and inhibition of myoblast fusion and myotube formation. These data suggest that neuregulins may regulate skeletal myogenesis in vivo and that this regulation is dependent on the state of differentiation of the myocytes.