The findings of this study suggest that RhoB is a modifier of adhesion and growth factor signals that are associated with cellular stress, in particular stresses associated with neoplastic transformation. A defect in MEF motility was detected. This defect might relate to inappropriate integrin trafficking at some level. RhoB has been suggested to have a specialized function in intracellular receptor trafficking (9
), and differences in β1 integrin mobility on gels suggestive of a difference in posttranslational modification were observed. We interpreted the defect in MEF motility to be conditional on stress, however, because (i) −/− mice developed in an apparently normal manner; (ii) −/− mice did not display any defects in wound healing, where motility defects would be expected to be manifested; and (iii) in vitro culture subjects MEFs to stress (for an example, see reference 19
). In support of some role in motility, RhoB has been implicated in the delamination of neural crest cells during chick development (18
). However, the mechanisms governing this role may have varied during evolution, insofar as stress appears to be a prerequisite for RhoB to influence cell motility in mouse cells.
Through studies of the antineoplastic properties of FTIs we have previously documented a specialized connection between RhoB function and neoplastic transformation. These studies indicated that FTIs act not by eliminating RhoB function but instead by elevating a geranylgeranylated RhoB isoform that negatively impacts the proliferation and survival of transformed cells (3
). This gain-of-function mechanism is compatible with the findings of this study, which argues that RhoB has a negative regulatory or modifier function in transformed cells. FTIs may to a large degree mediate their antitransforming effects by accentuating an intrinsic negative regulatory function of RhoB. Previous findings suggesting a positive role for Rho proteins in transformation were gained by the use of dominant inhibitory mutants of RhoA or RhoB (13
). These mutants broadly block endogenous Rho functions by competing for a variety of Rho exchange factors. By specifically eliminating RhoB we have shown that this Rho protein has a negative role rather than a positive role in transformation. A tumor-prone phenotype was revealed by the increased propensity of rhoB
null mice for DMBA-induced papilloma formation. The results were consistent with an effect on the kinetics of tumor initiation, but it would be premature to conclude that RhoB loss acts in this way without additional investigation. Ongoing “oncomouse” crosses will allow this issue to be assessed and possible tumor suppressor or modifier roles for RhoB to be examined further.
Our findings confirm previous evidence that RhoB attenuates growth factor responses in established cells (5
). A focus on TGF-β was stimulated by observations linking RhoB to this context-dependent regulator of transformed cell growth. TGF-β stimulates actin stress fiber formation in Ras-transformed cells in a manner that is associated with upregulation of RhoB and RhoA and that is reversed by the generalized Rho inhibitor C3 transferase (21
). Here we showed that although RhoB was dispensable for TGF-β-induced stress fiber formation in transformed cells, it sensitized the cells to this process, a result that argues for its participation in the TGF-β response. Similarly, while RhoB loss did not abolish the effects of TGF-β on transformed cell proliferation, it modified the response in an adhesion-dependent manner. While we did not identify the basis for these biological effects, preliminary results from gene hybridization experiments argue against the notion that there are differences in receptor levels in cells (N. Rane, unpublished observations). Taken together, these observations prompt further investigations of how RhoB may influence TGF-β signaling in cells, particularly in the context of transformation or other stress- or disease-associated states.