Mechanism of attenuation of muscle protein degradation induced by tumor necrosis factor-alpha and angiotensin II by beta-hydroxy-beta-methylbutyrate

Both tumor necrosis factor-alpha (TNF-alpha)/interferon-gamma (IFN-gamma) and angiotensin II (ANG II) induced an increase in total protein degradation in murine myotubes, which was completely attenuated by treatment with beta-hydroxy-beta-methylbutyrate (HMB; 50 microM). There was an increase in formation of reactive oxygen species (ROS) within 30 min, as well as an increase in the activity of both caspase-3 and -8, and both effects were attenuated by HMB. Moreover, inhibitors of caspase-3 and -8 completely attenuated both ROS formation and total protein degradation induced by TNF-alpha/IFN-gamma and ANG II. There was an increased autophosphorylation of double-stranded RNA-dependent protein kinase (PKR), which was attenuated by the specific caspase-3 and -8 inhibitors. Neither ROS formation or protein degradation occurred in myotubes expressing a catalytically inactive PKR variant, PKRDelta6, in response to TNF-alpha/IFN-gamma, compared with myotubes expressing wild-type PKR, although there was still activation of caspase-3 and -8. HMB also attenuated activation of PKR, suggesting that it was important in protein degradation. Formation of ROS was attenuated by rotenone, an inhibitor of the mitochondrial electron transport chain, nitro-l-arginine methyl ester, an inhibitor of nitric oxide synthase, and SB 203580, a specific inhibitor of p38 mitogen-activated protein kinase (p38 MAPK), which also attenuated total protein degradation. Activation of p38 MAPK by PKR provides the link to ROS formation. These results suggest that TNF-alpha/IFN-gamma and ANG II induce muscle protein degradation by a common signaling pathway, which is attenuated by HMB, and that this involves the initial activation of caspase-3 and -8, followed by autophosphorylation and activation of PKR, which then leads to increased ROS formation via activation of p38 MAPK. Increased ROS formation is known to induce protein degradation through the ubiquitin-proteasome pathway.

Mechanism of induction of muscle protein degradation by angiotensin II

Angiotensin I and II have been shown to directly induce protein degradation in skeletal muscle through an increased activity and expression of the ubiquitin-proteasome proteolytic pathway. This investigation determines the role of the nuclear transcription factor nuclear factor-κB (NF-κB) in this process. Using murine myotubes as a surrogate model system both angiotensin I and II were found to induce activation of protein kinase C (PKC), with a parabolic dose-response curve similar to the induction of total protein degradation. Activation of PKC was required for the induction of proteasome expression, since calphostin C, a highly specific inhibitor of PKC, attenuated both the increase in total protein degradation and in proteasome expression and functional activity increased by angiotensin II. PKC is known to activate I-κB kinase (IKK), which is responsible for the phosphorylation and subsequent degradation of I-κB. Both angiotensin I and II induced an early decrease in cytoplasmic I-κB levels followed by nuclear accumulation of NF-κB. Using an NF-κB luciferase construct this was shown to increase transcriptional activation of NF-κB regulated genes. Maximal luciferase expression was seen at the same concentrations of angiotensin I/II as those inducing protein degradation. Total protein degradation induced by both angiotensin I and II was attenuated by resveratrol, which prevented nuclear accumulation of NF-κB, confirming that activation of NF-κB was responsible for the increased protein degradation. These results suggest that induction of proteasome expression by angiotensin I/II involves a signalling pathway involving PKC and NF-κB. © 2005 Elsevier Inc. All rights reserved.

Skeletal muscle atrophy, a link between depression of protein synthesis and increase in degradation

Both proteolysis-inducing factor (PIF) and angiotensin II have been shown to produce a depression in protein synthesis in murine myotubes concomitant with an increased phosphorylation of eukaryotic initiation factor 2 (eIF2α). Both PIF and angiotensin II were shown to induce autophosphorylation of the RNA-dependent protein kinase (PKR), and an inhibitor of this enzyme completely attenuated the depression in protein synthesis and prevented the induction of eIF2α phosphorylation. The PKR inhibitor also completely attenuated the increase in protein degradation induced by PIF and angiotensin II and prevented the increase in proteasome expression and activity. To confirm these results myotubes were transfected with plasmids that express either wild-type PKR, or a catalytically inactive PKR variant, PKRΔ6. Myotubes expressing PKRΔ6 showed no increase in eIF2α phosphorylation in response to PIF or angiotensin II, no depression in protein synthesis, and no increase in protein degradation or increase in proteasome expression. Induction of the ubiquitin-proteasome pathway by PIF and angiotensin II has been linked to activation of the transcription factor nuclear factor-κB (NF-κB). Inhibition of PKR prevented nuclear migration of NF-κB in response to both PIF and angiotensin II, by preventing degradation of the inhibitor protein I-κB. Phosphorylation of PKR and eIF2α was also significantly increased in the gastrocnemius muscle of weight losing mice bearing the MAC16 tumor, suggesting that a similar process may be operative in cancer cachexia. These results provide a link between the depression of protein synthesis in skeletal muscle and the increase in protein degradation. © 2007 by The American Society for Biochemistry and Molecular Biology, Inc.

NF-κB mediates proteolysis-inducing factor induced protein degradation and expression of the ubiquitin-proteasome system in skeletal muscle

Loss of skeletal muscle in cancer cachexia has a negative effect on both morbidity and mortality. The role of nuclear factor-κB (NF-κB) in regulating muscle protein degradation and expression of the ubiquitin-proteasome proteolytic pathway in response to a tumour cachectic factor, proteolysis-inducing factor (PIF), has been studied by creating stable, transdominant-negative, muscle cell lines. Murine C2C12 myoblasts were transfected with plasmids with a CMV promoter that had mutations at the serine phosphorylation sites required for degradation of I-κBα, an NF-κB inhibitory protein, and allowed to differentiate into myotubes. Proteolysis-inducing factor induced degradation of I-κBα, nuclear accumulation of NF-κB and an increase in luciferase reporter gene activity in myotubes containing wild-type, but not mutant, I-κBα, proteins. Proteolysis-inducing factor also induced total protein degradation and loss of the myofibrillar protein myosin in myotubes containing wild-type, but not mutant, plasmids at the same concentrations as those causing activation of NF-κB. Proteolysis-inducing factor also induced increased expression of the ubiquitin-proteasome pathway, as determined by 'chymotrypsin-like' enzyme activity, the predominant proteolytic activity of the β-subunits of the proteasome, protein expression of 20S α-subunits and the 19S subunits MSSI and p42, as well as the ubiquitin conjugating enzyme, E214k, in cells containing wild-type, but not mutant, I-κBα. The ability of mutant I-κBα to inhibit PIF-induced protein degradation, as well as expression of the ubiquitin-proteasome pathway, confirms that both of these responses depend on initiation of transcription by NF-κB. © 2005 Cancer Research UK.

Mechanism of the attenuation of proteolysis-inducing factor stimulated protein degradation in muscle by β-hydroxy-β-methylbutyrate

The leucine metabolite β-hydroxy-β-methylbutyrate (HMB) prevents muscle protein degradation in cancer-induced weight loss through attenuation of the ubiquitin-proteasome proteolytic pathway. To investigate the mechanism of this effect, the action of HMB on protein breakdown and intracellular signaling leading to increased proteasome expression by the tumor factor proteolysis-inducing factor (PIF) has been studied in vitro using murine myotubes as a surrogate model of skeletal muscle. A comparison has been made of the effects of HMB and those of eicosapentaenoic acid (EPA), a known inhibitor of PIF signaling. At a concentration of 50 μmol/L, EPA and HMB completely attenuated PIF-induced protein degradation and induction of the ubiquitin-proteasome proteolytic pathway, as determined by the "chymotrypsin-like" enzyme activity, as well as protein expression of 20S proteasome α- and β-subunits and subunit p42 of the 19S regulator. The primary event in PIF-induced protein degradation is thought to be release of arachidonic acid from membrane phospholipids, and this process was attenuated by EPA, but not HMB, suggesting that HMB might act at another step in the PIF signaling pathway. EPA and HMB at a concentration of 50 μmol/L attenuated PIF-induced activation of protein kinase C and the subsequent degradation of inhibitor κBα and nuclear accumulation of nuclear factor κB. EPA and HMB also attenuated phosphorylation of p42/44 mitogen-activated protein kinase by PIF, thought to be important in PIF-induced proteasome expression. These results suggest that HMB attenuates PIF-induced activation and increased gene expression of the ubiquitin-proteasome proteolytic pathway, reducing protein degradation.

Increased expression of the ubiquitin-proteasome pathway in murine myotubes by proteolysis-inducing factor (PIF) is associated with activation of the transcription factor NF-kappa B

Proteolysis-inducing factor (PIF), isolated from a cachexia-inducing murine tumour, has been shown to stimulate protein breakdown in C 2C12 myotubes. The effect was attenuated by the specific proteasome inhibitor lactacystin and there was an elevation of proteasome 'chymotrypsin-like' enzyme activity and expression of 205 proteasome α-subunits at concentrations of PIF between 2 and 16 nM. Higher concentrations of PIF had no effect. The action of PIF was attenuated by eicosapentaenoic acid (EPA) (50 μM). At a concentration of 4 nM, PIF induced a transient decrease in IκBα levels after 30 min incubation, while no effect was seen at 20 nM PIF. The level of IκBα, an NF-κB inhibitory protein, returned to normal after 60 min. Depletion of IκBα from the cytosol was not seen in myotubes pretreated with EPA, suggesting that the NF-κB/IκB complex was stabilised. At concentrations between 2 and 8 nM, PIF stimulated an increased nuclear migration of NF-κB, which was not seen in myotubes pretreated with EPA. The PIF-induced increase in chymotrypsin-like enzyme activity was also attenuated by the NF-κB inhibitor peptide SN50, suggesting that NF-κB may be involved in the PIF-induced increase in proteasome expression. The results further suggest that EPA may attenuate protein degradation induced by PIF, at least partly, by preventing NF-κB accumulation in the nucleus. © 2003 Cancer Research UK.

Activation of ATP-ubiquitin-dependent proteolysis in skeletal muscle in vivo and murine myoblasts in vitro by a proteolysis-inducing factor (PIF)

Loss of skeletal muscle is a major factor in the poor survival of patients with cancer cachexia. This study examines the mechanism of catabolism of skeletal muscle by a tumour product, proteolysis-inducing factor (PIF). Intravenous administration of PIF to normal mice produced a rapid decrease in body weight (1.55 ± 0.12 g in 24 h) that was accompanied by increased mRNA levels for ubiquitin, the Mr 14 000 ubiquitin carrier-protein, E2, and the C9 proteasome subunit in gastrocnemius muscle. There was also increased protein levels of the 20S proteasome core and 19S regulatory subunit, detectable by immunoblotting, suggesting activation of the ATP-ubiquitin-dependent proteolytic pathway. An increased protein catabolism was also seen in C2C12 myoblasts within 24 h of PIF addition with a bell-shaped dose-response curve and a maximal effect at 2-4 nM. The enhanced protein degradation was attenuated by anti-PIF antibody and by the proteasome inhibitors MG115 and lactacystin. Glycerol gradient analysis of proteasomes from PIF-treated cells showed an elevation in chymotrypsin-like activity, while Western analysis showed a dose-related increase in expression of MSSI, an ATPase that is a regulatory subunit of the proteasome, with a dose-response curve similar to that for protein degradation. These results confirm that PIF acts directly to stimulate the proteasome pathway in muscle cells and may play a pivotal role in protein catabolism in cancer cachexia. © 2001 Cancer Research Campaign.

Involvement of phosphoinositide 3-kinase and Akt in the induction of muscle protein degradation by proteolysis-inducing factor

In the present study the role of Akt/PKB (protein kinase B) in PIF- (proteolysis-inducing factor) induced protein degradation has been investigated in murine myotubes. PIF induced transient phosphorylation of Akt at Ser(473) within 30 min, which was attenuated by the PI3K (phosphoinositide 3-kinase) inhibitor LY294002 and the tyrosine kinase inhibitor genistein. Protein degradation was attenuated in myotubes expressing a dominant-negative mutant of Akt (termed DNAkt), compared with the wild-type variant, whereas it was enhanced in myotubes containing a constitutively active Akt construct (termed MyrAkt). A similar effect was observed on the induction of the ubiquitin-proteasome pathway. Phosphorylation of Akt has been linked to up-regulation of the ubiquitin-proteasome pathway through activation of NF-kappaB (nuclear factor kappaB) in a PI3K-dependent process. Protein degradation was attenuated by rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin), when added before, or up to 30 min after, addition of PIF. PIF induced transient phosphorylation of mTOR and the 70 kDa ribosomal protein S6 kinase. These results suggest that transient activation of Akt results in an increased protein degradation through activation of NF-kappaB and that this also allows for a specific synthesis of proteasome subunits.

Mechanism of muscle protein degradation in Cancer Cachexia

A protein-mobilising factor of estimated molecular weight 24 KDa (p24) was purified both from the cachexia-inducing MAC 16 tumour and the urine of cachectic cancer patients by a combination of ammonium sulphate precipitation and affinity chromatography using a monoclonal antibody developed against the murine material. Administration of p24 to non tumour-bearing mice caused a decrease in body weight 24 h after the first injection, which was attenuated by prior treatment with the monoclonal antibody. Loss of body weight was accompanied by an accelerated loss of skeletal muscle protein, as determined by the release of tyrosine from this tissue. This was associated with an increased release of PGE2 and both protein degradation and PGE2 release were attenuated by the monoclonal antibody. Loss of protein mass arose from both a decrease in the rate of protein synthesis and an elevation of protein breakdown; the latter due to an activation of the ubiquitin-proteasome proteolytic system. In isolated muscle, p24 was capable of promoting protein breakdown and this was also associated with increased PGE2 levels. Both tyrosine and PGE2 release, were inhibited by PGE2 inhibitors and a specific inhibitor of cPLA2. When added to muscle cells in culture, p24 caused an elevation in the rates of total and myofibrillar protein breakdown and a depression in the rate of protein synthesis which was inhabitable by short-term incubation in insulin, suggesting that p24 may inhibit protein synthesis by causing an arrest in the translational process.

Evidence that interaction between conserved residues in transmembrane helices 2, 3, and 7 are crucial for human VPAC1 receptor activation

The VPAC(1) receptor belongs to family B of G protein-coupled receptors (GPCR-B) and is activated upon binding of the vasoactive intestinal peptide (VIP). Despite the recent determination of the structure of the N terminus of several members of this receptor family, little is known about the structure of the transmembrane (TM) region and about the molecular mechanisms leading to activation. In the present study, we designed a new structural model of the TM domain and combined it with experimental mutagenesis experiments to investigate the interaction network that governs ligand binding and receptor activation. Our results suggest that this network involves the cluster of residues Arg(188) in TM2, Gln(380) in TM7, and Asn(229) in TM3. This cluster is expected to be altered upon VIP binding, because Arg(188) has been shown previously to interact with Asp(3) of VIP. Several point mutations at positions 188, 229, and 380 were experimentally characterized and were shown to severely affect VIP binding and/or VIP-mediated cAMP production. Double mutants built from reciprocal residue exchanges exhibit strong cooperative or anticooperative effects, thereby indicating the spatial proximity of residues Arg(188), Gln(380), and Asn(229). Because these residues are highly conserved in the GPCR-B family, they can moreover be expected to have a general role in mediating function.

Mechanism of activation of dsRNA-dependent protein kinase (PKR) in muscle atrophy

The role of Ca2+ in the activation of PKR (double-stranded-RNA-dependent protein kinase), which leads to skeletal muscle atrophy, has been investigated in murine myotubes using the cell-permeable Ca2+ chelator BAPTA/AM (1,2-bis (o-aminphenoxy) ethane-N,N,N',N'-tetraacetic acid tetra (acetoxymethyl) ester). BAPTA/AM effectively attenuated both the increase in total protein degradation, through the ubiquitin–proteasome pathway, and the depression of protein synthesis, induced by both proteolysis-inducing factor (PIF) and angiotensin II (Ang  II). Since both protein synthesis and degradation were attenuated this suggests the involvement of PKR. Indeed BAPTA/AM attenuated both the activation  (autophosphorylation) of PKR and the subsequent phosphorylation of eIF2a (eukaryotic initiation factor 2a) in the presence of PIF, suggesting the involvement of Ca2+ in this process. PIF also induced an increase in the activity of both caspases-3 and -8, which was attenuated by BAPTA/AM. The increase in caspase-3 and -8 activity was shown to be responsible for the activation of PKR, since the latter was completely attenuated by the specific caspase-3 and -8 inhibitors. These results suggest that Ca2+ is involved in the increase in protein degradation and decrease in protein synthesis by PIF and Ang II through activation of PKR by caspases-3 and -8.

No gender differences in brain activation during the N-back task:An fMRI study in healthy individuals

Gender differences have been well established in verbal and spatial abilities but few studies have examined if these differences also extend into the domain of working memory in terms of behavioural differences and brain activation. The conclusions that can be drawn from these studies are not clear cut but suggest that even though gender differences might not be apparent from behavioural measures, the underlying neural substrate associated with working memory might be different in men and women. Previous research suggests activation in a network of frontal and parietal regions during working memory tasks. This study aimed to investigate gender differences in patterns of brain activation during a verbal version of the N-back working memory task, which incorporates the effects of increased demands on working memory. A total of 50 healthy subjects, aged 18 to 58 years, that were equally split by gender were recruited matched for age, levels of education and ethnicity. All subjects underwent functional magnetic resonance imaging. We found that men and women performed equally well in terms of accuracy and response times, while using similar brain regions to the same degree. Our observations indicate that verbal working memory is not affected by gender at the behavioural or neural level, and support the findings of a recent meta-analysis by Hyde ([2005]: Sex Roles 53:717-725) that gender differences are generally smaller than intra-gender differences in many cognitive domains. © 2009 Wiley-Liss, Inc.

Investigation of the activation mechanism and structural characterization of the CGRP receptor

The calcitonin gene-related peptide (CGRP) receptor is an unusual G protein-coupled receptor (GPCR) in that it comprises the calcitonin receptor-like receptor (CLR), receptor activity modifying protein 1 (RAMP1) and the receptor component protein (RCP). The RAMP1 has two other homologues – RAMP2 and RAMP3. The endogenous ligand for this receptor is CGRP, a 37 amino acid neuropeptide that act as a vasodilator. This peptide has been implicated in the aetiology of health conditions such as inflammation, Reynaud’s disease and migraine. A clear understanding of the mode of activation of this receptor could be key in developing therapeutic agents for associated health conditions. Although the crystal structure of the N-terminal extracellular domain (ECD) of this receptor (in complex with an antagonist) has been published, the details of receptor-agonist interactions at this domain, and so ultimately the mechanism of receptor activation, are still unclear. Also, the C-terminus of the CLR (in the CGRP receptor), especially around the presumed helix 8 (H8) region, has not been well studied for its role in receptor signalling. This research project investigated these questions. In this study, certain residues making up the putative N-terminal ligand-binding core of the CLR (in the CGRP receptor) were mapped out and found to be crucial for receptor signalling. They included W69 and D70 of the WDG motif in family B GPCRs, as well as Y91, F92, D94 and F95 in loop 2 of CLR N-terminus. Also, F163 at the cytoplasmic end of TM1 and certain residues spanning H8 and associated C-terminal region of CLR were found to be required for CGRP receptor signalling. These residues were investigated by site-directed mutagenesis where they were mutated to alanine (or other residues in specific cases) and the effect of the mutations on receptor pharmacology assessed by evaluating cAMP production, cell surface expression, total cell expression and aCGRP-mediated receptor internalization. Moreover, the N-terminal ECDs of the CLR and RAMPs (RAMP1, RAMP2 and RAMP3) were produced in a yeast host strain (Pichia pastoris) for the purpose of structural interaction study by surface plasmon resonance (SPR). Following expression and purification, these receptor proteins were found to individually retain their secondary structures when analysed by circular dichroism (CD). Results were analysed and interpreted with the knowledge of the secretin family receptor paradigm. The research described in this thesis has produced novel data that contributes to a clearer understanding of CGRP receptor pharmacology. The study on CLR and RAMPs ECDs could be a useful tool in determining novel interacting GPCR partners of RAMPs.

Mechanism of attenuation of angiotensin-II-induced protein degradation by insulin-like growth factor-I (IGF-I)

Insulin-like growth factor-I (IGF-I) has been shown to attenuate protein degradation in murine myotubes induced by angiotensin II through downregulation of the ubiquitin-proteasome pathway, although the mechanism is not known. Angiotensin II is known to upregulate this pathway through a cellular signalling mechanism involving release of arachidonic acid, activation of protein kinase Cα (PKCα), degradation of inhibitor-κB (I-κB) and nuclear migration of nuclear factor-κB (NF-κB), and all of these events were attenuated by IGF-I (13.2 nM). Induction of the ubiquitin-proteasome pathway has been linked to activation of the RNA-activated protein kinase (PKR), since an inhibitor of PKR attenuated proteasome expression and activity in response to angiotensin II and prevented the decrease in the myofibrillar protein myosin. Angiotensin II induced phosphorylation of PKR and of the eukaryotic initiation factor-2 (eIF2) on the α-subunit, and this was attenuated by IGF-I, by induction of the expression of protein phosphatase 1, which dephosphorylates PKR. Release of arachidonic acid and activation of PKCα by angiotensin II were attenuated by an inhibitor of PKR and IGF-I, and the effect was reversed by Salubrinal (15 μM), an inhibitor of eIF2α dephosphorylation, as was activation of PKCα. In addition myotubes transfected with a dominant-negative PKR (PKRΔ6) showed no release of arachidonate in response to Ang II, and no activation of PKCα. These results suggest that phosphorylation of PKR by angiotensin II was responsible for the activation of the PLA2/PKC pathway leading to activation of NF-κB and that IGF-I attenuates protein degradation due to an inhibitory effect on activation of PKR. © 2007 Elsevier Inc. All rights reserved.

Role of reactive oxygen species in protein degradation in murine myotubes induced by proteolysis-inducing factor and angiotensin II

The antioxidants butylated hydroxytoluene (BHT, 1 mM) and d-α-tocopherol (10 μM) completely attenuated protein degradation in murine myotubes in response to both proteolysis-inducing factor (PIF) and angiotensin II (Ang II), suggesting that the formation of reactive oxygen species (ROS) plays an important role in this process. Both PIF and Ang II induced a rapid and transient increase in ROS formation in myotubes, which followed a parabolic dose-response curve, similar to that for total protein degradation. Antioxidant treatment attenuated the increase in expression and activity of the ubiquitin-proteasome proteolytic pathway by PIF and Ang II, by preventing the activation of the transcription factor nuclear factor-κB (NF-κB), through inhibition of phosphorylation of the NF-κB inhibitor protein (I-κB) and its subsequent degradation. ROS formation by both PIF and Ang II was attenuated by diphenyleneiodonium (10 μM), suggesting that it was mediated through the NADPH oxidase system. ROS formation was also attenuated by trifluoroacetyl arachidonic acid (10 μM), a specific inhibitor of cytosolic phospholipase A2, U-73122 (5 μM) and D609 (200 μM), inhibitors of phospholipase C and calphostin C (300 nM), a highly specific inhibitor of protein kinase C (PKC), all known activators of NADPH oxidase. Myotubes containing a dominant-negative mutant of PKC did not show an increase in ROS formation in response to either PIF or Ang II. The two Rac1 inhibitors W56 (200 μM) and NSC23766 (10 μM) also attenuated both ROS formation and protein degradation induced by both PIF and Ang II. Rac1 is known to mediate signalling between the phosphatidylinositol-3 kinase (PI-3K) product and NADPH oxidase, and treatment with LY24002 (10 μM), a highly selective inhibitor of PI-3K, completely attenuated ROS production in response to both PIF and Ang II, and inhibited total protein degradation, while the inactive analogue LY303511 (100 μM) had no effect. ROS formation appears to be important in muscle atrophy in cancer cachexia, since treatment of weight losing mice bearing the MAC16 tumour with d-α-tocopherol (1 mg kg- 1) attenuated protein degradation and increased protein synthesis in skeletal muscle. © 2007 Elsevier Inc. All rights reserved.