Although the cellular functions of mammalian Cdc42 has been well investigated by the dominate negative or constitutively active mutant expression approach in a variety of clonal cell lines, it has become clear that experimental limitations of the conventional methods could lead to erroneous interpretations. Recent studies of Cdc42 function using genetic knockout approach in ES and ES-derived fibroblastoid clones (Chen et al., 2000
; Czuchra et al., 2005
) raise concerns about the physiological significance of some of previous findings. Our present work demonstrating that Cdc42 is critically involved in actin filopodia formation, cell motility, directional migration, and cell growth and is required for the serum regulation of PAK1, GSK3β, MLC, ERK1/2, JNK, and NF-κB pathways in primary MEFs help affirm that Cdc42 indeed can play these roles in primary cells. The comparative studies of Cdc42 loss-of-activity and gain-of-activity primary MEFs further suggest that a balanced Cdc42 activity is important for proper signaling output and cell regulation.
In Swiss 3T3 fibroblast cells, it was proposed that a hierarchy of signaling cascade from Cdc42 to Rac1 to RhoA may be at work in mediating cell actin reorganization (Nobes and Hall, 1995
). Such cross-talk among the Rho GTPases is likely cell type and clonal dependent. In primary MEFs, Cdc42-deficiency leads to reduced Rac1 activity but normal RhoA activity under both serum-free and serum-stimulation conditions, whereas Cdc42GAP knockout cells display constitutively elevated Cdc42 activity without detectable alteration of Rac1 or RhoA activity (), suggesting that Cdc42 activity is necessary but not sufficient for Rac1 activation. Because most phenotypes of Cdc42−/−
cells cannot be rescued by expression of an active Rac1 mutant, Cdc42 appears to regulate cell behaviors including actin organization, adhesion, and directed migration in a Rac1 activity–independent manner. However, it remains possible that certain phenotypes we observed in the Cdc42−/−
cells are associated with other related Rho GTPase activities that are regulated by Cdc42.
Cdc42 regulation of actin polymerization and filopodia formation has been shown in a wide variety of mammalian cell types as well as in yeast, flies, and worms. Studies carried out in Cdc42-deficient ES cells provided supporting evidence of its essential role in actin polymerization and actin microfilament formation. However, recent studies in clonal fibroblastoid cells generated by differentiation and immortalization of ES cells showed normal formation of filopodia and lamellipodia in the absence of Cdc42, raising the possibility that Cdc42-related Rho GTPases such as Wrch2 or TC10 could be playing a redundant role. In our primary cell setting, MEFs did not form filopodia after bradykinin stimulation or at the leading edge after wound damage upon Cdc42 deletion, whereas spontaneous filopodia were abundant in Cdc42GAP−/−
cells (), suggesting that Cdc42 activity is necessary and sufficient for filopodia induction. One unexpected observation of the Cdc42−/−
cells is that no dorsal lamellipodia was formed in the absence of Cdc42 upon PDGF stimulation when peripheral lamellipodia was evident. Takenawa's group has shown that WAVE1 and WAVE2 play differential roles in dorsal and peripheral lamellipodia formation, with WAVE1 being essential for dorsal ruffling and WAVE2 key for peripheral ruffle formation (Suetsugu et al., 2003
). In this context, our data suggest that Cdc42 contributes to the regulation of WAVE1 or a related pathway in dorsal lamellipodia induction.
Cdc42 was found to regulate adhesion complex formation and cell migration (Nobes and Hall, 1999
). In macrophages inhibition of Cdc42 blocks chemotaxis toward a CSF gradient without affecting cell mobility (Ridley, 2001
). In Drosophila
Cdc42 loss of function does not affect the migration of peripheral glial cells (Sepp and Auld, 2003
). In our studies, Cdc42 deletion in primary MEFs causes abnormal cell spreading, reduced adhesion to fibronectin, defective mobility in wound healing, and decreased chemotaxis toward a serum gradient ( and ). Some of these effects may be related to impaired formation of filopodia and/or defective polarity of the cells. In the Cdc42 gain-of-activity Cdc42GAP−/−
MEFs, adhesion and directional migration were inhibited but motility in wound healing was not (), consistent with the notion that Cdc42 activity is involved in directional movement. We further found that the serum-regulated signaling components including PAK1, GSK3β, MLC, cofilin, and FAK were affected by Cdc42 deletion (), supporting previous findings that the Cdc42-PAK1-MLC/Cdc42-PAK1-cofilin pathways are important for actin reorganization, the Cdc42-GSK3β signaling axis is important for cell polarized migration, and the Cdc42 may regulate FAK activity in modulating adhesion (Etienne-Manneville and Hall, 2002
; Ridley, 2001
In Cdc42-deficient ES or ES-derived fibroblastoid cells, cell growth proceeds normally without mitotic defects (Chen et al., 2000
; Czuchra et al., 2005
). In primary MEFs, however, Cdc42 activity is critical for cell proliferation, as loss or gain of activity of Cdc42 affects cell cycle progression and/or survival (; Wang et al., 2005
). The cell growth defects of the Cdc42−/−
cells correlate with defects in ERK1/2, JNK, and/or p70S6K activity, and with a defect in transcriptional activation of NF-κB ( and ). Although our results are mostly consistent with previous reports in the literature where Cdc42 is known to be essential in eukaryotic cell growth and can regulate apoptosis and G1/S-phase transition under dominant mutant overexpression conditions, they provide the first genetic evidence that Cdc42 plays an important cell growth regulatory role in a mammalian primary cell setting.
Direct comparison of the proliferation properties of the Cdc42 loss- and gain-of-activity cell models leads to a few unexpected observations of Cdc42 signaling and function. First, both loss and gain of Cdc42 activities in MEFs cause cell growth inhibition. Loss of Cdc42 affects both the cell cycle G1/S-phase transition and cell survival, whereas gain of Cdc42 activity increases spontaneous apoptosis only (Wang et al., 2005
). Second, loss of Cdc42 dampens serum-induced PAK1 and JNK activities, whereas gain of Cdc42 activity promotes PAK1 and JNK activation. The PAK1-JNK axis appears to be the only pathway examined that show a “linear” tendency of regulation by Cdc42 activity in the two knockout cell models. Other related pathways such as ERK1/2, p38, or p70S6K show either no effect or disparate effect by Cdc42 or Cdc42GAP deletion (e.g., both Cdc42 and Cdc42GAP knockout cells show a decrease in p70S6K response, whereas Cdc42−/−
cells are deficient in ERK1/2 response but Cdc42GAP−/−
cells appear normal in ERK1/2 activity). Third, although Cdc42 activity is necessary but not sufficient for serum-induced NF-κB transcription, it is neither necessary nor sufficient for SRF transcription activation. It remains to be seen if most of these altered signaling pathways are involved in the growth phenotypes of the Cdc42 and Cdc42GAP knockout cells, because the elevated JNK activity in Cdc42GAP−/−
MEFs is responsible for increased cell apoptosis. Overall, our parallel examination of Cdc42−/−
MEF cells suggests that a balanced, or tightly regulated, Cdc42 activity is essential for proper signaling effects.
Given the multiple effector pathways implicated in Cdc42 signaling to the actin cytoskeleton and nucleus, including the PAK1 regulated MLC and MEK1 and the Par3/Par6/aPKC complex-mediated GSK3β, it was somewhat surprising that Cdc42-deficient ES cells exhibited normal phosphorylation patterns of mitogen- and stress-activated protein kinases and other related signaling kinases, including GSK3β, ERK1/2, JNK, p38, or AKT, and that Cdc42−/−
fibroblastoid cells also appeared normal in these signaling responses. In this context, our primary Cdc42 loss- or gain-of-activity MEFs display mostly similar pattern of signaling alterations as expected in the literature. One explanation of the discrepancies between our observations and the ES or ES-derived fibroblastoid studies may be the cell-type differences. Because Cdc42 conventional knockout mice die at the gastrulation stage, it is possible that Cdc42 is dispensable during early embryonic development and ES cell growth. Recent gene targeting studies implicating Cdc42 in keratinocyte stem cell differentiation (Wu et al., 2006
) and in hematopoietic stem cell quiescence maintenance (Yang et al., 2005
) further highlight a cell lineage–specific role of Cdc42. Another explanation could come from differences in primary and clonal cell genetic backgrounds. Cdc42−/−
MEFs are spontaneously immortalized and can grow similarly like WT MEFs (our unpublished observation), suggesting that Cdc42 is not essential for cell proliferation once immortalized, as is the case in the ES-derived fibroblastoid cell lines. These explanations may also apply to the differences between our results and previous observations in actin organization and cell migration (Czuchra et al., 2005
). Because Cdc42 is ubiquitously expressed and is capable of mediating signal transduction in multiple pathways, a major challenge in future studies is to define its cell-type– and stimulus-specific signaling mechanism and function in diverse cell settings under physiological conditions.