Transforming growth factor beta (TGFbeta) and Angiotensin II (AngII) signaling occurs through two distinct receptor superfamilies, the serine/threonine kinase and G protein-coupled receptors (GPCRs). Through diametric actions, TGFbeta and AngII regulate various biological responses, including cell proliferation and migration. Previously, we identified the G protein-coupled receptor kinase 2 (GRK2), which acts through a negative feedback loop mechanism to terminate Smad signaling. To investigate the impact of TGFbeta-induced GRK2 expression on GPCR signaling, we examined its effect on AngII signaling in vascular smooth muscle cells (VSMCs). We show that activation of the TGFbeta signaling cascade results in increased GRK2 expression levels, consequently inhibiting AngII-induced ERK phosphorylation and antagonizing AngII-induced VSMC proliferation and migration. The inhibitory effect of TGFbeta on AngII signaling occurs at the MEK-ERK interface and is abrogated when an anti-sense oligonucleotide directed against GRK2 is used. Thus, we conclude that TGFbeta signaling antagonizes AngII-induced VSMC proliferation and migration through the inhibition of ERK phosphorylation. GRK2 is a key factor in mediating this crosstalk.

Thesis (Ph. D.)--Virginia Commonwealth University, 2007. / Prepared for: Dept. of Physiology. Bibliography: leaves 158-171. Available online via the Internet.

La paroi, qui est une structure dynamique entourant chaque cellule végétale, joue un rôle primordial dans le développement des plantes et la transduction de signaux en réponse à des changements environnementaux et à des attaques par des pathogènes. Un des composants majeurs de la paroi primaire est la cellulose qui y est enchevêtrée avec d’autres polysaccharides tels que les hémicelluloses et les pectines. L’objectif de la thèse était de comprendre comment la synthèse de cellulose est coordonnée avec l’assemblage et le remodelage de la paroi au cours de la croissance. Au cours de ces vingt dernières années, de nombreuses molécules inhibant la synthèse de la cellulose (CBI) ont été identifiées et représentent de très bons outils pour disséquer la biologie cellulaire du remodelage pariétale. Ces CBI présentent différentes structures ce qui suggère qu’il existe plusieurs cibles pour perturber la biosynthèse de la cellulose. En étudiant 5 d’entre eux et nous avons pu confirmer qu’ils provoquaient une disparition ou une immobilisation des complexes de cellulose synthase (CSC) à la membrane plasmique. Par contre la caractérisation d’un nouveau CBI, l’Hypostuntine, a permis de montrer qu’il agissait différemment des CBI déjà décrits. L’Hypostuntine inhibe la croissance sans changement détectable des CSC au niveau de la membrane plasmique suggérant que l’Hypostuntine ne cible pas directement le CSC. Des études en microscopie électronique à transmission des parois de plantules traitées à l’HS ont permis de montrer que l’Hypostuntine interfère au niveau du mécanisme cellulaire qui coordonne la biosynthèse de la cellulose et l’assemblage d’une paroi extensible. Pour distinguer le rôle de ces six CBI étudiés, des tests de résistance croisés, des analyses de la composition pariétale et de voies de signalisation ont également été étudiées. Les résultats montrent que toutes ces réponses dépendent entre autres de THESEUS1, un récepteur like kinase de la famille des Catharanthus roseus récepteur kinases CrRLK1L mais suggèrent aussi la présence d’autres acteurs spécifiques induits par chaque CBI. / The cell wall, which is a dynamic structure surrounding each plant cell, plays a fundamental role in the plant development and in the signalling pathways in response to environmental changes and pathogen attacks. One of the major component of the primary cell wall is the cellulose polymer which is embedded between hemicelluloses and pectins. The question of the PhD was how the synthesis of cellulose is coordinated with the assembly and remodelling of the cell wall during cell expansion? During these last twenty years, a multitude of molecules that inhibit cellulose synthesis (CBI) have been identified and they represent powerful tools to dissect the cell biology of cell wall assembly. The large variation in chemical composition of the CBI suggests the existence of multiple targets for these molecules for perturbing the cellulose biosynthesis. I studied five of these molecules and confirmed that they promote a clearance or an immobilisation of the cellulose synthase complex (CSC) from the plasma membrane. The characterisation of the new molecule, named Hypostuntin, showed that it mode of action on CSC is different from the ones described for the other CBI. Hypostuntin inhibits cell growth without detectable changes in CSC activity. This suggests that the CSC is not the primary target of Hypostuntin. Transmission electron microscopy on developing cell walls suggests that Hypostuntin interferes with a process that coordinates cellulose synthesis with the assembly of an extensible wall. To ascertain the role of the six studied CBI, we performed tests of cross resistance, analysis of cell wall composition and signalling pathways. Our results show that the responses are THESEUS 1 dependent, a receptor belonging to the Catharanthus roseus receptor like kinase CrRLK1L but also suggest the presence of other actors specific of the molecules.

No description available.

No description available.

L’auto-incompatibilité est une barrière génétique qui permet à une plante de reconnaître et rejeter son propre pollen tout en acceptant le pollen d’individus moins apparentés d’un point de vue génétique. Chez les Brassicacées, l’auto-incompatibilité est contrôlée par un locus hautement polymorphe appelé le locus S, qui contient les déterminants mâle et femelle. Le stigmate exprime le déterminant femelle de l’auto-incompatibilité, S-LOCUS RECEPTOR KINASE (SRK). Chez Brassica oleracea, la localisation subcellulaire d’SRK est unique en son genre : le récepteur est localisé principalement au niveau des endosomes et dans une moindre mesure à la membrane plasmique.Nous avons étudié la fonction de la localisation endosomale de SRK chez Arabidopsis thaliana. Premièrement, nous avons réintroduit l’auto-incompatibilité chez Arabidopsis thaliana grâce à l’expression d’un allèle fonctionnel de SRK en provenance d’Arabidopsis lyrata (une espèce auto-incompatible). Deuxièmement, nous avons montré qu’un mutant perte de fonction de DYNAMIN-RELATED PROTEIN1A, une protéine requise pour l’endocytose, abolissait l’auto-incompatibilité. Nos résultats suggèrent que l’endocytose est requise pour l’auto-incompatibilité, et que SRK pourrait activer sa voie de signalisation depuis les endosomes. / Self-incompatibility is a genetic barrier by which a plant recognizes and rejects its own pollen while allowing pollen from more distantly related plants to germinate. In the Brassicacea family, it is controlled by a highly polymorphic locus called the S-locus, which contains the male and female determinants of self-incompatibility. The stigma expresses the female determinant of self-incompatibility, the plant receptor kinase (PRK) S-LOCUS RECEPTOR KINASE (SRK). In Brassica oleracea, SRK has a unique subcellular localization among PRK: the receptor is mostly localized in endosomes and to a lesser extent at the plasma membrane.We investigated the function of the endosomal localization of SRK in Arabidopsis thaliana. Firstly, we reintroduced self-incompatibility in Arabidopsis thaliana by expression of a functional SRK allele from Arabidopsis lyrata (a self-incompatible species). Secondly, we showed that a loss-of-function mutant of DYNAMIN-RELATED PROTEIN1A, a protein required for endocytosis, abolished self-incompatibility. Our results suggest that endocytosis is required for self-incompatibility, and that SRK may be signaling from endosomal compartments.

Orientador: Marcelo Augusto Christoffolete / Dissertação (mestrado) - Universidade Federal do ABC, Programa de Pós-Graduação em Biossistemas, 2012

Receptor kinases (RKs) are transmembrane proteins that have been shown to regulate an array of important processes in A. thaliana, including polar cell growth, plant reproduction, and many other plant growth processes. In this thesis, I examine RECEPTOR KINASE 7 (RK7) and FERONIA (FER), two closely related transmembrane RKs, and their effects on plant reproduction. The RK7 gene when knocked out (rk7) in conjunction with FER resulted in delayed plant growth, decreased seed yield, and a lower percentage of the seeds germinating as compared to the single FER knockout. Transgenic plants with GUS reporter driven by RK7 promoter and RK7 promoter expressed GFP-tagged RK7 (RK7-GFP) were generated to study, respectively, the expression property of the RK7 gene and characterize the location of the RK7 protein. RK7 expression increased in the papillary cells as a direct result of pollination. Transgenic plants with RK7-GFP showed that RK7 protein localizes to the plasma membrane of stigma cells and pollination induces prominent internalization of this protein. RK7 is also expressed during seedling growth. rk7 mutant seedlings had a much weaker physiological response to brassinosteroids than wild type plants, implicating an involvement of RK7 in brassinosteroid signaling. Taken together this data point to the importance of RK7 in plant growth and reproduction through its ability to receive and transduce signals.

Emerging trends in cell-therapy based tissue repair have focused on the renewable source of adult stem cells including human bone marrow-derived mesenchymal stromal cells (hMSCs). Due to immunomodulatory properties as well as a potential to differentiate into cells characteristic of all three germ layers, hMSCs provide a source of immature cells for utilization in cell-therapy based treatments. Marrow isolated adult multilineage inducible (MIAMI) cells are a homogeneous sub-population of hMSCs which maintain self-renewal potential during ex vivo expansion, in addition to efficiently undergoing trans-differentiation into neuron-like cells in vitro. Even though hMSCs have the potential to be used for neural tissue repair, the molecular mechanisms by which they are stimulated to become neuron-like cells have not been fully characterized. Therefore the work described herein focuses on the molecular mechanisms by which MIAMI cells undergo NT-3 dependent neuronal commitment. MIAMI cells express both the full length (FL-) and tyrosine kinase deficient (TKd-) isoforms of the NTRK3 receptor, the primary NT-3 receptor, at the protein level. NT-3 stimulation of MIAMI cells during neuronal commitment induced the phosphorylation of FL-NTRK3, degradation of TKd-NTRK3, downstream activation of the Mek1/2-Erk1/2 signaling cascade, and subsequent up-regulation of a limited number of pro-neuronal genes. These findings were verified using chemical inhibitors to block NTRK autophosphorylation (K252a) and Erk1/2 activation (U0126). TKd-NTRK3 is hypothesized to activate Rac1 upon NT-3 stimulation. Rac1 was found to suppress NT-3 stimulated Erk1/2 phosphorylation, as well as downstream gene expression, as determined using a Rac1 chemical inhibitor. Further characterization confirmed that Rac1b is the predominant Rac1 isoform in MIAMI cells. Rac1b siRNA mediated knock-down resulted in increased expression of the pro-neuronal genes NGN2, MAP2, NFH and NFL during NT-3 stimulation via regulation of Mek1/2-Erk1/2. Rac1b is also involved in NT-3 stimulated cell proliferation, as well as repression of CCND1 and CCNB1 mRNA expression. In an attempt to enhance neuronal differentiation of MIAMI cells, EGF and bFGF were used to pretreat MIAMI cells prior to NT-3 stimulated neuronal commitment. EGF/bFGF pretreatment increased NTRK3 and NTRK1 protein levels along with NT-3 stimulated Erk1/2 phosphorylation. In addition, bFGF versus EGF/bFGF pretreatment restricted the expression of the pro-neuronal transcription factors Ngn2 and Prox1 versus the neural stem cells self-renewal transcription factor Musashi-1, respectively. The culmination of this work provides a model for the NT-3 induced neuronal commitment of MIAMI cells in vitro, as well as insight into the neurogenic potential of MSCs for future applications in cell-therapy based tissue repair.

Self-incompatibility is a genetic barrier by which a plant recognizes and rejects its own pollen while allowing pollen from more distantly related plants to germinate. In the Brassicacea family, it is controlled by a highly polymorphic locus called the S-locus, which contains the male and female determinants of self-incompatibility. The stigma expresses the female determinant of self-incompatibility, the plant receptor kinase (PRK) S-LOCUS RECEPTOR KINASE (SRK). In Brassica oleracea, SRK has a unique subcellular localization among PRK: the receptor is mostly localized in endosomes and to a lesser extent at the plasma membrane.We investigated the function of the endosomal localization of SRK in Arabidopsis thaliana. Firstly, we reintroduced self-incompatibility in Arabidopsis thaliana by expression of a functional SRK allele from Arabidopsis lyrata (a self-incompatible species). Secondly, we showed that a loss-of-function mutant of DYNAMIN-RELATED PROTEIN1A, a protein required for endocytosis, abolished self-incompatibility. Our results suggest that endocytosis is required for self-incompatibility, and that SRK may be signaling from endosomal compartments.