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FJ
EXPRESS SUMMARY ARTICLE The Full-length version of this article is also available, published online September 7, 2005 as doi:10.1096/fj.05-4030fje. |
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* Inserm U461, Faculté de Pharmacie Paris-XI, Chatenay-Malabry, France;
¶ Inserm U533, Faculté des Sciences, Nantes, France; and
Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/Inserm/ULP, Illkirch, France
1 Correspondence: E-mail: j.bertoglio{at}cep.u-psud.fr
SPECIFIC AIMS
Rho GTPases are key regulators of many cellular functions including cytoskeleton organization, which is important for cell morphology and mobility, cell cycle progression and cytokinesis. It has recently been recognized that Rho GTPase activity is required for the specialized functions of the peripheral cells that act in the immune response such as antigen presenting cells and lymphocytes. Stimulation of T lymphocytes with interleukin-2 (IL-2) induces clonal expansion of antigen specific populations and provides a model to study cell cycle entry and cell cycle progression.
We have performed gene expression analysis in a model of human T lymphocytes that proliferate in response to IL-2. In addition to changes in genes relevant to cell cycling and to the antiapoptotic effects of IL-2, we specifically analyzed expression and variations of > 300 genes involved in Rho GTPase-signaling pathways.
PRINCIPAL FINDINGS
1. Detection of known or expected IL-2-regulated genes in Kit 225 cells
Kit 225 is a nontransformed CD4 positive human lymphocyte cell line that strictly depends on IL-2 for proliferation. When deprived of IL-2, Kit 225 cells become arrested in the G1 phase of the cell cycle and will undergo apoptosis after 72 h unless rescued by IL-2 addition. Total RNA was extracted from Kit 225 cells that had been either deprived of IL-2 for 48 h (time 0 control) or deprived and restimulated with IL-2 for 4 and 16 h in 3 independent experiments. Initial gene expression analysis was performed using a "Rho chip" that contained cDNA probes for 88 genes related to the Rho GTPase pathways. An additional series of experiments were then performed using Affymetrix DNA microarray HG-U133A, according to standardized procedures.
Of the 
18,000 human genes displayed on the HG-U133A chip, 9296 were expressed in Kit 225 cells under at least one of the culture conditions, based on the absolute calls determined by MAS software provided by Affymetrix for data analysis. Of these 9296 genes, 1423 were induced at least 2-fold by IL-2, with 3 different patterns (early, late, or transient induction) and 1372 were repressed with similarly early, late or transient patterns.
To assess the validity of our analysis, we first queried our data for genes involved in cell proliferation or apoptosis, including genes previously known or expected to be regulated by IL-2. IL-2 stimulation induced, or increased, expression of a number of genes involved in cell cycle progression and cell survival. In contrast, antiproliferative and cell death-promoting genes were repressed.
These results also illustrate, at the molecular level, that IL-2-deprived cells are arrested in the G1 phase of the cell cycle (with high p27 expression) and that upon IL-2 addition they synchronously reenter the cell cycle to reach the S-phase at 16–20 h poststimulation.
2. Expression analysis of Rho GTPases
The Rho GTPase family contains an estimated 20 different genes, of which the best functionally characterized are the Rho (A,B,C,G) the Rac (1,2,3) and Cdc42.
Of these, the only genes found to vary in response to IL-2 were Rac2 (increased) and RhoC (decreased).
3. Expression analysis of GEFs and GAPs
RhoGEFs represent a large family of genes that share a common structure invariably containing at least a plextrin homology (PH) domain and the signature Dbl homology (DH) domain. Data banks contain 75 DH/PH-containing putative RhoGEFs. Many of the corresponding proteins have never been characterized, or have only been assigned specificity for one or the other Rho GTPase in vitro, through biochemical approaches. With a few exceptions, the in vivo specificity of GEFs (or GAPs) remains to be established. Of the GEFs analyzed in this study, 16 showed significantly regulated expression in response to IL-2 (Table 1
, Fig. 1
).
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Two GEFs, Net1 and ARHGEF3 (also known as XPLN), that are structurally related were found to be increased in response to IL-2 with, however, different kinetics. Whereas ARHGEF3 is increased as soon as the 4 h time point and remains stable thereafter, Net1 mRNA appears initially decreased and increases dramatically at 16 h.
ARHGEF3/XPLN is so far a unique example of a RhoGEF with selectivity within the Rho family since it has been shown, both in vitro and in vivo, to activate RhoA and RhoB but not RhoC. The rapid increase in ARHGEF3 expression when cells reenter the cell cycle may thus reflect a requirement for early activation of RhoA in the absence of RhoC activation. Little is known on the function of Net1 besides the oncogenic activity in NIH3T3 of a truncated protein and its nuclear localization, suggesting that its activity may be regulated by cellular compartmentalization. Our data suggest that in addition, Net1 may be expressed at specific stages of the cell cycle, as it is not detectable by Western blot neither in G1 cells nor at the G1/S transition. Differences in expression kinetics may reflect functional differences and it is tempting to speculate that ARHGEF3 might be required to activate RhoA early in the cell cycle, e.g., at the G1/S transition, whereas Net1 might be necessary to regulate later functions of RhoA during G2/M. Further experiments are required to specifically address these hypotheses.
Another GEF that we found to be induced late in the cell cycle is Ect2, an exchange factor that is active on all 3 Rho GTPases and was initially described as a Dbl family protein with transforming potential in NIH3T3 fibroblasts. A large body of evidence now clearly establishes that Ect2 (or its drosophila ortholog Pebble) is the critical exchange factor for regulating Cdc42 and RhoA during mitosis, as expression of dominant negative versions of Ect2, or siRNA depletion of Ect2, invariably resulted in failed cytokinesis.
Besides exchange factors, important regulators of the Rho pathways are the GAPs that stimulate GTP hydrolysis. In these experiments, mRNAs for several GAPs were found to be regulated in response to IL-2, the most significant variations being observed for ARHGAP12 (rapidly and strongly decreased) and for ARHGAP11A (rapidly and strongly increased). Unfortunately, like with most of the other GAPs identified in our experiments, these are rather ill-defined proteins.
Of special interest, however, is the case of RACGAP1, also known as MgcRacGap, a GAP for Rac that becomes phosphorylated by Aurora B during cell cycle progression to switch specificity toward RhoA. Recent publications have clearly shown that MgcRacGap acts in concert with Ect2 to regulate ingression of the cleavage furrow during cytokinesis. This is a complex mechanism, however, as Ect2 and MgcRacGap seem to be involved not only in regulating Rho late in mitosis but may also affect Cdc42 activity in metaphase. It is noteworthy that mRNA for MgcRacGap undergo variations that strictly parallel those of Ect2 mRNA, with an early decrease and a rebound at the 16 h time point (Table 1)
. Western blot analysis confirmed peak expression of the MgcRacGap protein in G2/M and thus dividing T lymphocytes, but presumably other cell types as well, coregulate these two proteins to achieve maximal expression at the time when function of these essential regulators is required.
CONCLUSIONS AND SIGNIFICANCE
We have shown here that expression of a number of components of the Rho GTPase pathways is regulated in proliferating human T lymphocytes. It is likely, however, that most of the data reported here may not be directly related to T lymphocytes or proliferation induced by IL-2, but may reflect general cell biology phenomena, in particular related to cell cycle progression.
Activation (GTP loading) of a given Rho GTPase results from coordinated signals targeting the proper GDI, activating and relocalizing GEFs to membranes, and/or down regulating the activity of GAPs. It has recently been proposed that which GEF is activated by a given stimulus selects the outcome of Rho activation. Some GEFs are able, through their multiple interaction domains, to act as scaffold proteins that assemble the pathways and recruit proper effector proteins. Then, how the GEF is selected becomes the critical question. Recruitment by the receptors, membrane translocation, and/or selective phosphorylation are all well described mechanisms that lead to selectivity in Rho activation. We propose that an additional, and possibly simpler way of choosing which GEF should be used at the top of a given pathway, is by regulating its expression. This is certainly obvious in the case of cell type- or tissue-specific expression of some GEFs. Our results suggest that regulated expression during cell cycle progression may be an important mechanism in assembling specific signal transduction cascades that need to be active at certain times during the cell cycle.
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FOOTNOTES
To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.05-4030fje;
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