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.

Mitochondrial superoxide anion radicals mediate induction of apoptosis in cardiac myoblasts exposed to chronic hypoxia

Both reactive oxygen species (ROS) and ATP depletion may be significant in hypoxia-induced damage and death, either collectively or independently, with high energy requiring, metabolically active cells being the most susceptible to damage. We investigated the kinetics and effects of ROS production in cardiac myoblasts, H9C2 cells, under 2%, 10% and 21% O2 in the presence or absence of apocynin, rotenone and carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone. H9C2 cells showed significant loss of viability within 30 min of culture at 2% oxygen which was not due to apoptosis, but was associated with an increase in protein oxidation. However, after 4 h, apoptosis induction was observed at 2% oxygen and also to a lesser extent at 10% oxygen; this was dependent on the levels of mitochondrial superoxide anion radicals determined using dihydroethidine. Hypoxia-induced ROS production and cell death could be rescued by the mitochondrial complex I inhibitor, rotenone, despite further depletion of ATP. In conclusion, a change to superoxide anion radical steady state level was not detectable after 30 min but was evident after 4 h of mild or severe hypoxia. Superoxide anion radicals from the mitochondrion and not ATP depletion is the major cause of apoptotic cell death in cardiac myoblasts under chronic, severe hypoxia.

Gravity spun polycaprolactone fibres for soft tissue engineering:interaction with fibroblasts and myoblasts in cell culture

Poly(ε-caprolactone) (PCL) fibres were produced by wet spinning from solutions in acetone under low shear (gravity flow) conditions. As-spun PCL fibres exhibited a mean strength and stiffness of 7.9 MPa and 0.1 GPa, respectively and a rough, porous surface morphology. Cold drawing to an extension of 500% resulted in increases in fibre strength (43 MPa) and stiffness (0.3 GPa) and development of an oriented, fibrillar surface texture. The proliferation rate of Swiss 3T3 mouse fibroblasts and C2C12 mouse myoblasts on as-spun, 500% cold-drawn and gelatin-modified PCL fibres was determined in cell culture to provide a basic measure of the biocompatibility of the fibres. Proliferation of both cell types was consistently higher on gelatin-coated fibres relative to as-spun fibres at time points below 7 days. Fibroblast growth rates on cold-drawn PCL fibres exceeded those on as-spun fibres but myoblast proliferation was similar on both substrates. After 1 day in culture, both cell types had spread and coalesced on the fibres to form a cell layer, which conformed closely to the underlying topography. The high fibre compliance combined with a potential for modifying the fibre surface chemistry with cell adhesion molecules and the surface architecture by cold drawing to enhance proliferation of fibroblasts and myoblasts, recommends further investigation of gravity-spun PCL fibres for 3-D scaffold production in soft tissue engineering. © 2005 Elsevier Ltd. All rights reserved.

Identification and characterization of a membrane receptor for proteolysis-inducing factor on skeletal muscle

Proteolysis-inducing factor (PIF) is a sulfated glycoprotein produced by cachexia-inducing tumors, which induces atrophy of skeletal muscle. PIF has been shown to bind specifically with high affinity (Kd, in nanomolar) to sarcolemma membranes from skeletal muscle of both the mouse and the pig, as well as murine myoblasts and a human muscle cell line. Ligand binding was abolished after enzymatic deglycosylation, suggesting that binding was mediated through the oligosaccharide chains in PIF. Chondroitin sulfate, but not heparan or dermatan sulfate, showed competitive inhibition (Kd, 1.1 × 10-7 mol/L) of binding of PIF to the receptor, suggesting an interaction with the sulfated oligosaccharide chains. Ligand blotting of [ 35S]PIF to triton solublized membranes from C2C 12 cells provided evidence for a binding protein of apparent M r of ∼40,000. Amino acid sequence analysis showed the PIF receptor to be a DING protein. Antisera reactive to a 19mer from the N-terminal amino acid residues of the binding protein attenuated protein degradation and activation of the ubiquitin-proteasome pathway induced by PIF in murine myotubes. In addition, the antisera was highly effective in attenuating the decrease in body weight in mice bearing the MAC16 tumor, with a significant increase in muscle wet weight due to an increase in the rate of protein synthesis, together with a reduction in protein degradation through attenuation of the increased proteasome expression and activity. These results confirm that the PIF binding protein has a functional role in muscle protein atrophy in cachexia and that it represents a potential new therapeutic target. ©2007 American Association for Cancer Research.

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.

Development of an in-vitro model system to investigate the mechanism of muscle protein catabolism induced by proteolysis-inducing factor

The mechanism of muscle protein catabolism induced by proteolysis-inducing factor, produced by cachexia-inducing murine and human tumours has been studied in vitro using C2C12 myoblasts and myotubes. In both myoblasts and myotubes protein degradation was enhanced by proteolysis-inducing factor after 24 h incubation. In myoblasts this followed a bell-shaped dose-response curve with maximal effects at a proteolysis-inducing factor concentration between 2 and 4 nM, while in myotubes increased protein degradation was seen at all concentrations of proteolysis-inducing factor up to 10 nM, again with a maximum of 4 nM proteolysis-inducing factor. Protein degradation induced by proteolysis-inducing factor was completely attenuated in the presence of cycloheximide (1 μM), suggesting a requirement for new protein synthesis. In both myoblasts and myotubes protein degradation was accompanied by an increased expression of the α-type subunits of the 20S proteasome as well as functional activity of the proteasome, as determined by the 'chymotrypsin-like' enzyme activity. There was also an increased expression of the 19S regulatory complex as well as the ubiquitin-conjugating enzyme (E214k), and in myotubes a decrease in myosin expression was seen with increasing concentrations of proteolysis-inducing factor. These results show that proteolysis-inducing factor co-ordinately upregulates both ubiquitin conjugation and proteasome activity in both myoblasts and myotubes and may play an important role in the muscle wasting seen in cancer cachexia. © 2002 Cancer Research UK.

Effect of a tumour-produced lipid-mobilizing factor on protein synthesis and degradation

Treatment of murine myoblasts, myotubes and tumour cells with a tumour-produced lipid mobilizing factor (LMF), caused a concentration-dependent stimulation of protein synthesis, within a 24 h period. There was no effect on cell number or [3H] thymidine incorporation, but a similar concentration-dependent stimulation of 2-deoxyglucose uptake. LMF produced an increase in intracellular cyclic AMP levels, which was linearly (r2 = 0.973) related to the increase in protein synthesis. The effect of LMF was attenuated by the adenylate cyclase inhibitor MDL12330A, and was additive with the stimulation produced by forskolin. Both propranolol (10 μM) and the specific β3-adrenergic receptor antagonist SR 59230A (10-5M), significantly reduced the stimulation of protein synthesis induced by LMF. Protein synthesis was also increased by 69% (P = 0.006) in soleus muscles of mice administered LMF, while there was a 26% decrease in protein degradation (P = 0.03). While LMF had no effect on the lysosomal enzymes, cathepsins B and L, there was a decrease in proteasome activity, as determined both by the 'chymotrypsin-like' enzyme activity, as well as expression of proteasome α-type subunits, determined by Western blotting. These results show that in addition to its lipid-mobilizing activity LMF also increases protein accumulation in skeletal muscle both by an increase in protein synthesis and a decrease in protein catabolism. © 2001 Cancer Research Campaign.

Novel biodegradable fibres for applications in tissue engineering and drug delivery

The preparation and characterisation of novel biodegradable polymer fibres for application in tissue engineering and drug delivery are reported. Poly(e-caprolactone) (PCL) fibres were produced by wet spinning from solutions in acetone under low shear (gravity flow) conditions. The tensile strength and stiffness of as-spun fibres were highly dependent on the concentration of the spinning solution. Use of a 6% w/v solution resulted in fibres having strength and stiffness of 1.8 MPa and 0.01 GPa respectively, whereas these values increased to 9.9 MPa and 0.1 GPa when fibres were produced from 20% w/v solutions. Cold drawing to an extension of 500% resulted in further increases in fibre strength (up to 50 MPa) and stiffness (0.3 GPa). Hot drawing to 500% further increased the fibre strength (up to 81 MPa) and stiffness (0.5 GPa). The surface morphology of as-spun fibres was modified, to yield a directional grooved pattern by drying in contact with a mandrel having a machined topography characterised by a peak-peak separation of 91 mm and a peak height of 30 mm. Differential scanning calorimetery (DSC) analysis of as-spun fibres revealed the characteristic melting point of PCL at around 58°C and a % crystallinity of approximately 60%. The biocompatibility of as-spun fibres was assessed using cell culture. The number of attached 3T3 Swiss mouse fibroblasts, C2C12 mouse myoblasts and human umbilical vein endothelial cells (HUVECs) on as-spun, 500% cold drawn, and gelatin coated PCL fibres were observed. The results showed that the fibres promoted cell proliferation for 9 days in cell culture and was slightly lower than on tissue culture plastic. The morphology of all cell lines was assessed on the various PCL fibres using scanning electron microscopy. The cell function of HUVECs growing on the as-spun PCL fibres was evaluated. The ability HUVECs to induce an immune response when stimulated with lipopolysaccaride (LPS) and thereby to increase the amount of cell surface receptors was assessed by flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR). The results showed that PCL fibres did not inhibit this function compared to TCP. As-spun PCL fibres were loaded with 1 % ovine albumin (OVA) powder, 1% OVA nanoparticles and 5% OVA nanoparticles by weight and the protein release was assessed in vitro. PCL fibres loaded with 1 % OVA powder released 70%, 1% OVA nanoparticle released 60% and the 5% OVA nanoparticle released 25% of their protein content over 28 days. These release figures did not alter when the fibres were subjected to lipase enzymatic degradation. The OVA released was examined for structural integrity by SDS-PAGE. This showed that the protein molecular weight was not altered after incorporation into the fibres. The bioactivity of progesterone was assessed following incorporation into PCL fibres. Results showed that the progesterone released had a pronounced effect on MCF-7 breast epithelial cells, inhibiting their proliferation. The PCL fibres display high fibre compliance, a potential for controlling the fibre surface architecture to promote contact guidance effects, favorable proliferation rate of fibroblasts, myoblasts and HUVECs and the ability to release pharmaceuticals. These properties recommended their use for 3-D scaffold production in soft tissue engineering and the fibres could also be exploited for controlled presentation and release of biopharmaceuticals such as growth factors.

Cellular signalling pathways involved in muscle atrophy in cancer cachexia

Cancer cachexia encompases severe weight loss, characterised by the debilitating atrophy of adipose and skeletal muscle mass. Skeletal muscle proteolysis in cancer cachexia is mediated by a sulphated glycoprotein with a relative molecular mass of 24kDa, termed Proteolysis-Inducing Factor (PIF). PIF induced a significant increase in protein degradation, peaking at 4.2nM PIF (p<0.001), ‘chymotrypsin-like’ activity of the proteasome (p<0.001) and increased expression of components of the ATP-ubiquitin dependent proteolytic pathway. This was attenuated in vitro by pre-incubation with the PKC inhibitor calphostin C (100µM) and NF-kB the inhibitors SN50 (18µM), curcumin (50µM) and resveratrol (30µM), 2 hours prior to the addition of PIF. In vivo studies found the IKK inhibitor resveratrol (1mg/kg) to be successful in attenuating protein degradation (p<0.001) and upregulation of ubiquitin-dependent proteolysis in MAC16 tumour bearing mice. C2C12 myoblasts transfected with mutant IkBα and PKCα inserts did not elicit a PIF-induced response, suggesting the importance of the transcription factor NF-kB and PKC  involvement in PIF signal transduction. 15(S)-HETE acts as an intracellular mediator of PIF and exerts an effect through the activation of PKC and subsequently IKK, which phosphorylates IkBα and allows NF-kB to migrate to the nucleus. This effect was negated with the PKC inhibitor calphostin C (300nM). A commercially produced PIF receptor antibody was raised in rabbits immunised with a peptide containing the partial N-terminal sequence of the PIF receptor. The PIF receptor antibody was successful in attenuating the PIF-induced increase in skeletal muscle catabolism and protein degradation in vitro at 10µg/ml (p<0.001) and 3.47mg/kg in vivo (p<0.001). The data suggest great potential in the development of this antibody as a therapy against cancer cachexia.

Second messenger pathways in the action of cachectic factors

Cachexia in cancer is characterised by progressive depletion of both adipose tissue stores and skeletal muscle mass. Two catabolic factors produced by cachexia-inducing tumours have the potential for inducing these changes in body composition: (i) proteolysis-inducing factor (PIF) which acts on skeletal muscle to induce both protein degradation and inhibit protein synthesis, (ii) lipid-mobilising factor (LMF), which has been shown to directly induce lipolysis in isolated epididymal murine white adipocytes. Administration of lipid-mobilising factor (LMF) to mice produced a specific reduction in carcass lipid with a tendency to increase non-fat carcass mass. Treatment of murine myoblasts, myotubes and tumour cells with tumour-produced LMF, caused concentration dependent stimulation of protein synthesis, within a 24hr period. It produced an increase in intracellular cyclic AMP levels, which was linearly related to the increase in protein synthesis. The observed effect was attenuated by pretreating cells with the adenylate cyclase inhibitor, MDL12330A and was additive with stimulation produced by forskolin. Both propranolol and a specific 3 adrenergic antagonist SR59230A, significantly reduced the stimulation of protein synthesis induced by LMF. LMF also affected protein degradation in vitro, as demonstrated by a reduction in proteasome activity, a key component of the ubiquitin-dependent proteolytic pathway. These effects were opposite to those produced by PIF which caused both a decrease in the rate of protein synthesis and an elevation on protein breakdown when incubated in vitro.Incubation of LMF with a fat cell line produced alterations in the levels of guanine-nucleotide binding proteins (G proteins). This was also evident in adipocyte plasma membranes isolated from mice bearing the tumour model of cachexia, MAC16 adenocarcinoma and from patients with cancer cachexia. Progression through the cachectic state induced an upregulation of stimulatory G proteins paralleled with a downregulation of inhibitory G proteins. These changes would contribute to the increased lipid mobilisation seen in cancer cachexia.

Role of Ca2+ in proteolysis-inducing factor (PIF)-induced atrophy of skeletal muscle

Proteolysis-inducing factor (PIF) induces muscle loss in cancer cachexia through a high affinity membrane bound receptor. This study investigates the mechanism by which the PIF receptor communicates to intracellular signalling pathways. C2C12 murine myoblasts were used as a model using PIF purified from MAC16 tumours. Calcium imaging was determined using fura-4-acetoxymethyl ester (Fura-4-AM). PIF induced a rapid rise in Ca2 +i, which was completely attenuated by a anti-receptor antibody, or peptides representing 20 mers of the N-terminus of the PIF receptor. Other agents catabolic for skeletal muscle including angiotensin II (AngII) tumour necrosis factor-a (TNF-a) and lipopolysaccharide (LPS) also induced a rise in Ca2 +i, but this was not attenuated by anti-PIF-receptor antibody. The rise in Ca2 +i induced by PIF and AngII was completely attenuated by the Zn2 + chelator D-myo-inositol-1,2,6-triphosphate, and this was reversed by administration of exogenous Zn2 +. The Ca2 +i rise induced by PIF was independent of the presence of extracellular Ca2 +, and attenuated by the Ca2 + pump inhibitor thapsigargin, suggesting that the Ca2 +i rise was due to release from intracellular stores. This rise in Ca2 +i induced by PIF was attenuated by both the phospholipase C inhibitor U73122 and 2-APB, an inhibitor of the inositol 1,4,5-triphosphate receptor, suggesting the involvement of a G-protein. Binding of the PIF to its receptor in skeletal muscle triggers a rise in Ca2 +i, which initiates a signalling cascade leading to a depression in protein synthesis, and an increase in protein degradation.