http://www.recercat.cat:80/handle/2072/48780 www.ub.edu Thu, 20 Jun 2013 08:37:42 GMT 2013-06-20T08:37:42Z http://www.recercat.cat:80/bitstream/id/26331/ http://www.recercat.cat:80/handle/2072/48780 http://www.recercat.cat:80/handle/2072/212072 Mutations in the cystic fibrosis gene in patients with congenital absence of the vas deferens Chillón, M.; Casals, T.; Mercier, Bernard; Bassas, Lluís; Lissens, Willy; Silber, Sherman; Romey, Marie Catherine; Ruiz Romero, Javier; Verlingue, Claudine; Claustres, Mireille; Nunes Martínez, Virginia; Férec, Claude; Estivill, Xavier, 1955- Background: Congenital bilateral absence of the vas deferens (CBAVD) is a form of male infertility in which mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene have been identified. The molecular basis of CBAVD is not completely understood. Although patients with cystic fibrosis have mutations in both copies of the CFTR gene, most patients with CBAVD have mutations in only one copy of the gene. Methods: To investigate CBAVD at the molecular level, we have characterized the mutations in the CFTR gene in 102 patients with this condition. None had clinical manifestations of cystic fibrosis. We also analyzed a DNA variant (the 5T allele) in a noncoding region of CFTR that causes reduced levels of the normal CFTR protein. Parents of patients with cystic fibrosis, patients with types of infertility other than CBAVD, and normal subjects were studied as controls. Results: Nineteen of the 102 patients with CBAVD had mutations in both copies of the CFTR gene, and none of them had the 5T allele. Fifty-four patients had a mutation in one copy of CFTR, and 34 of them (63 percent) had the 5T allele in the other CFTR gene. In 29 patients no CFTR mutations were found, but 7 of them (24 percent) had the 5T allele. In contrast, the frequency of this allele in the general population was about 5 percent. Conclusions: Most patients with CBAVD have mutations in the CFTR gene. The combination of the 5T allele in one copy of the CFTR gene with a cystic fibrosis mutation in the other copy is the most common cause of CBAVD. The 5T allele mutation has a wide range of clinical presentations, occurring in patients with CBAVD or moderate forms of cystic fibrosis and in fertile men. http://www.recercat.cat:80/handle/2072/212072 http://www.recercat.cat:80/handle/2072/212071 Mutation analysis in cystic fibrosis Gasparini, P.; Pignatti, P.F.; Novelli, G.; Dallapiccola, B.; Nunes Martínez, Virginia; Casals, T.; Estivill, Xavier, 1955-; Fernández, E.; Balassopoulou, A.; Loukopoulos, D.; Lavinha, J.; Simova, L.; Komel, R. The article by Lemna et al. (Feb. 1 issue)1 furthers the evaluation of the ΔF508 mutation, which is associated with some cases of cystic fibrosis. Although its real effect may be to help in documenting the substantial clinical variation that can occur among persons who possess the same small genetic deletion, the finding has encouraged calls for general screening... http://www.recercat.cat:80/handle/2072/212071 http://www.recercat.cat:80/handle/2072/212032 Gleevec, an Abl family inhibitor, produces a profound change in cell shape and migration (in press) Chen, Zaozao; Lessey, Elizabeth; Berginski, Matthew E.; Cao, Li; Li, Jonathan; Trepat Guixer, Xavier; Itano, Michelle; Gómez, Shawn M.; Kapustina, Maryna; Huang, Cai; Burridge, Keith; Truskey, George; Jacobson, Ken The issue of how contractility and adhesion are related to cell shape and migration pattern remains largely unresolved. In this paper we report that Gleevec (Imatinib), an Abl family kinase inhibitor, produces a profound change in the shape and migration of rat bladder tumor cells (NBTII) plated on collagen-coated substrates. Cells treated with Gleevec adopt a highly spread D-shape and migrate more rapidly with greater persistence. Accompanying this more spread state is an increase in integrin-mediated adhesion coupled with increases in the size and number of discrete adhesions. In addition, both total internal reflection fluorescence microscopy (TIRFM) and interference reflection microscopy (IRM) revealed a band of small punctate adhesions with rapid turnover near the cell leading margin. These changes led to an increase in global cell-substrate adhesion strength, as assessed by laminar flow experiments. Gleevec-treated cells have greater RhoA activity which, via myosin activation, led to an increase in the magnitude of total traction force applied to the substrate. These chemical and physical alterations upon Gleevec treatment produce the dramatic change in morphology and migration that is observed. http://www.recercat.cat:80/handle/2072/212032 http://www.recercat.cat:80/handle/2072/212031 Reinforcement versus Fluidization in Cytoskeletal Mechanoresponsiveness Krishnan, Ramaswamy; Park, Chan Young; Lin, Yu-Chun; Mead, Jere; Jaspers, Richard T.; Trepat Guixer, Xavier; Lenormand, Guillaume; Tambe, Dhananjay; Smolensky, Alexander V.; Knoll, Andrew H.; Butler, James P.; Fredberg, Jeffrey J. Every adherent eukaryotic cell exerts appreciable traction forces upon its substrate. Moreover, every resident cell within the heart, great vessels, bladder, gut or lung routinely experiences large periodic stretches. As an acute response to such stretches the cytoskeleton can stiffen, increase traction forces and reinforce, as reported by some, or can soften and fluidize, as reported more recently by our laboratory, but in any given circumstance it remains unknown which response might prevail or why. Using a novel nanotechnology, we show here that in loading conditions expected in most physiological circumstances the localized reinforcement response fails to scale up to the level of homogeneous cell stretch; fluidization trumps reinforcement. Whereas the reinforcement response is known to be mediated by upstream mechanosensing and downstream signaling, results presented here show the fluidization response to be altogether novel: it is a direct physical effect of mechanical force acting upon a structural lattice that is soft and fragile. Cytoskeletal softness and fragility, we argue, is consistent with early evolutionary adaptations of the eukaryotic cell to material properties of a soft inert microenvironment. http://www.recercat.cat:80/handle/2072/212031 http://www.recercat.cat:80/handle/2072/211942 Progressive Purkinje Cell Degeneration in tambaleante Mutant Mice Is a Consequence of a Missense Mutation in HERC1 E3 Ubiquitin Ligase Mashimo,T.; Hadjebi,O.; Amair Pinedo,F.; Tsurumi,T.; Langa,F.; Serikawa,T.; Sotelo,C.; Guénet,J.L.; Rosa López, José Luis The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domain shave been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a GuA transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N- terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology. http://www.recercat.cat:80/handle/2072/211942 http://www.recercat.cat:80/handle/2072/211941 Progressive Purkinje Cell Degeneration in tambaleante Mutant Mice Is a Consequence of a Missense Mutation in HERC1 E3 Ubiquitin Ligase Mashimo,T.; Hadjebi,O.; Amair Pinedo,F.; Tsurumi,T.; Langa,F.; Serikawa,T.; Sotelo,C.; Guénet,J.L.; Rosa López, José Luis The HERC gene family encodes proteins with two characteristic domains: HECT and RCC1-like. Proteins with HECT domain shave been described to function as ubiquitin ligases, and those that contain RCC1-like domains have been reported to function as GTPases regulators. These two activities are essential in a number of important cellular processes such as cell cycle, cell signaling, and membrane trafficking. Mutations affecting these domains have been found associated with retinitis pigmentosa, amyotrophic lateral sclerosis, and cancer. In humans, six HERC genes have been reported which encode two subgroups of HERC proteins: large (HERC1-2) and small (HERC3-6). The giant HERC1 protein was the first to be identified. It has been involved in membrane trafficking and cell proliferation/growth through its interactions with clathrin, M2-pyruvate kinase, and TSC2 proteins. Mutations affecting other members of the HERC family have been found to be associated with sterility and growth retardation. Here, we report the characterization of a recessive mutation named tambaleante, which causes progressive Purkinje cell degeneration leading to severe ataxia with reduced growth and lifespan in homozygous mice aged over two months. We mapped this mutation in mouse chromosome 9 and then performed positional cloning. We found a GuA transition at position 1448, causing a Gly to Glu substitution (Gly483Glu) in the highly conserved N- terminal RCC1-like domain of the HERC1 protein. Successful transgenic rescue, with either a mouse BAC containing the normal copy of Herc1 or with the human HERC1 cDNA, validated our findings. Histological and biochemical studies revealed extensive autophagy associated with an increase of the mutant protein level and a decrease of mTOR activity. Our observations concerning this first mutation in the Herc1 gene contribute to the functional annotation of the encoded E3 ubiquitin ligase and underline the crucial and unexpected role of this protein in Purkinje cell physiology. http://www.recercat.cat:80/handle/2072/211941 http://www.recercat.cat:80/handle/2072/211503 Integrin-Specific Mechanoresponses to Compression and Extension Probed by Cylindrical Flat-Ended AFM Tips in Lung Cells Acerbi, A.; Luque González, T.A.; Giménez Hidalgo, A.; Puig, M.; Reguart, N.; Farré Ventura, Ramon; Navajas Navarro, Daniel; Alcaraz Casademunt, Jordi Cells from lung and other tissues are subjected to forces of opposing directions that are largely transmitted through integrin-mediated adhesions. How cells respond to force bidirectionality remains ill defined. To address this question, we nanofabricated flat-ended cylindrical Atomic Force Microscopy (AFM) tips with ~1 µm2 cross-section area. Tips were uncoated or coated with either integrin-specific (RGD) or non-specific (RGE/BSA) molecules, brought into contact with lung epithelial cells or fibroblasts for 30 s to form focal adhesion precursors, and used to probe cell resistance to deformation in compression and extension. We found that cell resistance to compression was globally higher than to extension regardless of the tip coating. In contrast, both tip-cell adhesion strength and resistance to compression and extension were the highest when probed at integrin-specific adhesions. These integrin-specific mechanoresponses required an intact actin cytoskeleton, and were dependent on tyrosine phosphatases and Ca2+ signaling. Cell asymmetric mechanoresponse to compression and extension remained after 5 minutes of tip-cell adhesion, revealing that asymmetric resistance to force directionality is an intrinsic property of lung cells, as in most soft tissues. Our findings provide new insights on how lung cells probe the mechanochemical properties of the microenvironment, an important process for migration, repair and tissue homeostasis. http://www.recercat.cat:80/handle/2072/211503 http://www.recercat.cat:80/handle/2072/211299 Hexokinase 2, Glycogen Synthase and Phosphorylase Play a Key Role in Muscle Glycogen Supercompensation Irimia, JM.; Rovira, J.; Nielsen, J.; Guerrero, M.; Wojtaszewski, J.; Cussó Fresquet, M. Roser Background: Glycogen-depleting exercise can lead to supercompensation of muscle glycogen stores, but the biochemical mechanisms of this phenomenon are still not completely understood. Methods: Using chronic low-frequency stimulation (CLFS) as an exercise model, the tibialis anterior muscle of rabbits was stimulated for either 1 or 24 hours, inducing a reduction in glycogen of 90% and 50% respectively. Glycogen recovery was subsequently monitored during 24 hours of rest. Results: In muscles stimulated for 1 hour, glycogen recovered basal levels during the rest period. However, in those stimulated for 24 hours, glycogen was supercompensated and its levels remained 50% higher than basal levels after 6 hours of rest, although the newly synthesized glycogen had fewer branches. This increase in glycogen correlated with an increase in hexokinase-2 expression and activity, a reduction in the glycogen phosphorylase activity ratio and an increase in the glycogen synthase activity ratio, due to dephosphorylation of site 3a, even in the presence of elevated glycogen stores. During supercompensation there was also an increase in 59-AMP-activated protein kinase phosphorylation, correlating with a stable reduction in ATP and total purine nucleotide levels. Conclusions: Glycogen supercompensation requires a coordinated chain of events at two levels in the context of decreased cell energy balance: First, an increase in the glucose phosphorylation capacity of the muscle and secondly, control of the enzymes directly involved in the synthesis and degradation of the glycogen molecule. However, supercompensated glycogen has fewer branches. http://www.recercat.cat:80/handle/2072/211299 http://www.recercat.cat:80/handle/2072/211155 Genetic and genomic analysis modeling of germline c-MYC overexpression and cancer susceptibility Solé, Xavier; Hernández, Pilar; López de Heredia, Miguel; Armengol, Lluís; Rodríguez Santiago, Benjamín; Gómez, Laia; Maxwell, Christopher A.; Aguiló Lúcia, Fernando; Condom, E.; Abril, Jesús; Pérez Jurado, Luis; Estivill, Xavier, 1955-; Nunes Martínez, Virginia; Capellà, Gabriel; Gruber, Stephen B.; Moreno Aguado, Víctor; Pujana, Miguel Angel Background: Germline genetic variation is associated with the differential expression of many human genes. The phenotypic effects of this type of variation may be important when considering susceptibility to common genetic diseases. Three regions at 8q24 have recently been identified to independently confer risk of prostate cancer. Variation at 8q24 has also recently been associated with risk of breast and colorectal cancer. However, none of the risk variants map at or relatively close to known genes, with c-MYC mapping a few hundred kilobases distally. Results: This study identifies cis-regulators of germline c-MYC expression in immortalized lymphocytes of HapMap individuals. Quantitative analysis of c-MYC expression in normal prostate tissues suggests an association between overexpression and variants in Region 1 of prostate cancer risk. Somatic c-MYC overexpression correlates with prostate cancer progression and more aggressive tumor forms, which was also a pathological variable associated with Region 1. Expression profiling analysis and modeling of transcriptional regulatory networks predicts a functional association between MYC and the prostate tumor suppressor KLF6. Analysis of MYC/Myc-driven cell transformation and tumorigenesis substantiates a model in which MYC overexpression promotes transformation by down-regulating KLF6. In this model, a feedback loop through E-cadherin down-regulation causes further transactivation of c-MYC. Conclusion: This study proposes that variation at putative 8q24 cis-regulator(s) of transcription can significantly alter germline c-MYC expression levels and, thus, contribute to prostate cancer susceptibility by down-regulating the prostate tumor suppressor KLF6 gene. http://www.recercat.cat:80/handle/2072/211155 http://www.recercat.cat:80/handle/2072/210881 Control of hair cell excitability by vestibular primary sensory neurons. Brugeaud, Aurore; Travo, Cécile; Demêmes, Danielle; Lenoir, Marc; Llorens i Baucells, Jordi; Puel, Jean-Luc; Chabbert, Christian In the rat utricle, synaptic contacts between hair cells and the nerve fibers arising from the vestibular primary neurons form during the first week after birth. During that period, the sodium-based excitability that characterizes neonate utricle sensory cells is switched off. To investigate whether the establishment of synaptic contacts was responsible for the modulation of the hair cell excitability, we used an organotypic culture of rat utricle in which the setting of synapses was prevented. Under this condition, the voltage-gated sodium current and the underlying action potentials persisted in a large proportion of nonafferented hair cells. We then studied whether impairment of nerve terminals in the utricle of adult rats may also affect hair cell excitability. We induced selective and transient damages of afferent terminals using glutamate excitotoxicity in vivo. The efficiency of the excitotoxic injury was attested by selective swellings of the terminals and underlying altered vestibular behavior. Under this condition, the sodium-based excitability transiently recovered in hair cells. These results indicate that the modulation of hair cell excitability depends on the state of the afferent terminals. In adult utricle hair cells, this property may be essential to set the conditions required for restoration of the sensory network after damage. This is achieved via re-expression of a biological process that occurs during synaptogenesis. http://www.recercat.cat:80/handle/2072/210881 http://www.recercat.cat:80/handle/2072/210880 Base genética de la cistinuria, heterogeneidad genética Nunes Martínez, Virginia La cistinuria es una aminoaciduria que se transmite de forma autosómica rececesiva. Clínicamente se distinguen 3 formas de cistinuria, tipos I, II y III. Inicialmente identificamos un gen, implicado en el transporte de cistina y aminoácidos básicos, rBAT como responsable de esta enfermedad. Hasta la fecha se han identificado 22 mutaciotles en rBAT responsables de cistinuria. Mediante estudios de mutaciones y ligamiento genético en familias con cistinuria hemos podido demostrar que la enfermedad es heterogénea. Sólo la cistinuria de tipo I se debe a mutaciones en rBAT, mientras otros genes serán responsables de los tipos II y III. Actualmente estamos realizando estudios de exclusión genética en familias tipo no I, que nos permitirían asignar las regiones cromosómicas en las que se encuentran el gen o los genes responsables de cistinuria tipo II y/o III, paso previo para poder aislar y caracterizar dichos genes. http://www.recercat.cat:80/handle/2072/210880 http://www.recercat.cat:80/handle/2072/210810 Errors congènits del metabolisme (ECM). Pàmpols, T.; Arranz, J. A.; Artuch Iriberri, Rafael; Baiget, M.; Borja, F.; Briones, P.; Casals, T.; Chabás, A.; Coll, M. J.; Rio, E. del; Domínguez, C.; Estivill, Xavier, 1955-; Gallano, P.; Girós, M.; Martínez, M.; Maya, A.; Mila, M.; Margarit, E.; Nunes Martínez, Virginia; Oriola Ambrós, Josep La terminologia i el concepte d'Error Congènit del Metabolisme (ECM), van ser establerts per A. Garrod a principis de segle. Avui día sabem que estan causats per errors o mutacions en els gens. Degut a la naturalesa del nostre codi genètic, segons el qual les instruccions del DNA són traduïdes a un producte gènic, les proteïnes, que seran les encarregades d'executar-lo; les mutacions del DNA es tradueixen en proteïnes anòmales amb la corresponent... http://www.recercat.cat:80/handle/2072/210810 http://www.recercat.cat:80/handle/2072/210766 Mspl restriction fragment length polymorphism near exon 10 of cystic fibrosis (CFTR) gene. Bosch, A.; Kruyer, H.; Nunes Martínez, Virginia; Estivill, Xavier, 1955- Source/Description: The probe used is a 98 bp fragment amplified by PCR from a cDNA clone of the CFTR gene or from genomic DNA corresponding to exon 10, using two primers from this exon (1)... http://www.recercat.cat:80/handle/2072/210766 http://www.recercat.cat:80/handle/2072/210764 A polymorphic DNA probe from chromosome 7 (7q22) Casals, T.; Nunes Martínez, Virginia; Estivill, Xavier, 1955- Source/Description: p6a-l is a O.9 kb HindIII/BamHl genomic fragment subclone or cosmic cNX.6a in pUC13. cNX.6a was isolated from a non-methylated enriched library from the CMGT cell line Cll (1,2). http://www.recercat.cat:80/handle/2072/210764 http://www.recercat.cat:80/handle/2072/210763 PCR detection of the pKM.19/ScrfI RFLP (D7S23), a marker closely linked to the cystic fibrosis mutation Nunes Martínez, Virginia; Chillón, M.; Lench, N.; Ramsay, M.; Estivill, Xavier, 1955- Source: Description: pKM-19 is a 1.0 kb EcoRI human genomic fragment inserted in pUC13, that detects a Scrfl (CC/NGG) RFLP (1, 2). We report here the primer sequences suitable for the detection of this RFLP by PCR... http://www.recercat.cat:80/handle/2072/210763 http://www.recercat.cat:80/handle/2072/210765 Scrfl restriction fragment length polymorphism at the D7S23 locus (probe pKM.19), closely linked to cystic fibrosis Nunes Martínez, Virginia; Ramsay, M.; Casals, T.; Chillón, M.; Lench, N.; Schwartz, M.; Estivill, Xavier, 1955- Source/Description: pKM.19 is a 1.0 kb EcoRI genomic fragment in pUC13 (ref. 1,2). pPl was isolated independently but contains the same fragment as pKM.19 (ref. 3)... http://www.recercat.cat:80/handle/2072/210765 http://www.recercat.cat:80/handle/2072/210762 SSCP-polymorphism in intron 12 of the CFTR gene recognized by BclI Chillón, M.; Nunes Martínez, Virginia; Estivill, Xavier, 1955- Source/Description: SSCP analysis of intron 12 of the CFTR gene from PCR products showed an extra band in several DNA samples. Sequencing of the additional fragment extra band revealed a T- A change in the position 1898 + 152 of CFTR (Fig. 1). The change is a polymorphism which can be identified by SSCP or by BclI digestion... http://www.recercat.cat:80/handle/2072/210762 http://www.recercat.cat:80/handle/2072/210665 Reversible mitochondrial respiratory chain impairment during symptomatic hyperlactatemia associated with antiretroviral therapy Miró, Òscar; López, Sònia; Martínez, Esteban; Rodríguez Santiago, Benjamín; Blanco, José L.; Milinkovic, Ana; Miró Meda, José M.; Nunes Martínez, Virginia; Casademont, Jordi; Gatell, José M.; Cardellach, Francesc Direct evidence confirming the hypothesis that a dysfunction of the mitochondrial respiratory chain (MRC) underlies the pathogenesis of hyperlactatemia associated with highly active antiretroviral therapy (HAART) is scarce. We studied mitochondrial DNA (mtDNA) content and MRC function in the skeletal muscle of an HIV-infected patient during an episode of symptomatic hyperlactatemia. Skeletal muscle biopsy was performed during the episode when the patient was symptomatic and 3 months later when the patient was clinically recovered. Assessment of mitochondria was performed using histological, polarographic, spectrophotometrical, and Southern blot and real time PCR DNA quantification methods. The histological study disclosed extensive mitochondrial impairment in the form of ragged-red fibers or equivalents on oxidative reactions. These findings were associated with an increase in mitochondrial content and a decrease in both mitochondrial respiratory capacity and MRC enzyme activities. Mitochondrial DNA content declined to 53% of control values. Mitochondrial abnormalities had almost disappeared later when the patient became asymptomatic. Our findings support the hypothesis that MRC dysfunction stands at the basis of HAART-related hyperlactatemia. http://www.recercat.cat:80/handle/2072/210665 http://www.recercat.cat:80/handle/2072/210664 Respiratory chain dysfunction associated with multiple mitochondrial DNA deletions in antiretroviral therapy-related lipodystrophy. Miró, Òscar; Gómez, Montserrat; Pedrol, Enric; Cardellach, Francesc; Nunes Martínez, Virginia; Casademont i Pou, Jordi Highly-active antiretroviral therapy (HAART) can induce a characteristic lipodystrophy syndrome characterized by peripheral fat wasting and central adiposity, usually associated with hyperlipidaemia and insulin resistance [1,2]. Indirect data have led some authors to propose that mitochondrial dysfunction could play a role in this syndrome [3,4].To date, as recently outlined by Kakuda et al. [5] in this journal, HIV-infected patients developing lipodystrophy have not been studied for mitochondrial changes or respiratory chain capacity... http://www.recercat.cat:80/handle/2072/210664 http://www.recercat.cat:80/handle/2072/210597 TGF-β dependent regulation of oxygen radicals during transdifferentiation of activated hepatic stellate cells to myofibroblastoid cells Proell, Verena; Carmona Cuenca, Irene; Murillo, Miguel M.; Huber, Heidemarie; Fabregat Romero, Isabel; Mikulits, Wolfgang Background: The activation of hepatic stellate cells (HSCs) plays a pivotal role during liver injury because the resulting myofibroblasts (MFBs) are mainly responsible for connective tissue re-assembly. MFBs represent therefore cellular targets for anti-fibrotic therapy. In this study, we employed activated HSCs, termed M1-4HSCs, whose transdifferentiation to myofibroblastoid cells (named M-HTs) depends on transforming growth factor (TGF)-β. We analyzed the oxidative stress induced by TGF-β and examined cellular defense mechanisms upon transdifferentiation of HSCs to M-HTs. Results: We found reactive oxygen species (ROS) significantly upregulated in M1-4HSCs within 72 hours of TGF-β administration. In contrast, M-HTs harbored lower intracellular ROS content than M1-4HSCs, despite of elevated NADPH oxidase activity. These observations indicated an upregulation of cellular defense mechanisms in order to protect cells from harmful consequences caused by oxidative stress. In line with this hypothesis, superoxide dismutase activation provided the resistance to augmented radical production in M-HTs, and glutathione rather than catalase was responsible for intracellular hydrogen peroxide removal. Finally, the TGF-β/NADPH oxidase mediated ROS production correlated with the upregulation of AP-1 as well as platelet-derived growth factor receptor subunits, which points to important contributions in establishing antioxidant defense. Conclusion: The data provide evidence that TGF-β induces NADPH oxidase activity which causes radical production upon the transdifferentiation of activated HSCs to M-HTs. Myofibroblastoid cells are equipped with high levels of superoxide dismutase activity as well as glutathione to counterbalance NADPH oxidase dependent oxidative stress and to avoid cellular damage. http://www.recercat.cat:80/handle/2072/210597 http://www.recercat.cat:80/handle/2072/210596 Growth factor- and cytokine-driven pathways governing liver stemness and differentiation. Sánchez, Aránzazu; Fabregat Romero, Isabel Liver is unique in its capacity to regenerate in response to injury or tissue loss. Hepatocytes and other liver cells are able to proliferate and repopulate the liver. However, when this response is impaired, the contribution of hepatic progenitors becomes very relevant. Here, we present an update of recent studies on growth factors and cytokine-driven intracellular pathways that govern liver stem/progenitor cell expansion and differentiation, and the relevance of these signals in liver development, regeneration and carcinogenesis. Tyrosine kinase receptor signaling, in particular, c-Met, epidermal growth factor receptors or fibroblast growth factor receptors, contribute to proliferation, survival and differentiation of liver stem/progenitor cells. Different evidence suggests a dual role for the transforming growth factor (TGF)-β signaling pathway in liver stemness and differentiation. On the one hand, TGF-β mediates progression of differentiation from a progenitor stage, but on the other hand, it contributes to the expansion of liver stem cells. Hedgehog family ligands are necessary to promote hepatoblast proliferation but need to be shut off to permit subsequent hepatoblast differentiation. In the same line, the Wnt family and β-catenin/T-cell factor pathway is clearly involved in the maintenance of liver stemness phenotype, and its repression is necessary for liver differentiation during development. Collectively, data indicate that liver stem/progenitor cells follow their own rules and regulations. The same signals that are essential for their activation, expansion and differentiation are good candidates to contribute, under adequate conditions, to the paradigm of transformation from a pro-regenerative to a pro-tumorigenic role. From a clinical perspective, this is a fundamental issue for liver stem/progenitor cell-based therapies. http://www.recercat.cat:80/handle/2072/210596 http://www.recercat.cat:80/handle/2072/210595 Dysregulation of apoptosis in hepatocellular carcinoma cells Fabregat Romero, Isabel Hepatocellular carcinoma (HCC) is a major health problem, being the sixth most common cancer world-wide. Dysregulation of the balance between proliferation and cell death represents a pro-tumorigenic principle in human hepatocarcinogenesis. This review updates the recent relevant contributions reporting molecular alterations for HCC that induce an imbalance in the regulation of apoptosis. Alterations in the expression and/or activation of p53 are frequent in HCC cells, which confer on them resistance to chemotherapeutic drugs. Many HCCs are also insensitive to apoptosis induced either by death receptor ligands, such as FasL or TRAIL, or by transforming growth factor-beta (TGF-beta). Although the expression of some pro-apoptotic genes is decreased, the balance between death and survival is dysregulated in HCC mainly due to overactivation of anti-apoptotic pathways. Indeed, some molecules involved in counteracting apoptosis, such as Bcl-XL, Mcl-1, c-IAP1, XIAP or survivin are over-expressed in HCC cells. Furthermore, some growth factors that mediate cell survival are up-regulated in HCC, as well as the molecules involved in the machinery responsible for cleavage of their pro-forms to an active peptide. The expression and/or activation of the JAK/STAT, PI3K/AKT and RAS/ERKs pathways are enhanced in many HCC cells, conferring on them resistance to apoptotic stimuli. Finally, recent evidence indicates that inflammatory processes, as well as the epithelial-mesenchymal transitions that occur in HCC cells to facilitate their dissemination, are related to cell survival. Therefore, therapeutic strategies to selectively inhibit anti-apoptotic signals in liver tumor cells have the potential to provide powerful tools to treat HCC. http://www.recercat.cat:80/handle/2072/210595 http://www.recercat.cat:80/handle/2072/210594 Alzheimer's disease modifies progenitor cell expression of monoamine oxidase B in the subventricular zone Pugliese, Marco; Rodríguez Allué, Manuel José; Gimeno-Bayón J.; Pujadas, L.; Billett, E.E.; Wells, C.; Mahy Gehenne, Josette Nicole In the adult brain, progenitor cells remaining in the sub-ventricular zone (SVZ) are frequently identified as glial fibrillary acidic protein (GFAP)-positive cells that retain attributes reminiscent of radial glia. Because the very high expression of monoamine oxidase B (MAO-B) in the subventricular area has been related to epithelial and astroglial expression, we sought to ascertain whether it was also expressed by progenitor cells of human control and Alzheimer"s disease (AD) patients. In the SVZ, epithelial cells and astrocyte-like cells pre-sented rich MAO-B activity and immunolabeling. Nes-tin-positive cells were found in the same area, showing a radial glia-like morphology. When coimmunostaining and confocal microscopy were performed, most nestin-positive cells showed MAO-B activity and labeling. The increased progenitor activity in SVZ proposed for AD patients was confirmed by the positive correlation between the SVZ nestin/MAO-B ratio and the progres- sion of the disease. Nestin/GFAP-positive cells, devoid of MAO-B, can represent a distinct subpopulation of an earlier phase of maturation. This would indicate that MAO-B expression takes place in a further step of nes-tin/GFAP-positive cell differentiation. In the early AD stages, the discrete MAO-B reduction, different from the severe GFAP decrease, would reflect the capacity of this population of MAO-B-positive progenitor cells to adapt to the neurodegenerative process. http://www.recercat.cat:80/handle/2072/210594 http://www.recercat.cat:80/handle/2072/210541 Overexpression of guanylate cyclase activating protein 2 in rod photoreceptors in vivo leads to morphological changes at the synaptic ribbon. López del Hoyo, Natalia; Fazioli, Lucrezia; López Begines, Santiago; Fernández Sánchez, Laura; Cuenca, Nicolás; Llorens i Baucells, Jordi; De la Villa, Pedro; Méndez, Ana Guanylate cyclase activating proteins are EF-hand containing proteins that confer calcium sensitivity to retinal guanylate cyclase at the outer segment discs of photoreceptor cells. By making the rate of cGMP synthesis dependent on the free intracellular calcium levels set by illumination, GCAPs play a fundamental role in the recovery of the light response and light adaptation. The main isoforms GCAP1 and GCAP2 also localize to the synaptic terminal, where their function is not known. Based on the reported interaction of GCAP2 with Ribeye, the major component of synaptic ribbons, it was proposed that GCAP2 could mediate the synaptic ribbon dynamic changes that happen in response to light. We here present a thorough ultrastructural analysis of rod synaptic terminals in loss-of-function (GCAP1/GCAP2 double knockout) and gain-of-function (transgenic overexpression) mouse models of GCAP2. Rod synaptic ribbons in GCAPs−/− mice did not differ from wildtype ribbons when mice were raised in constant darkness, indicating that GCAPs are not required for ribbon early assembly or maturation. Transgenic overexpression of GCAP2 in rods led to a shortening of synaptic ribbons, and to a higher than normal percentage of club-shaped and spherical ribbon morphologies. Restoration of GCAP2 expression in the GCAPs−/− background (GCAP2 expression in the absence of endogenous GCAP1) had the striking result of shortening ribbon length to a much higher degree than overexpression of GCAP2 in the wildtype background, as well as reducing the thickness of the outer plexiform layer without affecting the number of rod photoreceptor cells. These results indicate that preservation of the GCAP1 to GCAP2 relative levels is relevant for maintaining the integrity of the synaptic terminal. Our demonstration of GCAP2 immunolocalization at synaptic ribbons at the ultrastructural level would support a role of GCAPs at mediating the effect of light on morphological remodeling changes of synaptic ribbons. http://www.recercat.cat:80/handle/2072/210541 http://www.recercat.cat:80/handle/2072/210484 Mouse hepatic oval cells require Met-dependent PI3K to impair TGF-β-Induced oxidative stress and apoptosis. Martínez Palacián, Adoración; Del Castillo, Gaelle; Suárez Causado, Amileth; García Álvaro, María; De la Morena Frutos, Diego; Fernández, Margarita; Roncero, Cesáreo; Fabregat Romero, Isabel; Herrera, Blanca; Sánchez, Aránzazu We have previously shown that oval cells harboring a genetically inactivated Met tyrosine kinase (Met−/− oval cells) are more sensitive to TGF-β-induced apoptosis than cells expressing a functional Met (Metflx/flx), demonstrating that the HGF/Met axis plays a pivotal role in oval cell survival. Here, we have examined the mechanism behind this effect and have found that TGF-β induced a mitochondria-dependent apoptotic cell death in Metflx/flx and Met−/− oval cells, associated with a marked increase in levels of the BH3-only proteins Bim and Bmf. Bmf plays a key role during TGF-β-mediated apoptosis since knocking down of BMF significantly diminished the apoptotic response in Met-/- oval cells. TGF-β also induced oxidative stress accompanied by NADPH oxidase 4 (Nox4) mRNA up-regulation and decreased protein levels of antioxidant enzymes. Antioxidants inhibit both TGF-β-induced caspase 3 activity and Bmf up-regulation, revealing an oxidative stress-dependent Bmf regulation by TGF-β. Notably, oxidative stress-related events were strongly amplified in Met−/− oval cells, emphasizing the critical role of Met in promoting survival. Pharmacological inhibition of PI3K did impair HGF-driven protection from TGF-β-induced apoptosis and increased sensitivity of Metflx/flx oval cells to TGF-ß by enhancing oxidative stress, reaching apoptotic indices similar to those obtained in Met−/− oval cells. Interestingly, both PI3K inhibition and/or knockdown itself resulted in caspase-3 activation and loss of viability in Metflx/flx oval cells, whereas no effect was observed in Met−/− oval cells. Altogether, results presented here provide solid evidences that both paracrine and autocrine HGF/Met signaling requires PI3K to promote mouse hepatic oval cell survival against TGF-β-induced oxidative stress and apoptosis. http://www.recercat.cat:80/handle/2072/210484 http://www.recercat.cat:80/handle/2072/210483 NADPH oxidase NOX4 mediates stellate cell activation and hepatocyte cell death during liver fibrosis development. Sancho, Patrícia; Mainez Villoro, Jessica; Crosas Molist, Eva; Roncero, César; Fernández Rodriguez, Conrado M.; Pinedo, Fernando; Huber, Heidemarie; Eferl, Robert; Mikulits, Wolfgang; Fabregat Romero, Isabel A role for the NADPH oxidases NOX1 and NOX2 in liver fibrosis has been proposed, but the implication of NOX4 is poorly understood yet. The aim of this work was to study the functional role of NOX4 in different cell populations implicated in liver fibrosis: hepatic stellate cells (HSC), myofibroblats (MFBs) and hepatocytes. Two different mice models that develop spontaneous fibrosis (Mdr2−/−/p19ARF−/−, Stat3Δhc/Mdr2−/−) and a model of experimental induced fibrosis (CCl4) were used. In addition, gene expression in biopsies from chronic hepatitis C virus (HCV) patients or non-fibrotic liver samples was analyzed. Results have indicated that NOX4 expression was increased in the livers of all animal models, concomitantly with fibrosis development and TGF-β pathway activation. In vitro TGF-β-treated HSC increased NOX4 expression correlating with transdifferentiation to MFBs. Knockdown experiments revealed that NOX4 downstream TGF-β is necessary for HSC activation as well as for the maintenance of the MFB phenotype. NOX4 was not necessary for TGF-β-induced epithelial-mesenchymal transition (EMT), but was required for TGF-β-induced apoptosis in hepatocytes. Finally, NOX4 expression was elevated in patients with hepatitis C virus (HCV)-derived fibrosis, increasing along the fibrosis degree. In summary, fibrosis progression both in vitro and in vivo (animal models and patients) is accompanied by increased NOX4 expression, which mediates acquisition and maintenance of the MFB phenotype, as well as TGF-β-induced death of hepatocytes. http://www.recercat.cat:80/handle/2072/210483 http://www.recercat.cat:80/handle/2072/210421 ROS production is essential for the apoptotic function of E2F1 in pheochromocytoma and neuroblastoma cell lines Espada, Lilia; Meo-Evoli, Nathalie; Sancho, Patrícia; Real, Sebastian; Fabregat Romero, Isabel; Ambrosio Viale, Santiago; Tauler Girona, Albert In this study we demonstrate that accumulation of reactive oxygen species (ROS) is essential for E2F1 mediated apoptosis in ER-E2F1 PC12 pheochromocytoma, and SH-SY5Y and SK-N-JD neuroblastoma stable cell lines. In these cells, the ER-E2F1 fusion protein is expressed in the cytosol; the addition of 4-hydroxytamoxifen (OHT) induces its translocation to the nucleus and activation of E2F1target genes. Previously we demonstrated that, in ER-E2F1 PC12 cells, OHT treatment induced apoptosis through activation of caspase-3. Here we show that caspase-8 activity did not change upon treatment with OHT. Moreover, over-expression of Bcl-xL arrested OHT-induced apoptosis; by contrast, over-expression of c-FLIP, did not have any effect on OHT-induced apoptosis. OHT addition induces BimL expression, its translocation to mitochondria and activation of Bax, which is paralleled by diminished mitochondrial enrichment of Bcl-xL. Treatment with a Bax-inhibitory peptide reduced OHT-induced apoptosis. These results point out the essential role of mitochondria on the apoptotic process driven by E2F1. ROS accumulation followed E2F1 induction and treatment with the antioxidant N-acetylcysteine, inhibited E2F1-induced Bax translocation to mitochondria and subsequent apoptosis. The role of ROS in mediating OHT-induced apoptosis was also studied in two neuroblastoma cell lines, SH-SY5Y and SK-N-JD. In SH-SY5Y cells, activation of E2F1 by the addition of OHT induced ROS production and apoptosis, whereas over-expression of E2F1 in SK-N-JD cells failed to induce either response. Transcriptional profiling revealed that many of the genes responsible for scavenging ROS were down-regulated following E2F1-induction in SH-SY5Y, but not in SK-N-JD cells. Finally, inhibition of GSK3β blocked ROS production, Bax activation and the down regulation of ROS scavenging genes. These findings provide an explanation for the apparent contradictory role of E2F1 as an apoptotic agent versus a cell cycle activator. http://www.recercat.cat:80/handle/2072/210421 http://www.recercat.cat:80/handle/2072/210420 Cell fusion reprogramming leads to a specific hepatic expression pattern during mouse bone marrow derived hepatocyte formation In Vivo Quintana Bustamante, Oscar; Grueso, Esther; García Escudero, Ramón; Arza, Elvira; Álvarez Barrientos, Alberto; Fabregat Romero, Isabel; García Bravo, María; Meza, Néstor W.; Segovia, José C. The fusion of bone marrow (BM) hematopoietic cells with hepatocytes to generate BM derived hepatocytes (BMDH) is a natural process, which is enhanced in damaged tissues. However, the reprogramming needed to generate BMDH and the identity of the resultant cells is essentially unknown. In a mouse model of chronic liver damage, here we identify a modification in the chromatin structure of the hematopoietic nucleus during BMDH formation, accompanied by the loss of the key hematopoietic transcription factor PU.1/Sfpi1 (SFFV proviral integration 1) and gain of the key hepatic transcriptional regulator HNF-1A homeobox A (HNF-1A/Hnf1a). Through genome-wide expression analysis of laser captured BMDH, a differential gene expression pattern was detected and the chromatin changes observed were confirmed at the level of chromatin regulator genes. Similarly, Tranforming Growth Factor-β1 (TGF-β1) and neurotransmitter (e.g. Prostaglandin E Receptor 4 [Ptger4]) pathway genes were over-expressed. In summary, in vivo BMDH generation is a process in which the hematopoietic cell nucleus changes its identity and acquires hepatic features. These BMDHs have their own cell identity characterized by an expression pattern different from hematopoietic cells or hepatocytes. The role of these BMDHs in the liver requires further investigation. http://www.recercat.cat:80/handle/2072/210420 http://www.recercat.cat:80/handle/2072/210375 Long-term treatment with insulin induces apoptosis in brown adipocytes: role of oxidative stress Porras, Almudena; Zuluaga, Susana; Valladares, Amparo; Alvárez, Alberto M.; Herrera, Blanca; Fabregat Romero, Isabel; Benito, Manuel Trying to define the precise role played by insulin regulating the survival of brown adipocytes, we have used rat fetal brown adipocytes maintained in primary culture. The effect of insulin on apoptosis and the mechanisms involved were assessed. Different from the known effects of insulin as a survival factor, we have found that long-term treatment (72 h) with insulin induces apoptosis in rat fetal brown adipocytes. This process is dependent on the phosphatidylinositol 3-kinase/mammalian target of rapamycin/p70 S6 kinase pathway. Short-term treatment with the conditioned medium from brown adipocytes treated with insulin for 72 h mimicked the apoptotic effect of insulin. During the process, caspase 8 activation, Bid cleavage, cytochrome c release, and activation of caspases 9 and 3 are sequentially produced. Treatment with the caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (Z-VAD), prevents activation of this apoptotic cascade. The antioxidants, ascorbic acid and superoxide dismutase, also impair this process of apoptosis. Moreover, generation of reactive oxygen species (ROS), probably through reduced nicotinamide adenine dinucleotide phosphate oxidases, and a late decrease in reduced glutathione content are produced. According to this, antioxidants prevent caspase 8 activation and Bid cleavage, suggesting that ROS production is an important event mediating this process of apoptosis. However, the participation of uncoupling protein-1, -2, and -3 regulating ROS is unclear because their levels remain unchanged upon insulin treatment for 72 h. Our data suggest that the prolonged hyperinsulinemia might cause insulin resistance through the loss of brown adipose tissue. http://www.recercat.cat:80/handle/2072/210375 http://www.recercat.cat:80/handle/2072/209846 Delayed mTOR inhibition with low dose of everolimus reduces TGFβ expression, attenuates proteinuria and renal damage in the renal mass reduction model Kurdián, Melania; Herrero Fresneda, Immaculada; Lloberas Blanch, Núria; Giménez Bonafé, Pepita; Coria, Virginia; Grande, María T.; Boggia, José; Malacrida, Leonel; Torras, Joan; Arévalo, Miguel A.; González-Martínez, Francisco; López-Novoa, José M.; Grinyo Boira, Josep M.; Noboa, Oscar The immunosuppressive mammalian target of rapamycin (mTOR) inhibitors are widely used in solid organ transplantation, but their effect on kidney disease progression is controversial. mTOR has emerged as one of the main pathways regulating cell growth, proliferation, differentiation, migration, and survival. The aim of this study was to analyze the effects of delayed inhibition of mTOR pathway with low dose of everolimus on progression of renal disease and TGFβ expression in the 5/6 nephrectomy model in Wistar rats. http://www.recercat.cat:80/handle/2072/209846