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Originally published In Press as doi:10.1074/jbc.M603508200 on May 11, 2006

J. Biol. Chem., Vol. 281, Issue 27, 18652-18659, July 7, 2006
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Genetic Deletion of Rac1 GTPase Reveals Its Critical Role in Actin Stress Fiber Formation and Focal Adhesion Complex Assembly*

Fukun Guo, Marcella Debidda, Linda Yang, David A. Williams, and Yi Zheng1

From the Division of Experimental Hematology, Children's Hospital Research Foundation, University of Cincinnati, Cincinnati, Ohio 45229

Received for publication, April 12, 2006 , and in revised form, May 10, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Rac1 is an intracellular signal transducer regulating a variety of cell functions. Previous studies by overexpression of dominant-negative or constitutively active mutants of Rac1 in clonal cell lines have established that Rac1 plays a key role in actin lamellipodia induction, cell-matrix adhesion, and cell anoikis. In the present studies, we have examined the cellular behaviors of Rac1 gene-targeted primary mouse embryonic fibroblasts (MEFs) after Cre recombinase-mediated deletion of Rac1 gene. Rac1-null MEFs became contracted and elongated in morphology and were defective in lamellipodia formation, cell spreading, cell-fibronectin adhesion, and focal contact formation in response to platelet-derived growth factor or serum. Unexpectedly, deletion of Rac1 also abolished actin stress fibers in the cells without detectable alteration of endogenous RhoA activity. Although the expression and/or activation status of focal adhesion complex components such as Src, FAK, and vinculin were not affected by Rac1 deletion, the number and size of adhesion plaques were significantly reduced, and the molecular complex between Src, FAK, and vinculin was dissembled in Rac1-null cells. Overexpression of an active RhoA mutant or ROK failed to rescue the stress fiber and adhesion plaque defects of the Rac1-null cells. Although Rac1 deletion caused a significant reduction in phospho-PAK1, -AKT, and -ERK under serum stimulation, reconstitution of active PAK1, but not AKT or MEK1, was able to rescue the actin cytoskeleton and adhesion phenotypes of the Rac1-deficient cells. Furthermore, Rac1 deletion led to a marked increase in spontaneous apoptosis that could be rescued by active PAK1, AKT, or MEK1 expression. Our results obtained from gene-targeted primary MEFs indicate that Rac1 is essential not only for lamellipodia induction but also for the RhoA-regulated actin stress fiber and focal adhesion complex formation and that Rac1 is involved in cell survival regulation through anoikis-dependent as well as -independent mechanisms.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Rac1 is a member of the Rho GTPase family of intracellular signal transducers. It cycles between an inactive GDP-bound form and an active GTP-bound form under tight regulation and controls a variety of cellular functions through multiple downstream effector pathways (1-5). Previous studies have implicated Rac1 as one of the key signaling components controlling actin cytoskeleton organization in mammalian cells (5). In particular, Rac1 has been shown to mediate lamellipodia formation and membrane ruffling in response to serum and PDGF2 stimulation (6). In addition, Rac1 activity appears to be essential in regulating cell-extracellular matrix adhesion and cell migration (7-10), and the Rac1-mediated adhesion to integrin may also be critical for cell anoikis regulation (11-12).

The molecular mechanism underlying Rac1-mediated actin cytoskeleton reorganization and adhesion has been a subject of intensive studies. In fibroblast cells, Rac1 is thought to regulate lamellipodia formation and membrane ruffling through p21-activated kinase 1 (PAK1)-dependent and -independent pathways (13-15), whereas another Rho family GTPase, RhoA, controls the actin stress fiber and focal adhesion plaque assembly at least partly mediated through Rho kinase (ROK, also termed ROCK) (16-18). Rac1 seems to act upstream of RhoA during actin cytoskeleton reorganization (6, 16, 19) and has been suggested to be an important regulator of adhesion contacts of the cells to integrin (20-22). Although it is well documented that Rac1 can regulate phosphatidylinositol-3 kinase/AKT as well as ERK kinase pathways through PAK1, which may in turn affect cell adhesion complex formation or complex stability (23-27), the definitive causal relationship between Rac1 activity and the adhesion contact components such as FAK, paxillin, p130cas, vinculin, and Src remains unclear (28). Whether Rac1 regulates cell survival exclusively through the anoikis-related mechanism has also not been causally demonstrated (11, 12).

To date, most cell biological studies of Rac1 GTPase were carried out by overexpression of constitutively active and/or dominant-negative mutants in clonal cells. Although this approach has pioneered a number of key discoveries including the critical involvement of Rac1 in lamellipodia formation and cell adhesion, it has several limitations given the recent realization of abundant signaling cross-talk between Rac1 and other Rho GTPases as well as cell clonal variability (2-4). On the one hand, the Rac1 dominant-negative mutant works by sequestering the upstream guanine nucleotide exchange factor family that includes over 80 members in the human or mouse genome, many of which are known capable of serving multiple Rho GTPases (2). Because of its noncatalytic nature, an excessive amount of expression of the dominant-negative mutant is typically needed for effectively sequestering the guanine nucleotide exchange factors to block endogenous Rac1 activity, which could impact on the function of other Rho GTPases (29). On the other hand, overexpression of the constitutively GTP-bound Rac1 mutant may activate a number of common effectors shared between Rac1 and other Rho GTPases (e.g. PAKs, IQGAPs, and IRSp53, Ref. 4), causing Rac1-irrelevant functional outcomes (30, 31). Since a balanced GTP binding/GTP hydrolysis cycle of Rac1 is required for effective signal flow (32), the overexpressed mutants in cells may also introduce artifacts by locking the small GTPase in one conformation at a fixed intracellular location. Furthermore, because Rac1 activity and its regulated cell functions can be affected by genomic changes that occur during in vitro cell cloning and passage, it is possible that the functional outcomes of Rac1 mutant expression depend on the clonal history of a given cell type. It is thus highly desirable to use genetic approaches in primary cells to assess Rac1 cell functions and the signaling requirements.

In the present studies, we have utilized conditional gene-targeted Rac1 loxp/loxp mice to generate Rac1-deficient primary mouse embryonic fibroblasts (MEFs) to assess and re-examine the requirement of Rac1 in actin cytoskeleton organization, cell-extracellular matrix adhesion, and survival in a primary cell context without possible "side effects" of Rac1 dominant mutant expression. We show that in addition to the previously implicated role in regulating lamellipodia formation, Rac1 is also essential for actin stress fiber and focal adhesion formation downstream of or in parallel to RhoA and ROK activities. The effector PAK1 is at least partially responsible for the Rac1-mediated actin and adhesion effects. Our results further suggest that Rac1 is involved in cell survival regulation through anoikis-dependent as well as -independent mechanisms.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
DNA Constructs—Retroviral constructs expressing Rac1, an active RhoA mutant RhoA-Leu-63, and ROK were generated by ligating the corresponding cDNA fragments into the BamHI and EcoRI sites in frame with the nucleotides encoding a three-hemagglutinin (HA)3 tag at the 5' end of the retroviral vector MIEG3 that expresses enhanced green fluorescent protein (EGFP) bicistronically (33-35). A constitutively active PAK1 mutant, Pak1 T423E, was supplied by Dr. J. Chernoff (Fox Chase Cancer Center, Philadelphia, PA) and used for PCR amplification and insertion into MIEG3 vector for retroviral expression. A constitutively active AKT1 mutant containing N-terminal Src myristoylation sequences was cloned into MiT vector with internal ribosome entry site-Thy1.1 (kindly supplied by Dr. David Hildeman, Cincinnati Children's Research Foundation, Cincinnati, OH). The adenoviral construct expressing MEK1 was a generous gift from Dr. Jeff Molkentin (Cincinnati Children's Research Foundation).

Cell Culture and Generation of Rac1-null Mouse Embryonic Fibroblasts (MEFs)—Conditional Rac1loxp/loxp mice in C57Bl/6 background were generated as described previously (36). The flox allele contains two loxP sites flanking exon 1 (Fig. 1A). Primary MEFs were isolated from decapitated embryonic day 11.5-13.5 mouse embryos of the Rac1wt/wt and Rac1loxp/loxp genotypes. Cells were cultured in Dulbecco's modified Eagle's medium containing L-glutamine, 10% fetal bovine serum, non-essential amino acids, beta-mercaptoethanol, and gentamicin. All tissue culture materials were obtained from Invitrogen. To generate Rac1-null alleles, Rac1loxp/loxp MEFs were infected twice with low titer adenoviral Cre recombinase (adeno-Cre) (kindly supplied by Dr. Jeff Molkentin, Cincinnati Children's Research Foundation) to achieve complete removal of Rac1 alleles without detectable toxicity or with retrovirus derived from an MIEG3 vector expressing Cre/yellow fluorescent protein.

Retrovirus Infection—Recombinant retroviruses were produced using the ecotropic Phoenix packaging cell system (35, 36). Primary MEFs were infected with the retroviruses and harvested 48-72 h after infection. EGFP- and/or yellow fluorescent protein-positive cells were isolated by fluorescence-activated cell sorting (FACS). For MEFs expressing AKT1 together with Thy1.1, the cells were incubated with phosphatidylethanolamine-conjugated Thy1.1 antibody (Pharmingen) followed by FACS sorting of phosphatidylethanolamine-positive cells.

Immnofluorescence—Primary MEFs were seeded onto coverslips at 2 x 104 cells/slip density. The cells were fixed with 3.7% formaldehyde and permeabilized with 0.1% Triton X-100. The F-actin structure of the cells was visualized by rhodamine-conjugated phalloidin (Molecular Probes) staining. Vinculin was visualized by anti-vinculin monoclonal antibody (Sigma) followed by rhodamine-conjugated anti-mouse secondary antibody (Molecular Probes) labeling (35). For the co-staining of F-actin, phospho-FAK, and vinculin, the cells were incubated with anti-phospho-FAK monoclonal antibody (BD Transduction Laboratories) and anti-vinculin polyclonal antibody (Sigma) followed by rhodamine-conjugated phalloidin, fluorescein isothiocyanate-conjugated anti-rabbit secondary antibody (Molecular Probes), and Cy5-conjugated anti-mouse (Molecular Probes) labeling. The cells were visualized by using a conventional Axiovert microscope (Carl Zeiss) or a confocal fluorescence microscope (Zeiss LSM 510).

Immnunoprecipitation and Immunoblotting—Focal adhesion complex formation was examined by immunoprecipitation and Western blotting. Briefly, the cells were lysed, and the protein contents of the cell lysates were normalized by the Bradford method. An equal amount of proteins was incubated with anti-Src monoclonal antibody (Upstate Biotechnology) at 4 °C overnight. The immunocomplex was captured by protein A/G agarose bead (Santa Cruz Biotechnology) at 4 °C after a 2-h incubation. The immunoprecipitates were washed with phosphate-buffered saline and subjected to Western blot analysis.

For Western blot, immunoprecipitates or cell lysates were separated by 10% SDS-polyacrylamide gel electrophoresis. The expression or activation (phosphorylation) of Rac1, FAK, HA-tagged RhoA-L63 and ROK, RhoA, Src, PAK1, AKT1, and ERK1/2 was probed by using corresponding antibodies. Antibodies against Rac1 and FAK were purchased from BD Transduction Laboratories. The anti-HA antibody was obtained from Sigma. The anti-RhoA antibody was from Santa Cruz Biotechnology, and anti-phospho-Src antibody was from Upstate Biotechnology. All other antibodies used for Western blot including antibodies against phospho-PAK1, PAK1, phospho-AKT1, AKT1, phospho-ERK1/2, and ERK1/2 were purchased from Cell Signaling Technology.

Cell Spreading Assay—Cells were trypsinized, resuspended in Leibovitz L-15 medium (Invitrogen) containing 10% FBS, L-glutamine, non-essential amino acids, beta-mercaptoethanol and gentamicin, and plated onto 10 µg/ml fibronectin-coated coverglasses. Immediately after plating, the cells were covered with mineral oil (Sigma) and placed on a microscope stage in a live cell incubator at 37 °C (In Vivo Technologies). Cell spreading was imaged on an Axiovert microscope (Carl Zeiss) equipped with an Orca-C4742-80 camera (Hamamatsu photonix) controlled by the Openlab software (Improvision).

Cell-Fibronectin Adhesion—2 x 104 of the cells were plated onto 10 µg/ml fibronectin-coated 96-well plates. At different time points, the unattached cells were washed away with Dulbecco's-modified phosphate-buffered saline. The attached cells were trypsinized and quantified.

Endogenous RhoA Activity Assay—GST-Rhotekin containing the RhoA-interactive domain of Rhotekin was used to probe the endogenous RhoA-GTP activity as described previously (34).

Cell Apoptosis Analysis—The apoptotic cells were measured by Cy5-conjugated annexin V staining followed by FACS analysis on a FACS-Caliber machine (33).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Generation of Rac1-/- Mouse Embryonic Fibroblasts—Primary MEFs were prepared from embryonic day 11.5-13.5 mouse embryos of the Rac1loxp/loxp and Rac1wt/wt genotypes and were used before passage 5. To achieve Rac1 allele deletion, Rac1loxp/loxp MEFs were infected with adenovirus expressing the Cre recombinase (adeno-Cre) or with retrovirus expressing Cre (Fig. 1A). Two days after infection, Rac1 expression in the cells was analyzed by Western blotting of the cell lysates with an anti-Rac1 antibody from BD Transduction Laboratories. This antibody appears to be highly specific toward endogenous Rac1 since it did not detect Rac2 or Rac3 in multiple blood lineages or the forebrain of mice where Rac1 gene was specifically targeted (data not shown). As shown in Fig. 1C, typically 90-95% of endogenous Rac1 in the MEFs was readily removed by the adeno-Cre or retroviral Cre infection protocol. To better complement the Rac1-/- cell studies, control cells that express WT Rac1 containing an N-terminal (HA)3 tag were also generated by transduction of Rac1loxp/loxp cells with retroviruses derived from an MIEG3 vector expressing Rac1/EGFP bicistronically (Fig. 1B). Infection of the Rac1 reconstituted MEFs with adeno-Cre or retroviral Cre resulted in deletion of endogenous Rac1 and produced the Rac1-/-/Rac1 MEFs. The amount of reconstituted (HA)3-Rac1 in the Rac1-/-/Rac1 cells was comparable with that of endogenous Rac1 as judged by Western blotting analysis (Fig. 1C). Since both adeno-Cre and retroviral Cre expression achieved similar effects in Rac1 protein deletion and cell morphologic/actin/adhesion changes of the Rac1loxp/loxp cells, we describe only the adeno-Cre-mediated Rac1 gene targeting results in the following experiments.

Deletion of Rac1 Disrupts F-actin Structures and Causes Delayed Cell Spreading and Cell-Fibronectin Adhesion—Previous cell line-based, Rac1 dominant mutant overexpression studies have shown that Rac1 is a pivotal regulator of lamellipodia and membrane ruffle formation (5, 6). To examine the effect of Rac1 deletion on cell actin cytoskeleton organization, we stained F-actin of Rac1-deficient MEFs with rhodamine-conjugated phalloidin. Rac1 deletion led to a contracted, elongated cell morphology that lacks detectable F-actin structure other than cortical actin (Fig. 2). The cells failed to respond to FBS or PDGF to form lamellipodia (Fig. 2). The Rac1-/- cells also lacked any detectable actin stress fibers in the presence of FBS (Fig. 2). In contrast, similar to WT cells, the Rac1-/- cells reconstituted by Rac1 expression (Rac1-/-/Rac1) showed abundant lamellipodia and actin stress fibers under similar conditions. These observations indicate that Rac1 regulates actin cytoskeleton assembly including both the lamellipodia and the stress fibers structures to impact on cell morphology.

Rac1 has been implicated in cell spreading and cell-extracellular matrix adhesion (7, 8, 28, 37). To examine whether deletion of Rac1 affects cell spreading and cell-fibronectin interaction, wild type and Rac1-/- MEFs were plated onto fibronectin-coated coverglasses and were monitored for morphologic changes during the time course of cell attachment or were plated onto fibronectin-coated 96-well plates to allow adhesion. As shown in Fig. 3A, WT MEFs spread quickly on fibronectin within 21 min. In contrast, Rac1-deficient cells were significantly delayed in spreading such that few cells spread after 30 min on the fibronectin surface. The Rac1-/- cells eventually displayed a uniform spindle-like, contracted morphology after overnight plating (Fig. 2; data not shown). Consistent with the observed spreading defects, Rac1-/- cells showed impaired adhesion to fibronectin in the presence of a variety of stimuli including FBS, PDGF, epidermal growth factor, and insulin-like growth factor (Fig. 3B). The effect of Rac1 loss on cell-fibronectin adhesion at a shorter time point (e.g. 30 min after plating) was more severe than that at a longer time point (e.g. 2 h after plating). This phenotype was rescued by reintroduction of wild type Rac1 into Rac1-/- cells (Fig. 3B). These results demonstrate a role of Rac1 in regulating the kinetics of cell spreading and integrin-mediated cell adhesion.


Figure 1
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  		FIGURE 1.
Conditional knock-out of Rac1 and generation of Rac1-/- and Rac1-reconstituted Rac1-/-/Rac1 MEFs. A, the conditional Rac1 flox allele contains two loxp sites flanking exon 1 of Rac1 gene. The exon 1 encodes the first 28 amino acids of Rac1 that include critical sequences required for GTP binding. B, the retroviral construct expressing HA-tagged Rac1 and EGFP is depicted. C, treatment of the Rac1loxp/loxp MEFs by adeno-Cre effectively inhibited Rac1 expression, and infection of the Rac1loxp/loxp MEFs containing retrovirus expressing HA-Rac1 with adeno-Cre resulted in the replacement of endogenous Rac1 with exogenous HA-Rac1 as revealed by an anti-Rac1 Western blot.

 
Rac1 Deletion Disrupts Focal Adhesion Complex Formation—Rac1 has been suggested to be involved in the proper distribution and activation of a number of components of cell adhesion contacts (7, 28). To examine whether Rac1 deficiency affects adhesion complex formation, we carried out Western blotting analysis to determine the expression and complex formation properties of a few adhesion molecules and performed immunofluorescence studies to examine the adhesion plaque distribution of the Rac1-/- cells. As shown in Fig. 4A, Rac1 deletion did not affect expression or activity of Src, FAK, and vinculin. However, the loss of Rac1 prevented Src, FAK, and vinculin from forming a stable molecular complex, which is known to be important for steady state cell focal adhesion. Immunofluorescence staining of phospho-FAK, vinculin, and actin provided additional evidence that the absence of Rac1 caused a significant decrease in phospho-FAK and vinculin co-localization to form recognizable focal adhesion plaques in the presence of either FBS or PDGF (Fig. 4B). This effect was associated with the loss of F-actin structures within the cell body. These results suggest that although Rac1 is not required for FAK or Src activation, it is critical for focal adhesion molecular complex assembly and functional adhesion plaque formation. These data help further clarify the functional relationship between Rac1 and FAK.


Figure 2
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  		FIGURE 2.
Deletion of Rac1 leads to contraction in cell morphology and serum and PDGF insensitivity in actin reorganization. 2 x 104 of WT, Rac1-/-, or Rac1-reconstituted Rac1-/-/Rac1 MEFs was plated onto coverglass and serum-starved overnight and then stimulated with 10% fetal bovine serum or 10 nM PDGF for 15 min. The cells were fixed in 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, and stained for F-actin by using rhodamine-conjugated phalloidin.

 
Rac1 Is Required for RhoA Regulated Stress Fiber Formation—Rac1 was previously shown to act upstream of RhoA to regulate cell actin reorganization and adhesion in fibroblasts (6, 16). In particular, active RhoA and its effector ROK are thought to be necessary and sufficient to induce actin stress fiber and focal adhesion plaque formation regardless of Rac1 activity status (16, 18). The lack of detectable actin stress fibers and focal adhesion plaques in Rac1-/- cells prompted us to investigate whether the endogenous activity of RhoA in the cells was down-regulated due to Rac1 deletion. Overexpression of a constitutively active RhoA mutant (RhoA-L30) (Fig. 5A) or the RhoA effector ROK (Fig. 6C) in Rac1-null cells did not generate stress fibers. Further, the active RhoA-GTP species in Rac1-deficient cells was at a similar level as that in wild type cells (Fig. 5B), suggesting that the loss of stress fiber formation of Rac1-/- cells is not due to RhoA activity alteration. Western blot analysis showed that the exogenous RhoA-L63 or ROK was efficiently expressed in the Rac1-null cells (Figs. 5C and 6B). These results indicate that Rac1 is required for RhoA and ROK-regulated actin stress fiber formation. It is possible that RhoA functions either upstream or in parallel to Rac1 in the regulation of stress fiber formation.

Active PAK1, but Not AKT or ERK, Rescues the Loss of Actin Stress Fiber and Focal Adhesion Plaque Phenotypes of Rac1-deficient Cells—Rac1 is thought to engage multiple effector pathways, including the PAK1, AKT, and ERK1/2 kinases, to regulate actin cytoskeleton and cell-extracellular matrix adhesion, as suggested by a body of literature derived from dominant mutant overexpression studies (23-27). To begin to understand the molecular mechanisms underlying Rac1-mediated actin reorganization and focal adhesion plaque formation, we next examined the endogenous PAK1, AKT, and ERK1/2 activities in Rac1-deficient cells by immunoblotting of phospho-PAK1, -Akt, and -Erk1/2. As shown in Fig. 6A, Rac1 deletion attenuated phospho-PAK1, -AKT, and -ERK1/2 levels without affecting PAK1, AKT, and ERK1/2 expressions, suggesting that Rac1 indeed regulates these intracellular kinase activities. To see whether the altered PAK1, AKT, or ERK1/2 activity, as well as the RhoA effector ROK, in the Rac1-/- cells is responsible for the actin structural and cell-fibronectin adhesion phenotypes, we introduced exogenously an active PAK1 mutant, PAK1 T423E, ROK, an N-terminal myristoylated active AKT mutant, or the ERK activator MEK1 into the Rac1-/- cells by retrovirus or adenovirus infection. Western blot experiments confirm that these kinases were readily expressed and activated in the cells (Fig. 6B). Among the four kinases, active PAK1, but not ROK, AKT, or MEK1, was able to rescue the actin structures including stress fibers in ~60% of the Rac1-/- cells (Fig. 6C). The effect of these kinases on actin organization is mimicked by their ability (or lack of ability) to rescue the cell-fibronectin adhesion phenotype (Fig. 7A) and the vinculin-marked adhesion plaques in Rac1-/- cells (Fig. 7B) as only active PAK1 was capable of rescuing Rac1 knock-out cells from the slowed adhesion kinetics and the loss of adhesion complex formation. Interestingly, the active PAK1 mutant had no effect on the AKT activation status in either wild type or Rac1-null MEFs, whereas the active AKT mutant led to PAK1 activation, suggesting that AKT activity may positively regulate PAK1 (Fig. 6B). Moreover, the active PAK1 mutant does not appear to affect ERK1/2 activity, whereas the active AKT expression was able to down-regulate ERK1/2 activity in our system (Fig. 6B). Taken together, these results suggest that PAK1, but not ROK, AKT, or ERK1/2, is necessary and sufficient for Rac1-mediated actin cytoskeleton regulation and adhesion. The relationship among these kinases downstream of Rac1 is likely complex in mediating signals to actin reorganization and/or cell adhesion.


Figure 3
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  		FIGURE 3.
Deletion of Rac1 causes delayed cell spreading and defective cell-fibronectin adhesion. A, deletion of Rac1 led to slowed kinetics in cell spreading on fibronectin. WT and Rac1-/- cells were trypsinized, resuspended in the Leibovitz L-15 medium, and plated onto 10 µg/ml fibronectin-coated coverglasses. The cell morphologies were imaged using an Axiovert microscope at the indicated time points. B, Rac1 deletion inhibited cell-fibronectin adhesion. 2 x 104 of the serum-starved WT, Rac1-/-, or Rac1-/-/Rac1 cells in the presence or absence of 10% FBS (serum), 10 nM PDGF, 10 nm epidermal growth factor (EGF), or 10 nM insulin-like growth factor (IGF) was plated onto fibronectin-coated 96-well plates and incubated for 0.5 or 2 h. The attached cells were quantified and presented as the relative ratio to that of WT cells at 0.5 h time point.

 


Figure 4
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  		FIGURE 4.
Rac1 deletion impairs focal adhesion complex formation. A, WT or Rac1-/- cells were lysed and sequentially incubated with anti-Src antibody and protein A/G beads. The beads were precipitated by centrifugation. The whole cell lysate and anti-Src immunoprecipitates were subjected to Western blotting with the indicated antibodies. p-FAK, phospho-FAK; p-Src, phospho-Src. B, 2 x 104 of WT or Rac1-/- cells were cultured on coverglasses. The subconfluent cells were serum-starved overnight, stimulated with 10% FBS or 10 nM PDGF for 15 min, fixed in 3.7% formaldehyde, permeabilized with 0.1% Triton X-100, and co-stained for F-actin, phospho-FAK, and vinculin with rhodamine-conjugated phalloidin, anti-phospho-FAK monoclonal antibody, and anti-vinculin polyclonal antibody, respectively.

 


Figure 5
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  		FIGURE 5.
Rac1 regulates actin stress fiber formation independently of RhoA activity. A, expression of an active RhoA mutant failed to rescue the loss of actin stress fiber phenotype in Rac1-/- cells. WT and Rac1loxp/loxp MEFs were infected with retrovirus expressing EGFP together with RhoA-L63 that carries an N-terminal HA tag. EGFP-positive cells were collected by FACS and treated with adeno-Cre. The cells were stained for F-actin by using rhodamine-conjugated phalloidin. B, Rac1 deletion did not alter endogenous RhoA activity. Endogenous RhoA activity in WT or Rac1-/- cells was measured by an immobilized GST-Rhotekin pull-down assay. The amount of RhoA-GTP was detected by Western blotting of the co-precipitates with an anti-RhoA antibody. C, the expression of exogenous RhoA mutant in WT and Rac1-/- cells was confirmed by Western blotting of HA-RhoA-L63 with an anti-HA antibody.

 
Rac1 Regulates Anoikis-dependent and -independent Cell Survival through PAK1, AKT, and ERK1/2—Rac1 has been proposed to regulate cell survival through the adhesion-mediated anti-anoikis mechanism by a number of studies (11, 12). However, convincingly demonstrating such an effect in wild type adherent cells has been difficult because suspending the adherent cells may introduce additional stress that could induce cell apoptosis. Rac1 deletion in primary MEFs resulted in slowed cell growth (data not shown), an effect that might be attributed to cell cycle defect and/or apoptosis. To this end, we have examined the cell apoptosis index of Rac1-/- cells by annexin V staining and FACS analysis. As shown in Fig. 8A, Rac1 deficiency resulted in a significant increase in cell spontaneous apoptosis, an effect reversible by Rac1 reconstitution in Rac1-/-/Rac1 cells. Immunofluorescence staining of active caspase 3 further confirmed this phenotype (data not shown). These results may suggest a role of Rac1 in cell anoikis because Rac1 deletion reduced cell adhesion to fibronectin and the focal adhesion plaque formation. An examination of the active PAK1 mutant-, ROK-, active AKT1 mutant-, or MEK1-expressing Rac1-null cells revealed that not only the active PAK1 mutant was able to effectively suppress the apoptosis phenotype of the Rac1-/- cells, the active AKT1 mutant and MEK1, but not ROK, were also able to significantly inhibit cell apoptosis (Fig. 8B). Given the effects of these kinases on cell actin structure and adhesion phenotypes of the Rac1-/- cells, i.e. active PAK1 rescues the actin organization and adhesion defects of the Rac1-/- cells, whereas ROK, AKT1, and MEK1 were not effective (Figs. 6 and 7), these results indicate that Rac1 regulates both anoikis-dependent and anoikis-independent pathways to maintain cell survival.


Figure 6
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  		FIGURE 6.
A, effects of Rac1 deletion on PAK1, AKT, and ERK expression and activities. Rac1 was deleted by adeno-Cre treatment of Rac1loxp/loxp MEFs. WT and Rac1-/- MEFs were harvested and immunoblotted for phospho-PAK1 (p-PAK1), phospho-AKT (p-AKT), and phospho-ERK (p-ERK) by the respective antibodies. The levels of total PAK1, AK T, and ERK were blotted in parallel. B,WT and Rac1loxp/loxp MEFs were infected with retrovirus expressing EGFP together with ROK or a constitutively active PAK1 mutant, a retrovirus expressing a constitutive active AK T mutant together with Thy1.1, or an adenovirus expressing MEK1. The EGFP- or Thy1.1-positive cells were collected by FACS cell sorting and subsequently treated with adeno-Cre to delete Rac1. Cell lysates were probed with the indicated antibodies. The expression of exogenous ROK was blotted by an anti-HA antibody. C, expression of an active mutant of PAK1, but not ROK, AKT, or ERK, partially rescued the loss of actin stress fiber phenotype of Rac1-/- cells. The cells were stained for F-actin by using rhodamine-conjugated phalloidin.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we show that gene targeting of Rac1 in primary MEFs results in disruption of F-actin structures including lamellipodia and actin stress fiber, defects in cell spreading and cell-fibronectin adhesion complex formation, and increased cell spontaneous apoptosis. Significantly, we found that Rac1 does not depend on RhoA activity for actin stress fiber induction; rather, RhoA and its effector ROK appear to require functional Rac1 for the stress fiber induction and focal adhesion complex assembly. We further demonstrate Rac1 is essential for signaling pathways regulating PAK1, AKT, and ERK1/2 and show that active PAK1, but not AKT1 or MEK1, can rescue cell actin and adhesion phenotypes in the Rac1 knock-out cells. Meanwhile, active PAK1, AKT, and MEK1 all seem to be able to partly rescue the survival phenotype of Rac1-null cells. Although our finding that Rac1 is involved in serum and PDGF-mediated lamellipodia formation, cell spreading, cell-extracellular matrix adhesion, and cell survival is consistent with most published literature using the dominant-negative mutant of Rac1 in fibroblast cell lines, the results that Rac1 is essential for actin stress fiber formation and focal adhesion complex assembly downstream or in parallel to RhoA-ROK pathway is unexpected, and the reconstitution studies with PAK1, ROK, AKT1, or MEK1 help implicate their contributions (or lack of contribution) to Rac1-mediated actin structural regulation, adhesion, and survival functions.

Rac1 was previously shown to regulate focal complex formation, whereas RhoA mediates focal contact/adhesion formation in cell-extracellular matrix adhesion (7). Both focal complex and focal contact/adhesion utilize similar components to form an adhesion molecular complex, but the site of the Rac1-mediated focal complex is localized at the edges of lamellipodia, whereas the site of RhoA-mediated focal contact/adhesion is at the anchoring site of stress fibers (7). Two landmark studies published over a decade ago suggested a signaling dogma that multiple growth factors including LPA and serum can activate RhoA and the RhoA-dependent actin stress fiber formation independently from Rac1 (6, 16). Down-regulation of RhoA activity resulted in a loss of actin stress fiber assembly induced by active Rac1 or PDGF (16), prompting the proposal that at least in fibroblasts, Rac1 either acts upstream of RhoA or is not involved in actin stress fiber and focal adhesion formation (5, 7). Our results showing that removal of Rac1 in primary MEFs abolishes actin stress fiber and focal adhesion plaques as well as the lamellipodia structure under both PDGF and serum stimulation conditions indicate that Rac1 plays an essential role in the assembly of both types of the actin structures, i.e. lamellipodia and stress fiber. Further, because reconstitution of an active RhoA mutant or the previously implicated actin stress fiber inducer ROK (18) could not rescue the loss of actin stress fiber phenotype of the Rac1-null cells, Rac1 appears to transduce a permissive signal for cell actin organization and functions either downstream from or in parallel to the RhoA-ROK signal axis in stress fiber and focal contact/adhesion complex formation. The differences between our findings and previously reported observations may reflect cell clonal variations (e.g. primary versus immortalized clones) and/or the dosage and specificity of dominant-negative Rac1/RhoA mutants versus Rac1-specific gene targeting.


Figure 7
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  		FIGURE 7.
Active PAK1, but not ROK, AKT1, or ERK, partially rescues the adhesion and focal complex phenotypes of the Rac1-/- cells. A, 2 x 104 of the indicated cells were plated on 10 µg/ml fibronectin-coated 96-well plates. After a 30-min incubation, the cells were washed three times with phosphate-buffered saline. The attached cells were trypsinized and counted. The results are expressed as the fold of adherent cells to that of WT MEFs. B, WT or Rac1-/- cells expressing the indicated kinases were stained for vinculin by anti-vinculin monoclonal antibody to reveal the focal adhesion complex.

 
PAK1 has been implicated as one of the downstream effectors for both Rac1 and Cdc42 (4), and its role in Rac1-mediated actin restructuring has been controversial (13-15). We found that loss of Rac1 results in reduced PAK1 activity without affecting endogenous Cdc42 activity,3 suggesting that PAK1 is a bona fide effector of Rac1. Reconstitution of an active PAK1 mutant into the Rac1-null cells can rescue cell morphology, actin organizations including stress fiber formation, and cell adhesion, strongly supporting a role of PAK1 in the Rac1-regulated actin cytoskeleton reorganization and cell adhesion. It will be interesting to see how the loss of Rac1 affects additional effector pathways implicated for actin polymerization such as the WAVE1/WAVE2 regulators of membrane ruffling (38) and to further define whether active PAK1 reconstitution in the Rac1-null cells also affects the LIM kinase/myosin light chain kinase signaling axis related to actomyosin function (39, 40). Given the closely associated functional outcomes of actin reorganization and cell adhesion complex formation, it is possible that these two functions regulated by Rac1 are tightly linked and may not be separable from one another.

The relationship between Rac1 and AKT is complex since AKT has been suggested to function both upstream and downstream of Rac1, relating to the cell actin cytoskeleton and migration properties (41-43). We found that Rac1 deletion decreases AKT activity, consistent with Rac1 acting upstream of AKT. However, AKT does not seem to be involved in Rac1-mediated actin reorganization because reconstitution of an active AKT mutant failed to affect F-actin structure in Rac1-null cells. Similarly, the active AKT mutant was unable to rescue focal adhesion plaque formation and cell-fibronectin adhesion of Rac1-deficient cells, corroborating with previous observations that AKT is not involved in connective tissue growth factor-induced actin cytoskeleton disassembly and loss of focal adhesion (44).

It remains controversial whether and how Rac1 regulates ERK1/2 activity. An early report showed that constitutively active Rac1 could activate c-Jun N-terminal kinase (JNK) and p38 but not ERK1/2 among mitogen-activated protein (MAP) kinases (45). In contrast, a number of other reports proposed a role of Rac1 in regulating ERK1/2, either activating or suppressing their activities (11, 46). Our results that Rac1 deletion led to decreased ERK activity in response to serum indicate that Rac1 mediates signaling, leading to ERK1/2 activation in primary MEFs. However, MEK1 expression does not affect actin cytoskeleton and adhesion properties of Rac1-null cells, suggesting that Rac1-ERK pathway is not involved in cytoskeleton organization and focal adhesion complex formation in primary MEFs as it was suggested in normal rat kidney cells (17). Our results further indicate possible differences between clonal and primary cells and/or the dominant mutants and gene targeting approaches.

In the debate of the relationship among PAK1, AKT, and ERK1/2, our reconstitution studies in wild type and Rac1-null MEFs also help address a number of related issues. AKT has been reported to act either upstream or downstream of PAK1 (47, 48), whereas PAK1 may or may not regulate ERK1/2 activation depending on the nature of specific stimuli (23, 47, 49). Further, AKT could either activate or suppress ERK activity (11, 50-52). In the context of primary MEFs, we found that introduction of an active PAK1 mutant into both wild type and Rac1-null MEFs had no effect on the AKT activation status, whereas introduction of an active AKT mutant into wild type and Rac1-null MEFs led to PAK1 activation, consistent with AKT positively regulating PAK1. Moreover, the active PAK1 does not appear to alter ERK1/2 activity, whereas active AKT suppresses ERK1/2 activity. Clearly, much remains to be sorted out about the contribution and interplays among these and other Rac1-effector pathways in the regulation of cell actin organization and adhesion, and this needs to be considered in the cell type and clonality context as well.


Figure 8
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  		FIGURE 8.
Rac1 deletion induces cell apoptosis that can be partially inhibited by expression of active PAK1, AKT1, or the ERK activator MEK1. A, WT, Rac1-/- and Rac1-/-/Rac1 cells were harvested by trypsinization and analyzed by FACS for apoptotic cell population following annexin V staining. B, WT or Rac1-/- cells reconstituted with the indicated kinases were assayed for apoptosis cell population by annexin V staining and FACS.

 
Finally, our current studies help shed light on the role of Rac1 in the anoikis-dependent and anoikis-independent mode of cell survival regulation. Both the cell adhesion rescue-defective active AKT mutant and the MEK1 and the adhesion rescue-effective PAK1 were able to at least partly suppress the apoptosis phenotype of Rac1-null cells, implicating a role of Rac1 in regulating both the adhesion-mediated and the adhesion-independent mode of survival. Given the intertwined relationship among PAK1, AKT, and ERK1/2 downstream of Rac1, it is likely that a balanced act among these and other Rac1-regulated signaling components are required to mediate Rac1 signaling in anoikis-dependent and -independent mechanisms.

Note Added in Proof—While this manuscript was in press, a related paper on the role of Rac1 in cell migration using Rac1 knockout fibroblast was published (Vidali, L., Chen, F., Cichetti, G., Ohta, Y., and Kwiatkowski, D. J. (2006) Mol. Biol. Cell 17, 2377-2390).


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants GM 53943, GM 60523, and DK062757. 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

1 To whom correspondence should be addressed: Yi Zheng, Experimental Hematology, Children's Hospital Research Foundation, 3333 Burnet Ave., Cincinnati, OH 45229. Tel.: 513-636-0595; Fax: 513-636-3768; E-mail: yi.zheng{at}chmcc.org.

2 The abbreviations used are: PDGF, platelet-derived growth factor; EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorting; FBS, fetal bovine serum; MEF, mouse embryonic fibroblast; PAK1, p21-activated kinase 1; ERK, extracellular signal-regulated kinase; HA, hemagglutinin; WT, wild type. Back

3 F. Guo and Y. Zheng, unpublished observation. Back


   ACKNOWLEDGMENTS
 
We thank Dr. David J. Kwiakowski (Harvard Medical School, Boston, MA) for providing the Rac1loxp/loxp mice. We are in debt to Drs. Bastiano Sanna and Jeff Molkentin (Children's Hospital Research Foundation, Cincinnati, OH) for the Cre recombinase and MEK1 expressing adenoviruses and to Dr. David Hilderman (Children's Hospital Research Foundation) for the retrovirus expressing active AKT1 and Thy1.1.



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