Myogenin, MyoD and IGF-I regulate muscle mass but not fiber-type conversion during resistance training in rats

The purpose of this study was to test the hypothesis that skeletal muscle adaptations induced by long-term resistance training (RT) are associated with increased myogenic regulatory factors (MRF) and insulin-like growth factor-I (IGF-I) mRNA expression in rats skeletal muscle. Male Wistar rats were divided into 4 groups: 8-week control (C8), 8-week trained (T8), 12-week control (C12) and 12-week trained (T12). Trained rats were submitted to a progressive RT program (4 sets of 10-12 repetitions at 65-75% of the 1RM, 3 day/week), using a squat-training apparatus with electric stimulation. Muscle hypertrophy was determined by measurement of muscle fiber cross-sectional area (CSA) of the muscle fibers, and myogenin, MyoD and IGF-I mRNA expression were measured by RT-qPCR. A hypertrophic stabilization occurred between 8 and 12 weeks of RT (control-relative % area increase, T8: 29% vs. T12: 35%; p>0.05) and was accompanied by the stabilization of myogenin (control-relative % increase, T8: 44.8% vs. T12: 37.7%, p>0.05) and MyoD (control-relative % increase, T8: 22.9% vs. T12: 22.3%, p>0.05) mRNA expression and the return of IGF-I mRNA levels to the baseline (control-relative % increase, T8: 30.1% vs. T12: 1.5%, p<0.05). Moreover, there were significant positive correlations between the muscle fiber CSA and mRNA expression for MyoD (r=0.85, p=0.0001), myogenin (r=0.87, p=0.0001), and IGF-I (r=0.88, p=0.0001). The significant (p<0.05) increase in myogenin, MyoD and IGF-I mRNA expression after 8 weeks was not associated with changes in the fiber-type frequency. In addition, there was a type IIX/D-to-IIA fiber conversion at 12 weeks, even with the stabilization of MyoD and myogenin expression and the return of IGF-I levels to baseline. These results indicate a possible interaction between MRFs and IGF-I in the control of muscle hypertrophy during long-term RT and suggest that these factors are involved more in the regulation of muscle mass than in fiber-type conversion. © Georg Thieme Verlag KG Stuttgart · New York.

Produção leiteira em cabras da raça Saanen: influência dos hormônios cortisol e IGF-I

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Nitric oxide and brazilian propolis combined accelerates tissue repair by modulating cell migration, cytokine production and collagen deposition in experimental leishmaniasis

The fact that drugs currently used in the treatment of Leishmania are highly toxic and associated with acquired resistance has promoted the search for new therapies for treating American tegumentary leishmaniasis (ATL). In this study, BALB/c mice were injected in the hind paw with Leishmania (Leishmania) amazonensis and subsequently treated with a combination of nitric oxide (NO) donor (cis-[Ru(bpy)(2)imN(NO)](PF6)(3)) (Ru-NO), given by intraperitoneal injection, and oral Brazilian propolis for 30 days. Ru-NO reached the center of the lesion and increased the NO level in the injured hind paw without lesion exacerbation. Histological and immunological parameters of chronic inflammation showed that this combined treatment increased the efficacy of macrophages, determined by the decrease in the number of parasitized cells, leading to reduced expression of proinflammatory and tissue damage markers. In addition, these drugs in combination fostered wound healing, enhanced the number of fibroblasts, pro-healing cytokines and induced collagen synthesis at the lesion site. Overall, our findings suggest that the combination of the NO donor Ru-NO and Brazilian propolis alleviates experimental ATL lesions, highlighting a new therapeutic option that can be considered for further in vivo investigations as a candidate for the treatment of cutaneous leishmaniasis.

Influence of Insulin-like Growth Factor I (IGF-I) on the survival and the in vitro development of caprine preantral follicles

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tamoxifen Subcellular Localization; Observation of Cell-Specific Cytotoxicity Enhancement by Inhibition of Mitochondrial ETC Complexes I and III

Recently, a nongenomic cytotoxic component of the chemotherapeutic agent tamoxifen (TAM) has been identified that predominantly triggers mitochondrial events. The present study delineates the intracellular fate of TAM and studies its interaction with a spectrum of cell homeostasis modulators primarily relevant to mitochondria. The subcellular localization of TAM was assessed by confocal fluorescence microscopy. The effect of the modulators on TAM cytotoxicity was assessed by standard MTT assays. Our findings show that in estrogen receptor positive MCF7 breast adenocarcinoma cells and DU145 human prostate cancer cells, TAM largely accumulates in the mitochondria and endoplasmic reticulum, but not lysosomes. Our results further demonstrate that in MCF7, but not in DU145 cells, mitochondrial electron transport chain complex I and III inhibitors exacerbate TAM toxicity with an order of potency of myxothiazol = stigmatellin > rotenone > antimycin A, suggesting a cell-specific cytotoxic interplay between mitochondrial complex I and III function and TAM action.

Protein Disulfide Isomerase Is Required for Platelet-derived Growth Factor-induced Vascular Smooth Muscle Cell Migration, Nox1 NADPH Oxidase Expression, and RhoGTPase Activation

Vascular Smooth Muscle Cell (VSMC) migration into vessel neointima is a therapeutic target for atherosclerosis and postinjury restenosis. Nox1 NADPH oxidase-derived oxidants synergize with growth factors to support VSMC migration. We previously described the interaction between NADPH oxidases and the endoplasmic reticulum redox chaperone protein disulfide isomerase (PDI) in many cell types. However, physiological implications, as well as mechanisms of such association, are yet unclear. We show here that platelet-derived growth factor (PDGF) promoted subcellular redistribution of PDI concomitant to Nox1-dependent reactive oxygen species production and that siRNA-mediated PDI silencing inhibited such reactive oxygen species production, while nearly totally suppressing the increase in Nox1 expression, with no change in Nox4. Furthermore, PDI silencing inhibited PDGF-induced VSMC migration assessed by distinct methods, whereas PDI overexpression increased spontaneous basal VSMC migration. To address possible mechanisms of PDI effects, we searched for PDI interactome by systems biology analysis of physical protein-protein interaction networks, which indicated convergence with small GTPases and their regulator RhoGDI. PDI silencing decreased PDGF-induced Rac1 and RhoA activities, without changing their expression. PDI co-immunoprecipitated with RhoGDI at base line, whereas such association was decreased after PDGF. Also, PDI co-immunoprecipitated with Rac1 and RhoA in a PDGF-independent way and displayed detectable spots of perinuclear co-localization with Rac1 and RhoGDI. Moreover, PDI silencing promoted strong cytoskeletal changes: disorganization of stress fibers, decreased number of focal adhesions, and reduced number of RhoGDI-containing vesicular recycling adhesion structures. Overall, these data suggest that PDI is required to support Nox1/redox and GTPase-dependent VSMC migration.

Testosterone Induces Vascular Smooth Muscle Cell Migration by NADPH Oxidase and c-Src-Dependent Pathways

Testosterone has been implicated in vascular remodeling associated with hypertension. Molecular mechanisms underlying this are elusive, but oxidative stress may be important. We hypothesized that testosterone stimulates generation of reactive oxygen species (ROS) and migration of vascular smooth muscle cells (VSMCs), with enhanced effects in cells from spontaneously hypertensive rats (SHRs). The mechanisms (genomic and nongenomic) whereby testosterone induces ROS generation and the role of c-Src, a regulator of redox-sensitive migration, were determined. VSMCs from male Wistar-Kyoto rats and SHRs were stimulated with testosterone (10(-7) mol/L, 0-120 minutes). Testosterone increased ROS generation, assessed by dihydroethidium fluorescence and lucigenin-enhanced chemiluminescence (30 minutes [SHR] and 60 minutes [both strains]). Flutamide (androgen receptor antagonist) and actinomycin D (gene transcription inhibitor) diminished ROS production (60 minutes). Testosterone increased Nox1 and Nox4 mRNA levels and p47phox protein expression, determined by real-time PCR and immunoblotting, respectively. Flutamide, actinomycin D, and cycloheximide (protein synthesis inhibitor) diminished testosterone effects on p47phox. c-Src phosphorylation was observed at 30 minutes (SHR) and 120 minutes (Wistar-Kyoto rat). Testosterone-induced ROS generation was repressed by 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day]pyrimidin-4-amine (c-Src inhibitor) in SHRs and reduced by apocynin (antioxidant/NADPH oxidase inhibitor) in both strains. Testosterone stimulated VSMCs migration, assessed by the wound healing technique, with greater effects in SHRs. Flutamide, apocynin, and 3-(4-chlorophenyl) 1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-day] pyrimidin-4-amine blocked testosterone-induced VSMCs migration in both strains. Our study demonstrates that testosterone induces VSMCs migration via NADPH oxidase-derived ROS and c-Src-dependent pathways by genomic and nongenomic mechanisms, which are differentially regulated in VSMCs from Wistar-Kyoto rats and SHRs. (Hypertension. 2012; 59: 1263-1271.). Online Data Supplement

Transcriptional regulation of human mu-opioid receptor gene: functional characterization of activating and inhibitory transcription factors

The organization of the nervous and immune systems is characterized by obvious differences and striking parallels. Both systems need to relay information across very short and very long distances. The nervous system communicates over both long and short ranges primarily by means of more or less hardwired intercellular connections, consisting of axons, dendrites, and synapses. Longrange communication in the immune system occurs mainly via the ordered and guided migration of immune cells and systemically acting soluble factors such as antibodies, cytokines, and chemokines. Its short-range communication either is mediated by locally acting soluble factors or transpires during direct cell–cell contact across specialized areas called “immunological synapses” (Kirschensteiner et al., 2003). These parallels in intercellular communication are complemented by a complex array of factors that induce cell growth and differentiation: these factors in the immune system are called cytokines; in the nervous system, they are called neurotrophic factors. Neither the cytokines nor the neurotrophic factors appear to be completely exclusive to either system (Neumann et al., 2002). In particular, mounting evidence indicates that some of the most potent members of the neurotrophin family, for example, nerve growth factor (NGF) and brainderived neurotrophic factor (BDNF), act on or are produced by immune cells (Kerschensteiner et al., 1999) There are, however, other neurotrophic factors, for example the insulin-like growth factor-1 (IGF-1), that can behave similarly (Kermer et al., 2000). These factors may allow the two systems to “cross-talk” and eventually may provide a molecular explanation for the reports that inflammation after central nervous system (CNS) injury has beneficial effects (Moalem et al., 1999). In order to shed some more light on such a cross-talk, therefore, transcription factors modulating mu-opioid receptor (MOPr) expression in neurons and immune cells are here investigated. More precisely, I focused my attention on IGF-I modulation of MOPr in neurons and T-cell receptor induction of MOPr expression in T-lymphocytes. Three different opioid receptors [mu (MOPr), delta (DOPr), and kappa (KOPr)] belonging to the G-protein coupled receptor super-family have been cloned. They are activated by structurallyrelated exogenous opioids or endogenous opioid peptides, and contribute to the regulation of several functions including pain transmission, respiration, cardiac and gastrointestinal functions, and immune response (Zollner and Stein 2007). MOPr is expressed mainly in the central nervous system where it regulates morphine-induced analgesia, tolerance and dependence (Mayer and Hollt 2006). Recently, induction of MOPr expression in different immune cells induced by cytokines has been reported (Kraus et al., 2001; Kraus et al., 2003). The human mu-opioid receptor gene (OPRM1) promoter is of the TATA-less type and has clusters of potential binding sites for different transcription factors (Law et al. 2004). Several studies, primarily focused on the upstream region of the OPRM1 promoter, have investigated transcriptional regulation of MOPr expression. Presently, however, it is still not completely clear how positive and negative transcription regulators cooperatively coordinate cellor tissue-specific transcription of the OPRM1 gene, and how specific growth factors influence its expression. IGF-I and its receptors are widely distributed throughout the nervous system during development, and their involvement in neurogenesis has been extensively investigated (Arsenijevic et al. 1998; van Golen and Feldman 2000). As previously mentioned, such neurotrophic factors can be also produced and/or act on immune cells (Kerschenseteiner et al., 2003). Most of the physiologic effects of IGF-I are mediated by the type I IGF surface receptor which, after ligand binding-induced autophosphorylation, associates with specific adaptor proteins and activates different second messengers (Bondy and Cheng 2004). These include: phosphatidylinositol 3-kinase, mitogen-activated protein kinase (Vincent and Feldman 2002; Di Toro et al. 2005) and members of the Janus kinase (JAK)/STAT3 signalling pathway (Zong et al. 2000; Yadav et al. 2005). REST plays a complex role in neuronal cells by differentially repressing target gene expression (Lunyak et al. 2004; Coulson 2005; Ballas and Mandel 2005). REST expression decreases during neurogenesis, but has been detected in the adult rat brain (Palm et al. 1998) and is up-regulated in response to global ischemia (Calderone et al. 2003) and induction of epilepsy (Spencer et al. 2006). Thus, the REST concentration seems to influence its function and the expression of neuronal genes, and may have different effects in embryonic and differentiated neurons (Su et al. 2004; Sun et al. 2005). In a previous study, REST was elevated during the early stages of neural induction by IGF-I in neuroblastoma cells. REST may contribute to the down-regulation of genes not yet required by the differentiation program, but its expression decreases after five days of treatment to allow for the acquisition of neural phenotypes. Di Toro et al. proposed a model in which the extent of neurite outgrowth in differentiating neuroblastoma cells was affected by the disappearance of REST (Di Toro et al. 2005). The human mu-opioid receptor gene (OPRM1) promoter contains a DNA sequence binding the repressor element 1 silencing transcription factor (REST) that is implicated in transcriptional repression. Therefore, in the fist part of this thesis, I investigated whether insulin-like growth factor I (IGF-I), which affects various aspects of neuronal induction and maturation, regulates OPRM1 transcription in neuronal cells in the context of the potential influence of REST. A series of OPRM1-luciferase promoter/reporter constructs were transfected into two neuronal cell models, neuroblastoma-derived SH-SY5Y cells and PC12 cells. In the former, endogenous levels of human mu-opioid receptor (hMOPr) mRNA were evaluated by real-time PCR. IGF-I upregulated OPRM1 transcription in: PC12 cells lacking REST, in SH-SY5Y cells transfected with constructs deficient in the REST DNA binding element, or when REST was down-regulated in retinoic acid-differentiated cells. IGF-I activates the signal transducer and activator of transcription-3 (STAT3) signaling pathway and this transcription factor, binding to the STAT1/3 DNA element located in the promoter, increases OPRM1 transcription. T-cell receptor (TCR) recognizes peptide antigens displayed in the context of the major histocompatibility complex (MHC) and gives rise to a potent as well as branched intracellular signalling that convert naïve T-cells in mature effectors, thus significantly contributing to the genesis of a specific immune response. In the second part of my work I exposed wild type Jurkat CD4+ T-cells to a mixture of CD3 and CD28 antigens in order to fully activate TCR and study whether its signalling influence OPRM1 expression. Results were that TCR engagement determined a significant induction of OPRM1 expression through the activation of transcription factors AP-1, NF-kB and NFAT. Eventually, I investigated MOPr turnover once it has been expressed on T-cells outer membrane. It turned out that DAMGO induced MOPr internalisation and recycling, whereas morphine did not. Overall, from the data collected in this thesis we can conclude that that a reduction in REST is a critical switch enabling IGF-I to up-regulate human MOPr, helping these findings clarify how human MOPr expression is regulated in neuronal cells, and that TCR engagement up-regulates OPRM1 transcription in T-cells. My results that neurotrophic factors a and TCR engagement, as well as it is reported for cytokines, seem to up-regulate OPRM1 in both neurons and immune cells suggest an important role for MOPr as a molecular bridge between neurons and immune cells; therefore, MOPr could play a key role in the cross-talk between immune system and nervous system and in particular in the balance between pro-inflammatory and pro-nociceptive stimuli and analgesic and neuroprotective effects.

Cell death mechanisms triggered by monoclonal antibodies against CD99 in Ewing sarcoma: Cross-talk between MDM2, IGF-1R and Ras/MAPK

CD99 is a 32 kDa transmembrane protein whose high expression characterizes Ewing sarcoma (ES), a very aggressive pediatric bone tumor. In addition to its diagnostic value, CD99 has therapeutic potential since it leads to rapid and massive ES cell death when engaged with specific antibodies. Here a novel mechanism of cell death triggered via CD99 is shown, leading, ultimately, to the appearance of macropinocytotic vescicles. Anti-CD99 mAb 0662 induces MDM2 ubiquitination and degradation, which causes not only a p53 reactivation but also the IGF-1R induction and its subsequent internalization; CD99 results internalized together with IGF-1R inside endosomes, but then the two molecules display a different sorting: CD99 is degraded, while IGF-1R is recycled on the surface, causing, as a final step, the up-regulation of RAS-MAPK. High-expressing CD99 mesenchymal stem cells show mild Ras induction but no p53 activation and escape cell death, but in presence of EWS/FLI1 mesenchymal stem cells expressing CD99 show a stronger Ras induction and a p53 reactivation, leading to a significant cell death rate. We propose that CD99 triggering in a EWS/FLI1-driven oncogenetic context creates a synergy between RAS upregulation and p53 activation in ES cells, leading to cell death. Moreover, our data rule out possible concerns on toxicity related to the broad CD99 expression in normal tissues and provide the rationale for the therapeutic use of anti-CD99 MAbs in the clinic.

Sphingosine kinase 1 is critically involved in nitric oxide-mediated human endothelial cell migration and tube formation

Sphingosine kinases (SKs) convert sphingosine to sphingosine 1-phosphate (S1P), which is a bioactive lipid that regulates a variety of cellular processes including proliferation, differentiation and migration.

Critical roles for Rac GTPases in T-cell migration to and within lymph nodes

Naive T cells continuously recirculate between secondary lymphoid tissue via the blood and lymphatic systems, a process that maximizes the chances of an encounter between a T cell and its cognate antigen. This recirculation depends on signals from chemokine receptors, integrins, and the sphingosine-1-phosphate receptor. The authors of previous studies in other cell types have shown that Rac GTPases transduce signals leading to cell migration and adhesion; however, their roles in T cells are unknown. By using both 3-dimensional intravital and in vitro approaches, we show that Rac1- and Rac2-deficient T cells have multiple defects in this recirculation process. Rac-deficient T cells home very inefficiently to lymph nodes and the white pulp of the spleen, show reduced interstitial migration within lymph node parenchyma, and are defective in egress from lymph nodes. These mutant T cells show defective chemokine-induced chemotaxis, chemokinesis, and adhesion to integrin ligands. They have reduced lateral motility on endothelial cells and transmigrate in-efficiently. These multiple defects stem from critical roles for Rac1 and Rac2 in transducing chemokine and sphingosine-1-phosphate receptor 1 signals leading to motility and adhesion.

Congenital hypothyroidism in a kitten resulting in decreased IGF-I concentration and abnormal liver function tests

A 7-month-old male kitten was presented with chronic constipation and retarded growth. Clinical examination revealed disproportional dwarfism with mild skeletal abnormalities and a palpable thyroid gland. The presumptive diagnosis of congenital hypothyroidism was confirmed by low serum total thyroxine (tT(4)) concentration prior to and after the administration of thyroid stimulation hormone (TSH), increased endogenous TSH concentration and abnormal thyroid scintigraphic scan. The kitten had abnormal liver function tests and decreased insulin-like growth factor 1 (IGF-1) concentration, both of which returned to normal in correspondence with an improvement of the clinical signs after 6 weeks of thyroxine therapy. Congenital hypothyroidism is a rare disease that may present with considerable variation in clinical manifestation. In cases in which clinical signs are ambiguous, disorders such as portosystemic shunt and hyposomatotropism have to be taken into account as differential diagnosis. As hypothyroidism may be associated with abnormal liver function tests and low IGF-1 concentrations, test results have to be interpreted carefully.

Differential requirement for ROCK in dendritic cell migration within lymphatic capillaries in steady-state and inflammation

Dendritic cell (DC) migration via lymphatic vessels to draining lymph nodes (dLNs) is crucial for the initiation of adaptive immunity. We imaged this process by intravital microscopy (IVM) in the ear skin of transgenic mice bearing red-fluorescent vasculature and yellow-fluorescent DCs. DCs within lymphatic capillaries were rarely transported by flow, but actively migrated within lymphatics and were significantly faster than in the interstitium. Pharmacologic blockade of the Rho-associated protein kinase (ROCK), which mediates nuclear contraction and de-adhesion from integrin ligands, significantly reduced DC migration from skin to dLNs in steady-state. IVM revealed that ROCK blockade strongly reduced the velocity of interstitial DC migration, but only marginally affected intralymphatic DC migration. By contrast, during tissue inflammation, ROCK blockade profoundly decreased both interstitial and intralymphatic DC migration. Inhibition of intralymphatic migration was paralleled by a strong up-regulation of ICAM-1 in lymphatic endothelium, suggesting that during inflammation ROCK mediates de-adhesion of DC-expressed integrins from lymphatic-expressed ICAM-1. Flow chamber assays confirmed an involvement of lymphatic-expressed ICAM-1 and DC-expressed ROCK in DC crawling on lymphatic endothelium. Overall, our findings further define the role of ROCK in DC migration to dLNs and reveal a differential requirement for ROCK in intralymphatic DC crawling during steady-state and inflammation.