Distinct roles of two structurally closely related focal adhesion proteins, alpha-parvins and beta-parvins, in regulation of cell morphology and survival

Proteins at cell-extracellular matrix adhesions (e.g. focal adhesions) are crucially involved in regulation of cell morphology and survival. We show here that CH-ILKBP/actopaxin/alpha-parvin and affixin/beta-parvin (abbreviated as alpha- and beta-parvin, respectively), two structurally closely related integrin-linked kinase (ILK)-binding focal adhesion proteins, are co-expressed in human cells. Depletion of alpha-parvin dramatically increased the level of beta-parvin, suggesting that beta-parvin is negatively regulated by alpha-parvin in human cells. Loss of PINCH-1 or ILK, to which alpha- and beta-parvin bind, significantly reduced the activation of Rac, a key signaling event that controls lamellipodium formation and cell spreading. We were surprised to find that loss of alpha-parvin, but not that of beta-parvin, markedly stimulated Rac activation and enhanced lamellipodium formation. Overexpression of beta-parvin, however, was insufficient for stimulation of Rac activation or lamellipodium formation, although it was sufficient for promotion of apoptosis, another important cellular process that is regulated by PINCH-1, ILK, and alpha-parvin. In addition, we show that the interactions of ILK with alpha- and beta-parvin are mutually exclusive. Overexpression of beta-parvin or its CH(2) fragment, but not a CH(2) deletion mutant, inhibited the ILK-alpha-parvin complex formation. Finally, we provide evidence suggesting that inhibition of the ILK-alpha-parvin complex is sufficient, although not necessary, for promotion of apoptosis. These results identify Rac as a downstream target of PINCH-1, ILK, and parvin. Furthermore, they demonstrate that alpha- and beta-parvins play distinct roles in mammalian cells and suggest that the formation of the ILK-alpha-parvin complex is crucial for protection of cells from apoptosis.     

A novel integrin-linked kinase-binding protein, affixin, is involved in the early stage of cell-substrate interaction

Focal adhesions (FAs) are essential structures for cell adhesion, migration, and morphogenesis. Integrin-linked kinase (ILK), which is capable of interacting with the cytoplasmic domain of beta1 integrin, seems to be a key component of FAs, but its exact role in cell-substrate interaction remains to be clarified. Here, we identified a novel ILK-binding protein, affixin, that consists of two tandem calponin homology domains. In CHOcells, affixin and ILK colocalize at FAs and at the tip of the leading edge, whereas in skeletal muscle cells they colocalize at the sarcolemma where cells attach to the basal lamina, showing a striped pattern corresponding to cytoplasmic Z-band striation. When CHO cells are replated on fibronectin, affixin and ILK but not FA kinase and vinculin concentrate at the cell surface in blebs during the early stages of cell spreading, which will grow into membrane ruffles on lamellipodia. Overexpression of the COOH-terminal region of affixin, which is phosphorylated by ILK in vitro, blocks cell spreading at the initial stage, presumably by interfering with the formation of FAs and stress fibers. The coexpression of ILK enhances this effect. These results provide evidence suggesting that affixin is involved in integrin-ILK signaling required for the establishment of cell-substrate adhesion.     

Inhibition of Rac1 promotes BMP-2-induced osteoblastic differentiation

Small G proteins of the Rho family are pivotal regulators of several signaling networks. The Ras homolog family (Rho) and one of its targets, Rho-associated protein kinase (ROCK), participate in a wide variety of biological processes, including bone formation. A previous study has demonstrated that the ROCK inhibitor Y-27632 enhanced bone formation induced by recombinant human bone morphogenetic protein-2 (BMP-2) in vivo and in vitro. However, the effect of other Rho family members, such as Ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division cycle 42 (Cdc42), on bone formation remains unknown. In this study, we investigated whether Rac1 also participates in BMP-2-induced osteogenesis. Expression of a dominant-negative mutant of Rac1 enhanced BMP-2-induced osteoblastic differentiation in C2C12 cells, whereas a constitutively active mutant of Rac1 attenuated that effect. Knockdown of T-lymphoma invasion and metastasis 1 (Tiam1), a Rac-specific guanine nucleotide exchange factor, enhanced BMP-2-induced alkaline phosphatase activity. Further, we demonstrated that BMP-2 stimulated Rac1 activity. These results indicate that the activation of Rac1 attenuates osteoblastic differentiation in C2C12 cells.     

Constitutive overexpression of the integral membrane protein Itm2A enhances myogenic differentiation of C2C12 cells

Integral membrane protein 2A (Itm2A) is a transmembrane protein belonging to a family composed of at least two other members, Itm2B and Itm2C, all of them having a different expression pattern. The protein serves as a marker for early stages in chondrogenesis and T-cell development. Itm2A is also highly expressed in skeletal muscle. In order to understand the role of Itm2A in muscle development, we constitutively overexpressed exogenous Itm2A in C2C12 myoblast cells. Several clones expressing high levels of Itm2a were isolated and characterized. Overexpression was associated with enhanced tube formation and the appearance of multinuclear cells. Gene expression analysis demonstrated that muscle creatin kinase was upregulated in the presence of exogenous Itm2A. Interestingly, proliferation rates were not altered in the undifferentiated myoblast C2C12 cells. These results demonstrate that overexpression of Itm2a in C2C12 enhances myogenic differentiation in vitro.     

Intracellular signal transduction pathways induced by leptin in C2C12 cells

As experimental evidence suggests that leptin may have direct effects on peripheral tissues, we investigated some of the transductional molecules induced by leptin in C2C12 cells. In immunoprecipitation experiments using anti-p85 antibodies (a regulatory subunit of phosphatidylinositol-3-kinase; PI3K), we observed a significant increase in PI3K activity. Immunoblot analyses showed that Akt, GSK3, ERK1, ERK2, and p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation significantly increased after leptin treatment. Protein kinase C (PKC)-zeta was also activated by leptin, as documented by an immunocomplex kinase assay and immunoblotting experiments. The treatment of C2C12 cells with Wortmannin before leptin administration inhibited induction of the phosphorylation of ERKs (extracellular signal-regulated kinases) but not that of p38 MAPK, whereas pre-treatment with a PKC-zeta inhibitor partially decreased ERK phosphorylation. Taken together, our in vitro results further support the hypothesis that leptin acts acutely on skeletal muscle tissue through some of the components of insulin signalling, including PKC-zeta.     

Activity-dependent synaptogenesis: regulation by a CaM-kinase kinase/CaM-kinase I/betaPIX signaling complex

Neuronal activity augments maturation of mushroom-shaped spines to form excitatory synapses, thereby strengthening synaptic transmission. We have delineated a Ca(2+)-signaling pathway downstream of the NMDA receptor that stimulates calmodulin-dependent kinase kinase (CaMKK) and CaMKI to promote formation of spines and synapses in hippocampal neurons. CaMKK and CaMKI form a multiprotein signaling complex with the guanine nucleotide exchange factor (GEF) betaPIX and GIT1 that is localized in spines. CaMKI-mediated phosphorylation of Ser516 in betaPIX enhances its GEF activity, resulting in activation of Rac1, an established enhancer of spinogenesis. Suppression of CaMKK or CaMKI by pharmacological inhibitors, dominant-negative (dn) constructs and siRNAs, as well as expression of the betaPIX Ser516Ala mutant, decreases spine formation and mEPSC frequency. Constitutively-active Pak1, a downstream effector of Rac1, rescues spine inhibition by dnCaMKI or betaPIX S516A. This activity-dependent signaling pathway can promote synapse formation during neuronal development and in structural plasticity.     

Lactate regulates autophagy through ROS-mediated activation of ERK1/2/m-TOR/p-70S6K pathway in skeletal muscle

The role of autophagy and lysosomal degradation pathway in the regulation of skeletal muscle metabolism was previously studied. However, underlying molecular mechanisms are poorly understood. L-lactate which is utilized as an energetic substrate by skeletal muscle can also augment genes expression related to metabolism and up-regulate those being responsive to reactive oxygen species (ROS). Since ROS is the most important regulator of autophagy in skeletal muscle, we tested if there is a link between cellular lactate metabolism and autophagy in differentiated C2C12 myotubes and the gastrocnemius muscle of male wistar rats. C2C12 mouse skeletal muscle was exposed to 2, 6, 10, and 20 mM lactate and evaluated for lactate autophagic effects. Lactate dose-dependently increased autophagy and augmented ROS generation in differentiated C2C12 myotubes. The autophagic effect of lactate deterred in N-acetylcysteine presence (NAC, a ROS scavenger) indicated lactate regulates autophagy with ROS participation. Lactate-induced up-regulation of extracellular signal-regulated kinase 1/2 (ERK1/2) through ROS was required to regulate the autophagy by lactate. Further analysis about ERK1/2 up- and downstream indicated that lactate regulates autophagy through ROS-mediated the activation of ERK1/2/mTOR/p70S6K pathway in skeletal muscle. The in vitro effects of lactate on autophagy also occurred in the gastrocnemius muscle of male Wistar rats. In conclusion, we provided the lactate-associated regulation evidence of autophagy in skeletal muscle by activating ROS-mediated ERK1/2/mTOR/p70S6K pathway. Since the increase in cellular lactate concentration is a hallmark of energy deficiency, the results provide insight into a skeletal muscle mechanism to fulfill its enhanced energy requirement.     

Bortezomib inhibits C2C12 growth by inducing cell cycle arrest and apoptosis

Proteosome inhibitors such as bortezomib (BTZ) have been used to treat muscle wasting in animal models. However, direct effect of BTZ on skeletal muscle cells has not been reported. In the present study, our data showed that C2C12 cells exhibited a dose-dependent decrease in cell viability in response to increasing concentrations of BTZ. Consistent with the results of cell viability, Annexin V/PI analysis showed a significant increase in apoptosis after exposing the cells to BTZ for 24 h. The detection of cleaved caspase-3 further confirmed apoptosis. The apoptosis induced by BTZ was associated with reduced expression of p-ERK. Cell cycle analysis revealed that C2C12 cells underwent G2/M cell cycle arrest when incubated with BTZ for 24 h. Furthermore, BTZ inhibited formation of multinucleated myotubes. The inhibition of myotube formation was accompanied by decreased expression of Myogenin. Our data suggest that BTZ induces cell death and inhibits differentiation of C2C12 cells at clinically relevant doses.     

Cortisone and dexamethasone inhibit myogenesis by modulating the AKT/mTOR signaling pathway in C2C12

Myogenesis occurs in both the prenatal and postnatal periods and the prenatal myogenesis is related to the postnatal myogenesis and the incidence of disease later in life. Glucocorticoids used as therapeutic agents for many diseases, but cause adverse effects on muscle homeostasis, including defects in fetal muscle development. The action of glucocorticoids on differentiated skeletal muscle was well studied, but their effects on myotube formation have not been well investigated. Dexamethasone (DEX) and cortisone (COR), two synthetic therapeutic glucocorticoids, suppress myotube formation in C2C12 cells. Both COR and DEX attenuated myotube formation through modulation of myogenic regulatory factors. In addition, they affected the IGF/PI3K/AKT/mTOR signaling pathway, resulting in increased proteolytic protein (atrogin-1 and MURF1) for muscle degradation and decreased ribosomal S6 phosphorylation. The current results conclude that COR and DEX inhibit myotube formation in C2C12 cells by modulating both the myogenic program via MRFs and protein metabolism via IGF/PI3K/AKT/mTOR signaling pathway.     

Ginsenoside Rb1 exerts antidiabetic action on C2C12 muscle cells by leptin receptor signaling pathway

Context: Ginsenoside Rb1 improves insulin sensitivity and glucose uptake in muscle cells via different signaling pathways; however, it is not clear that it has any effect on leptin signaling in skeletal muscle.

Objectives: The aim of this study was to investigate the effect of ginsenoside Rb1 on leptin receptors expression and main signaling pathways of leptin (STAT3, PI3 kinase and ERK kinase) in C2C12 skeletal muscle cells.

Materials and methods: C2C12 myotubes were incubated with various concentrations of Rb1 (0.1, 1 and 10?μM) for different incubation times (1–12?h). Leptin receptors expression and GLUT-4 translocation were analyzed using realtime PCR and western blot analyses, respectively. PI3 and ERK kinases were blocked using their specific inhibitors (wortmannin and PD98059) in the presence and absence of RB1 to determine the main signaling pathway related to leptin receptor activation in C2C12 cells.

Results: Rb1 could maximally stimulate both leptin receptors (OBRa and OBRb) mRNA and protein expression and phosphorylation of STAT3, PI3K and ERK2 in C2C12 myotubes at 10?μM for 3?h. Rb1 induced GLUT4 translocation was inhibited by the silencing of OBRb mRNA, demonstrated that glucose uptake was mediated via leptin receptor activation. GLUT4 recruitment to the cell surface induced by Rb1 was inhibited by wortmannin, an inhibitor of PI3K in combination with OBRb siRNA, but not by PD98059 an ERK2 kinase-1 inhibitor, indicating that GLUT4 translocation induced by Rb1 was associated with the leptin receptor upregulation and subsequent activation of PI3K.

Conclusions: Our results suggest that Rb1 promote translocation of GLUT4 by upregulation of leptin receptors and activation of PI3K.     

Expression of basic fibroblast growth factor results in the decrease of myostatin mRNA in murine C2C12 myoblasts

During the development and regeneration of skeletal muscle,many growth factors,such asbasic fibroblast growth factor (bFGF,FGF-2) and myostatin,have been shown to play regulating roles.bFGF contributes to promote proliferation and to inhibit differentiation of skeletal muscle,whereas myostatinplays a series of contrasting roles.In order to elucidate whether the expression of bFGF has any relationshipwith the expression of myostatin in skeletal muscle cells,we constructed a eukaryotic expression vector forthe expression of exogenous bFGF in murine C2C12 myoblasts.Quantitative RT-PCR assays indicated thatwith the increase of the expression of exogenous bFGF gene,the expression of endogenous myostatin genewas suppressed at mRNA level and protein level.     

Emerging role of ILK and ELMO2 in the integration of adhesion and migration pathways

Integrins and their associated proteins are essential components of the cellular machinery that modulates adhesion and migration. In particular, integrin-linked kinase (ILK), which binds to the cytoplasmic tail of β1 integrins, is required for migration in a variety of cell types. We previously identified engulfment and motility 2 (ELMO2) as an ILK-binding protein in epidermal keratinocytes. Recently, we investigated the biological role of the ILK/ELMO2 complexes, and found that they exist in the cytoplasm. ILK/ELMO2 species are recruited by active RhoG to the plasma membrane, where they induce Rac1 activation and formation of lamellipodia at the leading edge of migrating cells. A large number of growth factors and cytokines induce keratinocyte migration. However, we found that formation of RhoG/ELMO2/ILK complexes occurs selectively upon stimulation by epidermal growth factor, but not by transforming growth factor-β1 or keratinocyte growth factor. Herein we discuss the relevance of these complexes to our understanding of the molecular mechanisms involved in cell migration, as well as their potential functions in morphogenesis and tissue regeneration following injury.     

Increased MAPK signaling during in vitro muscle wounding

Regeneration of skeletal muscle upon injury is a complex process, involving activation of satellite cells, followed by migration, fusion, and regeneration of damaged myofibers. Previous work concerning the role of the mitogen activated protein (MAP) kinase signaling pathways in muscle injury comes primarily from studies using chemically induced wounding. The purpose of this study was to test the hypothesis that physical injury to skeletal muscle cells in vitro activates the MAP kinase signaling pathways. We demonstrate that extracellular signal regulated kinases (ERKs) 1, 2, and p38 are rapidly and transiently activated in response to injury in C2C12 cells, and are primarily localized to cells adjacent to the wound bed. Culture medium from wounded cells is able to stimulate activation of p38 but not ERK in unwounded cells. These results suggest that both ERK and p38 are involved in the response of muscle cells to physical injury in culture, and reflect what is seen in whole tissues in vivo.     

Emerging role of ILK and ELMO2 in the integration of adhesion and migration pathways

Integrins and their associated proteins are essential components of the cellular machinery that modulates adhesion and migration. In particular, integrin-linked kinase (ILK), which binds to the cytoplasmic tail of β1 integrins, is required for migration in a variety of cell types. We previously identified engulfment and motility 2 (ELMO2) as an ILK-binding protein in epidermal keratinocytes. Recently, we investigated the biological role of the ILK/ELMO2 complexes, and found that they exist in the cytoplasm. ILK/ELMO2 species are recruited by active RhoG to the plasma membrane, where they induce Rac1 activation and formation of lamellipodia at the leading edge of migrating cells. A large number of growth factors and cytokines induce keratinocyte migration. However, we found that formation of RhoG/ELMO2/ILK complexes occurs selectively upon stimulation by epidermal growth factor, but not by transforming growth factor-β1 or keratinocyte growth factor. Herein we discuss the relevance of these complexes to our understanding of the molecular mechanisms involved in cell migration, as well as their potential functions in morphogenesis and tissue regeneration following injury.     

Selective androgen receptor modulator,S42 has anabolic and anti-catabolic effects on cultured myotubes

We previously identified a novel selective androgen receptor modulator, S42, that does not stimulate prostate growth but has a beneficial effect on lipid metabolism. S42 also increased muscle weight of the levator ani in orchiectomized Sprague–Dawley rats. These findings prompted us to investigate whether S42 has a direct effect on cultured C2C12 myotubes. S42 significantly lowered expression levels of the skeletal muscle ubiquitin ligase (muscle atrophy-related gene), atrogin1 and Muscle RING-Finger Protein 1(MuRF1) in C2C12 myotubes, as determined by real time PCR. Phosphorylation of p70 S6 kinase (p70S6K), an essential factor for promoting protein synthesis in skeletal muscle, was significantly increased by S42 to almost the same extent as by insulin, but this was significantly prevented by treatment with rapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1). However, phosphorylation of Akt, upstream regulator of mTORC1, was not changed by S42. S42 did not increase insulin-like growth factor 1 (Igf1) mRNA levels in C2C12 myotubes. These results suggest that S42 may have an anabolic effect through activation of mTORC1–p70S6K signaling, independent of IGF-1-Akt signaling and may exert an anti-catabolic effect through inhibition of the degradation pathway in cultured C2C12 myotubes.     

Protein kinase Cδ promotes proliferation and induces malignant transformation in skeletal muscle

In this paper, we investigated the isoform‐specific roles of certain protein kinase C (PKC) isoforms in the regulation of skeletal muscle growth. Here, we provide the first intriguing functional evidence that nPKCδ (originally described as an inhibitor of proliferation in various cells types) is a key player in promoting both in vitro and in vivo skeletal muscle growth. Recombinant overexpression of a constitutively active nPKCδ in C2C12 myoblast increased proliferation and inhibited differentiation. Conversely, overexpression of kinase‐negative mutant of nPKCδ (DN‐nPKCδ) markedly inhibited cell growth. Moreover, overexpression of nPKCδ also stimulated in vivo tumour growth and induced malignant transformation in immunodeficient (SCID) mice whereas that of DN‐nPKCδ suppressed tumour formation. The role of nPKCδ in the formation of rhabdomyosarcoma was also investigated where recombinant overexpression of nPKCδ in human rhabdomyosarcoma RD cells also increased cell proliferation and enhanced tumour formation in mouse xenografts. The other isoforms investigated (PKCα, β, ε) exerted only minor (mostly growth‐inhibitory) effects in skeletal muscle cells. Collectively, our data introduce nPKCδ as a novel growth‐promoting molecule in skeletal muscles and invite further trials to exploit its therapeutic potential in the treatment of skeletal muscle malignancies.     

Basic fibroblast growth factor stimulates activation of Rac1 through a p85 betaPIX phosphorylation-dependent pathway

In a previous study (Shin, E. Y., Shin, K. S., Lee, C. S., Woo, K. N., Quan, S. H., Soung, N. K., Kim, Y. G., Cha, C. I., Kim, S. R., Park, D., Bokoch, G. M., and Kim, E. G. (2002) J. Biol. Chem. 277, 44417-44430) we reported that phosphorylation of p85 betaPIX, a guanine nucleotide exchange factor (GEF) for Rac1/Cdc42, is a signal for translocation of the PIX complex to neuronal growth cones and is associated with basic fibroblast growth factor (bFGF)-induced neurite outgrowth. However, the issue of whether p85 betaPIX phosphorylation affects GEF activity on Rac1/Cdc42 is yet to be explored. Here we show that Rac1 activation occurs in a p85 betaPIX phosphorylation-dependent manner. A GST-PBD binding assay reveals that Rac1 is activated in a dose- and time-dependent manner in PC12 cells in response to bFGF. Inhibition of ERK or PAK2, the kinases upstream of p85 betaPIX in the bFGF signaling, prevents Rac1 activation, suggesting that phosphorylation of p85 betaPIX functions upstream of Rac1 activation. To directly address this issue, transfection studies with wild-type and mutant p85 betaPIX (S525A/T526A, a non-phosphorylatable form) were performed. Expression of mutant PIX markedly inhibits both bFGF- and nerve growth factor (NGF)-induced activation of Rac1, indicating that phosphorylation of p85 betaPIX is responsible for activation of this G protein. Both wild-type and mutant p85 betaPIX displaying negative GEF activity (L238R/L239S) are similarly recruited to growth cones, suggesting that Rac1 activation is not essential for translocation of the PIX complex (PAK2-p85 betaPIX-Rac1). However, expression of mutant p85 betaPIX (L238R/L239S) results in retraction of the pre-existing neurites. Our results provide evidence that bFGF- and NGF-induced phosphorylation of p85 betaPIX mediates Rac1 activation, which in turn regulates cytoskeletal reorganization at growth cones, but not translocation of the PIX complex.     

betaPak-interacting exchange factor-mediated Rac1 activation requires smgGDS guanine nucleotide exchange factor in basic fibroblast growth factor-induced neurite outgrowth

Neuritogenesis requires active actin cytoskeleton rearrangement in which Rho GTPases play a pivotal role. In a previous study (Shin, E. Y., Woo, K. N., Lee, C. S., Koo, S. H., Kim, Y. G., Kim, W. J., Bae, C. D., Chang, S. I., and Kim, E. G. (2004) J. Biol. Chem. 279, 1994-2004), we demonstrated that betaPak-interacting exchange factor (betaPIX) guanine nucleotide exchange factor (GEF) mediates basic fibroblast growth factor (bFGF)-stimulated Rac1 activation through phosphorylation of Ser-525 and Thr-526 at the GIT-binding domain (GBD). However, the mechanism by which this phosphorylation event regulates the Rac1-GEF activity remained elusive. We show here that betaPIX binds to Rac1 via the GBD and also activates the GTPase via an associated GEF, smgGDS, in a phosphorylation-dependent manner. Notably, the Rac1-GEF activity of betaPIX persisted for an extended period of time following bFGF stimulation, unlike other Rho GEFs containing the Dbl homology domain. We demonstrate that C-PIX, containing proline-rich, GBD, and leucine zipper domains can interact with Rac1 via the GBD in vitro and in vivo and also mediated bFGF-stimulated Rac1 activation, as determined by a modified GEF assay and fluorescence resonance energy transfer analysis. However, nonphosphorylatable C-PIX (S525A/T526A) failed to generate Rac1-GTP. Finally, betaPIX is shown to form a trimeric complex with smgGDS and Rac1; down-regulation of smgGDS expression by short interfering RNA causing significant inhibition of betaPIX-mediated Rac1 activation and neurite outgrowth. These results provide evidence for a new and unexpected mechanism whereby betaPIX can regulate Rac1 activity.     

Involvement of p38 MAPK-mediated signaling in the calpeptin-mediated suppression of myogenic differentiation and fusion in C2C12 cells

Calpeptin inhibits myoblast fusion by inhibiting the activity of calpain. However, the mechanism by which calpeptin inhibits myogenesis is not completely understood. This study examined how calpeptin affects the expression of the myogenic regulatory factors (MRFs) and the phosphorylation of p38 mitogen-activated protein kinase (MAPK) in differentiating C2C12 myoblasts. Consistent with previous reports, calpeptin inhibited the induction of μ-calpain and the formation of myotubes in these cells. In particular, calpeptin inhibited the expression of the early and mid differentiation markers including MyoD, Myf5, myogenin, and MRF4 as well as the expression of the late markers such as troponin T and myosin heavy chain (MyHC). Calpeptin also suppressed the phosphorylation of p38 MAPK in C2C12 cells. SB203580, a specific p38 inhibitor, prevented the expression of the muscle-specific markers and their fusion into myotubes in these cells, which was further accelerated in the presence of calpeptin. These findings suggest that calpeptin inhibits the myogenesis of skeletal muscle cells by down-regulating the MRFs and involving p38 MAPK signaling.