A widely conserved protein Rof inhibits transcription termination factor Rho and promotes Salmonella virulence program.

Transcription is crucial for the expression of genetic information and its efficient and accurate termination is required for all living organisms. Rho-dependent termination could rapidly terminate unwanted premature RNAs and play important roles in bacterial adaptation to changing environments. Although Rho has been discovered for about five decades, the regulation mechanisms of Rho-dependent termination are still not fully elucidated. Here we report that Rof is a conserved antiterminator and determine the cryogenic electron microscopy structure of Rho-Rof antitermination complex. Rof binds to the open-ring Rho hexamer and inhibits the initiation of Rho-dependent termination. Rof's N-terminal α-helix undergoes conformational changes upon binding with Rho, and is key in facilitating Rof-Rho interactions. Rof binds to Rho's primary binding site (PBS) and excludes Rho from binding with PBS ligand RNA at the initiation step. Further in vivo analyses in Salmonella Typhimurium show that Rof is required for virulence gene expression and host cell invasion, unveiling a physiological function of Rof and transcription termination in bacterial pathogenesis.

Rho-dependent termination enables cellular pH homeostasis.

The termination factor Rho, an ATP-dependent RNA translocase, preempts pervasive transcription processes, thereby rendering genome integrity in bacteria. Here, we show that the loss of Rho function raised the intracellular pH to >8.0 in Escherichia coli. The loss of Rho function upregulates tryptophanase-A (TnaA), an enzyme that catabolizes tryptophan to produce indole, pyruvate, and ammonia. We demonstrate that the enhanced TnaA function had produced the conjugate base ammonia, raising the cellular pH in the Rho-dependent termination defective strains. On the other hand, the constitutively overexpressed Rho lowered the cellular pH to about 6.2, independent of cellular ammonia levels. Since Rho overexpression may increase termination activities, the decrease in cellular pH could result from an excess H+ ion production during ATP hydrolysis by overproduced Rho. Furthermore, we performed in vivo termination assays to show that the efficiency of Rho-dependent termination was increased at both acidic and basic pH ranges. Given that the Rho level remained unchanged, the alkaline pH increases the termination efficiency by stimulating Rho's catalytic activity. We conducted the Rho-mediated RNA release assay from a stalled elongation complex to show an efficient RNA release at alkaline pH, compared to the neutral or acidic pH, that supports our in vivo observation. Whereas acidic pH appeared to increase the termination function by elevating the cellular level of Rho. This study is the first to link Rho function to the cellular pH homeostasis in bacteria. IMPORTANCE The current study shows that the loss or gain of Rho-dependent termination alkalizes or acidifies the cytoplasm, respectively. In the case of loss of Rho function, the tryptophanase-A enzyme is upregulated, and degrades tryptophan, producing ammonia to alkalize cytoplasm. We hypothesize that Rho overproduction by deleting its autoregulatory DNA portion increases termination function, causing excessive ATP hydrolysis to produce H+ ions and cytoplasmic acidification. Therefore, this study is the first to unravel a relationship between Rho function and intrinsic cellular pH homeostasis. Furthermore, the Rho level increases in the absence of autoregulation, causing cytoplasmic acidification. As intracellular pH plays a critical role in enzyme function, such a connection between Rho function and alkalization will have far-reaching implications for bacterial physiology.

Structural basis of the transcription termination factor Rho engagement with transcribing RNA polymerase from Thermus thermophilus.

Transcription termination is an essential step in transcription by RNA polymerase (RNAP) and crucial for gene regulation. For many bacterial genes, transcription termination is mediated by the adenosine triphosphate-dependent RNA translocase/helicase Rho, which causes RNA/DNA dissociation from the RNAP elongation complex (EC). However, the structural basis of the interplay between Rho and RNAP remains obscure. Here, we report the cryo-electron microscopy structure of the Thermus thermophilus RNAP EC engaged with Rho. The Rho hexamer binds RNAP through the carboxyl-terminal domains, which surround the RNA exit site of RNAP, directing the nascent RNA seamlessly from the RNA exit to its central channel. The ß-flap tip at the RNA exit is critical for the Rho-dependent RNA release, and its deletion causes an alternative Rho-RNAP binding mode, which is irrelevant to termination. The Rho binding site overlaps with the binding sites of other macromolecules, such as ribosomes, providing a general basis of gene regulation.

Transcriptional pause extension benefits the stand-by rather than catch-up Rho-dependent termination.

Transcriptional pause is essential for all types of termination. In this single-molecule study on bacterial Rho factor-dependent terminators, we confirm that the three Rho-dependent termination routes operate compatibly together in a single terminator, and discover that their termination efficiencies depend on the terminational pauses in unexpected ways. Evidently, the most abundant route is that Rho binds nascent RNA first and catches up with paused RNA polymerase (RNAP) and this catch-up Rho mediates simultaneous releases of transcript RNA and template DNA from RNAP. The fastest route is that the catch-up Rho effects RNA-only release and leads to 1D recycling of RNAP on DNA. The slowest route is that the RNAP-prebound stand-by Rho facilitates only the simultaneous rather than sequential releases. Among the three routes, only the stand-by Rho's termination efficiency positively correlates with pause duration, contrary to a long-standing speculation, invariably in the absence or presence of NusA/NusG factors, competitor RNAs or a crowding agent. Accordingly, the essential terminational pause does not need to be long for the catch-up Rho's terminations, and long pauses benefit only the stand-by Rho's terminations. Furthermore, the Rho-dependent termination of mgtA and ribB riboswitches is controlled mainly by modulation of the stand-by rather than catch-up termination.

Identification of a Rho-Dependent Termination Site In Vivo Using Synthetic Small RNA.

Rho promotes Rho-dependent termination (RDT) at the Rho-dependent terminator, producing a variable-length region without secondary structure at the 3' end of mRNA. Determining the exact RDT site in vivo is challenging, because the 3' end of mRNA is rapidly removed after RDT by 3'-to-5' exonuclease processing. Here, we applied synthetic small RNA (sysRNA) to identify the RDT region in vivo by exploiting its complementary base-pairing ability to target mRNA. Through the combined analyses of rapid amplification of cDNA 3' ends, primer extension, and capillary electrophoresis, we could precisely map and quantify mRNA 3' ends. We found that complementary double-stranded RNA (dsRNA) formed between sysRNA and mRNA was efficiently cleaved by RNase III in the middle of the dsRNA region. The formation of dsRNA appeared to protect the cleaved RNA 3' ends from rapid degradation by 3'-to-5' exonuclease, thereby stabilizing the mRNA 3' end. We further verified that the signal intensity at the 3' end was positively correlated with the amount of mRNA. By constructing a series of sysRNAs with close target sites and comparing the difference in signal intensity at the 3' end of wild-type and Rho-impaired strains, we finally identified a region of increased mRNA expression within the 21-bp range, which was determined as the RDT region. Our results demonstrated the ability to use sysRNA as a novel tool to identify RDT regions in vivo and expand the range of applications of sysRNA. IMPORTANCE sysRNA, which was formerly widely employed, has steadily lost popularity as more novel techniques for suppressing gene expression come into existence because of issues such as unstable inhibition effect and low inhibition efficiency. However, it remains an interesting topic as a regulatory tool due to its ease of design and low metabolic burden on cells. Here, for the first time, we discovered a new method to identify RDT regions in vivo using sysRNA. This new feature is important because since the discovery of the Rho protein in 1969, specific identification of RDT sites in vivo has been difficult due to the rapid processing of RNA 3' ends by exonucleases, and sysRNA might provide a new approach to address this challenge.

Intrinsic and Rho-dependent termination cooperate for efficient transcription termination at 3' untranslated regions.

The intrinsic, and the Rho-dependent mechanisms of transcription termination are conserved in bacteria. Generally, the two mechanisms have been illustrated as two independent pathways occurring in the 3' ends of different genes with contrasting requirements to halt RNA synthesis. However, a majority of intrinsic terminators terminate transcription inefficiently leading to transcriptional read-through. The unwanted transcription in the downstream region beyond the terminator would have undesired consequences. To prevent such transcriptional read-through, bacteria must have evolved ways to terminate transcription more efficiently at or near the termination sites. We describe the participation of both the mechanisms, where intrinsic terminator and Rho factor contribute to prevent transcriptional read-through. Contribution from both the termination processes is demonstrated at the downstream regions of the genes both in vitro and in vivo in mycobacteria. Distinct patterns of cooperation between the two modes of termination were observed at the 3' untranslated regions of the genes to ensure efficient termination. We demonstrate similar mode of operation between the two termination processes in Escherichia coli suggesting a likely prevalence of this cooperation across bacteria. The reporter system developed to assess the Rho - intrinsic termination collaboration in vivo for mycobacteria and E. coli can readily be applied to other bacteria.

In vivo regulation of bacterial Rho-dependent transcription termination by the nascent RNA.

Bacterial Rho is a RNA-dependent ATPase that functions in the termination of transcription. The in vivo nature of the bacterial Rho-dependent terminators, as well as the mechanism of the Rho-dependent termination process, are not fully understood. Here, we measured the in vivo termination efficiencies of 72 Rho-dependent terminators in Escherichia coli by systematically performing qRT-PCR analyses of cDNA prepared from mid-log phase bacterial cultures. We found that these terminators exhibited a wide range of efficiencies, and many behaved differently in vivo compared to the predicted or experimentally determined efficiencies in vitro. Rho-utilization sites (rut sites) present in the RNA terminator sequences are characterized by the presence of C-rich/G-poor sequences or C > G bubbles. We found that weaker terminators exhibited a robust correlation with the properties (size, length, density, etc.) of these C > G bubbles of their respective rut sites, while stronger terminators lack this correlation, suggesting a limited role of rut sequences in controlling in vivo termination efficiencies. We also found that in vivo termination efficiencies are dependent on the rates of ATP hydrolysis as well as Rho-translocation on the nascent RNA. We demonstrate that weaker terminators, in addition to having rut sites with diminished C > G bubble sizes, are dependent on the Rho-auxiliary factor, NusG, in vivo. From these results, we concluded that in vivo Rho-dependent termination follows a nascent RNA-dependent pathway, where Rho-translocation along the RNA is essential and rut sequences may recruit Rho in vivo, but Rho-rut binding strengths do not regulate termination efficiencies.

Cryo-EM structure of transcription termination factor Rho from Mycobacterium tuberculosis reveals bicyclomycin resistance mechanism.

The bacterial Rho factor is a ring-shaped motor triggering genome-wide transcription termination and R-loop dissociation. Rho is essential in many species, including in Mycobacterium tuberculosis where rho gene inactivation leads to rapid death. Yet, the M. tuberculosis Rho [MtbRho] factor displays poor NTPase and helicase activities, and resistance to the natural Rho inhibitor bicyclomycin [BCM] that remain unexplained. To address these issues, we solved the cryo-EM structure of MtbRho at 3.3 Šresolution. The MtbRho hexamer is poised into a pre-catalytic, open-ring state wherein specific contacts stabilize ATP in intersubunit ATPase pockets, thereby explaining the cofactor preference of MtbRho. We reveal a leucine-to-methionine substitution that creates a steric bulk in BCM binding cavities near the positions of ATP γ-phosphates, and confers resistance to BCM at the expense of motor efficiency. Our work contributes to explain the unusual features of MtbRho and provides a framework for future antibiotic development.

Yes-associated protein reacts differently in vascular smooth muscle cells under different intensities of mechanical stretch.

Vascular smooth muscle cells (VSMCs) are stromal cells of the vascular wall and are continually exposed to mechanical signals. The loss of VSMCs is closely related to the occurrence of many vascular diseases, such as aortic aneurysms and aortic dissection. The proliferation and apoptosis of VSMCs are mechanically stimulated. Yes-associated protein (YAP), one of the core components of the Hippo pathway, plays a key role in the response of VSMCs to mechanical signals. In this study, we tested the impact of different intensities of mechanical stretch on the proliferation and apoptosis of VSMCs, as well as YAP. We tested VSMCs' proliferation and apoptosis and YAP reaction via immunocytochemistry, western blotting, CCK-8 and flow cytometric analysis. We found that 10% elongation could increase the phosphorylation of YAP and prevent it from entering the nucleus, as well as inhibit cell proliferation and promote apoptosis. However, 15% elongation reduced YAP phosphorylation and promoted its nuclear entry, thereby promoting cell proliferation and inhibiting apoptosis. Accordingly, YAP knockdown suppressed the phenotype of VMSCs induced by 15% elongation. Taken together, YAP regulates proliferation and apoptosis of VSMCs differently under different intensity of mechanical stretch. Mechanical stretch with appropriate intensity can promote the proliferation and inhibit apoptosis of VSMCs by activating YAP.

Rho-dependent transcription termination: a revisionist view.

Rho is a hexameric bacterial RNA helicase, which became a paradigm of factor-dependent transcription termination. The broadly accepted ("textbook") model posits a series of steps, wherein Rho first binds C-rich Rho utilization (rut) sites on nascent RNA, uses its ATP-dependent translocase activity to catch up with RNA polymerase (RNAP), and either pulls the transcript from the elongation complex or pushes RNAP forward, thus terminating transcription. However, this appealingly simple mechano-chemical model lacks a biological realism and is increasingly at odds with genetic and biochemical data. Here, we summarize recent structural and biochemical studies that have advanced our understanding of molecular details of RNA recognition, termination signaling, and RNAP inactivation in Rho-dependent transcription termination, rebalancing the view in favor of an alternative "allosteric" mechanism. In the revised model, Rho binds RNAP early in elongation assisted by the cofactors NusA and NusG, forming a pre-termination complex (PTC). The formation of PTC allows Rho to continuously sample nascent transcripts for a termination signal, which subsequently traps the elongation complex in an inactive state prior to its dissociation.

A Large Insertion Domain in the Rho Factor From a Low G + C, Gram-negative Bacterium is Critical for RNA Binding and Transcription Termination Activity.

Rho-dependent termination of transcription (RDTT) is a critical regulatory mechanism specific to bacteria. In a subset of species including most Actinobacteria and Bacteroidetes, the Rho factor contains a large, poorly conserved N-terminal insertion domain (NID) of cryptic function. To date, only two NID-bearing Rho factors from high G + C Actinobacteria have been thoroughly characterized. Both can trigger RDTT at promoter-proximal sites or with structurally constrained transcripts that are unsuitable for the archetypal, NID-less Rho factor of Escherichia coli (EcRho). Here, we provide the first biochemical characterization of a NID-bearing Rho factor from a low G + C bacterium. We show that Bacteroides fragilis Rho (BfRho) is a bona fide RNA-dependent NTPase motor able to unwind long RNA:DNA duplexes and to disrupt transcription complexes. The large NID (~40% of total mass) strongly increases BfRho affinity for RNA, is strictly required for RDTT, but does not promote RDTT at promoter-proximal sites or with a structurally constrained transcript. Furthermore, the NID does not preclude modulation of RDTT by transcription factors NusA and NusG or by the Rho inhibitor bicyclomycin. Although the NID contains a prion-like Q/N-rich motif, it does not spontaneously trigger formation of ß-amyloids. Thus, despite its unusually large RNA binding domain, BfRho behaves more like the NID-less EcRho than NID-bearing counterparts from high G + C Actinobacteria. Our data highlight the evolutionary plasticity of Rho's N-terminal region and illustrate how RDTT is adapted to distinct genomic contents.

Functionally uncoupled transcription-translation in Bacillus subtilis.

Tight coupling of transcription and translation is considered a defining feature of bacterial gene expression1,2. The pioneering ribosome can both physically associate and kinetically coordinate with RNA polymerase (RNAP)3-11, forming a signal-integration hub for co-transcriptional regulation that includes translation-based attenuation12,13 and RNA quality control2. However, it remains unclear whether transcription-translation coupling-together with its broad functional consequences-is indeed a fundamental characteristic of bacteria other than Escherichia coli. Here we show that RNAPs outpace pioneering ribosomes in the Gram-positive model bacterium Bacillus subtilis, and that this 'runaway transcription' creates alternative rules for both global RNA surveillance and translational control of nascent RNA. In particular, uncoupled RNAPs in B. subtilis explain the diminished role of Rho-dependent transcription termination, as well as the prevalence of mRNA leaders that use riboswitches and RNA-binding proteins. More broadly, we identified widespread genomic signatures of runaway transcription in distinct phyla across the bacterial domain. Our results show that coupled RNAP-ribosome movement is not a general hallmark of bacteria. Instead, translation-coupled transcription and runaway transcription constitute two principal modes of gene expression that determine genome-specific regulatory mechanisms in prokaryotes.

Gene autoregulation by 3' UTR-derived bacterial small RNAs.

Negative feedback regulation, that is the ability of a gene to repress its own synthesis, is the most abundant regulatory motif known to biology. Frequently reported for transcriptional regulators, negative feedback control relies on binding of a transcription factor to its own promoter. Here, we report a novel mechanism for gene autoregulation in bacteria relying on small regulatory RNA (sRNA) and the major endoribonuclease, RNase E. TIER-seq analysis (transiently-inactivating-an-endoribonuclease-followed-by-RNA-seq) revealed ~25,000 RNase E-dependent cleavage sites in Vibrio cholerae, several of which resulted in the accumulation of stable sRNAs. Focusing on two examples, OppZ and CarZ, we discovered that these sRNAs are processed from the 3' untranslated region (3' UTR) of the oppABCDF and carAB operons, respectively, and base-pair with their own transcripts to inhibit translation. For OppZ, this process also triggers Rho-dependent transcription termination. Our data show that sRNAs from 3' UTRs serve as autoregulatory elements allowing negative feedback control at the post-transcriptional level.

Rho factor mediates flagellum and toxin phase variation and impacts virulence in Clostridioides difficile.

The intestinal pathogen Clostridioides difficile exhibits heterogeneity in motility and toxin production. This phenotypic heterogeneity is achieved through phase variation by site-specific recombination via the DNA recombinase RecV, which reversibly inverts the "flagellar switch" upstream of the flgB operon. A recV mutation prevents flagellar switch inversion and results in phenotypically locked strains. The orientation of the flagellar switch influences expression of the flgB operon post-transcription initiation, but the specific molecular mechanism is unknown. Here, we report the isolation and characterization of spontaneous suppressor mutants in the non-motile, non-toxigenic recV flg OFF background that regained motility and toxin production. The restored phenotypes corresponded with increased expression of flagellum and toxin genes. The motile suppressor mutants contained single-nucleotide polymorphisms (SNPs) in rho, which encodes the bacterial transcription terminator Rho factor. Analyses using transcriptional reporters indicate that Rho contributes to heterogeneity in flagellar gene expression by preferentially terminating transcription of flg OFF mRNA within the 5' leader sequence. Additionally, Rho is important for initial colonization of the intestine in a mouse model of infection, which may in part be due to the sporulation and growth defects observed in the rho mutants. Together these data implicate Rho factor as a regulator of gene expression affecting phase variation of important virulence factors of C. difficile.

Overexpression of microRNA-136-3p Alleviates Myocardial Injury in Coronary Artery Disease via the Rho A/ROCK Signaling Pathway.

OBJECTIVE: Coronary artery disease (CAD) is a cardiovascular disease that poses a fatal threat to human health, and the identification of potential biomarkers may help to delineate its pathophysiological mechanisms. Accumulating evidence has implicated microRNAs (miRNAs) in the pathogenesis and development of cardiovascular diseases. The present study aims to identify the expression of miRNA-136-3p (miR-136-3p) in CAD and further investigate its functional relevance in myocardial injury both in vitro and in vivo. METHODS: Initially, CAD models were induced in rats by high-fat diet and intraperitoneal injection of pituitrin. Next, the effect of overexpressed miR-136-3p on cardiac function and pathological damage in myocardial tissue, cardiomyocyte apoptosis, oxidative stress and inflammatory response were assessed in CAD rats. Rat cardiac microvascular endothelial cells (CMECs) were isolated and cultured by the tissue explant method, and the CMEC injury model was induced by homocysteine (HCY). The function of miR-136-3p in vitro was further evaluated. RESULTS: miR-136-3p was poorly expressed in the myocardial tissue of CAD rats and CMEC injury models. In vivo assays indicated that overexpressed miR-136-3p could improve cardiac function and alleviate pathological damage in myocardial tissue, accompanied by reduced oxidative stress and inflammatory response. Moreover,in vitro assays suggested that overexpression of miR-136-3p enhanced proliferation and migration while inhibiting apoptosis of HCY-stressed CMECs. Notably, we revealed that EIF5A2 was a target gene of miR-136-3p, and miR-136-3p inhibited EIF5A2 expression and activation of the Rho A/ROCK signaling pathway. CONCLUSION: In conclusion, the overexpression of miR-136-3p could potentially impede myocardial injury in vitro and in vivo in CAD through the blockade of the Rho A/ROCK signaling pathway, highlighting a potential miR-136-3p functional relevance in the treatment of CAD.

iterb-PPse: Identification of transcriptional terminators in bacterial by incorporating nucleotide properties into PseKNC.

Terminator is a DNA sequence that gives the RNA polymerase the transcriptional termination signal. Identifying terminators correctly can optimize the genome annotation, more importantly, it has considerable application value in disease diagnosis and therapies. However, accurate prediction methods are deficient and in urgent need. Therefore, we proposed a prediction method "iterb-PPse" for terminators by incorporating 47 nucleotide properties into PseKNC-Ⅰ and PseKNC-Ⅱ and utilizing Extreme Gradient Boosting to predict terminators based on Escherichia coli and Bacillus subtilis. Combing with the preceding methods, we employed three new feature extraction methods K-pwm, Base-content, Nucleotidepro to formulate raw samples. The two-step method was applied to select features. When identifying terminators based on optimized features, we compared five single models as well as 16 ensemble models. As a result, the accuracy of our method on benchmark dataset achieved 99.88%, higher than the existing state-of-the-art predictor iTerm-PseKNC in 100 times five-fold cross-validation test. Its prediction accuracy for two independent datasets reached 94.24% and 99.45% respectively. For the convenience of users, we developed a software on the basis of "iterb-PPse" with the same name. The open software and source code of "iterb-PPse" are available at https://github.com/Sarahyouzi/iterb-PPse.

Regulatory interplay between small RNAs and transcription termination factor Rho.

The largest and best studied group of regulatory small RNAs (sRNAs) in bacteria act by modulating translation or turnover of messenger RNAs (mRNAs) through base-pairing interactions that typically take place near the 5' end of the mRNA. This allows the sRNA to bind the complementary target sequence while the remainder of the mRNA is still being made, creating conditions whereby the action of the sRNA can extend to transcriptional steps, most notably transcription termination. Increasing evidence corroborates the existence of a functional interplay between sRNAs and termination factor Rho. Two general mechanisms have emerged. One mechanism operates in translated regions subjected to sRNA repression. By inhibiting ribosome binding co-transcriptionally, the sRNA uncouples translation from transcription, allowing Rho to bind the nascent RNA and promote termination. In the second mechanism, which functions in 5' untranslated regions, the sRNA antagonizes termination directly by interfering with Rho binding to the RNA or the subsequent translocation along the RNA. Here, we review the above literature in the context of other mechanisms that underlie the participation of Rho-dependent transcription termination in gene regulation. This article is part of a Special Issue entitled: RNA and gene control in bacteria edited by Dr. M. Guillier and F. Repoila.

Riboswitch regulation mechanisms: RNA, metabolites and regulatory proteins.

Riboswitches are RNA sensors that have been shown to modulate the expression of downstream genes by altering their structure upon metabolite binding. Riboswitches are unique among cellular regulators in that metabolite detection is strictly performed using RNA interactions with the sensed metabolite and in which no regulatory protein is needed to mediate the interaction. However, recent studies have shed light on riboswitch control mechanisms relying on protein regulators to harness metabolite binding for the mediation of gene expression, thereby increasing the range of cellular factors involved in riboswitch regulation. The interaction between riboswitches and proteins adds another level of evolutionary pressure as riboswitches must maintain key residues for metabolite detection, structural switching and protein binding sites. Here, we review regulatory mechanisms involving Escherichia coli riboswitches that have recently been shown to rely on regulatory proteins. We also discuss the implication of such protein-based riboswitch regulatory mechanisms for genetic regulation.

Hypoxia and EGF Stimulation Regulate VEGF Expression in Human Glioblastoma Multiforme (GBM) Cells by Differential Regulation of the PI3K/Rho-GTPase and MAPK Pathways.

Glioblastoma multiforme (GBM) is one of the most common and deadly cancers of the central nervous system (CNS). It is characterized by the presence of hypoxic regions, especially in the core, leading to an increase in vascularity. This increased vascularization is driven by the expression of the major angiogenic inducer VEGF and the indirect angiogenic inducer Epidermal growth factor (EGF), which stimulates VEGF expression. In this study, we examine the regulation of VEGF by both hypoxia and the EGF signaling pathway. We also examine the involvement of pathways downstream from EGF signaling, including the mitogen-activated protein kinase/extracellular regulated kinase (MAPK/ERK) pathway and the Phosphatidylinositol-3-kinase/RhoA/C (PI3K/RhoA/C) pathway in this regulation. Our results show that VEGF expression and secretion levels increase following either hypoxia or EGF stimulation, with the two stimuli signaling in parallel. We also observed an increase in ERK and protein kinase B (Akt) phosphorylation, in response to EGF stimulation, with kinetics that correlated with the kinetics of the effect on VEGF. Using pharmacological inhibitors against ERK and PI3K and small interfering RNAs (siRNAs) against RhoA and RhoC, we found that both the ERK and the PI3K/RhoA/C pathways have to cooperate in order to lead to an increase in VEGF expression, downstream from EGF. In response to hypoxia, however, only ERK was involved in the regulation of VEGF. Hypoxia also led to a surprising decrease in the activation of PI3K and RhoA/C. Finally, the decrease in the activation of these Rho-GTPases was found to be mediated through a hypoxia-driven overexpression of the Rho-GTPase GTPase activating protein (GAP), StarD13. Therefore, while under normoxic conditions, EGF stimulates the activation of both the PI3K and the MAPK pathways and the induction of VEGF, in glioblastoma cells, hypoxic conditions lead to the suppression of the PI3K/RhoA/C pathway and an exclusive switch to the MAPK pathway.