For this report, we have investigated the effects of RhoE on cellular responses by using RhoE-inducible cell lines. RhoE expression inhibited cell proliferation and induced a G1 arrest, and it also reduced cell transformation by oncogenic Ras and Raf. Despite the activation of signaling pathways known to control cyclin D1 expression, RhoE-expressing cells failed to synthesize the cyclin D1 protein. The expression of E7, E1A, or cyclin E, which eliminates the Rb pocket protein G1 checkpoint, was sufficient to rescue cell proliferation in RhoE-expressing cells. Interestingly, endogenous RhoE levels increased in response to the DNA-damaging agent cisplatin, suggesting that RhoE could contribute to a checkpoint arrest induced by specific stimuli.
We consistently observed that RhoE inhibited cell growth, leading to the accumulation of RhoE-expressing cells in G1
, both in asynchronously growing cells and in synchronized cells. Cell cycle progression in fibroblasts requires cell attachment to the extracellular matrix via integrin-containing adhesions (42
). Thus, the reduction in contractility and adhesion induced by RhoE could indirectly lead to cell cycle arrest, although ROCK-dependent stress fibers are not essential for cell proliferation (36
). However, the cytoskeletal effects elicited by RhoE were only transient, since between 12 and 24 h after RhoE expression was first detected, normal levels of stress fibers were observed and the cell morphology was similar to that of control cells. It should be noted that previous work describing the cytoskeletal effects induced by Rnd family members analyzed cell morphology at short times after protein or cDNA injection (13
). Since our cell cycle analysis was performed long after RhoE-expressing cells had reverted to a normal cytoskeletal phenotype, cell cycle inhibition in response to RhoE was not a consequence of its cytoskeletal effects. In agreement with this, we found that long-term RhoE expression did not compromise signaling events that are sensitive to cytoskeletal alterations and/or adhesion defects, such as ERK and FAK activation (18
The most significant difference in G1
regulators that we observed in RhoE-expressing cells was the lack of cyclin D1 and p21cip1
expression. RhoE-expressing cells also did not fully downregulate p27kip1
in response to serum, probably due to their failure to progress through the G1
/S transition, since p27kip1
degradation is an event triggered by cdk2-dependent phosphorylation late in G1
). The absence of p21cip1
is not likely to compromise cell proliferation (8
), but the lack of cyclin D1 in RhoE-expressing cells would prevent cdk4 activation and subsequent events in G1
phase progression. cdk4 is one of the kinases that contribute to pocket protein phosphorylation and functional inactivation, and in agreement with this, RhoE-expressing cells showed a significant inhibition in E2F transcriptional activity, as indicated by reporter assays and by the reduced expression of known E2F target genes. Thus, activation of the G1
-phase regulatory machinery was impaired in RhoE-expressing cells.
Cyclin D1 serves as a major signaling integrator of G1
progression, and its expression is tightly regulated by many signaling pathways, allowing extracellular signals to impinge on the core cell cycle machinery (9
). Surprisingly, neither the ERK pathway nor the PI3K/Akt pathway, two major pathways that modulate cyclin D1 transcriptional activation (37
) and protein stability (10
), appeared to be affected by RhoE. Similarly, we did not detect any differences in FAK and Rac activation, which have both been implicated in cyclin D1 expression (12
). It is possible that regulators further downstream on these pathways could be affected by RhoE. Interestingly, our data addressing the mechanisms underlying the lack of cyclin D1 showed that cyclin D1 transcriptional induction was not impaired by RhoE but that the production of cyclin D1 protein was inhibited.
Although the absence of cyclin D1 was the major alteration in the G1-phase machinery that we observed in RhoE-expressing cells, ectopic expression of cyclin D1 alone could not rescue S-phase entry, indicating that RhoE probably affects multiple targets to induce cell cycle arrest. However, the observation that E7, E1A, and cyclin E were all able to promote cell proliferation in RhoE-expressing cells confirmed that RhoE-mediated G1 arrest takes place prior to pocket protein hyperphosphorylation, as it can be overcome by signals that bypass the pRb-mediated restriction point.
We also analyzed changes in RhoE expression levels upon exposure to different stimuli in order to identify conditions in which RhoE could have a regulatory role in cell cycle progression. RhoE protein levels do not change throughout the cell cycle in NIH 3T3 cells, suggesting that RhoE does not regulate growth factor-induced cell cycle progression, although we cannot exclude the possibility that RhoE activity could be regulated through other mechanisms apart from altering expression levels. Interestingly, however, the induction of DNA damage with cisplatin, an antitumor agent that activates several checkpoint-related pathways leading to cell cycle arrest, induces an increase in RhoE levels. RhoE levels have also been reported to increase in response to UVB irradiation (21
), suggesting that RhoE may have a role in DNA damage-induced checkpoint control.
We also investigated whether the mechanisms that have been proposed to explain the cytoskeletal effects induced by RhoE, namely, ROCK I inhibition (35
) and p190RhoGAP activation (49
), could account for RhoE-mediated cell cycle arrest. The fact that RhoE induced cell cycle inhibition in the absence of visible cytoskeletal alterations suggests that the mechanisms underlying RhoE-induced cytoskeletal and proliferative effects could be independent. Since ROCK inhibition is compatible with cell proliferation (39
), we reasoned that ROCK I inhibition did not account for the RhoE-mediated cell cycle inhibition. In contrast, RhoA inhibition is known to induce cell cycle arrest (40
), and thus the RhoE-mediated effects on the cell cycle could be secondary to an inhibitory effect on RhoA. However, cell cycle inhibition induced by RhoA inhibition is characterized by different molecular events than RhoE-induced cell cycle arrest. Whereas RhoA (and ROCK) inhibition induces early cyclin D1 expression in NIH 3T3 cells (48
), cyclin D1 levels are undetectable in RhoE-expressing NIH 3T3 cells. Moreover, cell cycle arrest induced by RhoA inhibition is linked to increases in p21cip1
), whereas RhoE-expressing cells do not detectably express p21cip1
. Indeed, we did not detect any change in RhoA activation in RhoE-expressing cells, in contrast with a previous report (49
) in which mouse embryo fibroblasts transduced with Tat-fused RhoE showed decreased RhoA-GTP levels. It is conceivable that the effects observed by Wennerberg et al. are transient and/or only apparent upon the expression of very high levels of RhoE. Importantly, RhoA activation is not affected by RhoE in the context of mitogen-induced cell cycle entry, indicating that RhoE-induced cell cycle arrest is independent of its reported effect on RhoA. Consistent with these observations, constitutively active RhoA could not rescue cell growth in RhoE-expressing cells.
The ability of RhoE to inhibit focus formation induced by oncogenes such as Ras and Raf is likely to be a consequence of inhibiting cell cycle progression. Interestingly, RhoE has been shown to be upregulated in colon cancer cell lines in response to sulindac, a nonsteroidal anti-inflammatory drug with antiproliferative properties in these cells. At the time, none of the identified cDNAs that were upregulated in response to sulindac provided a plausible explanation for its antiproliferative effects (1
). Our data suggest that RhoE could contribute to the antiproliferative effects of nonsteroidal anti-inflammatory drugs. Conversely, estrogen treatment of prostatic stromal cells, a stimulus that has been implicated in prostate cancer progression, has been shown to induce RhoE repression, fueling the concept that RhoE may indeed have antitumoral effects (3
). Although these data collectively suggest a negative role for RhoE in cell proliferation and transformation, RhoE can also enhance cell migration through its cytoskeletal effects (13
) and could thereby contribute to cancer cell invasion. RhoE has indeed been shown to be important for morphological changes in the Raf-induced transformation of MDCK cells (14
). RhoE may therefore have either a positive or a negative role in tumorigenesis, depending on the cellular background. Cancer cells harbor many oncogenic alterations, such as mutations in the cyclin D/p16/pRb axis, which deregulate their cell cycle machinery (43
) and would override RhoE-imposed cell cycle arrest. These cells could benefit from a RhoE-mediated increase in invasiveness and motility. Interestingly, RhoE overexpression has been reported for a subclass of neuronal tumors, desmoplastic medulloblastomas (26
In conclusion, we have shown that RhoE can inhibit cell proliferation and transformation in addition to its known effects on the actin cytoskeleton, and we propose that the fine-tuning of RhoE function in cells contributes to the balanced coordination of cell proliferation and migration.