2-1 - HAL

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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.