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Originally published In Press as doi:10.1074/jbc.M407581200 on October 18, 2004

J. Biol. Chem., Vol. 279, Issue 52, 54896-54904, December 24, 2004
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G{alpha}13 Signals via p115RhoGEF Cascades Regulating JNK1 and Primitive Endoderm Formation*

Yi-Nan Lee{ddagger}, Craig C. Malbon§, and Hsien-yu Wang{ddagger}

From the {ddagger}Department of Physiology and Biophysics, Diabetes and Metabolic Diseases Research Center, State University of New York Stony Brook, Stony Brook, New York 11794-8661 and the §Department of Pharmacology, State University of New York Stony Brook, Stony Brook, New York 11794-8651

Received for publication, July 7, 2004 , and in revised form, October 18, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The heterotrimeric G-protein G13 mediates the formation of primitive endoderm from mouse P19 embryonal carcinoma cells in response to retinoic acid, signaling to the level of activation of c-Jun N-terminal kinase. The signal linkage map from MEKK1/MEKK4 to MEK1/MKK4 to JNK is obligate in this G{alpha}13-mediated pathway, whereas that between G{alpha}13 and MEKKs is not known. The overall pathway to primitive endoderm formation was shown to be inhibited by treatment with Clostridium botulinum C3 exotoxin, a specific inactivator of RhoA family members. Constitutively active G{alpha}13 was found to activate RhoA as well as Cdc42 and Rac1 in these cells. Although constitutively active Cdc42, Rac1, and RhoA all can activate JNK1, only the RhoA mutant was able to promote formation of primitive endoderm, mimicking expression of the constitutively activated G{alpha}13. Expression of the constitutively active mutant form of p115RhoGEF (guanine nucleotide exchange factor) was found to activate RhoA and JNK1 activities. Expression of the dominant negative p115RhoGEF was able to inhibit activation of both RhoA and JNK1 in response to either retinoic acid or the expression of a constitutively activated mutant of G{alpha}13. Expression of the dominant negative mutants of RhoA as well as those of either Cdc42 or Rac1, but not Ras, attenuated G{alpha}13-stimulated as well as retinoic acid-stimulated activation of all three of these small molecular weight GTPases, suggesting complex interrelationships among the three GTPases in this pathway. The formation of primitive endoderm in response to retinoic acid also could be blocked by expression of dominant negative mutants of RhoA, Cdc42, or Rac1. Thus, the signal propagated from G{alpha}13 to JNK requires activation of p115RhoGEF cascades, including p115RhoGEF itself, RhoA, Cdc42, and Rac1. In a concerted effort, RhoA in tandem with Cdc42 and Rac1 activates the MEKK1/4, MEK1/MKK4, and JNK cascade, thereby stimulating formation of primitive endoderm.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Elucidating the signal linkage map for the pathways that promote differentiation and development is a major goal of cell biology. Heterotrimeric G-proteins (G-proteins) occupy a central role in cell signaling, linking the most populous class of cell surface receptors to a smaller but diverse set of effectors that includes adenylylcyclases, ion channels, phospholipase C, Tec kinases, and members of the mitogen-activated protein kinase (MAPK)1 cascades (1). In the area of differentiation and development, G-proteins play essential roles (2). In mammals, as well as in Drosophila (3), Caenorhabditis elegans (4, 5), Xenopus (6), and zebrafish (7), G-proteins have been shown to play roles in development. Planar cell polarity (8), gastrulation movements (9), axonal guidance (10, 11), and the formation of primitive and parietal endoderm phenotypes from mouse embryonal cells (12) are examples of developmental processes mediated by G-proteins.

Prominent among the downstream cascades regulated by G-proteins are those composed from members of the families of MEKK, MEK, and MAPK (13, 14). The c-Jun N-terminal protein kinase (JNK) has been shown to occupy a critical place in the signal linkage maps for control of cellular differentiation, apoptosis, and planar cell polarity (15, 16). Various heterotrimeric G-protein subunits, including G{alpha}q (17-19), G{alpha}s (20), G{alpha}i1 (21), G{alpha}i2 (12, 21, 22), G{alpha}i3 (21), G{alpha}11 (23), G{alpha}12 (17, 24-26), G{alpha}13 (17, 18, 24, 26-29), G{alpha}16 (19), and G{beta}1/{gamma}2 (4, 5, 17), are known to activate MAPK cascades regulating JNKs. G{alpha}13 regulation of downstream signaling paths is responsive to activation by a variety of hormones, including endothelin-1 (17), thrombin (18), lysophosphatidic acid (11), polycystin-1 (30), catecholamines ({alpha}1-adrenergic) (31), serotonergic ligands (32), thromboxanes (31), angiotensin-II (33), CCK-A (34), and the morphogen retinoic acid (27, 28).

The P19 embryonal carcinoma cells were established from a mouse blastoderm and are multipotent cells capable of forming all three germ layers (35). The P19 cells respond to various agents that can promote differentiation to primitive endoderm (PE), mesoderm, and ectoderm as well as to neuron-like cells (36) and beating cardiac myocytes (37, 38). Low concentrations of the well known morphogen, retinoic acid, promote PE formation via a G{alpha}13-dependent pathway, highlighting a role of G{alpha}13 also observed in early mouse development (27, 28). Mice deficient in G{alpha}13 expression display vascular system defects and intrauterine death (39). Specific features of the signal linkage map from G{alpha}13 to PE formation have been elucidated, including obligate roles for activation of c-Jun, JNK1, MEK1 and MKK4, MEKK1, and MEKK4 (27-29). In the current work, we have elucidated the effector of G{alpha}13 as the p115RhoGEF that activates the small molecular weight GTPase RhoA. Interrupting signaling at the level of p115RhoGEF, RhoA, Cdc42, or Rac1 leads to a loss of RA signaling to JNK and failure of the embryonal P19 cells to form primitive endoderm.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture and Differentiation—The P19 embryonal carcinoma cells were purchased from the American Type Culture Collection (Rockville, MD). Both the stable transfectants and the wild-type clones were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum (Hyclone, Logan, UT) in a humidified atmosphere of 6% CO2. P19 cells cultured as monolayers on tissue culture plates in Dulbecco's modified Eagle's medium with 10% serum were induced to primitive endoderm by the addition of 10 nM all-trans retinoic acid (Sigma) for 2-4 days.

Plasmids and Transfections—The pCMV5 plasmid harboring the Q226L mutant form of G{alpha}13 was obtained from Dr. Alfred Gilman (Pharmacology, University of Texas Southwestern Medical School, Dallas, TX). For empty vector controls the pCDNA3 plasmid was employed. Expressed proteins were epitope-tagged either with the hemagglutinin antigen (HA-tagged) or with c-Myc (Myc-tagged). The HA-tagged versions of constitutively active p115-RhoGEF (pCMV5-HA-p115-{Delta}N{Delta} C-RhoGEF, residues 249-802) and of the dominant negative p115-RhoGEF (pCMV5-{Delta}DH-RhoGEF with excision of residues 466-547) were gifts from Dr. Gideon Bollag, ONYX Pharmaceuticals, Richmond, CA. The HA-tagged versions of the constitutively active Cdc42 (pCDNA3-HA-Cdc42Hs(Q61L)) and dominant negative Cdc42 (pCDNA3-HA-Cdc42Hs(T17N)) as well as the constitutively active Rac1 (pCDNA3-HA-Rac1(Q61L)) and dominant negative Rac1(pCDNA3-HA-Rac1(T17N)) plasmids were a gift from Dr. Richard A. Cerione (Department of Molecular Medicine, Cornell University, Ithaca, NY). The c-Myc-tagged version of the dominant negative RhoA (pCDNA3-Myc-RhoA(T19N)) plasmid was a gift from Dr. Dafna Bar-Sagi (Department of Molecular Genetics and Microbiology, SUNY-Stony Brook, Stony Brook, NY) and subsequently was engineered with three HA tags to replace the c-Myc tag. The constitutively active version of RhoA (pCDNA3-myc-RhoA(Q63L)) plasmid was a gift from Dr. Alan Hall (CRC Oncogene and Signal Transduction Group, Medical Research Council Laboratory for Molecular Cell Biology, University College London, UK). The assays of activated GTPases made use of pulldown assays using GST fusion proteins that contain domains that bind only the activated forms of each. The plasmids employed were the pGEX-GST-PAK1CRIB plasmid (harboring the Cdc42/Rac1 binding domain of PAK1, residues 70-149) and the pGEX-GST-RBD plasmid (harboring the RhoA binding domain of Rok{alpha}, residues 809-1062), both gifts from Dr. Lu-Hai Wang (Department of Microbiology, Mount Sinai School of Medicine, New York, NY). The P19 cells were transfected with one or more plasmids using Lipofectamine. Stably transfected P19 clones were selected in the presence of the neomycin analog, G418 (400 µg/ml).

Immunoblotting—Samples (10-50 µg of protein/lane) of total cell lysates were subjected to electrophoresis in SDS on 10% polyacrylamide gels. The resolved proteins were transferred electrophoretically to nitrocellulose blots. The blots were stained with primary antibodies and the immune complexes made visible by use of a second antibody coupled with horseradish peroxidase and developed using the enhanced chemiluminescence method. The antibodies were purchased from the following sources: anti-JNK1, anti-RhoA, and anti-Cdc42 antibodies from Santa Cruz Biotechnology (Santa Cruz, CA); anti-RAC1 antibody from Upstate Biotechnology (Lake Placid, NY); anti-HA antibodies from Roche Applied Science); and anti-cytokeratin endo A (PE marker) antibody (TROMA-1) from the Developmental Studies Hybridoma Bank (University of Iowa, Iowa City, IO).

Immunoprecipitation of JNK and Activity Assays—The immunoprecipitation reactions and solid-state assay of JNK were performed as detailed earlier (27) using the rGST-c-Jun amino-terminal fusion protein as substrate for JNK activity assay. The expression vector for rGST-c-Jun was generously provided by Dr. Roger Davis (Howard Hughes Medical Institute, University of Massachusetts Medical Center, Worcester, MA).

Assay of Activated RhoA, Rac1, and Cdc42—The assay was performed essentially as described earlier (40). The synthesis of the fusion polypeptides GST-RBD and GST-PAK1CRIB encoded in their respective plasmids was induced in Escherichia coli with 0.4 mM isopropyl-{beta}-galactopyranoside at 30 °C for 4 h and affinity-purified using 50% slurry of glutathione-Sepharose 4B (Amersham Biosciences) according to the batch purification method suggested by the manufacturer.

Indirect Immunofluorescence Staining of TROMA—The staining of the endoderm-specific marker antigen cytokeratin endo A by the monoclonal antibody TROMA-1 was performed as described (41). The P19 cells were cultured, stained, and subjected to analysis by epifluorescence microscopy as described previously. As the differentiated cells often grow from monolayers to whorls of cells with multiple layers, the indirect immunofluorescence and phase contrast images may not appear to be "in focus." This artifact is unavoidable under these conditions of differentiation.

Data Analysis—For all of the experiments reported, the data are compiled from at least three independent, replicate experiments performed on separate cultures on separate occasions with highly reproducible results. The indirect immunofluorescence and phase contrast images are of representative fields of interest.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
C. botulinum C3 Exotoxin Blocks RA-induced Formation of Primitive Endoderm—Treating P19 embryonal carcinoma (P19) cells with C. botulinum C3 exotoxin (C3) for 30 h or expression of C3 exotoxin in these cells blocked the ability of RA to promote transition of these embryonal cells to primitive endoderm, as established by assay of the expression of cytokeratin endo A (endo A), a marker protein specific for PE (Fig. 1A). Endo A expression, measured using the monoclonal antibody TROMA-1, increased more than 10-fold in response to RA, but this response was blocked by treatment with C3 exotoxin. Indirect immunofluorescent staining of endo A in P19 cells treated with RA in the absence (+RA) and the presence (+RA, +C3) of C3 exotoxin provides another demonstration that RhoA activity is essential for PE formation in response to RA (Fig. 1B). Analysis of the activity of another hallmark protein of PE formation, tissue plasminogen activator (tPA), also demonstrated the ability of C3 to attenuate the formation of PE in response to RA (Fig. 1C). The activation of JNK by RA treatment (27-29), an obligate step in RA-induced PE formation, was inhibited also by treating the cells with C3 exotoxin (Fig. 1D). Taken together these studies demonstrate RhoA to be an obligate element in the signaling from RA to activation of JNK and to formation of PE.



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  		FIG. 1.
C. botulinum C3 exotoxin blocks JNK activation and formation of primitive endoderm in response to retinoic acid. P19 cells were treated with or without the C3 exotoxin and then with retinoic acid for 4-6 days. Primitive endoderm formation was measured by expression of cytokeratin endo A using immunoblotting (A), by indirect immunofluorescent staining of P19 cells with the TROMA-1 monoclonal antibody (B), and by secretion of tissue plasminogen activator (C, tPA). Both retinoic acid-induced formation of primitive endoderm (A-C) and JNK1 activation (D) is blocked by C. botulinum C3 exotoxin. JNK1 activation was measured in whole cell lysates using GST-c-Jun as the substrate (D). The data presented are representative of separate experiments performed at least three times.

 
JNK1 Activation and PE Formation Are Stimulated by Expression of Constitutively Active RhoA—The formation of PE in response to RA can be mimicked in P19 cells by the transient expression of the constitutively active Q226L mutant of G{alpha}13 (QLG{alpha}13) (27, 28). The ability of the C3 exotoxin to block PE formation focused our attention on RhoA, the best known target of C3 exotoxin action. We examined the activation status of RhoA and two other GTPases, Cdc42 and Rac1, in P19 cells stimulated to form PE either by treatment with RA or by the transient expression of QLG{alpha}13 (Fig. 2). The activation status of RhoA, Cdc42, and Rac1 was probed using assays that specifically pull down only the activated forms of each GTPase. Treatment with RA resulted in robust activation of all three GTPases (Fig. 2A). Transient expression of activated QLG{alpha}13 mutant stimulated activation of all three GTPases, mimicking the action of RA (Fig. 2B). Treating cells with RA provokes an activation of JNK1 activity (Fig. 2C), as previously shown (27). We explored whether the activation of any one of the GTPases alone, like the treatment with RA (Fig. 2C), was sufficient to activate c-Jun N-terminal kinase. The expression of the constitutively active (CA) mutants of each of the GTPases, RhoA (Q63LRhoA), Cdc42 (Q61LCdc42), and Rac1 (Q61LRac1), provoked a strong activation of JNK1 activity, nearly equivalent to that stimulated in response to RA (Fig. 2C). Expression of the CA mutant of Ras (Q61LRas), in contrast, provoked no stimulation of JNK1 activity. The levels of expression of each of the GTPases in cells expressing QLG{alpha}13 or treated with RA were essentially unchanged (data not shown).



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  		FIG. 2.
Stimulation of primitive endoderm formation in P19 cells either by retinoic acid (RA) or by expression of constitutively activated Q226LG{alpha}13 leads to activation of RhoA, Cdc42, and Rac1, whereas primitive endoderm formation is stimulated only by expression of the constitutively activated mutant of RhoA. Activation of each of the GTPases was monitored in response to either treatment with retinoic acid (A) or expression of the constitutively activated mutant form of G{alpha}13 (B). JNK1 activity was measured in whole cell lysates from cells treated with retinoic acid or constitutively activated mutants of each GTPase (C). Primitive endoderm formation was measured by immunoblotting of cytokeratin endo A expression in whole cell lysates from cells treated with retinoic acid or those transiently expressing one constitutively activated mutant of each GTPase (D). Activation of JNK1 by RA was blocked by expression of the dominant negative (DN) mutants of the small molecular weight GTPases RhoA, Cdc42, and Rac1 (E).

 
The most interesting extension of this strategy was analysis of the ability of the expressed CA GTPases to stimulate the overall pathway and, like RA, promote the formation of primitive endoderm (Fig. 2D). Whereas expression of the CA-Cdc42, CA-Rac1, and CA-RhoA each led to robust activation of JNK1 activity (Fig. 2C), only the expression of CA-RhoA was able to promote the formation of primitive endoderm (Fig. 2D). Expression of CA-Ras also failed to stimulate the formation of PE. The formation of PE must require signals from RhoA in addition to JNK activation and those mediated by Cdc42 and/or Rac1. Furthermore, expression of DN-RhoA, DN-Cdc42, or DN-Rac1 blocks JNK activation in response to treatment of the cells with the morphogen RA (Fig. 2E). These observations suggest that activation of just a single GTPase, RhoA, is sufficient to mimic either the action of RA or the transient expression of the QLG{alpha}13 on the formation of PE.

Expression of p115RhoGEF Mutants Regulates JNK1 Activation and PE Formation—The exciting possibility was explored that the guanine nucleotide exchange factor for RhoA p115RhoGEF, known to be an effector of G{alpha}13, functions in the signaling from RA to PE formation. Constitutively active p115RhoGEF, lacking the N- and C-terminal regulatory domains (p115RhoGEF, 249-802), was expressed in cells, and formation of primitive endoderm (Fig. 3A) and the activity of JNK1 were assayed (Fig. 3B). Expression of CA-p115RhoGEF provokes the formation of primitive endoderm (Fig. 3A) and activation of JNK1 (Fig. 3B), just as did expression of QLG{alpha}13 (27, 28). Because guanine nucleotide exchange factors often regulate the activity of multiple GTPases, it was important to test whether the expression of the dominant negative mutant of RhoA (T19N mutant, DN-RhoA) attenuates the ability of the CA-p115RhoGEF to stimulate JNK1 activity (Fig. 3C). Expression of DN-RhoA sharply blocked the activation of JNK1 in response to CA-p115RhoGEF. Expression of either DN-Cdc42 (T17N mutant) or DN-Rac1 (T17N mutant) suppresses the ability of CA-p115RhoGEF to activate JNK1 (Fig. 3, D and E), whereas expression of DN-Ras has no effect on the activation of JNK1 in response to CA-p115RhoGEF (Fig. 3F).



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  		FIG. 3.
Expression of constitutively activated p115RhoGEF in P19 cells promotes the formation of primitive endoderm, mimicking the action of expression of QLG{alpha}13 or treatment with retinoic acid. Transient expression of constitutively activated p115RhoGEF stimulates formation of primitive endoderm (A) and activation of JNK1 (B) that is blocked by expression of DN-RhoA or treatment with C3 exotoxin (C), DN-Cdc42 (D), or DN-Rac1 (E), but not DN-Ras (F). Expression of DN-p115RhoGEF blocks JNK1 activation in response to stimulation by treatment with retinoic acid (G) or by expression of Q226LG{alpha}13 (H). The data presented are representative of at least three separate experiments. The values displayed in the bar graphs are mean values ± S.E. from three or more separate experiments.

 
RA treatment stimulates JNK1 activity, essential for the P19 embyronal cells to commit to a primitive endoderm phenotype. We explored whether the transient expression of DN-p115RhoGEF, lacking the DH domain residues ({Delta}466-547), would influence the ability of RA to activate JNK1. Expression of DN-p115RhoGEF, but not the expression of DN-PDZRho-GEF, blocks activation of JNK1 in response to RA (Fig. 3G). The expression of DN-p115RhoGEF similarly blocks the activation of JNK1 in response to expression of the constitutively active G{alpha}13, whereas expression of DN-PDZRhoGEF has no effect on the activation of JNK1 in response to expression of the constitutively active G{alpha}13 (Fig. 3H).

DN Mutants of RhoA, Cdc42, and Rac1 Block the Ability of Q226L G{alpha}13 to Activate All Three GTPases—The relationships among these small molecular weight GTPases in the signaling cascade from G{alpha}13 to JNK activation and to PE formation in response to RA were investigated. Cells expressing QLG{alpha}13 were transiently transfected with dominant negative mutant forms of RhoA, Cdc42, and Rac1 and the activities of the GTPases determined using the pulldown assays specific for each GTPase (Fig. 4). Expression of QLG{alpha}13 results in activation of all three GTPases, as shown earlier (Fig. 2B). Transient expression of the dominant negative mutant form of RhoA (DN-RhoA) blocked the activation of endogenous RhoA as well as the activation of Cdc42 and Rac1 in response to expression of QLG{alpha}13 (Fig. 4A). The ability of DN-RhoA to impact the activation of RhoA, Cdc42, and Rac1 suggests that Cdc42 and Rac1 could be downstream of RhoA in this pathway, because RhoA is the effector for p115RhoGEF. Expression of DN-Cdc42 blocks the ability of QLG{alpha}13 to active Cdc42, as expected. Surprisingly, the expression of DN-Cdc42 was found to also block the activation of RhoA and of Rac1 in response to QLG{alpha}13 (Fig. 4B). DN-Rac1 expression blocked the ability of QLG{alpha}13 to activate Rac1 but surprisingly was found to block also the activation of Cdc42 and RhoA in response to QLG{alpha}13 (Fig. 4C). Taking into consideration the fact that p115RhoGEF activates only RhoA, these data suggest that RhoA can signal to the other GTPases, whose activation appears to be obligate for signaling from QLG{alpha}13.



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  		FIG. 4.
Expression of dominant negative versions of RhoA, Cdc42, and Rac1 suppresses the ability of QLG{alpha}13 to stimulate activation of the GTPases. Transient expression of DN-RhoA (A), DN-Cdc42 (B), or DN-Rac1 (C) blocks Q226LG{alpha}13-stimulated activation of each of these three GTPases. The data presented are representative of at least three separate experiments.

 
Expression of DN Mutants of GTPases Blocks RA-stimulated Activation—We explored whether expression of DN mutants of GTPases altered GTPase activation in response to stimulation by the morphogen RA (Fig. 5). Much like the results of the assays performed with cells expressing QLG{alpha}13, the results obtained from RA-treated cells demonstrated that RA-stimulated GTPase activity can be suppressed for all three GTPases by the expression of DN-RhoA (Fig. 5A). Expression of DN-Cdc42 (Fig. 5B) and of DN-Rac1 (Fig. 5C) was found to block the ability of RA to activate all three of these GTPases. Thus, the activation of these GTPases in response to RA is mediated by the response of G{alpha}13 to p115RhoGEF and depends on the activation of RhoA as well as Cdc42 and Rac1.



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  		FIG. 5.
Expression of dominant negative versions of RhoA, Cdc42, and Rac1 suppresses the ability of retinoic acid to stimulate activation of the GTPases. Transient expression of DN-RhoA (A), DN-Cdc42 (B), or DN-Rac1 (C) blocks RA-stimulated activation of all three GTPases. The data presented are representative of at least three separate experiments.

 
Formation of Primitive Endoderm by RA Is Blocked by Expression of DN-RhoA, DN-Cdc42, or DN-Rac1—The roles of G{alpha}13, MEKK1/4, MEK1/MKK4, and JNK1 on RA-stimulated PE formation were established as essential through the use of dominant negative versions of each signaling element. To extend the current analysis from activation of GTPase and of JNK1, we explored the effects of the expression of the DN mutant forms of the GTPases on the ability of RA to promote the formation of primitive endoderm, using the expression of the PE marker protein cytokeratin endo A (Fig. 6). Expression of DN-RhoA suppresses the formation of PE in response to RA (Fig. 6A) as did the expression of the dominant negative forms of either Cdc42 or Rac1.



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  		FIG. 6.
Formation of primitive endoderm in P19 cells in response to retinoic acid is blocked by transient expression of DN-RhoA, DN-Cdc42, or DN-Rac1. Primitive endoderm formation was detected by immunoblotting of the PE marker cytokeratin endo A (A) or by indirect immunofluorescence of cells stained with the TROMA-1 monoclonal antibody (B). The data presented are representative of at least three separate experiments.

 
Using TROMA-1 monoclonal antibody against cytokeratin endo A to stain the fixed P19 cells, it was possible to perform indirect immunofluorescence and examine the effects of the dominant negative mutant forms of key signaling elements revealed in the current work (Fig. 6B). Positive staining of the P19 clones was strong for clones treated with RA alone but suppressed by expression of either DN-p115RhoGEF or DN-RhoA. Expression of DN-Cdc42 or DN-Rac1 again revealed the complex relationships among these three GTPases. Only one of these GTPases, RhoA, is known to be a direct effector for p115RhoGEF. The other GTPases, Cdc42 and Rac1, appear to play obligate, supporting roles in RA signaling to primitive endoderm formation. Further evidence demonstrating the ability of RhoA to signal downstream to Cdc42 and Rac1 in this signaling pathway was provided by additional experiments performed with C3 exotoxin that inactivated RhoA (Fig. 7). Treatment with C3 exotoxin leads to blockade of the activation of both Cdc42 and Rac1 in response to the expression of CA-p115RhoGEF.



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  		FIG. 7.
Treatment of P19 cells with C3 exotoxin blocks activation of both Cdc42 and Rac1 in response to the expression of constitutively activated p115RhoGEF (CA-p115). For details, see the legends to Figs. 1 and 3.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
The original proposal that G-proteins play important roles in cell differentiation and development (2) has been tested in a number of model systems. Studies performed in Drosophila (3), C. elegans (4), Xenopus (42), zebrafish (7), and more recently mammals (43, 44) clearly establish heterotrimeric G-proteins in signaling pathways controlling development. Results from gene disruption studies in mice establish the role of G-proteins (39, 43), but the analysis of downstream signaling from these extrinsic membrane proteins to nuclear events regulating transcription has been a difficult task. The mouse P19 embryonal cell is a well accepted model for study of differentiation and aspects of early mouse development (35), as these cells can be coaxed into primitive endoderm, parietal endoderm, visceral endoderm, neuron-like cells, and beating cardiac myocytes under the proper conditions. Retinoic acid is a morphogen that at low concentrations is capable of stimulating P19 cell differentiation to primitive endoderm (38). Earlier it was shown that this morphogen induces the expression of G{alpha}13 and that this expression, also observed in early stage mouse embryonic development, is obligate for the formation of PE (28). Expression of the constitutively active mutant of G{alpha}13 alone is sufficient, in the absence of the morphogen RA, to promote formation of PE (Fig. 8) (27, 28). At the level of transcription it was shown that phosphorylation of c-Jun by JNK1 was essential for G{alpha}13 signaling to PE formation, as expression of dominant negative JNK1 was able to block either RA-induced or QLG{alpha}13-induced PE formation (27). The JNK cascade from MEKK1/4 to MEK1/MKK4 to JNK1 was essential for G{alpha}13-mediated stimulation of PE formation (29), and upstream of MEKK were several possible signaling elements including the MEKK-regulator p21-activated kinase (PAK) (45) as well as the small molecular weight GTPases such as Rac1, Cdc42, and RhoA (46-48).



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  		FIG. 8.
Schematic of the regulation of primitive endoderm formation by retinoic acid in P19 embryonal cells: mediation by p115RhoGEF. The scheme displays the information on retinoic acid (morphogen)-stimulated formation of primitive endoderm (PE) from P19 embryonal stem cells. Constitutively active versions of signaling molecules whose expression is capable of stimulating PE formation are identified in green. Dominant negative mutant versions of signaling molecules whose expression effectively attenuates or blocks PE formation are shown in red. The flow of information from G{alpha}13 to activation of JNK1 is displayed in blue.

 
The goal of the current research was to detail the immediate downstream signaling from G{alpha}13 to the level of PAK and/or of the proximal end of the JNK cascade, i.e. MEKKs. MEKK1 and -4, which mediate RA-induced PE formation in these P19 cells (27-29), have been shown to specifically associate with Rac1 and Cdc42, and kinase-inactive mutants of MEKK1/4 can block Rac/Cdc42 stimulation of JNK activity (49). In some cells, expression of dominant negative mutants of MEKK1/4 do not affect PAK activation of JNK, revealing that some PAK-regulated JNK signaling can be independent of MEKKs (49). Thus our task was to focus on the signaling from G{alpha}13 to MEKK1/4. Immediately downstream of G{alpha}13/G{alpha}12 are several possible candidates, including Tec kinases (50) and several RhoGEFs, including p115RhoGEF (51), LARG (52), and PDZ-RhoGEF (53). Each of these RhoGEFs when expressed in the proper cellular context may be able to specifically activate RhoA in response to activation of G{alpha}13.

Our studies with the C3 exotoxin, making use of its ability to ADP ribosylate and to inactivate specifically RhoA, as well as its ability to suppress PE formation, pointed us to RhoA as an prime downstream suspect for G{alpha}13 regulation for study in this signaling paradigm (Fig. 8). The linkage between G{alpha}13 and RhoA was uncovered through a series of studies both in vitro and in vivo that demonstrated p115RhoGEF to be an effector of G{alpha}13 that can activate RhoA by virtue of its ability to enhance guanine nucleotide exchange of this GTPase (51, 54). Activated G{alpha}13 binds to an RGS-like domain on the N terminus of p115RhoGEF with the RGS domain acting like a GTPase-activating protein stimulating the GTPase activity of G{alpha}13, which, in turn, enhances the GEF activity of p115RhoGEF specifically (55, 56). Although several other GEFs have been shown able to enhance guanine nucleotide exchange of RhoA (57), e.g. Lbc RhoGEF, p190RhoGEF, PDZ-RhoGEF, and LARG, we have shown that expression of the dominant negative mutant of p115RhoGEF blocks G{alpha}13-mediated activation of JNK1 as well as of PE formation. PDZ-RhoGEF and LARG, like p115RhoGEF, each display the presence of an RGS-like sequence. These two RhoGEF are not likely involved in G{alpha}13-mediated activation of JNK and PE formation studied herein, because PDZRhoGEF has been shown to be unable to interact with G{alpha}13 (56). Furthermore, treatment of the cells with oligodeoxynucleotides antisense to Lbc-RhoGEF was without effect on RA-stimulated formation of primitive endoderm.2 These observations argue forcefully for a central role of this G{alpha}13-sensitive RhoA GEF in this signaling cascade.

Expression of a dominant negative mutant of p115RhoGEF, as well as of the DN-RhoA, was sufficient to block both RA- and QLG{alpha}13-stimulated activation of JNK1 and formation of PE, supporting the central tenet that p115RhoGEF acts as the effector of G{alpha}13. Expression of the dominant negative mutant of either Cdc42 or Rac1 blocked JNK activation, but expression of the constitutively active RhoA uniquely was able to activate JNK and promote PE formation, mimicking both QLG{alpha}13 and RA treatment. Study of the DN and CA mutant forms of RhoA, Cdc42, and Rac1 suggests that signaling is propagated from G{alpha}13 to p115RhoGEF to RhoA and on to Cdc42 and Rac1. Signaling by Cdc42 and Rac1, like that of RhoA, was essential for the propagation of the signals from G{alpha}13 to JNK1 activation and to PE formation. In contrast, expression of either CA-Cdc42 or CA-Rac1, unlike expression of CA-RhoA, was not sufficient to promote PE formation. These small molecular weight GTPases play prominent roles in signaling to the level of cell morphology, with Cdc42 stimulating filopodia production, whereas Rac1 stimulates formation of lamellipodia (58). Our understanding of how these changes in cell morphology are essential for G{alpha}13 signaling to the level of primitive endoderm formation remains to be explored. The recent observation that p115RhoGEF and RhoA can bind to MEKK, especially MEKK1 directly, might suggest an alternative pathway from G{alpha}13 to MEKK activation (55, 59). Expression of the dominant negative form of either Cdc42 or Rac1 blocked G{alpha}13-induced formation of PE, suggesting that direct action of RhoA on MEKK activity may not be sufficient to provoke PE formation in the absence of Cdc42 and Rac1 signaling.

Other studies contribute to understanding the regulation of small molecular weight GTPases in the signaling cascade from G{alpha}13 to JNK1 (47). As noted above, Cdc42 is known to regulate PAK5 and thereby MEKKs (60). In NF-{kappa}B activation, Rac1 has been shown to regulate MEKKs (61). The mixed lineage kinase MLK3 has been shown to modulate the signaling from both Cdc42 and Rac1 to the MAPK cascade (61). Other examples exist for G{alpha}13 signaling to effector pathways other than the MAPK cascade, e.g. G{alpha}13 regulation of the Na+-H+ exchanger NHE1 is regulated via RhoA and thereby Cdc42 (62).

The current studies reveal major facets of the linkage between G{alpha}13 and the formation of primitive endoderm by embryonal carcinoma cells (Fig. 8). Signals in this pathway are controlled by two interacting cascades: 1) the activation of p115RhoGEF cascade by G{alpha}13 to the level of RhoA, Rac1, and Cdc42 and 2) the activation of MEKK1/4 to the level of phosphorylation of c-Jun by JNK1. It is important to highlight that constitutive activation of only some of the elements in either of the two cascades can promote the formation of PE as RA does (Fig. 8). Expression of the CA-G{alpha}13 obviates the need for RA, as does expression of the CA-p115RhoGEF. Expression of CA-RhoA, but not the CA versions of Cdc42 or Rac1, provokes the formation of PE as RA does. In the MEKK to JNK cascade, expression of only CA-MEKK1 or CA-MEKK4 is able to promote PE formation. Expression of CA-MEK1/MKK4 or CA-JNK1, in sharp contrast, fails to stimulate the formation of PE and demonstrates the need for additional signals beyond simple activation of JNK1 to promote PE formation.

Expression of dominant negative mutants of some of the signaling elements downstream of G{alpha}13 are effective in blocking morphogen-induced PE formation (Fig. 8). Expression of DN-G{alpha}13, DN-p115RhoGEF, and any one of the GTPases studied herein blocks RA-induced PE formation. Expression of DN-MEKK4 or DN-JNK1 also blocks PE formation in response to RA. In sharp contrast, expression of DN-MEK1 or DN-MKK4 does not suppress the ability of RA to stimulate PE formation. This is the first report to elucidate the major facets of a G{alpha}13-mediated control of primitive endoderm formation, offering insights that may be useful in understanding signaling to later stages of development and terminal differentiation.


   FOOTNOTES
 
* This work was supported by grants from the National Institutes of Health (to C. C. M.) and by an award from the March of Dimes Foundation (to H. Y. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

To whom correspondence should be addressed. Tel.: 631-444-3489; Fax: 631-444-3432; E-mail: wangh{at}pharm.sunysb.edu.

1 The abbreviations used are: MAPK, mitogen-activated protein kinase; PE, primitive endoderm; RA, retinoic acid; HA, hemagglutinin; GST, glutathione S-transferase; tPA, tissue plasminogen activator; CA, constitutively active; DN, dominant negative; PAK, p21-activated kinase; JNK, c-Jun N-terminal protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; MEKK, MEK kinase; GEF, guanine nucleotide exchange factor. Back

2 Y.-N. Lee, C. C. Malbon, and H.-y. Wang, unpublished results. Back



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