Carlos Pizarro Wilson

Testosterone induces cardiomyocyte hypertrophy through mammalian target of rapamycin complex 1 pathway

Elevated testosterone concentrations induce cardiac hypertrophy but the molecular mechanisms are poorly understood. Anabolic properties of testosterone involve an increase in protein synthesis. The mammalian target of rapamycin complex 1 (mTORC1) pathway is a major regulator of cell growth, but the relationship between testosterone action and mTORC1 in cardiac cells remains unknown. Here, we investigated whether the hypertrophic effects of testosterone are mediated by mTORC1 signaling in cultured cardiomyocytes. Testosterone increases the phosphorylation of mTOR and its downstream targets 40S ribosomal protein S6 kinase 1 (S6K1; also known as RPS6KB1) and eukaryotic initiation factor 4E-binding protein 1 (4E-BP1). The S6K1 phosphorylation induced by testosterone was blocked by rapamycin and small interfering RNA to mTOR. Moreover, the hormone increased both extracellular-regulated kinase (ERK1/2) and protein kinase B (Akt) phosphorylation. ERK1/2 inhibitor PD98059 blocked the testosterone-induced S6K1 phosphorylation, whereas Akt inhibition (Akt-inhibitor-X) had no effect. Testosterone-induced ERK1/2 and S6K1 phosphorylation increases were blocked by either 1,2-bis(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethylester or by inhibitors of inositol 1,4,5-trisphosphate (IP(3)) pathway: U-73122 and 2-aminoethyl diphenylborate. Finally, cardiomyocyte hypertrophy was evaluated by, the expression of beta-myosin heavy chain, alpha-skeletal actin, cell size, and amino acid incorporation. Testosterone increased all four parameters and the increase being blocked by mTOR inhibition. Our findings suggest that testosterone activates the mTORC1/S6K1 axis through IP(3)/Ca(2+) and MEK/ERK1/2 to induce cardiomyocyte hypertrophy.

Regulation of cytoskeletal dynamics by redox signaling and oxidative stress: implications for neuronal development and trafficking

A proper balance between chemical reduction and oxidation (known as redox balance) is essential for normal cellular physiology. Deregulation in the production of oxidative species leads to DNA damage, lipid peroxidation and aberrant post-translational modification of proteins, which in most cases induces injury, cell death and disease. However, physiological concentrations of oxidative species are necessary to support important cell functions, such as chemotaxis, hormone synthesis, immune response, cytoskeletal remodeling, Ca2+ homeostasis and others. Recent evidence suggests that redox balance regulates actin and microtubule dynamics in both physiological and pathological contexts. Microtubules and actin microfilaments contain certain amino acid residues that are susceptible to oxidation, which reduces the ability of microtubules to polymerize and causes severing of actin microfilaments in neuronal and non-neuronal cells. In contrast, inhibited production of reactive oxygen species (ROS; e.g., due to NOXs) leads to aberrant actin polymerization, decreases neurite outgrowth and affects the normal development and polarization of neurons. In this review, we summarize emerging evidence suggesting that both general and specific enzymatic sources of redox species exert diverse effects on cytoskeletal dynamics. Considering the intimate relationship between cytoskeletal dynamics and trafficking, we also discuss the potential effects of redox balance on intracellular transport via regulation of the components of the microtubule and actin cytoskeleton as well as cytoskeleton-associated proteins, which may directly impact localization of proteins and vesicles across the soma, dendrites and axon of neurons.

Actin filaments—A target for redox regulation

Actin and its ability to polymerize into dynamic filaments is critical for the form and function of cells throughout the body. While multiple proteins have been characterized as affecting actin dynamics through noncovalent means, actin and its protein regulators are also susceptible to covalent modifications of their amino acid residues. In this regard, oxidation‐reduction (Redox) intermediates have emerged as key modulators of the actin cytoskeleton with multiple different effects on cellular form and function. Here, we review work implicating Redox intermediates in post‐translationally altering actin and discuss what is known regarding how these alterations affect the properties of actin. We also focus on two of the best characterized enzymatic sources of these Redox intermediates—the NADPH oxidase NOX and the flavoprotein monooxygenase MICAL—and detail how they have both been identified as altering actin, but share little similarity and employ different means to regulate actin dynamics. Finally, we discuss the role of these enzymes and redox signaling in regulating the actin cytoskeleton in vivo and highlight their importance for neuronal form and function in health and disease.

Testosterone increases GLUT4-dependent glucose uptake in cardiomyocytes

Testosterone exerts important effects in the heart. Cardiomyocytes are target cells for androgens, and testosterone induces rapid effects via Ca2+ release and protein kinase activation and long‐term effects via cardiomyocyte differentiation and hypertrophy. Furthermore, it stimulates metabolic effects such as increasing glucose uptake in different tissues. Cardiomyocytes preferentially consume fatty acids for ATP production, but under particular circumstances, glucose uptake is increased to optimize energy production. We studied the effects of testosterone on glucose uptake in cardiomyocytes. We found that testosterone increased uptake of the fluorescent glucose analog 2‐(N‐(7‐nitrobenz‐2‐oxa‐1, 3‐diazol‐4‐yl)amino)‐2‐deoxyglucose and [3H]2‐deoxyglucose, which was blocked by the glucose transporter 4 (GLUT4) inhibitor indinavir. Testosterone stimulation in the presence of cyproterone or albumin‐bound testosterone‐induced glucose uptake, which suggests an effect that is independent of the intracellular androgen receptor. To determine the degree of GLUT4 cell surface exposure, cardiomyocytes were transfected with the plasmid GLUT4myc‐eGFP. Subsequently, testosterone increased GLUT4myc‐GFP exposure at the plasma membrane. Inhibition of Akt by the Akt‐inhibitor‐VIII had no effect. However, inhibition of Ca2+/calmodulin protein kinase (CaMKII) (KN‐93 and autocamtide‐2 related inhibitory peptide II) and AMP‐activated protein kinase (AMPK) (compound C and siRNA for AMPK) prevented glucose uptake induced by testosterone. Moreover, GLUT4myc‐eGFP exposure at the cell surface caused by testosterone was also abolished after CaMKII and AMPK inhibition. These results suggest that testosterone increases GLUT4‐dependent glucose uptake, which is mediated by CaMKII and AMPK in cultured cardiomyocytes. Glucose uptake could represent a mechanism by which testosterone increases energy production and protein synthesis in cardiomyocytes. J. Cell. Physiol. 228: 2399–2407, 2013.

Exchange Protein Directly Activated by cAMP (EPAC) Regulates Neuronal Polarization through Rap1B

Acquisition of neuronal polarity is a complex process involving cellular and molecular events. The second messenger cAMP is involved in axonal specification through activation of protein kinase A. However, an alternative cAMP-dependent mechanism involves the exchange protein directly activated by cAMP (EPAC), which also responds to physiological changes in cAMP concentration, promoting activation of the small Rap GTPases. Here, we present evidence that EPAC signaling contributes to axon specification and elongation. In primary rat hippocampal neurons, EPAC isoforms were expressed differentially during axon specification. Furthermore, 8-pCPT, an EPAC pharmacological activator, and genetic manipulations of EPAC in neurons induced supernumerary axons indicative of Rap1b activation. Moreover, 8-pCPT-treated neurons expressed ankyrin G and other markers of mature axons such as synaptophysin and axonal accumulation of vGLUT1. In contrast, pharmacological inhibition of EPAC delayed neuronal polarity. Genetic manipulations to inactivate EPAC1 using either shRNA or neurons derived from EPAC1 knock-out (KO) mice led to axon elongation and polarization defects. Interestingly, multiaxonic neurons generated by 8-pCPT treatments in wild-type neurons were not found in EPAC1 KO mice neurons. Altogether, these results propose that EPAC signaling is an alternative and complementary mechanism for cAMP-dependent axon determination. SIGNIFICANCE STATEMENT This study identifies the guanine exchange factor responsible for Rap1b activation during neuronal polarization and provides an alternate explanation for cAMP-dependent acquisition of neuronal polarity.

Contribution of NADPH oxidase to the establishment of hippocampal neuronal polarity in culture

ABSTRACT Reactive oxygen species (ROS) produced by the NADPH oxidase (NOX) complex play important physiological and pathological roles in neurotransmission and neurodegeneration, respectively. However, the contribution of ROS to the molecular mechanisms involved in neuronal polarity and axon elongation is not well understood. In this work, we found that loss of NOX complex function altered neuronal polarization and decreased axonal length by a mechanism that involves actin cytoskeleton dynamics. These results indicate that physiological levels of ROS produced by the NOX complex modulate hippocampal neuronal polarity and axonal growth in vitro. Summary: NOX function is involved in the establishment of neuronal polarity.

Dissecting the role of redox signaling in neuronal development

The generation of abnormally high levels of reactive oxygen species (ROS) is linked to cellular dysfunction, including neuronal toxicity and neurodegeneration. However, physiological ROS production modulates redox‐sensitive roles of several molecules such as transcription factors, signaling proteins, and cytoskeletal components. Changes in the functions of redox‐sensitive proteins may be important for defining key aspects of stem cell proliferation and differentiation, neuronal maturation, and neuronal plasticity. In neurons, most of the studies have been focused on the pathological implications of such modifications and only very recently their essential roles in neuronal development and plasticity has been recognized. In this review, we discuss the participation of NADPH oxidases (NOXs) and a family of protein‐methionine sulfoxide oxidases, named molecule interacting with CasLs, as regulated enzymatic sources of ROS production in neurons, and describes the contribution of ROS signaling to neurogenesis and differentiation, neurite outgrowth, and neuronal plasticity.

A Feed-Forward Mechanism Involving the NOX Complex and RyR-Mediated Ca2+ Release During Axonal Specification

Physiological levels of ROS support neurite outgrowth and axonal specification, but the mechanisms by which ROS are able to shape neurons remain unknown. Ca2+, a broad intracellular second messenger, promotes both Rac1 activation and neurite extension. Ca2+ release from the endoplasmic reticulum, mediated by both the IP3R1 and ryanodine receptor (RyR) channels, requires physiological ROS levels that are mainly sustained by the NADPH oxidase (NOX) complex. In this work, we explore the contribution of the link between NOX and RyR-mediated Ca2+ release toward axonal specification of rat hippocampal neurons. Using genetic approaches, we find that NOX activation promotes both axonal development and Rac1 activation through a RyR-mediated mechanism, which in turn activates NOX through Rac1, one of the NOX subunits. Collectively, these data suggest a feedforward mechanism that integrates both NOX activity and RyR-mediated Ca2+ release to support cellular mechanisms involved in axon development. SIGNIFICANCE STATEMENT High levels of ROS are frequently associated with oxidative stress and disease. In contrast, physiological levels of ROS, mainly sustained by the NADPH oxidase (NOX) complex, promote neuronal development and axonal growth. However, the mechanisms by which ROS shape neurons have not been described. Our work suggests that NOX-derived ROS promote axonal growth by regulating Rac1 activity, a molecular determinant of axonal growth, through a ryanodine receptor (RyR)-mediated Ca2+ release mechanism. In addition, Rac1, one of the NOX subunits, was activated after RyR-mediated Ca2+ release, suggesting a feedforward mechanism between NOX and RyR. Collectively, our data suggest a novel mechanism that is instrumental in sustaining physiological levels of ROS required for axonal growth of hippocampal neurons.

Un vistazo general a los Principios Latinoamericanos de Derecho de los Contratos

espanolEste trabajo expone los resultados del grupo de investigacion que creo los Principios Latinoamericanos de Derecho de los Contratos. Explica la creacion del grupo de profesores investigadores que discutieron y redactaron el texto, presenta la metodologia y analiza los principales aspectos del texto, en lo relativo a la nocion del contrato, la formacion, el cumplimiento y el incumplimiento. EnglishThis paper seeks to show the results of the research group that created the Latin American Principles of Contract Law. It explains the creation of the group of research professors who discussed and drafted the text, clarifying the methodology and presenting the main aspects of the text, regarding the notion of contract, training, compliance and non-compliance.