Rho proteins belong to the small GTPases superfamily. They function as molecular switches that, in response to diverse stimuli, control key signaling and structural aspects of the cell. Although early studies proposed a role for Rho GTPases in cellular transformation, this effect was underestimated due to the fact that no genetic mutations affecting Rho-encoding genes were found in tumors. Recently, it has become evident that Rho GTPases participate in the carcinogenic process by either overexpression of some of the members of the family with oncogenic activity, downmodulation of other members with suggested tumor suppressor activity, or by alteration of upstream modulators or downstream effectors. Thus, alteration of the levels of expression of different members of the family of Rho GTPases has been detected in many types of human tumors leading to a great interest in the cellular effects elicited by these oncoproteins. This essay reviews the current evidence of dysregulation of Rho signaling by overexpression in human tumors.
Stats (signal transducers and activators of transcription) are latent cytoplasmic transcription factors that on a specific stimulus migrate to the nucleus and exert their transcriptional activity. Here we report a novel signaling pathway whereby RhoA can efficiently modulate Stat3 transcriptional activity by inducing its simultaneous tyrosine and serine phosphorylation. Tyrosine phosphorylation is exerted via a member of the Src family of kinases (SrcFK) and JAK2, whereas the JNK pathway mediates serine phosphorylation. Furthermore, cooperation of both tyrosine as well as serine phosphorylation is necessary for full activation of Stat3. Induction of Stat3 activity depends on the effector domain of RhoA and correlates with induction of both Src Kinase-related and JNK activities. Activation of Stat3 has biological implications. Coexpression of an oncogenic version of RhoA along with the wild-type, nontransforming Stat3 gene, significantly enhances its oncogenic activity on human HEK cells, suggesting that Stat3 is an essential component of RhoA-mediated transformation. In keeping with this, dominant negative Stat3 mutants or inhibition of its tyrosine or serine phosphorylation completely abrogate RhoA oncogenic potential. Taken together, these results indicate that Stat3 is an important player in RhoA-mediated oncogenic transformation, which requires simultaneous phosphorylation at both tyrosine and serine residues by specific signaling events triggered by RhoA effectors.
Rho GTPases are overexpressed in a variety of human tumors contributing to both tumor proliferation and metastasis. Recently, several studies demonstrate an essential role of transcriptional regulation in Rho GTPases-induced oncogenesis. Herein, we demonstrate that RhoA, Rac1, and Cdc42 promote the expression of cyclooxygenase-2 (COX-2) at the transcriptional level by a mechanism that is dependent on the transcription factor nuclear factor-B (NF-B), but not Stat3, a transcription factor required for RhoA-induced tumorigenesis. With respect to RhoA, this effect is dependent on ROCK, but not PKN. Treatment of RhoA-, Rac1-, and Cdc42-transformed epithelial cells with Sulindac and NS-398, two well-characterized nonsteroid antiinflammatory drugs (NSAIDs), results in growth inhibition as determined by cell proliferation assays. Accordingly, tumor growth of RhoA-expressing epithelial cells in syngeneic mice is strongly inhibited by NS-398 treatment. The effect of NSAIDs over RhoA-induced tumor growth is not exclusively dependent on COX-2 because DNA-binding of NF-B is also abolished upon NSAIDs treatment, resulting in complete loss of COX-2 expression. Finally, treatment of RhoA-transformed cells with Bay11-7083, a specific NF-B inhibitor, leads to inhibition of cell proliferation. We suggest that treatment of human tumors that overexpress Rho GTPases with NSAIDs and drugs that target NF-B could constitute a valid antitumoral strategy.
Rho proteins, members of the Ras superfamily of GTPases, are critical elements in signal transduction pathways governing cell proliferation and cell death. Different members of the family of human Rho GTPases, including RhoA, RhoC, and Rac1, participate in the regulation of apoptosis in response to cytokines and serum deprivation in different cell systems. Here, we have characterized the mechanism of apoptosis induced by Rac1 in NIH 3T3 cells. It requires protein synthesis and caspase-3 activity, but it is independent of the release of cytochrome c from mitochondria. Moreover, an increase in mitochondria membrane potential and the production of reactive oxygen species was observed. Rac1-induced apoptosis was related to the simultaneous increase in ceramide production and synthesis of FasL. Generation of FasL may be mediated by transcriptional regulation involving both c-Jun amino terminal kinase as well as nuclear factor-κB-dependent signals. None of these signals, ceramides or FasL, was sufficient to induce apoptosis in the parental cell line, NIH 3T3 cells. However, any of them was sufficient to induce apoptosis in the Rac1-expressing cells. Finally, inhibition of FasL signaling drastically reduced apoptosis by Rac1. Thus, Rac1 seems to induce apoptosis by a complex mechanism involving the generation of ceramides and the de novo synthesis of FasL. These results suggest that apoptosis mediated by Rac1 results from a signaling mechanism that involves biochemical and transcriptional events under control of Rac1.
Anticoagulant rodenticides are highly toxic compounds that are widely used for pest control of rodents, but that also may threaten the wildlife's health. This work aimed to assess the exposure to first- and second-generation anticoagulant rodenticides (ARs) in six birds of prey species from the Canary Islands (Spain). The concentrations of seven widely used ARs were determined by LC-MS/MS in 104 liver samples of six species of birds of prey (Buteo buteo, Accipiter nisus, Falco pelegrinoides, Falco tinnunculus, Asio otus, and Tyto alba). We determined that 61% of the livers had detectable residues of at least one AR. The most frequently detected AR was bromadiolone, which was detected in 60.3% of the positive cases. The detection frequencies of these compounds varied widely, depending on the species. More than 75% of the A. nisus, T. alba, and A. otus individuals had detectable rodenticide residues in the liver. However, F. tinnunculus exhibited the highest concentrations of AR, with median values above 100 ng/g w.w. We did not detect first-generation ARs in any of the samples. When grouped, nocturnal species exhibited higher AR concentrations than diurnal species (P<0.001). The residue levels were higher among small mammal-eaters than bird-eaters (P<0.01). While most animals exhibited no macroscopic signs of coagulation disorders, approximately 35% exceeded the threshold levels of toxicity, which suggests that these compounds could weaken these animals in their natural environment. In conclusion, the control of rodent populations by ARs suggests that these compounds will enter the food chain and thus threaten the vulnerable populations of raptors on the Canary Islands. Our findings require authorities to ban or strictly control the use of these rodenticides in the natural environment for the conservation of raptors and other predatory species.
The involvement of Rho GTPases in signal transduction pathways leading to transcription activation is one of the major roles of this family of GTPases. Thus, the identification of transcription factors regulated by Rho GTPases and the understanding of the mechanisms of their activation and its biological outcome are of great interest. Here, we provide evidence that Rho GTPases modulate Stat5a, a transcription factor of the family of signal transducers and activators of transcription. RhoA triggers tyrosine phosphorylation (Y696) of Stat5a via a JAK2-dependent mechanism and promotes DNA-binding activity of Stat5a. Tyrosine phosphorylation of Stat5a is also stimulated physiologically by lysophosphatidic acid (LPA) in a Rho-dependent manner. Simultaneously, RhoA reduces serine phosphorylation of Stat5a at both serine residues S726 and S780, resulting in a further increase of activity as defined by mutagenesis experiments. Furthermore, serine dephosphorylation of Stat5a by RhoA does not take place by down-modulation of either JNK1, MEK1, or p38 MAP kinases, as determined by transfection experiments or chemical inhibition of both MEK1, p38, and JNK serine kinases. Thus, RhoA regulates Stat5a via tyrosine phosphorylation and via a yet to be determined novel down-modulating pathway that involves serine dephosphorylation. Finally, we provide evidence for a role of Stat5a in RhoA-induced epithelial-to-mesenchymal transition with concomitant increase in vimentin expression, E-cadherin down-regulation, and cell motility
Poisoning with agricultural chemicals is a major threat for wildlife all over the world. We have developed and validated an analytical scheme aimed to the identification and quantification of a wide range of pesticides in fresh liver from wildlife specimens that are routinely delivered to the forensic laboratories for toxicological investigation. The proposed method is comprised of a general solid-liquid extraction followed by purification steps and three complementary liquid or gas chromatographic analyses with triple quadrupole mass spectrometry detection. The developed methodology allows for the determination of 117 highly toxic pesticides in a variety of samples from wildlife poisoning incidents. The validity of the method has been demonstrated in samples from 98 real cases submitted to our laboratory between 2010 and 2012. This method allowed the identification and quantification of poison in 78 of 94 fresh liver samples from wild animals and was successfully used for the identification of pesticides in 35 of 46 non-liver samples. Therefore, the extraction and cleanup method with minor modifications and the potency of triple quadrupole mass spectrometry allow this method to be used to simultaneously detect and quantify or semi-quantify a majority of the most toxic pesticides in a variety of complex and degraded matrices.
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