Leucine limitation induces autophagy and activation of lysosome-dependent proteolysis in C2C12 myotubes through a mammalian target of rapamycin-independent signaling pathway

Loss of muscle mass usually characterizes different pathologies (sepsis, cancer, trauma) and also occurs during normal aging. One reason for muscle wasting relates to a decrease in food intake. This study addressed the role of leucine as a regulator of protein breakdown in mouse C2C12 myotubes and aimed to determine which cellular responses regulate the process. Determination of the rate of protein breakdown indicated that leucine is one key regulator of this process in myotubes because starvation for this amino acid is responsible for 30-40% of the total increase generated by total amino acid starvation. Leucine restriction rapidly accelerates the rate of protein breakdown (+11 to 15% (p < 0.001) after 1 h of starvation) in a dose-dependent manner. By using various inhibitors, evidence is provided that acceleration of protein catabolism results mainly from an induction of autophagy, activation of lysosome-dependent proteolysis, without modification of mRNA levels encoding the lysosomal cathepsins B, L, or D. Those results suggest that autophagy is an essential cellular response for increasing protein breakdown in muscle following food deprivation. Induction of autophagy precedes a decrease in global protein synthesis (-20% to -30% (p < 0.001)) that occurs after 3 h of leucine starvation. Inhibition of the mammalian target of rapamycin (mTOR) activity does not abolish the effect of leucine starvation and the level of phosphorylated ribosomal S6 protein is not affected by leucine withdrawal. These latter data provide clear evidence that the mTOR signaling pathway is not involved in the mediation of leucine effects on both protein synthesis and degradation in C2C12 myotubes.     

Akirin2 could promote the proliferation but not the differentiation of duck myoblasts via the activation of the mTOR/p70S6K signaling pathway

The Akirin gene family normally contains two members that are essential to myoblast differentiation. Noticeably, the avian Akirin gene family comprises only one gene (Akirin2), However, it remains unknown whether avian Akirin gene family still has the function of Akirin1; moreover, it is still unclear whether and how Akirin2 plays a role in myoblast proliferation and differentiation. Interestingly, the unexpected functions of duck Akirin2 were revealed in the present study. The Real-time PCR results showed that between 12 and 48 h during the process of duck myoblasts differentiation, the overexpression of Akirin2 did not significantly increase the expression of myogenic regulatory factors. Flow cytometry analysis revealed that the cell cycle transition was accelerated by Akirin2 overexpression. Moreover, the overexpression of Akirin2 did not influence the myotube formation. Strikingly, when duck myoblasts were cultured in the growth medium, the overexpression of Akirin2 significantly enhanced cell viability. Although the expression of cyclin-dependent proteins did not significantly increase after transfection, the expression of the mammalian targets of rapamycin (mTOR) and p70 S6 kinase (p70S6K) increased. Furthermore, the protein expression of phospho-p70S6K (Ser 417) also increased. However, when rapamycin and pEGFP-N1-Akirin2 plasmids were added together to the growth medium, the positive impact of Akirin2 on cell viability and the mRNA expression of mTOR and p70S6K were significantly blocked. Furthermore, the expression of phospho-mTOR (Ser 2448) and phospho-p70S6K (Ser 417) were also blocked. Taken together, these results could suggest that duck Akirin2 could promote myoblast proliferation via the activation of the mTOR/p70S6K signaling pathway.     

Beta-1,3 glucan sulfate, but not beta-1,3 glucan, induces the salicylic acid signaling pathway in tobacco and Arabidopsis

Sulfate substituents naturally occurring in biomolecules, such as oligosaccharides and polysaccharides, can play a critical role in major physiological functions in plants and animals. We show that laminarin, a beta-1,3 glucan with elicitor activity in tobacco (Nicotiana tabacum), becomes, after chemical sulfation, an inducer of the salicylic acid (SA) signaling pathway in tobacco and Arabidopsis thaliana. In tobacco cell suspensions, the oxidative burst induced by the laminarin sulfate PS3 was Ca2+ dependent but partially kinase independent, whereas laminarin triggered a strickly kinase-dependent oxidative burst. Cells treated with PS3 or laminarin remained fully responsive to a second application of laminarin or PS3, respectively, suggesting two distinct perception systems. In tobacco leaves, PS3, but not laminarin, caused electrolyte leakage and triggered scopoletin and SA accumulation. Expression of different families of Pathogenesis-Related (PR) proteins was analyzed in wild-type and mutant tobacco as well as in Arabidopsis. Laminarin induced expression of ethylene-dependent PR proteins, whereas PS3 triggered expression of ethylene- and SA-dependent PR proteins. In Arabidopsis, PS3-induced PR1 expression was also NPR1 (for nonexpressor of PR genes1) dependent. Structure-activity analysis revealed that (1) a minimum chain length is essential for biological activity of unsulfated as well as sulfated laminarin, (2) the sulfate residues are essential and cannot be replaced by other anionic groups, and (3) moderately sulfated beta-1,3 glucans are active. In tobacco, PS3 and curdlan sulfate induced immunity against Tobacco mosaic virus infection, whereas laminarin induced only a weak resistance. The results open new routes to work out new molecules suitable for crop protection.     

PI3P signaling regulates receptor sorting but not transport in the endosomal pathway

While evidence is accumulating that phosphoinositide signaling plays a crucial role in growth factor and hormone receptor down-regulation, this signaling pathway has also been proposed to regulate endosomal membrane transport and multivesicular endosome biogenesis. Here, we have followed the fate of the down-regulated EGF receptor (EGFR) and bulk transport (fluid phase) markers in the endosomal pathway in vivo and in vitro. We find that bulk transport from early to late endosomes is not affected after inhibition of the phosphatidylinositol-3-phosphate (PI3P) signaling pathway, but that the EGFR then remains trapped in early endosomes. Similarly, we find that hepatocyte growth factor-regulated tyrosine kinase substrate (Hrs) is not directly involved in bulk solute transport, but is required for EGFR sorting. These observations thus show that transport and sorting can be uncoupled in the endosomal pathway. They also show that PI3P signaling does not regulate the core machinery of endosome biogenesis and transport, but controls the sorting of down-regulated receptor molecules in early endosomes via Hrs.     

Sirt2 induces C2C12 myoblasts proliferation by activation of the ERK1/2 pathway

Sirtuins are Class III histone deacetylases （HDACs） that have emerged as important regulators of diverse biological processes, and comprised of seven members （Sirtl to Sirt7） [1]. Sirt2 predominantly resides in the cytoplasm,     

Sialic acid inhibits agrin signaling in C2 myotubes

Acetylcholine receptor (AChR) clustering is an early event in neuromuscular synapse formation that is commonly studied using muscle cell culture. Motor neuron-derived agrin induces the postsynaptic tyrosine phosphorylation of both a muscle-specific kinase (MuSK) and the AChR beta-subunit. These phosphorylation events are required for AChR clustering, suggesting an agrin-driven signaling pathway. Both the phosphorylation events and AChR clustering can also be induced by neuraminidase, an enzyme that cleaves sialic acid from glycoconjugates, suggesting that neuraminidase is able to activate the agrin signaling pathway. A postulated signal for postsynaptic differentiation at sites of nerve-muscle contact during vertebrate development is the enzymatic removal of basal lamina components. We show here that bath-applied sialic acid has an effect directly opposite that of agrin or neuraminidase. Sialic acid not only decreases AChR clustering but also diminishes the tyrosine phosphorylation of MuSK and the AChR beta-subunit signal-transduction events normally driven by agrin. However, sialic acid does not prevent agrin-binding molecules from colocalizing with the decreased number of AChR clusters that do form, suggesting that sialic acid is acting to inhibit the agrin signaling pathway downstream of agrin binding to the muscle cell membrane. We propose a regulatory role for sialic acid in the signal transduction events of neuromuscular synapse formation, in which agrin or neuraminidase can overcome this sialic acid repression, resulting in the clustering of AChRs and other postsynaptic molecules.     

Fenofibrate but not fenofibric acid inhibits 11beta-hydroxysteroid dehydrogenase 1 in C2C12 myotubes

Microtubules (MTs) play an important role in cell division, and their functions are regulated by a set of microtubule-associated proteins (MAPs). Tubulin polymerization promoting protein family member 3 (TPPP3), also known as p20, is a new member of the tubulin polymerization promoting protein (TPPP) family. Previous studies have demonstrated that TPPP3 specifically binds to MTs and positively regulates MTs assembly, which leads to significant ultrastructural alterations of the MTs network. However, the physiological function of TPPP3 is still largely unknown. In the present study, we showed that knockdown of endogenous TPPP3 by RNA interference (RNAi) suppressed cell proliferation and induced cell cycle arrest in HeLa cells. Furthermore, we showed that the depletion of TPPP3 caused mitotic abnormalities, such as the formation of multipolar spindles and chromosome segregation errors, which lead to apoptosis in HeLa cells. Our study suggested that TPPP3 played a crucial role in cell mitosis by regulating centrosomes amplification and/or spindles translocation processes.     

Adiponectin reduces lipid content in chicken myoblasts by activating AMPK signaling pathway

Studies in mammals have shown that adiponectin is secreted mainly by adipocytes, and it plays a crucial role in glucose and lipid metabolism in muscles. Clarifying the cross-talk role of adiponectin between adipose tissue and skeletal muscle tissue is very important for internal homeostasis. The glucose and lipid metabolism of chicken is different from that of mammals, and the role of adiponectin in chickens is unclear. Therefore, it is of great significance to study the effect and mechanism of adiponectin on lipid metabolism in chickens. In the present study, the regulating effect of adiponectin on lipid metabolism in chicken myoblasts was explored by adding a certain concentration of exogenous recombinant adiponectin. Results showed that adiponectin reduced intracellular lipid content, increasing the mRNA expression of adiponectin receptor and cellular uptake of glucose and fatty acids. In addition, adiponectin activated the 5′ adenosine monophosphate activated protein kinase (AMPK) signaling pathway. The above results suggested that adiponectin reduced intracellular lipid content, mainly by binding to adiponectin receptor, activating AMPK pathway, increasing cellular uptake of glucose and fatty acids and promoting lipid oxidation.     

The Na+/H+ antiport is activated by serum and phorbol esters in proliferating myoblasts but not in differentiated myotubes. Properties of the activation process

The properties of the Na+/H+ exchange system have been studied with 22Na+ uptake techniques at two stages of muscle development: proliferating myoblasts and differentiated myotubes. The characteristics of the interactions of the exchanger with external H+, with external Na+, and with amiloride or its more potent analogs are the same at both stages of development. Differences between the two stages of development concern: (i) the internal pH (pHi) dependence of the Na+/H+ exchanger, and (ii) the activation of the Na+/H+ exchanger by serum and phorbol ester which is observed in myoblasts but not in myotubes. Properties of the Na+/H+ exchanger in myoblasts after serum activation seem to be identical to those observed in myotubes with or without serum as if myotube formation stabilized a fully activated state of the exchanger. The activation of the myoblast Na+/H+ exchange system by serum is due to a shift of the pHi dependence towards alkaline pHi values and to an increase in the maximal activity of the Na+/H+ exchange system at acidic pH. Phorbol esters which are well-known activators of protein kinase C can only partially mimic the effects of serum on the Na+/H+ exchanger: they produce a shift of the pH dependence, but they do not increase the maximal activity at acidic pH.     

Changes in phosphofructokinase isozymes during development of myoblasts to myotubes

The regulation of phosphofructokinase during development of C2C12 myoblasts to myotubes was investigated. Enzyme activity was markedly increased during myogenic development. The increase was observed when enzyme activity was measured under optimal conditions and was not due to changes in the allosteric kinetic properties of the enzyme. Immunoprecipitation of phosphofructokinase from [35S]methionine-labeled myogenic cells revealed that equal amounts of liver and muscle isozymes are present in myoblasts, while in myotubes there was a much higher level of the muscle isozyme. These results were confirmed using an immunoblotting technique. The increase in the level of muscle isozyme in myotubes is due to an increase in the rate of synthesis of the muscle isozyme and occurs in spite of a measurably small increase in its degradation rate. Northern blot analysis using a synthetic oligonucleotide probe showed a 25-fold increase in the level of muscle phosphofructokinase mRNA in myotubes. The conclusion is drawn that the increase in muscle isozyme in myotubes during myogenesis is due to an increase in its mRNA level.     

NaF induces early differentiation of murine bone marrow cells along the granulocytic pathway but not the monocytic or preosteoclastic pathway in vitro

The stimulatory effects of sodium fluoride (NaF) on bone formation have been explained solely by its activation of osteoblasts. However, whether and how NaF acts on the osteoclast lineage is poorly understood. We previously found that NaF differentiates HL-60 cells to granulocytic cells. To further test this action, we have employed here primary cultures of progenitor cells derived from murine bone marrow. NaF at subtoxic concentrations (<0.5 mM) significantly up-regulated activities of several intracellular enzymes (lactate dehydrogenase, beta-glucuronidase, acid phosphatase), cellular reduction of nitroblue tetrazolium, and nitric oxide (NO) production; which are all accepted as general differentiation markers. NaF (<0.5 mM) also up-regulated granulocyte-specific markers (chloroacetate esterase, cell surface antigens [Mac-1, Gr-1]) but not any of the monocyte-specific markers (nonspecific esterase, cell surface antigens [F4/80, MOMA-2]). Although other general differentiation markers (phagocytosis, adhesion, appearance, nuclear:cytoplasmic ratio) were not appreciably influenced by NaF, essentially in support of our previous data from HL-60 cells, the present findings suggest that NaF induces early differentiation of bone marrow hemopoietic progenitor cells along the granulocytic pathway but not the monocytic pathway that is linked to osteoclast formation. Therefore, in addition to its potent stimulatory effects on osteoblastic bone formation, NaF applied to patients with osteoporosis could be expected to indirectly reduce osteoclastic bone resorption.     

Functional complementation by wheat eIF2alpha in the yeast GCN2-mediated pathway

Translational control by specific eIF2alpha phosphorylation on serine 51 has been characterized in all eukaryotes with the significant exception of plants. In order to evaluate the capability of plant eIF2alpha to functionally control translation, the wild type (51S) and a nonphosphorylatable mutant (51A) of wheat eIF2alpha were expressed in a yeast genetic system. Expression of either wheat protein did not handicap growth under conditions that repress the eIF2alpha phosphorylation pathway. However, under conditions that induce specific eIF2alpha phosphorylation only strains expressing wheat 51S were able to grow between 2 and 4 days. Growth was dependent upon activity of yeast eIF2alpha kinase GCN2 and resulted in the increased translation of GCN4. The association between plant eIF2alpha and yeast eIF2B is supported by their specific coimmunoprecipitation from transgenic yeast cells. These data support the similarity among eukaryotic translational initiation processes and strengthen the concept that plants may contain an eIF2alpha phosphorylation pathway.     

Bacterial lipopolysaccharide induces expression of ABCA1 but not ABCG1 via an LXR-independent pathway

Two ATP-binding cassette transporter proteins, ABCA1 and ABCG1, may mediate an active efflux of cellular cholesterol and phospholipids. They are ubiquitously expressed and are subject to regulation by cholesterol loading or by treatment with agents that activate the nuclear hormone receptor LXR. Earlier studies in both primates and non-primates reported that treatment with endotoxin (bacterial lipopolysaccharide, LPS) reduces plasma levels of HDL cholesterol. To determine if such HDL reduction correlates with a change in ABCA1 or ABCG1 expression, their expressions were measured in THP-1 monocytes and mice treated with LPS. LPS treatment leads to a rapid, dose-dependent increase of ABCA1 but not ABCG1 mRNA expression. Analysis of mouse livers showed that LPS treatment decreases expression of CYP7A, another target gene of LXR. When THP-1 cells were transfected with the ABCA1 promoter construct (-928 to +101 bp), promoter activity was significantly increased by treatment of 22(R)-hydroxycholesterol but not by LPS. Together, these studies show that LPS regulates ABCA1 expression through an LXR-independent mechanism. Further studies showed that treatment with Rhodobacter sphaeroiders LPS, an LPS antagonist, or PD169316, a specific p38 MAP kinase inhibitor, prevented the induction of ABCA1 by LPS. Therefore, this suggests that both transport of LPS from the plasma membrane to an intracellular site and activation of p38 MAP kinase are involved in the LPS-mediated induction of ABCA1.     

Metabolic stress induces the lysosomal degradation of neuropilin-1 but not neuropilin-2

The neuropilins-1 and -2 (NRP1 and NRP2) function as receptors for both the semaphorins and vascular endothelial growth factor. In addition to their contribution to the development of the nervous system, NRP1 and NRP2 have been implicated in angiogenesis and tumor progression. Given their importance to cancer and endothelial biology and their potential as therapeutic targets, an important issue that has not been addressed is the impact of metabolic stress conditions characteristic of the tumor microenvironment on their expression and function. Here, we demonstrate that hypoxia and nutrient deprivation stimulate the rapid loss of NRP1 expression in both endothelial and carcinoma cells. NRP2 expression, in contrast, is maintained under these conditions. The lysosomal inhibitors chloroquine and bafilomycin A1 prevented the loss of NRP1 expression, but proteasomal inhibitors had no effect. The hypothesis that NRP1 is degraded by autophagy is supported by the findings that its expression is lost rapidly in response to metabolic stress, prevented with 3-methyladenine and induced by rapamycin. Targeted depletion of NRP2 using small hairpin RNA revealed that NRP2 can function in the absence of NRP1 to mediate endothelial tube formation in hypoxia. Studies aimed at assessing NRP function and targeted therapy in cancer and angiogenesis should consider the impact of metabolic stress.