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Endocrinology Vol. 139, No. 6 2892-2899
Copyright © 1998 by The Endocrine Society

ARTICLES

Mutant {alpha}-Subunit of the G Protein G12 Activates Proliferation and Inhibits Differentiation of 3T3-F442A Preadipocytes1

Delphine Denis-Henriot, Philippe de Mazancourt, Mireille Morot and Yves Giudicelli

Laboratoire de Biochimie de la Faculté de Médecine Paris-Ouest, INSERM CJF 94–02, Université René Descartes Paris V (D.D.-H., P.d.M., M.M., Y.G.), Paris; Hôpital de Poissy (D.D.-H., M.M., Y.G.), F78303 Poissy; and Hôpital R. Poincaré (P.d.M.), F92380 Garches, France

Address all correspondence and requests for reprints to: Dr. Philippe de Mazancourt, Laboratoire de Biochimie et Biologie Moléculaire, Hôpital R. Poincaré, F92380 Garches, France.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
We studied the G protein {alpha}-subunit G{alpha}12 in various tissues and cell lines. Significant amounts of G{alpha}12 were detected by immunoblots in liver, chromaffin cells, RINm5F cells, 3T3-F442A cells, and preadipocytes, but not in adipocytes, sperm, kidney, NB2A cells, or brain. To study the role of G{alpha}12 in adipose tissue differentiation, the preadipocyte cell line 3T3-F442A was transfected with wild-type G{alpha}12 or a constitutively activated mutant of G{alpha}12. Stable expression of the activated mutant of G{alpha}12 stimulated cell growth and inhibited preadipocyte differentiation. In contrast, wild-type G{alpha}12 overexpression inhibited preadipocyte differentiation, without any effect on cell proliferation. The role of G{alpha}12 on the Raf/MEK/mitogen-activating protein kinase (MAPK) cascade was studied. In confluent preadipocytes, expression of the activated mutant of G{alpha}12 induced an increase in B-Raf expression, but no change in MAPK activity. Differentiation was associated with a decrease in MAPK activity in control 3T3-F442A cells. Wild-type G{alpha}12 overexpression prevented the decrease in MAPK activity and induced MEK1, but not B-Raf, expression. Moreover, the activated mutant of G{alpha}12 induced an increase in MAPK activity and in the expression of both MEK1 and B-Raf. These data indicate that the activated mutant of G{alpha}12 stimulates the proliferation of 3T3-F442A preadipocytes, possibly through an increase in B-Raf expression, independently of the MEK/MAPK pathway, but prevents differentiation, probably through an increase in MEK1 expression and MAPK activity.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
G PROTEINS are heterotrimeric GTP-binding proteins that bind and exchange GDP for GTP as a result of their interaction with specific activated receptors (1, 2, 3, 4, 5). The subunits of G proteins are designated G{alpha} and Gß{gamma}. The G{alpha} subunits bind and hydrolyze GTP. Gß{gamma} and GTP-bound forms of G{alpha} subunits regulate the activity of specific downstream effectors until GTP is hydrolyzed to GDP and Gß{gamma} and G{alpha} reassociates. Point mutations analogous to ras-activating mutations impair the intrinsic guanosine triphosphatase activity of G{alpha} subunits, leading to persistent activation of downstream effectors in the absence of receptor activation. When constitutively activated, G{alpha} subunits induce various alterations in cell growth, ranging from activation of proliferation to neoplastic transformation in model systems (6, 7, 8, 9, 10, 11, 12). To date, 16 genes coding for 20 distinct G{alpha} subunits have been identified (5).

According to similarities in the deduced amino acid sequence, G{alpha} subunits can be divided into four classes. One of these classes is formed by G{alpha}12 and G{alpha}13 (13). G{alpha}12 has been found in all tissues examined to date (13, 14). A downstream effector molecule directly interacting with G{alpha}12 has yet to be identified. G{alpha}12, Gs{alpha}, and Gq{alpha} are involved in regulation of the mitogen-activated protein kinase (MAPK) cascade (12, 15, 16). The MAPK cascade plays an important role in the regulation of cell growth (17) and differentiation (18). MAPK (ERK1 and ERK2) activation requires dual phosphorylation on both tyrosine and threonine residues by MAPK kinase, also referred to as MEK (MAPK/ERK kinase). MEK1 and MEK2 are themselves positively regulated by phosphorylation on serine threonine residues by Raf proteins (19), which translocate to the plasma membrane once activated (20, 21).

When confluent, cells from the stromavascular fraction of adipose tissue in primary culture differentiate into adipocytes under appropriate culture conditions (22, 23). We recently showed that on differentiation of preadipocytes, G{alpha}12 expression decreases, evoking the possibility that G{alpha}12 activates preadipocyte proliferation and/or negatively controls preadipocyte differentiation (24). The constitutively activated mutant form of G{alpha}12 increases the growth rate and induces neoplastic transformation of NIH-3T3 and Rat-1 fibroblasts (11, 12). To investigate the possibility that G{alpha}12 regulates the proliferation and differentiation of preadipocytes, we stably transfected 3T3-F442A preadipocytes with G{alpha}12 or constitutively activated G{alpha}12 (Q229L), and tested for their effects on cell proliferation and differentiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell cultures
3T3-F442A preadipocytes were grown in DMEM supplemented with 10% FCS, 33 µM biotin, 100 IU/ml penicillin, and 0.1 mg/ml streptomycin until confluence and passaged every 4–6 days. For induction of differentiation, media were supplemented at confluence with 0.1 mM isobutylmethylxanthine (IBMX) and 25 nM dexamethasone. After 48 h, media were changed to Ham’s F-12 medium supplemented with 10% FCS, 15 nM insulin, 33 µM biotin, 100 IU/ml penicillin, and 0.1 mg/ml streptomycin and were then changed every other day. Cultures were harvested at confluence or after differentiation (usually 13–18 days after plating), either for immunoblotting or MAPK activity assays.

Vectors and transfection
Wild-type G{alpha}12 (G{alpha}12 wt), mutant G{alpha}12-Q229L (constitutively activated) and G{alpha}12-G228A (dominant inhibitory) complete open reading frame were cloned into the NotI site of pZipNeoSV(X) (pZN) expression vector as described previously (11). The pZN vector is a pBR322 derivative (BamHI-, Tets, Ampr) with Moloney leukemia virus long terminal repeats flanking the simian virus 40 origin of replication. The pZN vector contains a dominant selectable marker, neo, that confers resistance to geneticin (G418). 3T3-F442A cells (a subclone of the 3T3 mouse embryo cell line) were transfected with the lipofectin reagent (Life Technologies, Gaithersburg, MD) containing 1 µg vector (or 1 µg pZN alone as a control) in the absence of FCS. After 15 h at 37 C, vector-containing DMEM media were supplemented with 10% FCS. Selection of transfected cells was achieved with geneticin (G418, Life Technologies) treatment 48 h after transfection, at 500 µg/ml for 3 weeks. Clones were pooled, and aliquots were frozen in dimethylsulfoxide. Every aliquot was discarded after 8–10 passages, because 3T3-F442A cells progressively loose the ability to differentiate. For every aliquot, a 2-week treatment with geneticin (500 µg/ml) was started 4–6 days after thawing. All of the experiments described below were performed on 2–4 different aliquots of transfected cells. The efficiency of transfection was controlled by immunoblot analysis of confluent transfected 3T3-F442A preadipocytes as described below.

[3H]Thymidine incorporation
3T3-F442A preadipocytes were seeded at 104 cells/cm2 in 24-well plates, washed for 1 h in DMEM after 24 h, and exposed to DMEM containing 10% FCS and 1 µCi/well [3H]thymidine. At various time points, the wells were washed three times with 154 mM NaCl. Cells were lysed for 5 min in the presence of 1% SDS. Nucleic acids were then precipitated by 10% trichloroacetic acid for 45 min at 4 C, filtered on GF/C filters (Whatman, Clifton, NY), and counted.

Cell counting in cell growth experiments
Cells were grown in 96-well plates for the indicated times, fixed in 1% glutaraldehyde for 20 min at room temperature, washed with water, and stored at 4 C until all plates were collected. Fixed cells were then stained in 200 mM MES buffer containing 200 mM phosphoric acid, 200 mM formic acid, and 0.1% crystal violet (pH 6.0) for 15 min at room temperature. Wells were washed twice with water, then crystal violet was eluted with 100 µl 10% acetic acid, and optical densities were measured at 590 nm with a microplate spectrophotometer. Standard curves were established by counting cells after trypsinization with a hemocytometer.

Glycerol-3-phosphate dehydrogenase (GPDH) activity assay
3T3-F442A preadipocytes were sonicated at 4 C (three blasts of 10 sec; VibraCell 72434 (Bioblock, Strasbourg, France); setting, 40%) in 50 mM Tris, 1 mM EDTA, 250 mM sucrose, and 1 mM dithiothreitol, pH 7.5, at 4 C. The homogenates were centrifuged at 100,000 x g for 60 min at 4 C, and the resulting supernatants were used for GPDH assays by following NADH disappearance at 340 nm during enzyme-catalyzed dihydroxyacetone phosphate reduction (25, 26). Activities were normalized to the protein content of the 100,000 x g supernatant.

Cytosol and membrane preparation and immunoblotting
Rat preadipocyte membranes were (24), bovine brain cholate extract (27), rat liver membranes (28), rat adipocyte membranes (29), RINm5F membranes (30), and chromaffin cell fractions (31) were described previously. The 100,000 x g pellet containing the membrane fraction and the 100,000 x g supernatant containing the cytosolic fraction from 3T3-F442A, obtained as described above, were resuspended in 100 mM Tris, pH 6.8, containing 10 mM MgCl2 and used for immunoblot studies as previously described (32). Briefly, membranes and cytosols were diluted 1:1 in 2 x Laemmli’s buffer (33) containing 10% ß-mercaptoethanol and resolved on an SDS-polyacrylamide gel (11% acrylamide and 0.08% bisacrylamide). Proteins were then transferred to polyvinylidene difluoride membranes (125 mA, 20 h), blocked with Tris-buffered saline containing 0.05% Tween-20 (TTBS) and 2.5% gelatin for 2 h at room temperature, and immunoblotted (20 h at room temperature) with the antibodies in TTBS containing 2.5% gelatin. Polyvinylidene difluoride membranes were washed in TTBS. The antibody-antigen complexes were detected with either the ECL method (Amersham, Arlington Heights, IL) or 125I-labeled protein A for 2 h at room temperature in TTBS (0.2 µCi/ml) containing 2.5% gelatin, washed as described above, and exposed to Hyperfilm (Amersham) at -70 C. The protein concentration was determined by the amido black method (34) before loading onto the gel.

Anti-G{alpha}12, anti-Raf1, anti-MEK1, anti-B-Raf, and anti-MEK kinase antibodies were obtained from Santa Cruz Biotechnology (Santa Cruz, CA) and were used on 100,000 x g pellets or supernatants from 3T3-F442A cells at 0.5, 0.5, 0.1, 0.4, and 0.2 µg/ml, respectively. Anti-MEK2, anti-MEK1, anti-MEK2, anti-ERK1, and anti-ERK2 antibodies were obtained from Transduction Laboratories (Lexington, KY) and used at 0.125, 1, 0.25, and 0.05 µg/ml, respectively. The specificity of the signals was studied with peptide saturation experiments, as described in Results.

MAPK assay
MAPK activity was determined as follows. Briefly, confluent 3T3-F442A preadipocytes or 3T3-F442A preadipocytes exposed for 2 weeks to differentiation-inducing medium were sonicated at 4 C in 40 mM ß-glycerophosphate buffer (pH 7.3) containing 0.75 mM EGTA, 0.5 mM dithiothreitol, 1 mM phenylmethylsulfonylfluoride, 0.15 mM sodium vanadate, 10 µg/ml leupeptin, 5 µg/ml aprotinin, and 5 µg/ml trypsin inhibitor. Cytosolic extracts were obtained by centrifugation (100,000 x g for 30 min at 4 C), and frozen at -80 C. Cytosolic extracts were assayed for MAPK activity using the p42/p44 MAP kinase enzyme assay system (Amersham).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Expression of G{alpha}12 in various cells and cell lines
Initial studies based on RT-PCR showed a widespread distribution of G{alpha}12 messenger RNA (13). However, we recently showed that adipocytes express only small amounts of G{alpha}12 (24). Antibodies raised against the carboxyl-terminal decapeptide of the predicted G{alpha}12 gene product were used to examine the amount of G{alpha}12 in membrane fractions of various cells and cell lines (Fig. 1Go). Membranes from NIH-3T3 transfected with G{alpha}12 Q229L were used as a positive control (11). We detected G{alpha}12 in rat liver, RINm5F, bovine chromaffin cells (Fig. 1Go), and confluent rat preadipocytes in primary culture (Fig. 2Go). In contrast, we observed marginal or no staining in isolated adipocytes (Figs. 1Go and 2Go). No staining was observed in human sperm, rat kidney, Nb2A cells, bovine brain cholate extract, chromaffin granules, or heart. We also observed two bands of higher mol wt in chromaffin cell membranes. Although these bands disappeared in control peptide-saturating experiments, we do not yet know whether they are specific.



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  		Figure 1. Immunoblot analysis of membranes from various tissues and cell lines with antibodies specific for G{alpha}12. Affinity-purified antibody was used as described in Materials and Methods. Rainbow molecular mass markers (Amersham; right) are, from top to bottom, 45 and 30 kDa. Hyperfilms (Amersham) were exposed for 24 h at -70 C. Membranes used for immunoblot analysis were as follows: 75 µg rat kidney membranes, 200 µg human sperm membranes, 75 µg NB2A cell membranes, 75 µg rat liver membranes, 50 µg RINm5F cell membranes, 10 µg bovine brain cholate extract, 75 µg bovine chromaffin granules, 75 µg bovine chromaffin cell membranes, 75 µg rat heart membranes, 75 µg rat adipocyte membranes, 75 µg NIH-3T3 cell membranes, and 75 µg G{alpha}12Q229L-transfected NIH-3T3 cell membranes.

 


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  		Figure 2. Immunoblot analysis of membranes from pZipNeoSV(X)-transfected 3T3-F442A preadipocytes (ZN), G{alpha}12 wt-transfected 3T3-F442A preadipocytes (WT), G{alpha}12 Q229L-transfected 3T3-F442A preadipocytes (QL), confluent rat preadipocytes in primary culture (pread), and adipocytes prepared by collagenase digestion (adipo) with antibodies specific for G{alpha}12. Affinity-purified antibodies were used as described in Materials and Methods. Rat adipocytes and preadipocyte membranes were prepared as previously described (24). Hyperfilms (Amersham) were exposed for 24 h at -70 C.

 
Transfection of 3T3-F442A preadipocytes
Transfection of 3T3-F442A preadipocytes with pZNSV(X) vector, G{alpha}12 wt, or G{alpha}12Q229L (constitutive activation) induced the appearance of G418-resistant colonies after 3 weeks. In contrast, we could not obtain colonies in G{alpha}12 G228A (dominant inhibitory)-transfected cells.

Expression of G{alpha}12 in transfected cells
Anti-G{alpha}12 antibodies were used to examine the expression of G{alpha}12 in membrane fractions of transfected G418-resistant cells. As shown in Fig. 2Go, the antibodies detected a 45-kDa band in vector-transfected 3T3-F442A confluent preadipocytes. A stronger signal was detected in G{alpha}12 wt and G{alpha}12 Q229L transfectants.

Growth studies
Cultures transfected with G{alpha}12 Q229L were readily distinguishable from parental, control, or G{alpha}12 wt-transfected cells. Whereas control cells grew in a monolayer and showed a fusiform morphology, G{alpha}12 Q229L-transfected cells were rounded, with smaller nuclei, and foci formation was constantly observed 2 weeks after confluence in DMEM containing 10% FCS. No focus-forming activity was seen in vector- or G{alpha}12 wt-transfected cells (see Table 1Go).


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  		Table 1. Focus forming activity, saturation density, [3H]thymidine incorporation and doubling time in cells

 
Saturation density studies
Cells were grown to confluence in 10% FCS. After another 4 days, cells were trypsinized and counted. Cells expressing G{alpha}12 Q229L and G{alpha}12 wt reached a higher saturation density (see Table 1Go).

Detailed growth curves were established for cells expressing G{alpha}12 wt, G{alpha}12 Q229L, or pZN vector alone in the presence of 1% and 10% serum. Cells expressing G{alpha}12 Q229L had a significantly faster growth in the presence of either 1% or 10% serum (Fig. 3Go).



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  		Figure 3. Growth of 3T3-F442A preadipocytes in the presence of 10% FCS (A) and 1% FCS (B). •, pZipNeoSV(X)-transfected 3T3-F442A preadipocytes (ZN): {blacktriangleup}, G{alpha}12 wt-transfected 3T3-F442A preadipocytes (WT); {blacksquare}, G{alpha}12 Q229L-transfected 3T3-F442A preadipocytes (QL). Quadruplicate wells (96-well plates) were counted as described in Materials and Methods. This experiment is representative of four for 10% FCS and two for 1% FCS.

 
Doubling times were computed by linear regression analysis of the logarithmic values of cell number in the initial exponential phase of cell growth. Doubling times in 10% serum were significantly shorter for cells expressing G{alpha}12 Q229L (Table 1Go).

Thymidine incorporation in the presence of 10% serum was significantly higher in G{alpha}12 Q229L transfectants. In contrast, cells expressing G{alpha}12 wt incorporated [3H]thymidine at the same low rate as control transfectants (Table 1Go).

Effects on differentiation
When differentiation was induced as described in Materials and Methods, the adipocyte phenotype was acquired within 2 weeks in 3T3-F442A preadipocytes, as appreciated by both the appearance of lipid droplets (data not shown) and an increase in GPDH activity (data not shown). Similar data were obtained with 3T3-F442A preadipocytes transfected with pZN vector alone. In contrast, accumulation of lipid droplets was significantly reduced in G{alpha}12wt-transfected cells and was suppressed in G{alpha}12 Q229L-transfected cells (Fig. 4Go). GPDH activity, which normally appears when differentiation occurs, was significantly reduced in G{alpha}12wt-transfected preadipocytes and was comparable to that in confluent undifferentiated preadipocytes in G{alpha}12 Q229L-transfected cells (Fig. 5Go).



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  		Figure 4. Hemalun Oil Red-O staining of 3T3-F442A preadipocytes 14 days after induction of differentiation. A, pZipNeoSV(X)-transfected cells (pZN); B, G{alpha}12 wt-transfected 3T3-F442A preadipocytes (WT); C, G{alpha}12 Q229L-transfected 3T3-F442A preadipocytes (QL). Initial magnification, x20.

 


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  		Figure 5. GPDH activities in confluent 3T3-F442A preadipocytes and 3T3-F442A preadipocytes exposed to differentiation medium for 2 weeks. ZN, pZipNeoSV(X)-transfected 3T3-F442A preadipocytes; WT, G{alpha}12 wt-transfected 3T3-F442A preadipocytes; QL, G{alpha}12 Q229L-transfected 3T3-F442A preadipocytes. Results are the mean ± SEM from four different experiments.

 
Effects on MAPK activity
MAPK activity was determined on cytosolic extracts from confluent cells and cells exposed for 2 weeks to differentiation-inducing medium. As shown in Fig. 6Go, MAPK was not significantly modified by overexpression of either G{alpha}12 wt or G{alpha}12 Q229L in confluent cells. Differentiation was associated with a decrease in MAPK activity in pZN vector-transfected cells (Fig. 6Go) and in untransfected 3T3-F442A cells as well (data not shown). Overexpression of G{alpha}12 wt prevented the decrease in MAPK activity, whereas overexpression of G{alpha}12 Q229L induced a significant increase in MAPK activity when cells were exposed to differentiation-inducing medium.



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  		Figure 6. MAPK activities in confluent 3T3-F442A preadipocytes and 3T3-F442A preadipocytes exposed to differentiation medium for 2 weeks. ZN, pZipNeoSV(X)-transfected 3T3-F442A preadipocytes; WT, G{alpha}12 wt-transfected 3T3-F442A preadipocytes; QL, G{alpha}12 Q229L-transfected 3T3-F442A preadipocytes. Results are the mean ± SEM from three different experiments performed in duplicate.

 
Effects on ERK1 and ERK2 expression
ERK1 (p44 MAPK) expression was constant whatever the transfected cells and whatever the differentiation status. No variation in ERK2 (p42 MAPK) expression could be observed in pZN-, G{alpha}12wt-, and G{alpha}12 Q229L-transfected cells, but a marked decrease in ERK2 expression was found in cells exposed to the differentiation-inducing medium (Fig. 7Go).



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  		Figure 7. Effect of G{alpha}12 subunit transfection on ERK1 and ERK2 expression. Immunoblot analysis of cytosol extracts (15 µg) from confluent cells (left) or cells exposed to differentiation-inducing medium (right). pZipNeoSV(X)-transfected 3T3-F442A preadipocyte (ZN), G{alpha}12 wt-transfected 3T3-F442A preadipocyte (WT), and G{alpha}12 Q229L-transfected 3T3-F442A preadipocyte (QL) cytosol extracts were studied with antibodies specific for ERK1 and ERK2. Positive controls were human fibroblasts lysate (F; 10 µg) and cytosols extracted from kidney (K; 30 µg) for ERK1, and brain cytosols (B; 15 µg) for ERK2. This figure is representative of at least three different experiments.

 
Effects on MEK1 and MEK2 expression
Immunoblot analysis with anti-MEK1 antibody was performed on the cytosolic fraction of transfected 3T3-F442A cells. As shown in Fig. 8Go (top panel), similar amounts of MEK1 were detected in cytosols from all confluent cells studied. In contrast, MEK1 expression was increased in G{alpha}12 wt- and G{alpha}12 Q229L-transfected cells exposed to differentiation-inducing medium compared with that in pZN-transfected cells (284 ± 108% and 393 ± 83%, respectively; n = 6). MEK2 expression was constant whatever the transfected cells and whatever the differentiation status (Fig. 8Go, lower panel). The lower band observed with anti-MEK2 antibodies in differentiated G{alpha}12 wt- and G{alpha}12 Q229L-transfected cells may reflect cross-reactivity with MEK1. This was confirmed using an antibody reacting with both MEK1 and MEK2 (Fig. 8Go, middle panel).



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  		Figure 8. Effect of G{alpha}12 subunit transfection on MEK1 and MEK2 expression: immunoblot analysis of cytosol extracts (15 µg) from confluent cells (left) or cells exposed to differentiation-inducing medium (right). pZipNeoSV(X)-transfected 3T3-F442A preadipocyte (ZN), G{alpha}12 wt-transfected 3T3-F442A preadipocyte (WT), and G{alpha}12 Q229L-transfected 3T3-F442A preadipocyte (QL) cytosol extracts were studied with antibodies specific for MEK1 (50 µg cytosol extract), MEK2 (15 µg cytosol extract), or MEK1 plus MEK2 (15 µg cytosol extract). This figure is representative of at least three different experiments.

 
Effects on Raf1, B-RAF, and MEK kinase expression
Raf1 expression was analyzed by immunoblots on cytosol and membrane fractions (Fig. 9Go), and the specificity of the signal was confirmed in peptide control experiments (not shown). Similar amounts of Raf1 were found in cytosols from all confluent cells (96 ± 22% and 104 ± 45% in G{alpha}12 wt and G{alpha}12 Q229L, respectively, compared with pZN; n = 6). Raf1 was also present in the membrane fraction, and transfections did not induce any significant difference in expression (85 ± 16% and 89 ± 8% in G{alpha}12 wt and G{alpha}12 Q229L, respectively; n = 6). When cells were exposed to differentiation-inducing medium for 2 weeks, the amount of Raf1 was decreased in cytosols (49 ± 22%, 57 ± 20%, and 31 ± 24% in differentiated pZN-, G{alpha}12 wt-, and G{alpha}12 Q229L-transfected cells compared with confluent pZN cells; n = 6). After exposure to differentiation-inducing medium, Raf1 was still detected in the membrane fractions, and the signals were comparable in differentiated pZN-, G{alpha}12 wt-, and G{alpha}12 Q229L-transfected 3T3-F442A cells.



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  		Figure 9. Effect of G{alpha}12 subunit transfection on Raf-1 and B-Raf expression; immunoblot analysis of cytosol and membrane extracts (15 µg) from confluent cells (left) or cells exposed to differentiation-inducing medium (right). pZipNeoSV(X)-transfected 3T3-F442A preadipocyte (ZN), G{alpha}12 wt-transfected 3T3-F442A preadipocyte (WT), and G{alpha}12 Q229L-transfected 3T3-F442A preadipocyte (QL) extracts were studied with antibodies specific for Raf-1 (50 µg extract) or B-Raf (15 µg extract). This figure is representative of at least three different experiments.

 
B-Raf expression in cytosol was similar in pZN- and G{alpha}12 wt-transfected cells; B-Raf was present in cytosols from confluent and differentiated cells, but was not detected in the membrane fractions. B-Raf expression was increased in cytosols from G{alpha}12 Q229L-transfected cells compared with those from pZN- and G{alpha}12 wt-transfected cells (16 ± 10-fold compared with differentiated pZN cytosols; n = 3). In contrast to pZN- and G{alpha}12 wt-transfected cells, significant amounts of B-Raf were found in the membrane fractions of G{alpha}12 Q229L-transfected cells both in confluent cells and after 2 weeks in the differentiation-inducing medium. The specificity of the signal was confirmed with peptide control experiments (not shown). A 95-kDa isoform of B-Raf was detected in control tissues (brain) but was not present in 3T3-F442A cells (not shown).

G{alpha}12 stimulates the JNK pathway in COS-7 cells (35). Therefore and because MEK kinase is involved in this pathway (36, 37), MEK kinase expression was also studied, but was undetectable in our cells. A-Raf is a Raf-1 isoform highly expressed in kidney and urogenital tissue. A-Raf was not detected in 3T3-F442A cells (data not shown).


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The distribution of G{alpha}12 was originally described based on RT-PCR analysis of messenger RNA and showed a widespread distribution (13). The presence of G{alpha}12 has now been detected by immunological analysis in various tissues and cell lines (14). In this report we could demonstrate the presence of G{alpha}12 in a variety of tissues, including liver and adrenal. We also found G{alpha}12 immunoreactivity in 3T3-F442A preadipocytes. G{alpha}12 was found in normal rat preadipocytes (24) and in Rat1, mouse 3T3, and NIH-3T3 fibroblasts (11, 12). We report here high levels of G{alpha}12 expression in RINm5F insulinoma cells, as previously reported (14). We could not detect G{alpha}12 in chromaffin granules, which contain other G{alpha} subunits regulating a K+ channel (31). No staining of G{alpha}12 was seen in rat kidney and heart membranes, human sperm, NB2a cells, and bovine brain cholate extract. These data indicate that G{alpha}12, if present in these tissues, is expressed in quantities below the detection threshold for this antibody.

Confluent rat preadipocytes from the stroma vascular fraction in primary culture displayed a high content of G{alpha}12. This amount decreased when differentiation occurred, leading to marginal or no staining in in vitro differentiated preadipocytes or intact adipocytes isolated by collagenase digestion (24). These observations allow us to postulate that G{alpha}12 may be involved in the regulation of the adipogenic process.

To study the functions of G{alpha}12, we transfected 3T3-F442A preadipocytes with wild-type, constitutively activated, and dominant inhibitory G{alpha}12 or with vector alone. We were unable to obtain any clone with G{alpha}12-G228A (dominant inhibitory) transfected 3T3-F442A cells. We do not know whether this is due to failure of transfection. Alternatively, the possibility is still open that G{alpha}12 exerts some crucial roles in the control of proliferation, and that expression of a dominant inhibitory G{alpha}12 protein is lethal to the cell.

Our data demonstrate that constitutively activated G{alpha}12 highly transforms 3T3-F442A preadipocytes, as indicated by focus formation, saturation density study, and increased proliferation and DNA synthesis. This is consistent with previous reports on transfected NIH-3T3 (11, 38) or Rat-1 fibroblasts (12). However, no significant effect on doubling time or [3H]thymidine incorporation was seen with wild-type transfected 3T3-F442A cells, although saturation density was increased. It is possible that G{alpha}12 overexpression is not sufficient to induce an effect, or that the serum lot we used to induce growth did not contain enough ligand for the receptors that normally activate G{alpha}12. These ligands are thrombin in 1321N1 astroglial cells (39) and platelets (14), and thromboxane A2 in platelets (14).

The absence of differentiation observed in G{alpha}12 Q229L-transfected 3T3-F442A preadipocytes could be the result of the deregulated proliferation caused by the transforming capabilities of the mutant. However, the following points are in favor of a direct role of G{alpha}12 in the differentiation control: 1) G{alpha}12 Q229L-induced enhancement of MAPK activity is likely to directly inactivate peroxisome proliferator-activated receptor-{gamma} and thus to inhibit differentiation (40); and 2) G{alpha}12-wt-transfected cells have growth properties similar to those of control cells, but have decreased differentiation ability.

MAPK activity was not modified in confluent cells, due to the expression of G{alpha}12 wt- or G{alpha}12 Q229L. This was correlated with the expression of the two isoforms of p44 and p42 MAPK (ERK1 and ERK2), which was unchanged in control, G{alpha}12 wt-transfected, and G{alpha}12 Q229L-transfected confluent cells. These data indicate that the effects of G{alpha}12 Q229L on cell proliferation probably involve another signaling pathway. In this respect, Prasad et al. showed that G{alpha}12 activates Ras and JNK, but not the MAPK pathway, in NIH-3T3 cells (38). It was also shown by Post et al. that G{alpha}12 activates activating protein-1-mediated gene expression through a MAPK-independent pathway (41).

After 2 weeks in the differentiation-inducing medium, a time sufficient to induce the adipocyte phenotype in control cells, MAPK activity was half reduced in control cells and was associated with a decrease in ERK2 (p42), but not ERK1 (p44), expression. In contrast, MAPK activity was increased in G{alpha}12 wt- and G{alpha}12 Q229L-transfected cells compared with that in pZN-transfected cells. This increase in MAPK activity was more marked in G{alpha}12 Q229L-transfected cells, suggesting that the activated G{alpha}12 contributes to the effect we report here. However, these altered activities were not associated with any change in p44 and p42 MAPK (ERK1 and ERK2) expression in G{alpha}12 wt- or G{alpha}12 Q229L-transfected cells compared with that in control cells exposed to differentiation-inducing medium. As MAPK activities are the same in pZN-, G{alpha}12 wt-, and G{alpha}12 Q229L-transfected cells when pZN-transfected cells are confluent, this supports the idea that the MAPK differences observed when pZN-transfected cells are differentiated are a result and not a cause of the differentiation process.The stimulation of MAPK activity due to G{alpha}12 wt or G{alpha}12 Q229L that we report here differs from the data reported by Voyno-Yasenetsakaya et al. indicating that G{alpha}12 inhibits the ERK pathway (35), but the latter study was performed on transiently transfected COS-7 cells, and the effect we report here is seen only after 2 weeks in the differentiation-inducing medium.

G{alpha}12 has been reported to activate the JNK pathway (35, 38, 42), which, in turn, modulates gene transcription (43). We decided to study whether any modification of expression of upstream activators of MAPK could account for the increased MAPK activity we report here. In contrast to MEK2, whose expression was unaltered, we observed an increase in MEK1 expression in G{alpha}12 wt- and G{alpha}12 Q229L-transfected cells after exposure to differentiation inducing-medium, but not in confluent cells. This increased MEK1 expression might explain the increased MAPK activity after exposure to differentiation-inducing medium. We also observed an increase in B-Raf expression in G{alpha}12 wt- or G{alpha}12 Q229L-transfected cells both in confluent cells and after exposure to differentiation-inducing medium. These changes probably reflect the G{alpha}12 effect (either wild type or constitutively activated) on B-Raf and MEK1 expression. As the effects on MEK1 expression and MAPK activity are seen only after exposure to differentiation-inducing medium, the latter change might be due to the combined effect of G{alpha}12 and some factor present in the culture medium. Such a need for a growth factor was reported by Voyno-Yasenetsakaya et al. (12); G{alpha}12 Q229L-transfected Rat-1 fibroblasts displayed an increase in MAPK activity only in the presence of epidermal growth factor. This might be the case in preadipocytes as well.

These data also suggest that B-Raf might stimulate the proliferation process in preadipocytes, as previously reported in hematopoietic cells (44). As a matter of fact, the increased expression of B-Raf associated with the accelerated proliferation rate seen in 3T3-F442A preadipocytes after G{alpha}12 wt or G{alpha}12 Q229L transfection could lead to high amounts of B-Raf in the membrane fraction, which is believed to be its active compartment. Because MAPK was unaltered in confluent cells, we conclude that the effects of G{alpha}12 on B-Raf and proliferation described herein are unrelated to MAPK.

When G{alpha}12-Q229L-transfected cells were exposed for 2 weeks to the differentiation medium, we also observed a decrease in Raf-1 expression. This could result from Raf-1 down-regulation induced by cAMP, as reported in PC12 cells (45).

Protein kinase A activates B-raf, which, in turn, induces MAPK activity (45), although contradictory reports exist (46). We cannot rule out a role for protein kinase A in B-Raf activation. However, in G{alpha}12 Q229L-transfected cells, increased B-Raf expression was observed before IBMX-induced differentiation. These data suggest that the IBMX-induced cAMP rise is not obligatory for B-Raf activation by G{alpha}12 Q229L.

In conclusion, our data show that G{alpha}12 stimulates the proliferation and inhibits the differentiation of 3T3-F442A preadipocytes. Changes in B-Raf and MEK1 levels seem to be responsible for the proliferative and antidifferentiation activities of G{alpha}12, respectively. The effect of G{alpha}12 on the distal step of the cascade (i.e. increased MAPK activity but not amount), might be a consequence of the increased expression of some of the cascade components, namely MEK1.


   Acknowledgments
 
We thank Dr. S. J. Gutkind for the gift of the constructs.


   Footnotes
 
1 This work was supported by INSERM (CJF 94–02), the University Paris V, the Comité des Yvelines de la Ligue Contre le Cancer, and a grant from the Comité des Yvelines de la Ligue Contre le Cancer (to D.D.-H.). Back

Received September 8, 1997.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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