Copper Does Not Induce Tenogenic Differentiation but Promotes Migration and Increases Lysyl Oxidase Activity in Adipose-Derived Mesenchymal Stromal Cells.

BACKGROUND: Copper belongs to the essential trace metals that play a key role in the course of cellular processes maintaining the whole body's homeostasis. As there is a growing interest in transplanting mesenchymal stromal cells (MSCs) into the site of injury to improve the regeneration of damaged tendons, the purpose of the study was to verify whether copper supplementation may have a positive effect on the properties of human adipose tissue-derived MSCs (hASCs) which potentially can contribute to improvement of tendon healing. RESULTS: Cellular respiration of hASCs decreased with increasing cupric sulfate concentrations after 5 days of incubation. The treatment with CuSO4 did not positively affect the expression of genes associated with tenogenesis (COL1α1, COL3α1, MKX, and SCX). However, the level of COL1α1 protein, whose transcript was decreased in comparison to a control, was elevated after a 5-day exposition to 25 µM CuSO4. The content of the MKX and SCX protein in hASCs exposed to cupric sulfate was reduced compared to that of untreated control cells, and the level of the COL3α1 protein, whose transcript was decreased in comparison to a control, was elevated after a 5-day exposition to 25 µM CuSO4. The content of the MKX and SCX protein in hASCs exposed to cupric sulfate was reduced compared to that of untreated control cells, and the level of the COL3. CONCLUSION: Copper sulfate supplementation can have a beneficial effect on tendon regeneration not by inducing tenogenic differentiation, but by improving the recruitment of MSCs to the site of injury, where they can secrete growth factors, cytokines and chemokines, and prevent the effects of oxidative stress at the site of inflammation, as well as improve the stabilization of collagen fibers, thereby accelerating the process of tendon healing.

Interleukin-6 affects pacsin3, ephrinA4 expression and cytoskeletal proteins in differentiating primary skeletal myoblasts through transcriptional and post-transcriptional mechanisms.

Interleukin (IL)-6 is a proinflammatory cytokine released in injured and contracting skeletal muscles. In this study, we examined cellular expression of proteins associated with cytoskeleton organization and cell migration, chosen on the basis of microRNA profiling, in rat primary skeletal muscle cells (RSkMC) treated with IL-6 (1 ng/ml) for 11 days. MiRNA microarray analysis and qRT-PCR revealed increased expression of miR-154-3p and miR-338-3p in muscle cells treated with IL-6. Pacsin3 was downregulated post-transcriptionally by IL-6, but not by IGF-I. Ephrin4A protein was increased both in IL-6- and IGF-I-treated myocytes. IL-6, but not IGF-I, stimulated migratory ability of RSkMC, examined in wound healing assay. Alpha-actinin protein was slightly augmented in RSKMC treated with IL-6, similarly to IGF-I. IL-6, but not IGF-I, upregulated desmin in differentiating RSkMC. IL-6 supplementation caused accumulation of alpha-actinin and desmin in near-nuclear area of muscle cells, which was manifested by increased ratio: mean near-nuclear fluorescence/mean peripheral cytoplasm fluorescence of these proteins. We concluded that IL-6, a known proinflammatory cytokine and a physical activity-associated myokine, acting during differentiation of primary skeletal muscle cells, alters expression of nonmuscle-specific miRNAs. This cytokine causes differential effects on pacsin-3 and ephrinA4, through post-transcriptional inhibition and stimulation, respectively. IL-6-exerted modifications of cytoskeletal proteins in muscle cells include both transcriptional (desmin and dynein heavy chain 5) and post-transcriptional activation (alpha-actinin). Moreover, IL-6 augments near-nuclear distribution of cytoskeletal proteins, alpha-actinin and desmin and promotes migration of myocytes. Such effects suggest that IL-6 plays a role during skeletal muscle regeneration, acting through mechanisms independent of regulation of myogenic program.

Interleukin-8 enhances myocilin expression, Akt-FoxO3 signaling and myogenic differentiation in rat skeletal muscle cells.

Interleukin (IL)-8 is released both in visceral adipose tissue and in contracting skeletal muscles. In this study, we examined cellular pathways associated with muscle hypertrophy, chosen on the basis of microRNA profiling, in differentiating rat primary skeletal muscle cells (RSkMC) treated with IL-8 (1 ng/ml) for 11 days. IL-8 increased myocilin expression, Akt phosphorylation, FoxO3 dispersion throughout the cytoplasm, and reduced FoxO3 level. IL-8 decreased the expression of atrogin and MuRF1 and increased myotube length and diameter. We concluded that IL-8 present in extracellular environment of myoblasts induced to differentiation stimulates expression of myocilin, a protein important for skeletal muscle hypertrophy. This phenomenon was associated with: (a) activation of myogenic transcription, (b) increased phosphorylation and activation of PKB/Akt, leading to (c) cytoplasm distribution and degradation of a transcription factor FoxO3, (d) decreased expression of gene markers of proteolysis, atrogin and Murf1, and (e) increased myotube length and diameter. In this regard, IL-8 affects skeletal muscle cells similarly to IGF-I and can be considered as a potent anticatabolic factor for skeletal muscle.

Mechanisms of oat (Avena sativa L.) acclimation to phosphate deficiency.

BACKGROUND: Deficiency of available forms of phosphorus is common in most soils and causes reduction of crop plants growth and yield. Recently, model plants responses to phosphate (Pi) deficiency have been intensively studied. However, acclimation mechanisms of cereals like oat (Avena sativa L.), to low Pi stress remains not fully understood. Oat plants have been usually cultured on poor soils, with a low nutrient content, but their responses to such conditions are not well known, therefore the main goal of the study was to investigate the mechanisms that enable oat plants to grow under low Pi conditions. METHODS: Four oat cultivars (A. sativa, cv. Arab, Krezus, Rajtar and Szakal) were grown for three weeks in a nutrient media with various P sources: inorganic-KH2PO4 (control), organic-phytate (PA) and with no phosphate (-P). The effects of Pi deficiency on the level of P, oat growth parameters, intensity of photosynthesis, plant productivity, root exudation ability, localization, activity and isoforms of acid phosphatases, enzymes involved in Pi mobilization, were estimated. In addition, the effect of mycorrhization on plant growth was also observed. RESULTS: All studied oat cultivars grown on Pi-deficient media had significantly decreased Pi content in the tissues. Pi deficiency caused inhibition of shoot growth, but generally it did not affect root elongation; root diameter was decreased, root/shoot ratios increased, whereas PA plants showed a similar growth to control. Photosynthesis rate and productivity parameters decreased under low Pi nutrition, however, sugar content generally increased. Studied oat cultivars did not respond to low Pi via increased exudation of carboxylates from the roots, as pH changes in the growth media were not observed. Pi starvation significantly increased the activity of extracellular and intracellular acid phosphatases (APases) in comparison to the control plants. Three major APase isoforms were detected in oat tissues and the isoform pattern was similar in all studied conditions, usually with a higher level of one of the isoforms under Pi starvation. Generally no significant effects of mycorrhizal colonization on growth of oat cultivars were observed. DISCUSSION: We postulated that acid phosphatases played the most important role in oat cultivars acclimation to Pi deficiency, especially extracellular enzymes involved in Pi acquisition from soil organic P esters. These APases are mainly located in the epidermis of young roots, and may be released to the rhizosphere. On the other hand, intracellular APases could be involved in fast Pi remobilization from internal sources. Our study showed that oat, in contrast to other plants, can use phytates as the sole source of P. The studied oat cultivars demonstrated similar acclimation mechanisms to Pi deficiency, however, depending on stress level, they can use different pools of acid phosphatases.

Insulin-dependent cytoplasmic distribution of Rab4a in mouse adipocytes is inhibited by interleukin-6, -8, and -15.

The purpose of the study was to examine the effect of interleukins, IL-6, IL-8, and IL-15, on insulin-mediated redistribution of Rab4a, an early endosome marker, in mouse 3T3-L1 adipocytes. The interleukins did not affect cell viability; however, cell number was slightly but significantly higher in cultures exposed to IL-8 and IL-15. IL-8 and IL-15 decreased lipid storage in adipocytes, whereas IL-6 had no effect. Rab4A showed cytoplasmic localization, and in control unstimulated adipocytes it was found primarily nearby nucleus, that was supported by cellular fluorescence distribution profile, and by calculated indices, that is, high percentage of near-nuclear area fluorescence and a low mean peripheral cytoplasmic fluorescence/mean near-nuclear fluorescence ratio. Insulin stimulation (100 nmol/l, 30 min) altered the cytoplasmic localization of Rab4a in control adipocytes, which was manifested by its redistribution towards plasma membrane. This effect of insulin was prevented in adipocytes exposed to IL-6, IL-8, or IL-15. We concluded that insulin-dependent Rab4a redistribution, probably reflecting stimulation of vesicle-mediated transport, is inhibited in adipocytes subjected to differentiation in the presence of IL-6, IL-8, or IL-15. Such alterations may be involved in the mechanisms contributing to development of insulin resistance associated with inflammation; however, further studies in this field are required.

Palmitate exerts opposite effects on proliferation and differentiation of skeletal myoblasts.

The purpose of the study was to examine mechanisms controlling cell cycle progression/arrest and differentiation of mouse C2C12 myoblasts exposed to long-chain saturated fatty acid salt, palmitate. Treatment of proliferating myoblasts with palmitate (0.1 mmol/l) markedly decreased myoblast number. Cyclin A and cyclin D1 levels decreased, whereas total p21 and p21 complexed with cyclin-dependent kinase-4 (cdk4) increased in myoblasts treated with palmitate. In cells induced to differentiation addition of palmitate augmented the level of cyclin D3, the early (myogenin) and late (α-actinin, myosin heavy chain) markers of myogenesis, and caused an increase of myotube diameter. In conclusion, exposure to palmitate inhibits proliferation of myoblasts through a decrease in cyclin A and cyclin D1 levels and an increase of p21-cdk4 complex formation; however, it promotes cell cycle exit, myogenic differentiation and myotube growth.

Insulin-Like Growth Factor-I Increases Laminin, Integrin Subunits and Metalloprotease ADAM12 in Mouse Myoblasts.

The extracellular matrix (ECM) is considered a part of the myogenesis signaling mechanism. we hypothesized that insulin-like growth factor-I (IGF-I) modifies ECM during differentiation of mouse C2C12 cells. The myogenic effect of IGF-I (30 nmol/l) was manifested by increased myogenin and myosin heavy chain (MyHC) levels as well as fusion index (2.6 times over control) on the 3rd day of differentiation. IGF-I markedly augmented laminin, but not fibronectin. Cellular contents of integrin α3, α5 and ß1 during 3-day differentiation increased in the presence of IGF-I. Treatment with IGF-I increased the expression of the long form of metalloprotease ADAM12 (100 kDa) in myocytes. In conclusion: i) IGF-I caused an increase of laminin, integrin α3 and ß1 in C2C12 myogenic cells that can be secondary to stimulation of myogenesis; ii) IGF-I augmented integrin α5 and ADAM12 levels, suggesting a role of this growth factor in determination of the pool of reserve cells during myogenesis.

Transcriptional regulation of important cellular processes in skeletal myogenesis through interferon-γ.

The purpose of the present study was to investigate the effect of interferon (IFN)-γ on the transcriptomic profile of differentiating mouse C2C12 myogenic cells. Global gene expression was evaluated using whole mouse genome oligonucleotide microarrays, and the results were validated through real-time PCR. IFN-γ (1 ng/mL) increased myoblast proliferation but decreased cell respiration and myosin heavy chain content and slightly decreased the fusion index in differentiating C2C12 cell cultures. The genes upregulated through IFN-γ were involved in cell cycle; regulation of cell proliferation; programmed cell death; chemotaxis; and cytokine, growth factor, and peptidase activity, whereas the genes downregulated through IFN-γ primarily contributed to the regulation of transcription, cell-cell signaling, nitrogen compound biosynthesis, ser/thr protein kinase signaling, and regulation of the Wnt pathway. In conclusion, IFN-γ affects the expression of numerous genes associated with the regulation of several processes in myogenesis. The effects of IFN-γ on cellular transcription include (1) alteration of cytokine/growth factor expression, promoting cell proliferation and migration but inhibiting differentiation, (2) impairment of pro-myogenic transcription, (3) disruption of cell adhesion and sarcolemma/cytoskeleton organization, and (4) increased peptidase activity leading to enhanced proteolysis and apoptosis.

[Developmental programming of metabolic diseases--a review of studies on experimental animal models]. / Programowanie rozwojowe chorób metabolicznych--przeglad wyników badan na zwierzecych modelach doswiadczalnych.

Growth and development in utero is a complex and dynamic process that requires interaction between the mother organism and the fetus. The delivery of macro--and micronutrients, oxygen and endocrine signals has crucial importance for providing a high level of proliferation, growth and differentiation of cells, and a disruption in food intake not only has an influence on the growth of the fetus, but also has negative consequences for the offspring's health in the future. Diseases that traditionally are linked to inappropriate life style of adults, such as type 2 diabetes, obesity, and arterial hypertension, can be "programmed" in the early stage of life and the disturbed growth of the fetus leads to the symptoms of the metabolic syndrome. The structural changes of some organs, such as the brain, pancreas and kidney, modifications of the signaling and metabolic pathways in skeletal muscles and in fatty tissue, epigenetic mechanisms and mitochondrial dysfunction are the basis of the metabolic disruptions. The programming of the metabolic disturbances is connected with the disruption in the intrauterine environment experienced in the early and late gestation period. It causes the changes in deposition of triglycerides, activation of the hormonal "stress axis" and disturbances in the offspring's glucose tolerance. The present review summarizes experimental results that led to the identification of the above-mentioned links and it underlines the role of animal models in the studies of this important concept.

[Interactions of proliferation and differentiation signaling pathways in myogenesis]. / Interakcje szlaków sygnalowych proliferacji i róznicowania w miogenezie

The commitment of myogenic cells in skeletal muscle differentiation requires earlier irreversible interruption of the cell cycle. At the molecular level, several key regulators of the cell cycle have been identified: cyclin-dependent kinases and their cyclins stimulate the cell cycle progress and its arrest is determined by the activity of cdk inhibitors (Cip/Kip and INK protein families) and pocket protein family: Rb, p107 and p130. The biological activity of cyclin/cdk complexes allows the successive phases of the cell cycle to occur. Myoblast specialization, differentiation and fusion require the activity of myogenic regulatory factors, which include MyoD, myogenin, Myf5 and MRF4. MyoD and Myf5 play a role in muscle cell specialization, myogenin controls the differentiation process, whereas MRF4 is involved in myotube maturation. The deregulation of the cell cycle leads to uncontrolled proliferation, which antagonizes the functions of myogenic factors and it explains the lack of differentiation-specific gene expression in dividing cells. Conversely, the myogenic factor MyoD seems to cooperate with cell cycle inhibitors leading to inhibition of cell cycle progress and commitment to the differentiation process. The hypophosphorylated form of Rb and cdk inhibitors play an important role in permanent arrest of the cell cycle in differentiated myotubes. Furthermore, cyclin/cdk complexes not only regulate cell division by phosphorylation of several substrates, but may also control other cellular processes such as signal transduction, differentiation and apoptosis. Beyond regulating the cell cycle, Cip/Kip proteins play an important role in cell death, transcription regulation, cell fate determination, cell migration and cytoskeletal dynamics. The article summarizes current knowledge concerning the interactions of intracellular signaling pathways controlling crucial stages of fetal and regenerative myogenesis.

[Systemic and local mechanisms leading to cachexia in cancer]. / Ogólnoustrojowe i miejscowe mechanizmy prowadzace do kacheksji w chorobach nowotworowych.

Cachexia is a multifactorial syndrome of atrophy of skeletal muscle and adipose tissue, resulting in progressive loss of body weight associated with low quality of life and poor prognosis in cancer. Studies on experimental animal models and observations on patients have shown that the soluble factors secreted by tumor cells and tissues of the patient can participate in regulation of the wasting process. Cachexia is often accompanied by anorexia, which is caused by predominance of signals inhibiting appetite in the hypothalamus, such as release of proopiomelanocortin and anorexigenic action of proinflammatory cytokines (IL-1α, IL-1ß, IL-6, TNF-α). Cachexia is also accompanied by extensive metabolic changes consisting of increase of resting energy expenditure and disturbance of carbohydrate, protein and lipid metabolism. Increased expression of protein uncoupling phosphorylation leads to increased thermogenesis in skeletal muscle. Tumor tissue hypoxia caused by its growth beyond blood vessels activates the transcription factor HIF-1, which results in increase in glycolysis, and leads to lactic acid accumulation and activation of the energy inefficient Cori cycle. Loss of fat tissue is caused by increase of lipolysis induced by lipid-mobilizing factor (LMF) and proinflammatory cytokines. Skeletal muscle wasting in cachexia is caused by a reduction of protein synthesis at the stage of initiation and elongation of translation and the simultaneous increase of protein degradation via ubiquitin-dependent and lysosomal pathways. The main mediators of skeletal muscle wasting in cancer are proteolysis-inducing factor (PIF), proinflammatory cytokines, and angiotensin II acting through increased levels of reactive oxygen species (ROS) and nuclear factor NF-κB activation, as well as glucocorticoid activated FOXO transcription factors and myostatin. Understanding of the complexity of the interaction of factors produced by the tumor and the patient's body may form the basis for the development of effective treatments for cachexia in cancer and other pathological conditions.

[Maternal obesity and the development of skeletal muscle in offspring--fetal origin of metabolic disorders]. / Matczyna otylosc a rozwój miesni szkieletowych u potomstwa--plodowe pochodzenie zaburzen metabolicznych.

Suboptimal fetal environments due to inadequate maternal nutrition, obesity, inflammation or gestational diabetes expose the fetus to humoral cues that alter metabolism and growth parameters leading to metabolic disturbances later in life. The fetal stage is crucial for the development of skeletal muscle, a tissue playing an important role in metabolism. Maternal obesity induces inflammation in the fetus causing modifications in the development of fetal skeletal muscle. Changes in the normal course of myogenesis may arise through several mechanisms: changes in WNT/ß-catenin signaling pathway, decreased AMPK activity evoked by TNF-α, increased activity of NF-κB in response to inflammation, which leads to a decrease in myogenic factor MyoD, and increased expression of TGF ß1. Modification in fetal development associated with maternal obesity is attributed to epigenetic changes. Polyunsaturated fatty acids supplied in the diet did affect the development of insulin-sensitive tissues during both the fetal and postnatal period. The specific phenotype of skeletal muscle fibers may play a role in the development of obesity, i.e. fiber phenotype I (slow, oxidative) may protect against obesity and insulin resistance. Exploring the mechanisms of direct impact of maternal obesity on the development of tissues in the offspring may help to reduce the occurrence of metabolic diseases in later life.

Metabolic pathways of the camptothecin analog AR-67.

7-tert-Butyldimethylsilyl-10-hydroxycamptothecin (AR-67; also known as DB-67) is a novel lipophilic camptothecin analog in early-phase anticancer clinical trials. In support of these studies, we evaluated the metabolism of AR-67 in vitro and identified potential metabolites in patient samples. The lactone form of AR-67 was found to be preferentially metabolized over AR-67 carboxylate in human microsomes. Subsequently, the lactone form was tested as a substrate in a panel of CYP450 and UDP-glucuronosyltransferase (UGT) enzymes known to metabolize the majority of clinically approved molecules. AR-67 was metabolized by CYP3A5, CYP3A4, CYP1A1, and CYP1A2, in order of activity. Extrahepatic UGT1A8 and UGT1A7 possessed at least 6-fold higher metabolizing activity than UGT1A1 and other UGT enzymes tested. CYP1A1 and UGT1A7 displayed Michaelis-Menten kinetics, whereas CYP3A4, CYP3A5, and UGT1A8 displayed kinetics consistent with substrate inhibition. Chromatographic analysis of representative patient plasma and urine samples demonstrated the presence of AR-67 glucuronides and oxidized products in the urine but only in very minimal amounts. We conclude that limited in vivo metabolism of AR-67 by UGT1A1 may partly explain the absence of AR-67 glucuronides in plasma and hypothesize that UGT1A8- and CYP3A-mediated biotransformation within the gastrointestinal epithelium may provide protective mechanisms against AR-67 gastrointestinal toxicity.