STAT3 Activation in Skeletal Muscle Links Muscle Wasting and the Acute Phase Response in Cancer Cachexia

Cachexia, or weight loss despite adequate nutrition, significantly impairs quality of life and response to therapy in cancer patients. In cancer patients, skeletal muscle wasting, weight loss and mortality are all positively associated with increased serum cytokines, particularly Interleukin-6 (IL-6), and the presence of the acute phase response. Acute phase proteins, including fibrinogen and serum amyloid A (SAA) are synthesized by hepatocytes in response to IL-6 as part of the innate immune response. To gain insight into the relationships among these observations, we studied mice with moderate and severe Colon-26 (C26)-carcinoma cachexia.Moderate and severe C26 cachexia was associated with high serum IL-6 and IL-6 family cytokines and highly similar patterns of skeletal muscle gene expression. The top canonical pathways up-regulated in both were the complement/coagulation cascade, proteasome, MAPK signaling, and the IL-6 and STAT3 pathways. Cachexia was associated with increased muscle pY705-STAT3 and increased STAT3 localization in myonuclei. STAT3 target genes, including SOCS3 mRNA and acute phase response proteins, were highly induced in cachectic muscle. IL-6 treatment and STAT3 activation both also induced fibrinogen in cultured C2C12 myotubes. Quantitation of muscle versus liver fibrinogen and SAA protein levels indicates that muscle contributes a large fraction of serum acute phase proteins in cancer.These results suggest that the STAT3 transcriptome is a major mechanism for wasting in cancer. Through IL-6/STAT3 activation, skeletal muscle is induced to synthesize acute phase proteins, thus establishing a molecular link between the observations of high IL-6, increased acute phase response proteins and muscle wasting in cancer. These results suggest a mechanism by which STAT3 might causally influence muscle wasting by altering the profile of genes expressed and translated in muscle such that amino acids liberated by increased proteolysis in cachexia are synthesized into acute phase proteins and exported into the blood

GDF-15 is abundantly expressed in plexiform lesions in patients with pulmonary arterial hypertension and affects proliferation and apoptosis of pulmonary endothelial cells

<p>Abstract</p> <p>Background</p> <p>Growth-differentiation factor-15 (GDF-15) is a stress-responsive, transforming growth factor-β-related cytokine, which has recently been reported to be elevated in serum of patients with idiopathic pulmonary arterial hypertension (IPAH). The aim of the study was to examine the expression and biological roles of GDF-15 in the lung of patients with pulmonary arterial hypertension (PAH).</p> <p>Methods</p> <p>GDF-15 expression in normal lungs and lung specimens of PAH patients were studied by real-time RT-PCR and immunohistochemistry. Using laser-assisted micro-dissection, GDF-15 expression was further analyzed within vascular compartments of PAH lungs. To elucidate the role of GDF-15 on endothelial cells, human pulmonary microvascular endothelial cells (HPMEC) were exposed to hypoxia and laminar shear stress. The effects of GDF-15 on the proliferation and cell death of HPMEC were studied using recombinant GDF-15 protein.</p> <p>Results</p> <p>GDF-15 expression was found to be increased in lung specimens from PAH patients, com-pared to normal lungs. GDF-15 was abundantly expressed in pulmonary vascular endothelial cells with a strong signal in the core of plexiform lesions. HPMEC responded with marked upregulation of GDF-15 to hypoxia and laminar shear stress. Apoptotic cell death of HPMEC was diminished, whereas HPMEC proliferation was either increased or decreased depending of the concentration of recombinant GDF-15 protein.</p> <p>Conclusions</p> <p>GDF-15 expression is increased in PAH lungs and appears predominantly located in vascular endothelial cells. The expression pattern as well as the observed effects on proliferation and apoptosis of pulmonary endothelial cells suggest a role of GDF-15 in the homeostasis of endothelial cells in PAH patients.</p

Quadrupling Muscle Mass in Mice by Targeting TGF-ß Signaling Pathways

Myostatin is a transforming growth factor-ß family member that normally acts to limit skeletal muscle growth. Mice genetically engineered to lack myostatin activity have about twice the amount of muscle mass throughout the body, and similar effects are seen in cattle, sheep, dogs, and a human with naturally occurring loss-of-function mutations in the myostatin gene. Hence, there is considerable interest in developing agents capable of inhibiting myostatin activity for both agricultural and human therapeutic applications. We previously showed that the myostatin binding protein, follistatin, can induce dramatic increases in muscle mass when overexpressed as a transgene in mice. In order to determine whether this effect of follistatin results solely from inhibition of myostatin activity, I analyzed the effect of this transgene in myostatin-null mice. Mstn−/− mice carrying a follistatin transgene had about four times the muscle mass of wild type mice, demonstrating the existence of other regulators of muscle mass with similar activity to myostatin. The greatest effect on muscle mass was observed in offspring of mothers homozygous for the Mstn mutation, raising the possibility that either myostatin itself or a downstream regulator may normally be transferred from the maternal to fetal circulations. These findings demonstrate that the capacity for increasing muscle growth by manipulating TGF-ß signaling pathways is much more extensive than previously appreciated and suggest that muscle mass may be controlled at least in part by a systemic mode of action of myostatin

The C313Y Piedmontese mutation decreases myostatin covalent dimerisation and stability

<p>Abstract</p> <p>Background</p> <p>Myostatin is a key negative regulator of muscle growth and development, whose activity has important implications for the treatment of muscle wastage disorders. Piedmontese cattle display a double-muscled phenotype associated with the expression of C313Y mutant myostatin. <it>In vivo</it>, C313Y myostatin is proteolytically processed, exported and circulated extracellularly but fails to correctly regulate muscle growth. The C313Y mutation removes the C313-containing disulphide bond, an integral part of the characteristic TGF-β cystine-knot structural motif.</p> <p>Results</p> <p>Here we present <it>in vitro </it>analysis of the structure and stability of the C313Y myostatin protein that reveals significantly decreased covalent dimerisation for C313Y myostatin accompanied by a loss of structural stability compared to wild type. The C313Y myostatin growth factor, processed from full length precursor protein, fails to inhibit C2C12 myoblast proliferation in contrast to wild type myostatin. Although structural modeling shows the substitution of tyrosine causes structural perturbation, biochemical analysis of additional disulphide mutants, C313A and C374A, indicates that an intact cystine-knot motif is a major determinant in myostatin growth factor stability and covalent dimerisation.</p> <p>Conclusions</p> <p>This research shows that the cystine-knot structure is important for myostatin dimerisation and stability, and that disruption of this structural motif perturbs myostatin signaling.</p

Myostatin Inhibition in Muscle, but Not Adipose Tissue, Decreases Fat Mass and Improves Insulin Sensitivity

Myostatin (Mstn) is a secreted growth factor expressed in skeletal muscle and adipose tissue that negatively regulates skeletal muscle mass. Mstn−/− mice have a dramatic increase in muscle mass, reduction in fat mass, and resistance to diet-induced and genetic obesity. To determine how Mstn deletion causes reduced adiposity and resistance to obesity, we analyzed substrate utilization and insulin sensitivity in Mstn−/− mice fed a standard chow. Despite reduced lipid oxidation in skeletal muscle, Mstn−/− mice had no change in the rate of whole body lipid oxidation. In contrast, Mstn−/− mice had increased glucose utilization and insulin sensitivity as measured by indirect calorimetry, glucose and insulin tolerance tests, and hyperinsulinemic-euglycemic clamp. To determine whether these metabolic effects were due primarily to the loss of myostatin signaling in muscle or adipose tissue, we compared two transgenic mouse lines carrying a dominant negative activin IIB receptor expressed specifically in adipocytes or skeletal muscle. We found that inhibition of myostatin signaling in adipose tissue had no effect on body composition, weight gain, or glucose and insulin tolerance in mice fed a standard diet or a high-fat diet. In contrast, inhibition of myostatin signaling in skeletal muscle, like Mstn deletion, resulted in increased lean mass, decreased fat mass, improved glucose metabolism on standard and high-fat diets, and resistance to diet-induced obesity. Our results demonstrate that Mstn−/− mice have an increase in insulin sensitivity and glucose uptake, and that the reduction in adipose tissue mass in Mstn−/− mice is an indirect result of metabolic changes in skeletal muscle. These data suggest that increasing muscle mass by administration of myostatin antagonists may be a promising therapeutic target for treating patients with obesity or diabetes

Caffeic Acid Phenethyl Ester Causes p21Cip1 Induction, Akt Signaling Reduction, and Growth Inhibition in PC-3 Human Prostate Cancer Cells

Caffeic acid phenethyl ester (CAPE) treatment suppressed proliferation, colony formation, and cell cycle progression in PC-3 human prostate cancer cells. CAPE decreased protein expression of cyclin D1, cyclin E, SKP2, c-Myc, Akt1, Akt2, Akt3, total Akt, mTOR, Bcl-2, Rb, as well as phosphorylation of Rb, ERK1/2, Akt, mTOR, GSK3α, GSK3β, PDK1; but increased protein expression of KLF6 and p21Cip1. Microarray analysis indicated that pathways involved in cellular movement, cell death, proliferation, and cell cycle were affected by CAPE. Co-treatment of CAPE with chemotherapeutic drugs vinblastine, paclitaxol, and estramustine indicated synergistic suppression effect. CAPE administration may serve as a potential adjuvant therapy for prostate cancer

Muscle wasting in chronic kidney disease: the role of the ubiquitin proteasome system and its clinical impact

Muscle wasting in chronic kidney disease (CKD) and other catabolic diseases (e.g. sepsis, diabetes, cancer) can occur despite adequate nutritional intake. It is now known that complications of these various disorders, including acidosis, insulin resistance, inflammation, and increased glucocorticoid and angiotensin II production, all activate the ubiquitin–proteasome system (UPS) to degrade muscle proteins. The initial step in this process is activation of caspase-3 to cleave the myofibril into its components (actin, myosin, troponin, and tropomyosin). Caspase-3 is required because the UPS minimally degrades the myofibril but rapidly degrades its component proteins. Caspase-3 activity is easily detected because it leaves a characteristic 14kD actin fragment in muscle samples. Preliminary evidence from several experimental models of catabolic diseases, as well as from studies in patients, indicates that this fragment could be a useful biomarker because it correlates well with the degree of muscle degradation in dialysis patients and in other catabolic conditions

Activin signaling as an emerging target for therapeutic interventions

After the initial discovery of activins as important regulators of reproduction, novel and diverse roles have been unraveled for them. Activins are expressed in various tissues and have a broad range of activities including the regulation of gonadal function, hormonal homeostasis, growth and differentiation of musculoskeletal tissues, regulation of growth and metastasis of cancer cells, proliferation and differentiation of embryonic stem cells, and even higher brain functions. Activins signal through a combination of type I and II transmembrane serine/threonine kinase receptors. Activin receptors are shared by multiple transforming growth factor-β (TGF-β) ligands such as myostatin, growth and differentiation factor-11 and nodal. Thus, although the activity of each ligand is distinct, they are also redundant, both physiologically and pathologically in vivo. Activin receptors activated by ligands phosphorylate the receptor-regulated Smads for TGF-β, Smad2 and 3. The Smad proteins then undergo multimerization with the co-mediator Smad4, and translocate into the nucleus to regulate the transcription of target genes in cooperation with nuclear cofactors. Signaling through receptors and Smads is controlled by multiple mechanisms including phosphorylation and other posttranslational modifications such as sumoylation, which affect potein localization, stability and transcriptional activity. Non-Smad signaling also plays an important role in activin signaling. Extracellularly, follistatin and related proteins bind to activins and related TGF-β ligands, and control the signaling and availability of ligands

Anchoring skeletal muscle development and disease: the role of ankyrin repeat domain containing proteins in muscle physiology

The ankyrin repeat is a protein module with high affinity for other ankyrin repeats based on strong Van der Waals forces. The resulting dimerization is unusually resistant to both mechanical forces and alkanization, making this module exceedingly useful for meeting the extraordinary demands of muscle physiology. Many aspects of muscle function are controlled by the superfamily ankyrin repeat domain containing proteins, including structural fixation of the contractile apparatus to the muscle membrane by ankyrins, the archetypical member of the family. Additionally, other ankyrin repeat domain containing proteins critically control the various differentiation steps during muscle development, with Notch and developmental stage-specific expression of the members of the Ankyrin repeat and SOCS box (ASB) containing family of proteins controlling compartment size and guiding the various steps of muscle specification. Also, adaptive responses in fully formed muscle require ankyrin repeat containing proteins, with Myotrophin/V-1 ankyrin repeat containing proteins controlling the induction of hypertrophic responses following excessive mechanical load, and muscle ankyrin repeat proteins (MARPs) acting as protective mechanisms of last resort following extreme demands on muscle tissue. Knowledge on mechanisms governing the ordered expression of the various members of superfamily of ankyrin repeat domain containing proteins may prove exceedingly useful for developing novel rational therapy for cardiac disease and muscle dystrophies