Transcript 2-1 - HAL
INTRODUCTION Catecholamines act through a set of specific a2 and b-adrenoceptors. Whereas the role of catecholamines in the control of white adipocyte metabolism (lipolysis)has been well established, their invlvement in preadipocyte proliferation and differentiation remains poorly understood. We previously demonstrated that a2A-adrenoceptor-stimulation increases in a Gi/Godependent pathway preadipocytes proliferation (Bouloumie et al./J.B.C/269/3025430259/1994). This effect was associated with rapid and transient tyrosine phosphorylation of the p44 and p42 mitogen-activated protein kinases (MAPK). a2-adrenergic activation of the ras/MAPK pathway is mediated by the bg-subunits of the heterotrimeric Gi/Go proteins. In preadipcytes, stimulation of a2-adrenoceptors is also associated with striking Gi/Godependent rearrangement of actin cytoskeleton. This is characterized by a rapid spreading of the cells on their growing substratum, the formation of actin stress fibers and the increase in the tyrosyl phosphorylation of the pp125 Focal Adhesion Kinase (pp125FAK). These cellular events are known to be tightly controlled by a GTPase belonging to the Ras superfamily, p21RhoA. AIM OF THE STUDY Considering the morphological changes generated by stimulation of the a2adrenoceptors stimulation in preadipocytes, we analysed (1) the involvement of p21RhoA in a2-adrenergic-dependent reorganization of actin cytoskeleton and tyrosyl phosphorylation of pp125FAK, (2) the existence of a functional coupling between a2adrenoceptors and p21RhoA and (3) the putative involvement of bg-subunits of G proteins in this coupling. This study was realized in a2AF2 preadipocytes, a cell clone derived from the 3T3F442A preadipose cell line stably expressing the human a2C10adrenergic receptor. % of retracted cells INFLUENCE OF C3 EXOENZYME ON a2-ADRENERGICDEPENDENT REGULATION OF a2AF2 PREADIPOCYTE MORPHOLOGY 80 60 40 * 20 0 - + Control - + UK C3 Influence of C3 exoenzyme on a2adrenergic-induced spreading and stress fibers formation. a2AF2 preadipocytes were treated (C3) or not (Control) with C3 exoenzyme (72h, 10mg/ml). After serum starvation, control and C3 exoenzyme-treated a2AF2 preadipocytes were exposed (+) or not (-) to 1 mM UK14304 for 15 min. (A) Cell spreading was measured by quantifying the proportion of refringent cells present in a field. Values represent the mean +/S.E of three separate experiments. (B) Actin filaments were visualized using fluorescein isothiocyanate-labeled phallooidin. INFLUENCE OF C3 EXOENZYME ON a2ADRENERGIC-DEPENDENT TYROSYL PHOSPHORYLATION OF PP125FAK AND ERK2 REGULATION IN a2AF2 PREADIPOCYTES B A ERK2* FAK - + - Control - % of maximum phosphorylation 40 20 + Control - + UK C3 - + C3 * 100 60 - + Control C3 80 0 ERK2 * 100 % of maximum phosphorylation + UK UK * 80 60 40 20 0 - + Control - + C3 a2AF2 were treated (C3) or not (Control) with C3 exoenzyme (72h, 10mg/ml). After serum starvation, control and C3 exoenzyme-treated a2AF2 preadipocytes were exposed (+) or not (-) to 1 mM UK14304 for 2 min. The level of tyrosyl phosphorylation of pp125FAK (A) and ERK2 (B) was determined after immunoprecipitation of tyrosyl-phosphorylated proteins andwestern-blot western-blot and quantified. ERK2 phosphorylation is associated with a shift (ERK2*). Values ALPHA2-ADRENERGIC STIMULATION PROMOTES GDP/GTP EXCHANGE ON RHOA IN a2AF2 PREADIPOCYTES B % GTP/(GTP+GDP) GDP GTP Control Origin 2’ 5’ 15’ * 60 * 50 40 * 30 20 10 0 Control A 2' 5' 15' UK UK [32P] labelled a2AF2 preadipocytes were exposed (UK) or not (control) to 1mM UK14304 for various time. The relative proportion of [32P] GTP and GDP present in rhoA immunoprecipitate was determined as described in Materials and Methods. (A) Representative experiment. (B) Quantification from three separate experiments. Values correspond to the mean +/- SEM. Comparison with the control was performed using Student’s t test : *, P<0.05. INFLUENCE OF THE STABLE TRANSFECTION OF THE COOH-TERMINAL DOMAIN OF bARK1 ON a2-ADRENERGIC-STIMULATED CELL SPREADING AND TYROSYL PHOSPHORYLATION OF PP125FAK, ERK2 A ERK2* B ERK2 C FAK 40 20 * Clone 50 Control 60 2’ 5’ UK 2’ 5’ FCS * 0 Control UK (15MIN) Clone 50 Control % of retracted cells 80 2’ 5’ UK 2’ 5’ FCS Clone 50 (a2AF2 preadipocytes stably transfected with bARK-CT polypeptide) were serum starved and exposed to 1mM UK14304 or FCS10% (foetal Calf Serum) treatment for time indicated. (A) Cell spreading was measured by quantifying the proportion of refringent cells present in a field. The level of tyrosyl phosphorylation of ERK2 (B) and pp125FAK (C) was determined after immunoprecipitation of tyrosyl-phosphorylated proteins andwestern-blot. Values correspond to the mean +/- SEM of three separate experiments.