C/EBPβ mediates tumour-induced ubiquitin ligase atrogin1/MAFbx upregulation and muscle wasting

Upregulation of ubiquitin ligase atrogin1/MAFbx and muscle wasting are hallmarks of cancer cachexia; however, the underlying mechanism is undefined. Here, we describe a novel signalling pathway through which Lewis lung carcinoma (LLC) induces atrogin1/MAFbx upregulation and muscle wasting. C2C12 myotubes treated with LLC-conditioned medium (LCM) rapidly activates p38 MAPK and AKT while inactivating FoxO1/3, resulting in atrogin1/MAFbx upregulation, myosin heavy chain loss, and myotube atrophy. The p38α/β MAPK inhibitor SB202190 blocks the catabolic effects. Upon activation, p38 associates with C/EBPβ resulting in its phosphorylation and binding to a C/EBPβ-responsive cis-element in the atrogin1/MAFbx gene promoter. The promoter activity is stimulated by LCM via p38β-mediated activation of the C/EBPβ-responsive cis-element, independent of the adjacent FoxO1/3-responsive cis-elements in the promoter. In addition, p38 activation is observed in the muscle of LLC tumour-bearing mice, and SB202190 administration blocks atrogin1/MAFbx upregulation and muscle protein loss. Furthermore, C/EBPβ(-/-) mice are resistant to LLC tumour-induced atrogin1/MAFbx upregulation and muscle wasting. Therefore, activation of the p38β MAPK-C/EBPβ signalling pathway appears a key component of the pathogenesis of LLC tumour-induced cachexia.     

Constitutive expression of insulin receptor substrate (IRS)-1 inhibits myogenic differentiation through nuclear exclusion of Foxo1 in L6 myoblasts

Insulin-like growth factors (IGFs) are well known to play essential roles in enhancement of myogenic differentiation. In this report we showed that initial IGF-I signal activation but long-term IGF-1 signal termination are required for myogenic differentiation. L6 myoblast stably transfected with myc-epitope tagged insulin receptor substrate-1, myc-IRS-1 (L6-mIRS1) was unable to differentiate into myotubes, indicating that IRS-1 constitutive expression inhibited myogenesis. To elucidate the molecular mechanisms underlying myogenic inhibition, IGF-I signaling was examined. IGF-I treatment of control L6 cells for 18 h resulted in a marked suppression of IGF-I stimulated IRS-1 association with the p85 PI 3-kinase and suppression of activation of Akt that correlated with a down regulation of IRS-1 protein. L6-mIRS1 cells, in contrast, had sustained high levels of IRS-1 protein following 18 h of IGF-I treatment with persistent p85 PI 3-kinase association with IRS-1, Akt phosphorylation and phosphorylation of the downstream Akt substrate, Foxo1. Consistent with Foxo1 phosphorylation, Foxo1 protein was excluded from the nuclei in L6-mIRS1 cells, whereas Foxo1 was localized in the nuclei in control L6 cells during induction of differentiation. In addition, L6 cells stably expressing a dominant-interfering form of Foxo1, Δ256Foxo1 (L6-Δ256Foxo1) were unable to differentiate into myotubes. Together, these data demonstrate that IGF-I regulation of Foxo1 nuclear localization is essential for the myogenic program in L6 cells but that persistent activation of IGF-1 signaling pathways results in a negative feedback to prevent myogenesis.     

Effects of dimethyl sulphoxide and dexamethasone on mRNA expression of myogenesis- and muscle proteolytic system-related genes in mouse myoblastic C2C12 cells

We examined the time course of mRNA expression of myogenic cell differentiation- and muscle proteolytic system-related genes in cultures of C2C12 cells during differentiation from myoblasts to myotubes. Furthermore, we treated C2C12 myotubes with dimethyl sulphoxide (DMSO) and dexamethasone (Dex), and examined changes in these mRNA levels. Myogenin (Myog), Atrogin1, forkhead box O1 (Foxo1) and Capn1 mRNA levels increased in C2C12 cells differentiating from myoblasts to myotubes, whereas Myf5 mRNA levels decreased. Although genes such as MRF4, Foxo3a, UbB, Capn1 and MuRF1 mRNAs in the myotubes were affected by DMSO exposure, mRNA levels of other genes were not markedly affected by exposure to 0.02% or 0.5% DMSO. Myf5, MRF4, Atrogin1, Foxo3 and MuRF1 mRNA levels were elevated by Dex at all time points, Cbl and Capn1 mRNA levels were significantly elevated by Dex at 8 h, and Myog mRNA levels were significantly elevated by Dex at 24 h. However, CtsH mRNA levels decreased significantly with Dex at 24 h. This study provides a useful database of gene profiles that are differentially expressed throughout myogenic cell differentiation and the muscle proteolytic system.     

Foxo1 represses expression of musclin, a skeletal muscle-derived secretory factor

Musclin is a novel skeletal muscle-derived secretory factor, whose mRNA level is markedly regulated by nutritional status. In the present study, we investigated the mechanism of musclin mRNA regulation by insulin. In C2C12 myocytes, insulin-induced upregulation of musclin mRNA was significantly decreased by treatment of phosphatidylinositol 3-kinase (PI3K) inhibitor, LY294002, and was abolished in C2C12 myocytes stably expressing a constitutively active Foxo1 (Foxo1-3A), suggesting the involvement of Foxo1 in the regulation of musclin mRNA. Promoter deletion analysis of musclin promoter revealed that the region of −303/−123 is important for the repression of promoter activity by Foxo1. Chromatin immunoprecipitation assay showed that Foxo1 bound to musclin promoter. Musclin mRNA level was markedly downregulated in gastrocnemius muscle of Foxo1 transgenic mice. Our results demonstrated that Foxo1 downregulates musclin mRNA expression both in vitro and in vivo, which should explain insulin-mediated upregulation of this gene in muscle cells.     

Insulin-like growth factor 1/insulin signaling activates androgen signaling through direct interactions of Foxo1 with androgen receptor

The androgen-androgen receptor (AR) system plays vital roles in a wide array of biological processes, including prostate cancer development and progression. Several growth factors, such as insulin-like growth factor 1 (IGF1), can induce AR activation, whereas insulin resistance and hyperinsulinemia are correlated with an elevated incidence of prostate cancer. Here we report that Foxo1, a downstream molecule that becomes phosphorylated and inactivated by phosphatidylinositol 3-kinase/Akt kinase in response to IGF1 or insulin, suppresses ligand-mediated AR transactivation. Foxo1 reduces androgen-induced AR target gene expressions and suppresses the in vitro growth of prostate cancer cells. These inhibitory effects of Foxo1 are attenuated by IGF1 but are enhanced when it is rendered Akt-nonphosphorylatable. Foxo1 interacts directly with the C terminus of AR in a ligand-dependent manner and disrupts ligand-induced AR subnuclear compartmentalization. Foxo1 is recruited by liganded AR to the chromatin of AR target gene promoters, where it interferes with AR-DNA interactions. IGF1 or insulin abolish the Foxo1 occupancy of these promoters. Of interest, a positive feedback circuit working locally in an autocrine/intracrine manner may exist, because liganded AR up-regulates IGF1 receptor expression in prostate cancer cells, presumably resulting in higher IGF1 signaling tension and further enhancing the functions of the receptor itself. Thus, Foxo1 is a novel corepressor for AR, and IGF1/insulin signaling may confer stimulatory effects on AR by attenuating Foxo1 inhibition. These results highlight the potential involvement of metabolic syndrome and hyperinsulinemia in prostate diseases and further suggest that intervention of IGF1/insulin-phosphatidylinositol 3-kinase-Akt signaling may be of clinical value for prostate diseases.     

IFN-gamma does not mimic the catabolic effects of TNF-alpha

Cachexia is common in chronic inflammatory diseases and is attributed, in part, to an elevation of circulating proinflammatory cytokines. TNF-alpha is the prototype in this category. IFN-gamma is also thought to play a role, but the evidence supporting this model is primarily indirect. To determine the direct effects of IFN-gamma stimulation on muscle cells, we selected key components of the procatabolic signaling pathways by which TNF-alpha stimulates protein loss. We tested two hypotheses: 1) IFN-gamma mimics TNF-alpha signaling by increasing intracellular oxidant activity and activating MAPKs and NF-kappaB and 2) IFN-gamma increases the expression of the ubiquitin ligases atrogin1/MAFbx and muscle-specific ring finger protein 1 (MuRF1). Results showed that treatment with IFN-gamma at 60 ng/ml increased Stat1 phosphorylation after 15 min, indicating receptor activation. IFN-gamma had no effect on cytosolic oxidant activity, as measured by 2',7'-dichlorofluorescein oxidation. Nor did IFN-gamma activate JNK, ERK1/2, or p38 MAPK, as assessed by Western blot. Treatment for up to 60 min did not decrease IkappaB-alpha protein levels, as measured by Western blot analysis, or the DNA binding activity of NF-kappaB, as measured by EMSA. After 6 h, IFN-gamma decreased Akt phosphorylation and increased atrogin1/MAFbx and MuRF1 mRNA. Daily treatment for up to 72 h did not alter adult fast-type myosin heavy chain content or the total protein-to-DNA ratio. These data show that responses of myotubes to IFN-gamma and TNF-alpha differ markedly and provide little evidence for a direct catabolic effect of IFN-gamma on muscle.     

S-allyl cysteine inhibits TNFα-induced skeletal muscle wasting through suppressing proteolysis and expression of inflammatory molecules

Background

Elevated levels of inflammatory molecules are key players in muscle wasting/atrophy leading to human morbidity. TNFα is a well-known pro-inflammatory cytokine implicated in the pathogenesis of muscle wasting under diverse clinical settings. S-allyl cysteine (SAC), an active component of garlic (Allium sativum), has established anti-oxidant and anti-inflammatory effects in various cell types. However, the impact of SAC on skeletal muscle pathology remains unexplored. Owing to the known anti-inflammatory properties of SAC, we investigated whether pre-treatment with SAC has a protective role in TNFα-induced atrophy in cultured myotubes.

Molecular mechanisms of insulin-like growth factor 1 promoted synthesis and retention of hyaluronic acid in porcine oocyte-cumulus complexes

The purpose of the present study was to elucidate signaling pathways by which insulin like-growth factor 1 (IGF1) promotes FSH-stimulated synthesis and retention of hyaluronic acid (HA) in pig oocyte-cumulus complexes (OCCs) cultured in serum-free medium. We found that IGF1 had no effects on FSH-stimulated production of cAMP and activation of protein kinase A in the OCCs. Immunoblotting with phospho-specific antibodies showed that FSH moderately phosphorylated v-akt murine thymoma viral oncogene homolog (AKT) and mitogen-activated kinase 3 and 1 (MAPK3/1) in cumulus cells. The exposure of OCCs to both FSH and IGF1 resulted in a significant (P < 0.05) increase in AKT and MAPK3/1 phosphorylation. An inhibitor of phosphoinositide-3-kinase (PIK3), LY 294002, significantly (P < 0.05) reduced the IGF1-enhanced phosphorylation of AKT, and inhibitors of AKT (SH6) and MAPK3/1 (U0126) significantly (P < 0.05) decreased the synthesis and retention of HA stimulated by concomitant exposure of OCCs to both FSH and IGF1. The IGF1-promoted synthesis of HA was not accompanied by an increase in the relative abundance of hyaluronan synthase 2 (HAS2) mRNA in the cumulus cells. We conclude that IGF1 promotes the FSH-stimulated synthesis and retention of HA in pig OCCs by PIK3/AKT- and MAPK3/1-dependent mechanisms.     

Experimental hyperthyroidism in rats increases the expression of the ubiquitin ligases atrogin‐1 and MuRF1 and stimulates multiple proteolytic pathways in skeletal muscle

Muscle wasting is commonly seen in patients with hyperthyroidism and is mainly caused by stimulated muscle proteolysis. Loss of muscle mass in several catabolic conditions is associated with increased expression of the muscle‐specific ubiquitin ligases atrogin‐1 and MuRF1 but it is not known if atrogin‐1 and MuRF1 are upregulated in hyperthyroidism. In addition, it is not known if thyroid hormone increases the activity of proteolytic mechanisms other than the ubiquitin–proteasome pathway. We tested the hypotheses that experimental hyperthyroidism in rats, induced by daily intraperitoneal injections of 100 µg/100 g body weight of triiodothyronine (T3), upregulates the expression of atrogin‐1 and MuRF1 in skeletal muscle and stimulates lysosomal, including cathepsin L, calpain‐, and caspase‐3‐dependent protein breakdown in addition to proteasome‐dependent protein breakdown. Treatment of rats with T3 for 3 days resulted in an approximately twofold increase in atrogin‐1 and MuRF1 mRNA levels. The same treatment increased proteasome‐, cathepsin L‐, and calpain‐dependent proteolytic rates by approximately 40% but did not influence caspase‐3‐dependent proteolysis. The expression of atrogin‐1 and MuRF1 remained elevated during a more prolonged period (7 days) of T3 treatment. The results provide support for a role of the ubiquitin–proteasome pathway in muscle wasting during hyperthyroidism and suggest that other proteolytic pathways as well may be activated in the hyperthyroid state. J. Cell. Biochem. 108: 963–973, 2009. © 2009 Wiley‐Liss, Inc.     

Effect of insoluble fibre on intestinal morphology and mRNA expression pattern of inflammatory,cell cycle and growth marker genes in a piglet model

The effects of insoluble dietary fibre differing in lignin content on intestinal morphology and mRNA expression was tested in an animal model of 48 weaned piglets. Engaged fibre sources were wheat bran (rich in cellulose and hemicellulose) and pollen from Chinese Masson pine (Pinus massoniana) (rich in lignin), respectively. The fibre sources were added to a basal diet as follows: no addition (control), 3.0% wheat bran, 1.27% pine pollen, and 2.55% pine pollen. The 12 animals of each feeding group were fed four experimental diets ad libitum for 37 days and were then slaughtered for retrieving tissue samples from stomach, jejunum, ileum, colon and mesenterial lymph nodes. Both fibre sources increased villus height of mucosa in jejunum (+10% on average) and ileum (+16% on average). Results of mRNA expression rates of inflammatory, cell cycle and growth marker genes (NFκB, TNFα, TGFβ, Caspase3, CDK4, IGF1) were specific to fibre source and tissue: wheat bran induced an up-regulation of NFκB in stomach and jejunum, as well as TNFα and TGFβ, and Caspase3 in jejunum. Pine pollen induced down regulation of NFκB, TNFα, TGFβ, Caspase3, CDK4 and IGF1 in the colon as well as up-regulation of NFκB and TGFβ in mesenterial lymph nodes. Finally, an overall data comparison based on a hierarchical cluster analysis showed a close relation between gene regulation in different gut sections and organs, as well as between small intestine morphology and zootechnical performance.     

KRASG12D- and BRAFV600E-Induced Transformation of Murine Pancreatic Epithelial Cells Requires MEK/ERK-Stimulated IGF1R Signaling

Mutation of KRAS is a common initiating event in pancreatic ductal adenocarcinoma (PDAC). Yet, the specific roles of KRAS-stimulated signaling pathways in the transformation of pancreatic ductal epithelial cells (PDEC), putative cells of origin for PDAC, remain unclear. Here, we show that KRAS(G12D) and BRAF(V600E) enhance PDEC proliferation and increase survival after exposure to apoptotic stimuli in a manner dependent on MEK/ERK and PI3K/AKT signaling. Interestingly, we find that activation of PI3K/AKT signaling occurs downstream of MAP-ERK kinase (MEK), and is dependent on the autocrine activation of the insulin-like growth factor (IGF) receptor (IGF1R) by IGF2. Importantly, IGF1R inhibition impairs KRAS(G12D)- and BRAF(V600E)-induced survival, whereas ectopic IGF2 expression rescues KRAS(G12D)- and BRAF(V600E)-mediated survival downstream of MEK inhibition. Moreover, we show that KRAS(G12D)- and BRAF(V600E)-induced tumor formation in an orthotopic model requires IGF1R. Interestingly, we show that while individual inhibition of MEK or IGF1R does not sensitize PDAC cells to apoptosis, their concomitant inhibition reduces survival. Our findings identify a novel mechanism of PI3K/AKT activation downstream of activated KRAS, illustrate the importance of MEK/ERK, PI3K/AKT, and IGF1R signaling in pancreatic tumor initiation, and suggest potential therapeutic strategies for this malignancy. Mol Cancer Res; 10(9); 1228-39. ?2012 AACR.     

TNF-alpha increases protein content in C2C12 and primary myotubes by enhancing protein translation via the TNF-R1, PI3K, and MEK

Recent evidence supports that TNF-alpha, long considered a catabolic factor, may also have a physiological function in skeletal muscle. The catabolic view, mainly based on correlative studies in human and in vivo animal models, was challenged by experiments with myoblasts, in which TNF-alpha induced differentiation. The biological effects of TNF-alpha in differentiated muscle, however, remain poorly understood. In the present study, we tested whether TNF-alpha has growth-promoting effects in myotubes, and we characterized the mechanisms leading to these effects. Treatment of C(2)C(12) myotubes with TNF-alpha for 24 h increased protein synthesis (PS) and enhanced cellular dehydrogenase activity by 22 and 26%, respectively, without changing cell numbers. These effects were confirmed in myotubes differentiated from primary rat myoblasts. TNF-alpha activated two signaling cascades: 1) ERK1/2 and its target eIF4E and 2) Akt and its downstream effectors GSK-3, p70(S6K), and 4E-BP1. TNF-alpha-induced phosphorylation of Akt, and ERK1/2 was inhibited by an antibody against TNF-alpha receptor 1 (TNF-R1). PD-98059 pretreatment abolished TNF-alpha-induced phosphorylation of ERK1/2 and eIF4E, whereas PS was only partially inhibited. LY-294002 completely abolished TNF-alpha-induced stimulation of PS as well as phosphorylation of Akt and its downstream targets GSK-3, p70(S6K), and 4E-BP1. Rapamycin inhibited TNF-alpha-induced phosphorylation of the mTOR C1 target p70(S6K) without altering TNF-alpha-induced PS and 4E-BP1 phosphorylation. In conclusion, our results provide evidence that TNF-alpha enhances PS in myotubes and that this is based on enhanced protein translation mediated by the TNF-R1 and PI3K-Akt and MEK-ERK signaling cascades.     

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.