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Originally published In Press as doi:10.1074/jbc.M406216200 on August 17, 2004

J. Biol. Chem., Vol. 279, Issue 44, 45824-45832, October 29, 2004
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GEFT, A Rho Family Guanine Nucleotide Exchange Factor, Regulates Neurite Outgrowth and Dendritic Spine Formation*

Brad Bryan{ddagger}, Vikas Kumar§, Lewis Joe Stafford{ddagger}, Yi Cai{ddagger}, Gangyi Wu§, and Mingyao Liu{ddagger}

From the {ddagger}Alkek Institute of Biosciences and Technology, and Department of Medical Biochemistry and Genetics, Texas A&M University System Health Science Center and the §Department of Molecular Physiology and Biophysics, Baylor College of Medicine, Houston, Texas 77030

Received for publication, June 3, 2004 , and in revised form, July 27, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The Rho family of small GTPases controls a wide range of cellular processes in eukaryotic cells, such as normal cell growth, proliferation, differentiation, gene regulation, actin cytoskeletal organization, cell fate determination, and neurite outgrowth. The activation of Rho-GTPases requires the exchange of GDP for GTP, a process catalyzed by the Dbl family of guanine nucleotide exchange factors. We demonstrate that a newly identified guanine nucleotide exchange factor, GEFT, is widely expressed in the brain and highly concentrated in the hippocampus, and the Purkinje and granular cells of the cerebellum. Exogenous expression of GEFT promotes dendrite outgrowth in hippocampal neurons, resulting in spines with larger size as compared with control spines. In neuroblastoma cells, GEFT promotes the active GTP-bound state of Rac1, Cdc42, and RhoA and increases neurite outgrowth primarily via Rac1. Furthermore, we demonstrated that PAK1 and PAK5, both downstream effectors of Rac1/Cdc42, are necessary for GEFT-induced neurite outgrowth. AP-1 and NF-{kappa}B, two transcriptional factors involved in neurite outgrowth and survival, were up-regulated in GEFT-expressing cells. Together, our data suggest that GEFT enhances dendritic spine formation and neurite outgrowth in primary neurons and neuroblastoma cells, respectively, through the activation of Rac/Cdc42-PAK signaling pathways.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Neurite outgrowth, a process responsible for neuronal patterning and connections, is crucial for the development of the nervous system (1). The regulation of neurite outgrowth is determined largely by the organization of the actin cytoskeleton in response to different environmental cues (2). The Rho family of small GTPases, which comprises the key regulators of the actin cytoskeleton (3), has been shown to mediate the morphological changes that are observed during neuronal development and plasticity such as neurite outgrowth, axonal guidance, and dendrite topology modifications (4–9). Members of the Rho family perform distinct roles in the regulation of the actin cytoskeleton. RhoA is responsible for the formation of focal adhesions and the assembly of actin stress fibers, and it has been found to inhibit the formation of neurite outgrowths (10, 11). Rac1 promotes the formation of membrane lamellae, whereas Cdc42 regulates the outgrowth of filopodia (12). Both Rac and Cdc42 positively regulate neurite outgrowth (13).

The Rho-GTPases function as molecular switches, cycling between GTP-bound forms and GDP-bound forms. They are active in the GTP-bound form, and hydrolysis of the GTP by their intrinsic GTPase activity returns them to the GDP-bound inactive state (14). The active/inactive states of these proteins are regulated by a variety of intracellular molecules, predominantly by two classes of proteins: GTPase-activating proteins and guanine nucleotide exchange factors (GEFs)1 (15). GTPase-activating proteins catalyze the intrinsic GTPase activity of the Rho proteins, thus inactivating them, whereas GEFs catalyze the exchange of GDP for GTP, thereby activating GTPases. The Rho GEFs are a class of enzymes with high specificity for Rho-GTPases and contain a Dbl homology domain of ~20 amino acids immediately followed by a pleckstrin homology domain of ~100 amino acids (16). Dbl homology domains interact directly with Rho-GTPases to catalyze guanine nucleotide exchange (17, 18). Pleckstrin homology domains promote the translocation of Dbl-related proteins to plasma membranes (19, 20), as well as participate directly in GTPase binding and regulation of GEF activity (21).

We recently identified GEFT as a new Rho-family-specific GEF that is highly expressed in the brain, heart, and skeletal muscle (22, 23). Unlike many other GEF proteins, GEFT comprises primarily the Dbl homology and pleckstrin homology domains only, with short N- and C-terminal sequences. Expression of GEFT has been shown to promote the formation of lamellipodia, actin microspikes, and filopodia in NIH3T3 cells via activation of the Rho family of small GTPases (22). A potential splice variant of GEFT, encoding the gene for p63RhoGEF, has also been identified and shown to be highly expressed in both the brain and heart, and it induces a RhoA-dependent stress fiber formation in several cell types (24, 25). Using primary hippocampal neurons and Neuro2a (N2A) neuroblastoma cell lines, we examined the role that GEFT plays in the regulation of dendritic spine morphogenesis and neurite outgrowth. In addition, we studied the molecular signaling mechanisms by which GEFT regulates dendritic spine morphogenesis and neurite outgrowth.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
DNA Constructs—The gene encoding human GEFT was subcloned from a pSPORT-6 vector into the HindIII and SalI sites of a pCMV-Tag2B vector (Stratagene), resulting in the plasmid pCMV-GEFT. RhoA, Cdc42, and Rac1 expression vectors were previously constructed (22). RhoA T19N, RhoA G14V, Cdc42 T17N, Cdc42 G12V, Rac1 T17V, and Rac1 G12V where obtained from the Guthrie cDNA resource Center. PAK1 constructs were kindly supplied by Dr. Jonathan Chernoff at Fox Chase Cancer Center. PAK5, PAK5 S573N/S602E, and PAK5 K478M were a generous donation from Audrey Minden (Columbia University).

Cell Culture, Transfection, and Differentiation—Dentate explants were cultured as previously described (26). Briefly, dentate gyrus and CA3 regions were isolated from the hippocampi of 2- to 4-day-old Sprague-Dawley rats, maintained in culture for 8–25 days on a Matrigel substrate (1:50 dilution, Collaborative Research), and plated on glass coverslips in medium supplemented with B-27 (Invitrogen). Proliferation of non-neuronal cells was prevented by the addition of 2 µM cytosine {beta}-D-arabinofuranoside (Sigma). The explants were transfected with pCMV-GEFT or vector at 7 days in vitro by calcium phosphate precipitation (27). Co-transfection with enhanced green fluorescent protein (GFP) was used to identified the transfected neurons and visualize the detailed morphology including dendritic spines. Neuro2A (N2A) cells were maintained in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (HyClone). Cell transfection was performed using LipofectAMINE (Invitrogen) according to the manufacturer's instructions. Cells were then allowed to grow for 48 h and assayed, or selected with G418 to produce stable cell lines. For each assay, empty vector was used as a control.

Neurite Outgrowth Measurements—To quantify dendrite morphology, the enhanced GFP-expressing neurons were imaged at 21 days in vitro using a high resolution Zeiss LSM 510 Meta system (Zeiss). To quantify the dendritic spines, a 50-µm image of the dendrite from primary or distal dendrites was viewed at high magnification (40x, numerical aperture of 1.4 with additional optic zoom of 2–3), and each individual spine present on the dendrites was manually traced onto an acetate sheet. The spines measured were selected from 30 randomly selected 50-µm dendritic segments, and the spine numbers measured were over 900 for control and 400 for GEFT. The drawings were then scanned into a computer and the maximum length, head width, and shape factor of each spine was calculated automatically with the Metamorph software and logged into Microsoft Excel. The limit set for considering dendritic outgrowth was filopodia greater than 4 µm without a bulbous head (28). Spines were defined as a headless protrusion 1–4 µm long or a headed protrusion of any length up to 4 µm. The cumulative percentage graphs include for the spine length, width, and area show the overall spread or distribution of the data for these parameters. A lower magnification (20x, numerical aperture of 0.75) was used to image the overall dendrite morphology. Whole cell morphology was traced onto an acetate sheet and scanned as above. Total dendritic branch length was calculated using Scion software, whereas tips were counted manually. The dendrite data shown are mean (±S.E.), and, using Student's unpaired t test, p < 0.001. Phase contrast images of N2A cells were viewed at x200 magnification, and images were captured on a charge-coupled device camera mounted on a Nikon Eclipse TS100 microscope using SPOT Advanced Imaging Software. Cells were scored for the percentage of cells expressing neurites, average number of neurites per cell, and average length of neurites. Cells with neurites were defined as cells that possessed at least one neurite of more than one-half the cell body diameter in length. The data presented are the mean of three individual transfected 10-cm2 dishes and are representative of at least three independent experiments. At least 250 cells per transfection were scored for neurite outgrowth. The N2A data shown are mean (±S.E.), and, using Student's unpaired t test, p < 0.05.

Immunofluorescence—Fluorescence images for primary rat neurons were captured using confocal microscopy using a high resolution Zeiss LSM 510 Meta system with sequential acquisition. A z series projection of ~7 to 15 images with 0.5- to 1-µm depth interval, each averaged two times was taken to cover the entire z dimension of the labeled neurons. N2A cells used for immunofluorescence were grown on 0.5% gelatin-coated glass coverslips. The cells were fixed (4% paraformaldehyde, 0.1% Triton X-100), blocked with 0.2% bovine serum albumin, and incubated with a monoclonal antibody against FLAG (M2 monoclonal, Sigma). Double-labeled immunostaining was carried out with the appropriate fluorochrome-conjugated secondary antibodies. Fluorescence images for N2A cells were captured at x400 on a charge-coupled device camera mounted on an Olympus inverted research microscope using Ultraview imaging software (Olympus, Inc.).

Transient Expression of Reporter Gene Assays—N2A cells were transfected using LipofectAMINE (Invitrogen) as described previously. Transfected cells were harvested after 48 h. The resulting cell lysates were analyzed for luciferase activity using enhanced chemiluminescence reagents from Promega, according to the manufacturer's instructions. The reporter constructs for the AP1-Luc and NF-{kappa}B-Luc were obtained from K. Guan (University of Michigan). The data presented are the mean of three individual transfected wells, and the experiments were performed at least three times.

GST Pull-down Assays—GTPase activation assays were performed by GST-p21 binding domain pull-down assays as described previously (29, 30). Briefly, cells transfected with GEFT or a control plasmid (pCMV-Tag2B) were washed and lysed on the dish in 50 mM Tris (pH 7.5), 500 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 10% glycerol, 10 mM MgCl2, 10 µg/ml leupeptin and aprotinin, and 1 mM phenylmethylsulfonyl fluoride. GTP-bound Rac and Cdc42 were pulled down using the GST-p21-binding domain of PAK1 immobilized on glutathione beads, whereas GTP-bound RhoA was pulled down using the GST-bound Rho-binding protein immobilized on glutathione beads. The amounts of active Rac1, Cdc42, and RhoA (GTP-bound form) were detected by Western blot using antibodies against Rac1, Cdc42, or RhoA.

PAK Kinase Assay—1 mg of total cell lysate was immunoprecipitated with anti-PAK antibody (Santa Cruz Biotechnology). Samples were resuspended in kinase buffer (30 mM HEPES (pH 7.5), 10 mM MgCl2, 2.5 mM EGTA, 1 mM dithiothreitol, 0.1 mM Na3VO4, 1 mM NaF) and mixed with MgCl2, ATP, myelin basic protein, and 10 µCi of [{gamma}-32P]ATP. The reaction was allowed to proceed and then loaded onto a 12% SDS-PAGE gel. The gel was dried and exposed to x-ray film overnight.

In Situ Hybridization—Brains from adult female mice were obtained and sectioned for slides. In situ hybridizations were performed using the Dig Wash and Detection Kit (Roche Diagnostics) according to the manufacturer's instructions.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
GEFT Is Highly Expressed in the Hippocampus, Granular Layer, and Purkinje Cells of the Adult Mouse Brain—To investigate the potential role of GEFT in neuronal cells and in brain development, we examined the expression of GEFT transcript using in situ hybridization on brain sections of adult mice. As shown in Fig. 1A, GEFT was found to be broadly expressed in all regions of the brain, however, significantly higher expression occurred in both the hippocampus (Fig. 1C) and the Purkinje cells and granular layer of the cerebellum (Fig. 1D). To confirm the specific expression of GEFT, a GEFT sense probe was used as a control in similar in situ hybridization assays. No positive staining was observed using the control sense probe (Fig. 1B).



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  		FIG. 1.
GEFT transcript is expressed highly in the hippocampus, granular cells, and Purkinje cells of the brain. In situ hybridization was performed on sagittally cut samples of adult mouse brains. A, x25 magnification of antisense GEFT mRNA binding to adult mouse brain. B, nonspecific binding using a sense mRNA probe for mouse GEFT was negligible. C, x100 magnification mouse GEFT mRNA expression in the hippocampus. D, x200 magnification of mouse GEFT mRNA expression in cerebellum. Scale bar = 100 µm.

 
GEFT Promotes Dendritic Spine Formation in Primary Hippocampus Neurons—Due to the high level of GEFT expression in the brain, specifically in the hippocampus, we sought to determine if GEFT might influence axonal outgrowth, dendrite spine formation, and dendritic arbor morphology by examining the effect of GEFT overexpression in rat dentate gyrus explant culture. GEFT significantly alters dendrite morphology in hippocampal neurons (Fig. 2, A and B), however, no noticeable difference was observed in axon length or branching (data not shown). To quantify the effect of GEFT on dendritic arbor complexity, we measured the total dendritic length and terminal tip number. As illustrated in Fig. 2C, there was a significant increase in terminal tip number (as a measure of dendritic branching) for GEFT transfected neurons (8.55 ± 0.77 tips/cell; control: 5.54 ± 0.27 tips/cell), further emphasizing its role in the regulation of dendritic outgrowth. However, the total dendrite length was found to be no different from the control cells (Fig. 2, D–F), suggesting that GEFT may primarily promote dendrite branching. In contrast to the subtle effects on overall dendritic morphology, overexpression of GEFT has more prominent effects on dendritic spines. As shown in Fig. 3, there was a significant increase in mature spine density for GEFT-transfected neurons (19.52 ± 1.59 spines/50-µm dendritic segment) as compared with control neurons (16.2 ± 0.67 spines/50-µm dendritic segment). In addition, the spines from the GEFT-transfected neurons were large in size as demonstrated by a significantly higher length (2.64 ± 0.07 µm), width (1.46 ± 0.02 µm), and area (2.25 ± 0.09 µm2) as compared with control spines (length, 1.85 ± 0.02 µm; width, 1.07 ± 0.01 µm; and area, 1.21 ± 0.01 µm2) (Fig. 3, E–G). Cumulative percentage graphs representing the overall spread or distribution of spine length, width, and area for vector and GEFT-transfected cells demonstrate very evidently an increase in spine length, width, and area for GEFT-transfected neurons (Fig. 3, H–J). Therefore, overexpression of GEFT in hippocampal neurons promotes a significant increment in mature spine density and larger spines with longer length, width, and area.



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  		FIG. 2.
GEFT increases the number of terminal dendritic tips in primary rat hippocampal neurons. The two panels demonstrate the GFP-transfected dentate gyrus neurons (A) and GFP-GEFT co-transfected neurons (B). The two graphs illustrate the quantification for dendritic length (C) and terminal tip number (D). The cells shown illustrate the prototype pattern of dendritic branching and length of control (E) and GEFT (F) expressing neurons. Scale bar = 50 µm. The p value for each experiment is <0.001.

 



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  		FIG. 3.
GEFT induces dendritic spine formation in primary rat hippocampal neurons. The two panels illustrate spine formation in GFP-transfected dentate gyrus neurons (A) and GFP-GEFT co-transfected neurons (B). The graphs illustrate mature spine number (C), filopodia number (D), dendritic spine length (E), width (F), and area (G). Cumulative percentage graphs are included for the spine length (H), width (I), and area (J), which show the overall spread or distribution of the data for these parameters. Scale bar = 20 µm (upper panel) and 5 µm (lower panel). The p value for each experiment is <0.001.

 
GEFT Induces Neurite Outgrowth in N2A Cells—To study more extensively the role of GEFT in the regulation of neurite outgrowth, we exogenously expressed this gene in a neuroblastoma cell line, N2A, and quantified the morphological changes in neurite outgrowth. FLAG-tagged GEFT was transiently expressed in N2A cells plated on gelatin-coated coverslips, and immunofluorescence was performed using an anti-FLAG antibody to examine the GEFT-mediated morphological changes in neurite outgrowth (Fig. 4A). Transfection efficiency was ~60%, allowing simultaneous observation of both transfected (bright staining) and non-transfected (nonspecific staining) cells. In non-transfected N2A cells, short neurites were present, but few in number. However, in N2A cells expressing GEFT, neurite outgrowths were significantly increased in number, length, and the degree of neuronal branching.



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  		FIG. 4.
GEFT induces neurite outgrowth in N2A neuroblastoma cells. A, a GEFT-FLAG fusion protein was transiently expressed in N2A cells. Immunostaining was performed to detect GEFT-FLAG. Neurite outgrowth was compared between transfected (bright cells) and untransfected cells (nonspecific staining). B, N2A cells were stably transfected with GEFT and detection of GEFT expression using anti-FLAG antibodies was performed. C and D, neurite outgrowth in stably transfected N2A cells expressing either vector control (C) or GEFT (D). The cells were grown for 48 h and observed under a phase contrast microscope. Scale bar = 250 µm.

 
Subsequently, stable cell lines were selected after transfection with either empty vector or GEFT. The expression of FLAG-tagged GEFT in the stable line was confirmed by Western blot analysis using an anti-FLAG antibody (Fig. 4B). We then quantified the morphology, number, and length of neurite outgrowths in N2A cells expressing empty vector or GEFT. Cells with neurites were defined as cells that possessed at least one neurite more than one-half the cell body diameter in length. Exogenous expression of GEFT in N2A cells induced significant changes in neurite morphology compared with control cells expressing vector only (Fig. 4, C and D). Control cells exhibited neurite outgrowths on 27.5 ± 8.2% of the cells (Fig. 5A), whereas GEFT-expressing cells exhibited neurite outgrowths on 49 ± 5.1% of the cells, an almost 2-fold increase for neurite outgrowth in GEFT-expressing cells compared with the control cell line (Fig. 5A). We further measured the average neurite length for control and GEFT-expressing cells (Fig. 5B). The average neurite length in control cells was 52 ± 4.2 µm. In the presence of exogenously expressed GEFT, the average neurite length increased to 103 ± 3.5 µm, resulting in a 2-fold increase in neurite length. In addition, we determined the average number of neurites per cell for vector and GEFT-expressing cells (Fig. 5C). Vector control cells exhibited 3.1 ± 0.6 neurites/cell, whereas GEFT-expressing cells exhibited 5.5 ± 0.9 neurites/cell. Therefore, our data suggest that expression of GEFT induces neurite outgrowth in N2A cells, including a strong increase in the number of cells exhibiting neurite processes, an increase in the number of neurites per cell, and an overall increase in the length of the neurite.



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  		FIG. 5.
GEFT expression increases the percentage of cells expressing neurites, the length of neurites, and the number of neurites per cell. N2A cells expressing either a control vector or GEFT were grown for 48 h and observed under a phase contrast microscope at x200 magnification. A, number of cells expressing neurites, neurite length (B), and the number of neurites per cell (C) were measured for both vector and GEFT cells. Cells with neurites were defined as cells that possessed at least one neurite more than one-half the cell body diameter in length. At least 250 cells were analyzed in each experiment, and the data are the means ± S.E. of triplicate experiments. The p value for each experiment is <0.05.

 
GEFT Induces Neurite Outgrowth via Activation of Rac1 in Neuronal Cells—It has been previously shown that exogenous expression of GEFT increases the active forms of both Rac1 and Cdc42 in NIH3T3 cells (22). To determine which Rho-GTPases mediate GEFT-induced neurite outgrowth of N2A cells in vivo, we compared the amount of GTP-bound forms (active status) of Rac1, Cdc42, and RhoA in N2A cells transfected with GEFT or a control plasmid. To determine the level of GTP-bound Rac1 and Cdc42 in the cells, we utilized a GST-PAK1 fusion protein containing the Rac1/Cdc42 binding domain as an affinity reagent in a GST pull-down assay. Pak1 is a downstream effector of Rac1 and Cdc42, and PAK1 binds only to the active GTP-bound forms of Rac1 and Cdc42 GTPases. To determine the level of GTP-bound Rho in the cells, we utilized a GST-Rho-binding protein fusion protein containing the Rho binding domain as an affinity reagent in a similar fashion as above. Transfection of GEFT in N2A cells increased the GTP-bound active forms of Rac1, Cdc42, and RhoA significantly when compared with the vector control (Fig. 6A). These data suggest that GEFT activates Rac1, Cdc42, and RhoA in the N2A cells by stimulating the guanine nucleotide exchange of the three GTPases. Although GEFT expression induces neuronal outgrowth, a process attributed to the activation of both Rac1 and Cdc42 and inhibited by RhoA, GEFT shows no specificity in catalyzing the GDP- to GTP-bound state for any Rho-GTPase in the N2A cells. It has previously been demonstrated that GEFT is most specific for Rac1 and Cdc42 in NIH3T3 cells, and multiple studies suggest that GEFT could regulate RhoA in a cell-type specific manner (22, 24, 25).



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  		FIG. 6.
GEFT activates the Rho family of small GTPases in N2A cells and promotes neurite outgrowth via Rac1. A, the amounts of activated Rac1 and Cdc42 were determined by GST pull-down assays using a GST-PAK1 domain. The amount of activated RhoA was determined by GST pull-down assays using GST-Rho-binding protein (RBP). Cells were transfected with GEFT or vector control, and lysates were incubated with either GST-PAK1 or GST-RBP. The collected fraction was analyzed using SDS-PAGE. The amounts of RhoA, Rac1, and Cdc42 were visualized by Western blot analysis using anti-RhoA-, anti-Rac1-, and anti-Cdc42-specific antibodies. Western blots were also performed to verify equal amounts of endogenous RhoA, Rac1, and Cdc42 expression. Number of neurites per cell (B) and neurite length (C) were measured for vector and GEFT N2A cells co-transfected with constitutively active (+) and dominant negative (–) forms of the RhoA, Rac1, and Cdc42. Cells with neurites were defined as cells that possessed at least one neurite more than one-half the cell body diameter in length. At least 250 cells were analyzed in each experiment, and the data are the means ± S.E. of triplicate experiments. p value for each experiment is <0.05.

 
Although GEFT may activate RhoA, Rac1, and Cdc42, it is essential to determine which of the Rho-GTPases acts as the effector for downstream induction of neurite outgrowth. Using constitutively active and dominant negative RhoA, Rac1, and Cdc42 mutants, we examined the downstream targets of GEFT in inducing neurite outgrowth by co-transfection of the mutants with GEFT. As shown in Fig. 6 (B and C), induction of neurite outgrowth (measured as number of neurites per cell (Fig. 6B) and neurite length (Fig. 6C)) in both vector and GEFT-expressing N2A cells was observed with the addition of the constitutive active mutants Rac1-G12V and Cdc42-G12V, respectively. As expected, in vector control cells, addition of Rac1-T17N and Cdc42-T17N dominant negative mutants blocked the induction of neurite outgrowths. In GEFT-expressing cells, co-transfection of Rac1 dominant negative mutant (T17N) significantly decreased neurite outgrowth compared with cells expressing GEFT alone or GEFT coexpressing Rac1-T17N, suggesting Rac1 plays a major role in GEFT-mediated neurite outgrowth. However, addition of Cdc42 dominant-negative mutant (Cdc42T17N) to GEFT-expressing cells showed no significant decrease in neurite outgrowth compared with that observed in GEFT cells. Inhibition of RhoA activity by transfection of dominant negative RhoA (T19N) resulted in increased neurite outgrowth in GEFT-expressing cells; however, addition of constitutively active RhoA (G14V) did not abrogate the effects of GEFT overexpression on neurite outgrowth. These data suggest that Rac1 plays the major role in GEFT-induced neurite outgrowth.

PAK1 and PAK5 Are Essential for GEFT-induced Neurite Outgrowth—Members of the mammalian p21-activated kinase (PAK) family of serine/threonine kinases constitute effectors for Rac1 and Cdc42, but not RhoA (31, 32). Two PAK family members, PAK1 and PAK5, have been implicated in neurite outgrowth. PAK1 kinase activity alone has been shown to be insufficient to induce neurite outgrowth, rather, the targeting of PAK1 to the plasma membrane induces the outgrowth of neurite-like structures in PC-12 cells (32). PAK5, a brain-specific PAK family member similar to Drosophila MBT ("mushroom body tiny") protein, has been shown to trigger both filopodium formation and neurite outgrowth in N1E-115 cells (32).

To investigate whether GEFT-mediated activation of Rac1 and Cdc42 triggers the activation of PAK, the kinase activity of PAK1 was assessed using myelin basic protein as a substrate (Fig. 7A). PAK1 activity was significantly increased in GEFT extracts (lane 2) compared with vector extracts (lane 1), suggesting that exogenous expression of GEFT up-regulates PAK1 activity.



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  		FIG. 7.
GEFT induction of neurite outgrowth is via PAK1 and PAK5. A, PAK kinase assays were performed with lysates from N2A cells expressing exogenous vector control (lane 1) or GEFT (lane 2) using myelin basic protein as a substrate for phosphorylation. Total Pak1 levels were verified using Western blots with an anti-Pak1 specific antibody. Number of neurites per cell (B) and neurite length (C) were measured for vector and GEFT N2A cells co-transfected with constitutively active (+) and dominant negative (–) forms of the PAK1 and PAK5. Cells with neurites were defined as cells that possessed at least one neurite more than one-half the cell body diameter in length. At least 250 cells were analyzed in each experiment, and the data are the means ± S.E. of triplicate experiments. p value for each experiment is <0.05.

 
To determine whether PAK1 and PAK5, both involved in regulating neurite outgrowth, mediate the promotion of GEFT-induced neurite outgrowth, N2A cells were co-transfected with PAK1 or PAK5 constitutively active and dominant negative mutants, along with GEFT or empty vector. As anticipated, addition of constitutively active PAK1-T423 or PAK5-S573N/S602E led to the induction of neurite outgrowth (as measured by number of neurites per cell (Fig. 7B) and length of neurites (Fig. 7C) in both vector and GEFT-expressing cells. Single addition of the dominant negative mutants, PAK1-K299R/H83L86L or PAK5-K478M, exhibited a major reduction in the levels of neurite outgrowth as compared with GEFT-expressing cells, with PAK5-K478M decreasing neurite outgrowth nearly to the level of the vector control. This suggests that the PAK1 dominant negative mutant alone is not sufficient to abolish GEFT-induced neurite induction. Expression of a PAK5 dominant negative mutant or co-transfection of PAK1 and PAK5 dominant negative mutants together in GEFT-expressing cells completely diminished neurite outgrowth to the level of the vector control, suggesting that PAK5 plays the major role in GEFT-induced neurite outgrowth.

GEFT Activates Rho Family-mediated Transcriptional Activities in Neurite Outgrowth—To further examine the signaling pathways activated by GEFT, we examined the ability of GEFT to stimulate transcription factors in the regulation of neurite outgrowth and nerve regeneration. It has been shown that activation of the Rho family of small GTPases leads to the MAPK-dependent activation of a number of transcriptional factors involved in cell proliferation and survival in a diverse number of cell types (33). Specifically, the AP-1 transcription factor, consisting of homo- or heterodimers of c-fos and c-jun, has been shown to be sufficient to induce neurite outgrowth in PC12 cells (34) and play a role in the initial regeneration of neurons after severe injury (35). In addition, the NF-{kappa}B signaling cascade has been found to be important for both neurite outgrowth and neuron survival (36). Using luciferase assays, we measured the effect of exogenously expressed GEFT on the activation of AP-1- and NF-{kappa}B-responsive promoters. N2A cells were transfected with luciferase reporter genes, together with the expression plasmids encoding RhoA, Rac1, and Cdc42. As shown in Fig. 8, expression of Rac1, Cdc42, and RhoA activates transcriptional activity of AP-1 and NF-{kappa}B, respectively, as judged by an increase in luciferase signal. Co-transfection of GEFT with Cdc42, Rac1, or RhoA synergistically increased both AP-1- and NF-{kappa}B-responsive promoter activity (Fig. 8, A and B) in our transcriptional reporter assays. These data suggest that GEFT mediates the Rho signaling pathways to regulate the transcriptional activities of AP1- and NF-{kappa}B-responsive promoters in neurite outgrowth and regeneration.



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  		FIG. 8.
GEFT activates AP-1 and NF-{kappa}B transcriptional activity. N2A cells were transfected with luciferase reporter plasmids, GEFT, and wild type forms of RhoA, Rac1, and Cdc42 as shown above. Luciferase assays were performed. Data shown are the average of three repeats and are the means ± S.E. of triplicate experiments. Similar experiments were performed at least three times.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The signal transduction pathways leading to the regulation of neurite outgrowth, including axon and dendrite development, have been subjected to intensive research in recent years. Considerable research has established that the Rho family of small GTPases is a key regulator of cytoskeleton structural changes following cell stimulation by growth factors (3). Cytoskeletal organization is a critical part of neuronal development, leading to the formation of various processes such as filopodia and lamellipodia, which play key roles in the guidance of neuronal growth cones toward attractive cues and away from repulsive cues and eventually leading to the formation of neurite outgrowths (37).

GEFT was initially identified as a potent oncogene in an enhanced retroviral mutagenesis screen for foci-forming genes in NIH3T3 cells (23). GEFT shows sequence homology to the Dbl family of GEFs and has been shown to activate the Rho family of small GTPases in both NIH3T3 and COS-7 cells, and dramatically promotes actin-cytoskeleton rearrangements with these cell types (22). This former study found that GEFT is highly expressed in brain tissue, suggesting the possibility of a role in neuronal differentiation and morphogenesis. Using in situ hybridization, we looked at the precise regions of the brain in which GEFT transcript is expressed. Expression was found ubiquitously throughout the brain; however, the highest level of expression was in the hippocampus as well as the Purkinje and granular cells of the cerebellum. The cell types in these regions play essential roles in correct neural function. The hippocampus is associated with the regulation of emotions, in the control of autonomic functions, and the transfer of information from short term memory to long term memory. Multiple lines of evidence suggest that the Rho family is important in hippocampal function (8, 38–40). Kalirin, a GEF homologous to GEFT in the Dbl homology and pleckstrin homology domains, has been shown to regulate dendritic spine formation in hippocampal neurons in response to ephrin stimulation (41). In addition, it has been demonstrated that the Rho family of small GTPases is activated by synaptic transmission during both low frequency stimulation and long term potentiation in the hippocampus (42). Within the cerebellum, granule cells function by sending excitatory bifurcate axons along the cerebellar cortex in the parallel fiber pathway. These axons make essential connections to the highly complex dendritic system of the Purkinje cells, which forms a monolayer around the granular layer of the cerebellum and functions as the sole output from the cerebellar cortex. The neuronal localization of GEFT transcript correlates well with recent data showing that the Rho family of GTPases is highly expressed within the hippocampus as well as the molecular and granular layers in the cerebellum (43).

Based on the data demonstrating that GEFT expression is high in the hippocampal region, and that previous data suggest that the Rho family is involved in neuronal plasticity (44, 45), we have tested and shown that GEFT functions in the regulation of dendritic development of primary rat hippocampal neurons. Transfection of GEFT into hippocampal neurons from rat dentate gyrus explant culture resulted in a significant increment in mature spine density, as well as larger spines with significantly longer length, width, and area. Whereas there was no increase in dendritic length in GEFT-expressing cells, there was a significant increase in terminal tip number upon GEFT expression. Furthermore, transfection of GEFT into N2A cells led to an induction in neurite outgrowth, including a strong increase in the number of cells exhibiting neurite processes, an increase in the number of neurites per cell, and an overall increase in the length of the neurite. These data suggest that GEFT is a potent regulator of dendritic and neurite outgrowth, and could potentially play a role in neuronal guidance and pathfinding.

Other regulators of the Rho family of small GTPases have been previously found to perform a role in the regulation of neurite outgrowth as well as alterations in the plasticity of pre-existing neuronal structures, including Tiam (the invasion-inducing T-lymphoma invasion and metastasis 1), Kalirin, and Trio (41, 46–50). Although we are unsure of the in vivo role of GEFT in neural function, we are currently addressing this issue with both transgenic and knockout mouse models.

Several studies have implicated the Rho family of GTPases and their downstream effectors in the regulation of neurite outgrowth. Rac1 and Cdc42 are established to serve stimulatory functions in neurite outgrowth, whereas RhoA appears to have an inhibitory function (50). We have shown that GEFT induces neurite outgrowth primarily via Rac1 activation. This is interesting considering that when using both GST-pull downs of activated Rho-GTPases and luciferase assays, GEFT appeared to strongly activate not only Rac1, but both Cdc42 and RhoA. However, to date no studies have examined the spatial relationship between GEFT and the Rho-GTPases, allowing the possibility that activation of specific GTPases occurs in discreet subcellular locations. In addition, although both Rac1 and Cdc42 are required for neurite formation in N2a cells, the former participates predominantly in lamellipodium formation and the latter in filopodium formation. Although both processes are important for neurite outgrowth, perhaps GEFT activity, in regard to neurite outgrowth, is most specific for Rac1-mediated lamellipodium formation. Our data have demonstrated that hippocampal neurons that express GEFT exhibit a reduction in the number of filopodia compared with the control cells. Perhaps this effect is mediated via GEFT activation of RhoA, because RhoA has been shown to inhibit filopodia formation (51). One of the downstream targets of Rac1 and Cdc42 is the PAK family of kinases. PAK1 is phosphorylated (active form) in the presence of exogenous GEFT. Both PAK1 and PAK5, especially PAK5, appear to play a positive role in the GEFT induction of neurite outgrowth, because addition of dominant negative PAK mutants completely inhibited GEFT-induced neurite outgrowth.

MAPK-regulated transcription factors (AP-1 and NF-{kappa}B) are capable of mediating the expression of genes involved in cell growth, proliferation, neurite outgrowth, and survival. Two such factors, NF-{kappa}B and AP-1, play important roles in the regulation of neurite outgrowth and regeneration. Overexpression of NF-{kappa}B has been associated with central nervous system lesions in a diverse array of diseases such as Parkinson's disease, Alzheimer's disease, AIDs dementia, and spinal cord injury (52–55). In addition, NF-{kappa}B has been shown to promote neuronal cell survival and to positively regulate neurite outgrowth (36). In fact, inhibition of NF-{kappa}B leads to a 50% reduction in neurite outgrowth in neuronal cells (36). Activation of AP-1, which is a homo- or heterodimer of c-fos and c-jun basic leucine zipper transcription factors, is sufficient to induce neurite outgrowth in PC12 cells (34). In addition, AP-1 binding to its promoter region is dramatically increased immediately following sciatic nerve injury and remains active while neurons are regenerating (35). We found that GEFT is capable of up-regulating the activities of both NF-{kappa}B and AP-1 transcription factors via activation of the Rho family of small GTPases. Perhaps GEFT could have an important role in processes involved in neurodegenerative diseases and nerve regeneration.

A variety of extracellular stimuli have been found to activate the Rho family of GTPases, including growth factors, cytokines, lysophosphatidic acid, interleukins, and matrix components via receptor tyrosine kinases, G protein-coupled receptors, and intergrin receptors, respectively. However, the mechanism by which different signaling pathways directly link the extracellular stimuli to the intracellular signal change is one of the key questions yet to be completely answered. This area has only recently become subject to considerable investigation, most notably with Kalirin, which regulates dendrite plasticity via activation of the EphrinB receptors, and Trio, which is involved in nerve growth factor-induced neurite outgrowth (49, 56). Our study did not investigate which extracellular signaling molecule leads to the activation of GEFT and its downstream effectors. However, future studies, specifically examining the activation of GEFT by extracellular signals, will address this level of regulation and help to shed light on this unknown. In addition, functional studies of GEFT in vivo and in neuronal development will be further characterized by GEFT transgenic and knockout mice, both of which are presently being undertaken in our laboratory.

In summary, we have characterized the neuronal role of GEFT, a new guanine nucleotide exchange factor specific for the Rho family of small GTPases, in spine formation and neurite outgrowth. GEFT is highly expressed in the hippocampus as well as the granule cells and Purkinje cells of the cerebellum, all of which depend on precise neural connections for cellular function. Overexpression of GEFT in both primary hippocampal neurons and neuroblastoma cell lines greatly enhances dendritic spine formation and neurite outgrowth, respectively. Neurite outgrowth induced by GEFT is mediated primarily via Rac1 and its downstream effector, PAK, and GEFT up-regulates key signaling cascades and transcription factors involved in neurite outgrowth and nerve regeneration.


   FOOTNOTES
 
* This work was supported by a grant from the National Institutes of Health, a Scientist Development Award from the American Heart Association, and a seed grant from Mission Connect, The Institute for Rehabilitation and Research Foundation to (to M. L.). 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: Alkek Institute of Biosciences and Technology, Texas A&M University System Health Science Center, 2121 W. Holcombe Blvd., Houston, TX 77030. Tel.: 713-677-7505; Fax: 713-677-7512; E-mail: mliu{at}tamu.edu.

1 The abbreviations used are: GEF, guanine nucleotide exchange factor; GFP, green fluorescent protein; PAK, p21-activated kinase; MAPK, mitogen-activated protein kinase; RBP, Rho-binding protein. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Jonathan Chernoff at Fox Chase Cancer Center for PAK1 constructs and Dr. Audrey Minden at Columbia University for PAK5 constructs. We also thank Dr. James F. Martin and Jennifer Selever in the Center for Cancer Biology and Nutrition for their help with mouse brain tissues and suggestions.



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 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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