Transcriptome analysis reveals lung-specific miRNAs associated with impaired mucociliary clearance induced by cigarette smoke in an in vitro human airway tissue model.

Exposure to cigarette smoke (CS) is strongly associated with impaired mucociliary clearance (MCC), which has been implicated in the pathogenesis of CS-induced respiratory diseases, such as chronic obstructive pulmonary diseases (COPD). In this study, we aimed to identify microRNAs (miRNAs) that are associated with impaired MCC caused by CS in an in vitro human air-liquid-interface (ALI) airway tissue model. ALI cultures were exposed to CS (diluted with 0.5 L/min, 1.0 L/min, and 4.0 L/min of clean air) from smoking five 3R4F University of Kentucky reference cigarettes under the International Organization for Standardization (ISO) machine smoking regimen, every other day for 1 week (a total of 3 days, 40 min/day). Transcriptome analyses of ALI cultures exposed to the high concentration of CS identified 5090 differentially expressed genes and 551 differentially expressed miRNAs after the third exposure. Genes involved in ciliary function and ciliogenesis were significantly perturbed by repeated CS exposures, leading to changes in cilia beating frequency and ciliary protein expression. In particular, a time-dependent decrease in the expression of miR-449a, a conserved miRNA highly enriched in ciliated airway epithelia and implicated in motile ciliogenesis, was observed in CS-exposed cultures. Similar alterations in miR-449a have been reported in smokers with COPD. Network analysis further indicates that downregulation of miR-449a by CS may derepress cell-cycle proteins, which, in turn, interferes with ciliogenesis. Investigating the effects of CS on transcriptome profile in human ALI cultures may provide not only mechanistic insights, but potential early biomarkers for CS exposure and harm.

Integration of transcriptome analysis with pathophysiological endpoints to evaluate cigarette smoke toxicity in an in vitro human airway tissue model.

Exposure to cigarette smoke (CS) is a known risk factor in the pathogenesis of smoking-caused diseases, such as chronic obstructive pulmonary diseases (COPD) and lung cancer. To assess the effects of CS on the function and phenotype of airway epithelial cells, we developed a novel repeated treatment protocol and comprehensively evaluated the progression of key molecular, functional, and structural abnormalities induced by CS in a human in vitro air-liquid-interface (ALI) airway tissue model. Cultures were exposed to CS (diluted with 0.5 L/min, 1.0 L/min, and 4.0 L/min clean air) generated from smoking five 3R4F University of Kentucky reference cigarettes under the International Organization for Standardization (ISO) machine smoking regimen, every other day for 4 weeks (3 days per week, 40 min/day). By integrating the transcriptomics-based approach with the in vitro pathophysiological measurements, we demonstrated CS-mediated effects on oxidative stress, pro-inflammatory cytokines and matrix metalloproteinases (MMPs), ciliary function, expression and secretion of mucins, and squamous cell differentiation that are highly consistent with abnormalities observed in airways of smokers. Enrichment analysis on the transcriptomic profiles of the ALI cultures revealed key molecular pathways, such as xenobiotic metabolism, oxidative stress, and inflammatory responses that were perturbed in response to CS exposure. These responses, in turn, may trigger aberrant tissue remodeling, eventually leading to the onset of respiratory diseases. Furthermore, changes of a panel of genes known to be disturbed in smokers with COPD were successfully reproduced in the ALI cultures exposed to CS. In summary, findings from this study suggest that such an integrative approach may be a useful tool for identifying genes and adverse cellular events caused by inhaled toxicants, like CS.

90-day nose-only inhalation toxicity study of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in Sprague-Dawley rats.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. Repeated dose inhalation toxicity data on NNK, particularly relevant to cigarette smoking, however, is surprisingly limited. Hence, there is a lack of direct information available on the carcinogenic and potential non-carcinogenic effects of NNK via inhalational route exposure. In the present study, the subchronic inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 23 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.2, 0.8, 3.2, or 7.8 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.0066, 0.026, 0.11, or 0.26 mg/L air) for 1 h/day for 90 consecutive days. Toxicity was evaluated by assessing body weights; food consumption; clinical pathology; histopathology; organ weights; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); tissue levels of the DNA adduct O6-methylguanine; blood and bone marrow micronucleus (MN) frequency; and bone marrow DNA strand breaks (comet assay). The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic lesions in the nose. Although the genotoxic biomarker O6-methylguanine was detected, genotoxicity from NNK exposure was negative in the MN and comet assays. The Lowest-Observed-Adverse-Effect-Level (LOAEL) was 0.8 mg/kg BW/day or 0.026 mg/L air of NNK for 1 h/day for both sexes. The No-Observed-Adverse-Effect-Level (NOAEL) was 0.2 mg/kg BW/day or 0.0066 mg/L air of NNK for 1 h/day for both sexes. The results of this study provide new information relevant to assessing the human exposure hazard of NNK.

In vitro dosimetry analyses for acrolein exposure in normal human lung epithelial cells and human lung cancer cells.

Establishing accurate dosimetry is important for assessing the toxicity of xenobiotics as well as for comparing responses between different test systems. In this study, we used acrolein as a model toxicant and defined the concentration-response relationships of the key adverse responses in normal human bronchial epithelial (NHBE) cells and human mucoepidermoid pulmonary carcinoma (NCI-H292) cells. Direct trace analysis of intracellular free acrolein is extremely challenging, if not impossible. Therefore, we developed a new method for indirectly estimating the intracellular uptake of acrolein. A 10-min treatment was employed to capture the rapid occurrence of the key alkylation reactions of acrolein. Responses, including protein carbonylation, GSH depletion, and GSH-acrolein (GSH-ACR) adduct formation, were all linearly correlated with acrolein uptake in both cell types. Compared to the NCI-H292 mucoepidermoid carcinoma cells, NHBE cells were more sensitive to acrolein exposure. Furthermore, results from the time-course studies demonstrated that depletion and conjugation of GSH were the primary adverse events and directly associated with the cytotoxicity induced by acrolein. In summary, these data suggest that cell susceptibility to acrolein exposure is closely associated with acrolein uptake and formation of GSH-ACR adducts. The dosimetric analysis presented in this study may provide useful information for computational modeling and risk assessment of acrolein using different test systems.

Smoke Chemistry, In Vitro Cytotoxicity, and Genotoxicity Demonstrates Enhanced Toxicity of Cigarillos Compared With Cigarettes.

There has been limited toxicity testing of cigarillos, including comparison to cigarettes. This study compared the smoke chemistry and the cytotoxic and genotoxic potential of 10 conventional cigarettes and 10 cigarillos based on the greatest market share. Whole smoke and total particulate matter (TPM) were generated using the Canadian Intense and International Organization for Standardization puffing protocols. Tobacco-specific nitrosamines, carbonyls, and polycyclic aromatic hydrocarbons were measured using gas chromatography-mass spectrometry. TPM smoke extracts were used for the in vitro assays. Cytotoxicity was assessed in human bronchial epithelial continuously cultured cell line cells using the neutral red uptake assay. Genotoxic potential was assessed using the micronucleus (human lung adenocarcinoma continuously cultured cell line cells), Ames, and thymidine kinase assays. TPM from all cigarillos tested was more cytotoxic than cigarettes. Micronucleus formation was significantly greater for cigarillos compared with cigarettes at the highest dose of TPM, with or without rat liver S9 fraction. In the Ames test +S9, both tobacco products exhibited significant dose-dependent increases in mutation frequency, indicating metabolic activation is required for genotoxicity. In the thymidine kinase assay +S9, cigarillos showed a significantly enhanced mutation frequency although both tobacco products were positive. The levels of all measured polycyclic aromatic hydrocarbons, tobacco-specific nitrosamines, and carbonyls (except acrolein) were significantly greater in cigarillos than cigarettes. The Canadian Intense puffing protocol demonstrated increased smoke constituent levels compared with International Organization for Standardization. Even though the gas vapor phase was not tested, the results of this study showed that under the tested conditions the investigated cigarillos showed greater toxicity than comparator cigarettes. This study found that there is significantly greater toxicity in the tested U.S. marketed cigarillos than cigarettes for tobacco constituent levels, cytotoxicity, and genotoxicity. These findings are important for understanding the human health toxicity from the use of cigarillos relative to cigarettes and for building upon knowledge regarding harm from cigarillos to inform risk mitigation strategies.

14-Day Nose-Only Inhalation Toxicity and Haber's Rule Study of NNK in Sprague-Dawley Rats.

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is one of the key tobacco-specific nitrosamines that plays an important role in human lung carcinogenesis. However, repeated inhalation toxicity data on NNK, which is more directly relevant to cigarette smoking, are currently limited. In the present study, the subacute inhalation toxicity of NNK was evaluated in Sprague Dawley rats. Both sexes (9-10 weeks age; 16 rats/sex/group) were exposed by nose-only inhalation to air, vehicle control (75% propylene glycol), or 0.8, 3.2, 12.5, or 50 mg/kg body weight (BW)/day of NNK (NNK aerosol concentrations: 0, 0, 0.03, 0.11, 0.41, or 1.65 mg/L air) for 1 h/day for 14 consecutive days. Toxicity was evaluated by assessing body and organ weights; food consumption; clinical pathology; histopathology observations; blood, urine, and tissue levels of NNK, its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), and their glucuronides (reported as total NNK, tNNK, and total NNAL, tNNAL, respectively); O6-methylguanine DNA adduct formation; and blood and bone marrow micronucleus frequency. Whether the subacute inhalation toxicity of NNK followed Haber's Rule was also determined using additional animals exposed 4 h/day. The results showed that NNK exposure caused multiple significant adverse effects, with the most sensitive endpoint being non-neoplastic histopathological lesions in the nose. The lowest-observed-adverse-effect level (LOAEL) was 0.8 mg/kg BW/day or 0.03 mg/L air for 1 h/day for both sexes. An assessment of Haber's Rule indicated that 14-day inhalation exposure to the same dose at a lower concentration of NNK aerosol for a longer time (4 h daily) resulted in greater adverse effects than exposure to a higher concentration of NNK aerosol for a shorter time (1 h daily).

Toxicokinetic and Genotoxicity Study of NNK in Male Sprague Dawley Rats Following Nose-Only Inhalation Exposure, Intraperitoneal Injection, and Oral Gavage.

The tobacco-specific nitrosamine NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone] is found in tobacco products and tobacco smoke. NNK is a potent genotoxin and human lung carcinogen; however, there are limited inhalation data for the toxicokinetics (TK) and genotoxicity of NNK in vivo. In the present study, a single dose of 5 × 10-5, 5 × 10-3, 0.1, or 50 mg/kg body weight (BW) of NNK, 75% propylene glycol (vehicle control), or air (sham control) was administered to male Sprague-Dawley (SD) rats (9-10 weeks age) via nose-only inhalation (INH) exposure for 1 h. For comparison, the same doses of NNK were administered to male SD rats via intraperitoneal injection (IP) and oral gavage (PO). Plasma, urine, and tissue specimens were collected at designated time points and analyzed for levels of NNK and its major metabolite 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL) and tissue levels of DNA adduct O6-methylguanine by LC/MS/MS. TK data analysis was performed using a non-linear regression program. For the genotoxicity subgroup, tissues were collected at 3 h post-dosing for comet assay analysis. Overall, the TK data indicated that NNK was rapidly absorbed and metabolized extensively to NNAL after NNK administration via the three routes. The IP route had the greatest systemic exposure to NNK. NNK metabolism to NNAL appeared to be more efficient via INH than IP or PO. NNK induced significant increases in DNA damage in multiple tissues via the three routes. The results of this study provide new information and understanding of the TK and genotoxicity of NNK.

Regulatory aspects of oncology drug safety evaluation: past practice, current issues, and the challenge of new drugs.

The drug development of new anti-cancer agents is streamlined in response to the urgency of bringing effective drugs to market for patients with limited life expectancy. FDA's regulation of oncology drugs has evolved from the practices set forth in Arnold Lehman's seminal work published in the 1950s through the current drafting of a new International Conference on Harmonization of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH) safety guidance for anti-cancer drug nonclinical evaluations. The ICH combines the efforts of the regulatory authorities of Europe, Japan, and the United States and the pharmaceutical industry from these three regions to streamline the scientific and technical aspects of drug development. The recent development of new oncology drug classes with novel mechanisms of action has improved survival rates for some cancers but also brings new challenges for safety evaluation. Here we present the legacy of Lehman and colleagues in the context of past and present oncology drug development practices and focus on some of the current issues at the center of an evolving harmonization process that will generate a new safety guidance for oncology drugs, ICH S9. The purpose of this new guidance will be to facilitate oncology drug development on a global scale by standardizing regional safety requirements.

Effects of cellular differentiation in human primary bronchial epithelial cells: Metabolism of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone.

Many of the toxicants in tobacco smoke undergo biotransformation in the lungs of smokers, both to reactive and to detoxified derivatives. Human air-liquid-interface (ALI) airway tissue models have emerged as an advanced in vitro model for evaluating the toxicity of inhaled substances; however, the metabolic potential of these cultures has not been evaluated extensively. In this study, we compared the metabolic activities of an ALI tissue model to the undifferentiated normal human primary bronchial epithelial (NHBE) cells from which it was derived. Measurement of the basal levels of gene expression for 84 phase I drug metabolism enzymes indicated that most genes were upregulated in ALI cultures compared to NHBE cells. Furthermore, the enzymatic activities of three cytochrome P450s involved in the bioactivation of tobacco-specific nitrosamines were higher in the ALI cultures, and the bioactivation of 4-(methylnitrosamine)-1-(3-pyridyl)-1-butanone (NNK), as measured by the formation of two of its major metabolites, i.e., keto acid and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), was significantly greater in the ALI cultures. Finally, NNK was a direct-acting genotoxicant in the ALI cultures, while the genotoxicity of NNK was detected in NHBE cells only in the presence of an exogenous liver S9 activation system. Taken together, our findings demonstrate the greater metabolic potential of well-differentiated ALI cultures than primary NHBE cells, supporting the potential use of ALI airway cultures as an alternative in vitro model for evaluating inhaled toxicants that require metabolic transformation.

Evaluation of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) mutagenicity using in vitro and in vivo Pig-a assays.

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) is a genotoxic carcinogen found in tobacco and tobacco smoke. Several in vitro and in vivo assays have been used for evaluating the genotoxicity of tobacco smoke and tobacco smoke constituents like NNK, yet it is not clear which in vitro assays are most appropriate for extrapolating the in vitro responses of these test agents to animal models and humans. The Pig-a gene mutation assay can be performed in vitro, in laboratory animals, and in humans, a potential benefit in estimating in vivo responses from in vitro data. In the current study we used Pig-a as a reporter of gene mutation both in vitro, in L5178Y/Tk+/- cells, and in vivo, in Sprague-Dawley rats. NNK significantly increased Pig-a mutant frequency in L5178Y/Tk+/- cells, but only at concentrations of 100 µg/ml and greater, and only in the presence of S9 activation. Pig-a mutations in L5178Y/Tk+/- cells were detected in 80% of the NNK-induced mutants, with the predominate mutation being G→A transition; vehicle control mutants contained deletions. In the in vivo study, rats were exposed to NNK daily for 90 days by inhalation, a common route of exposure to NNK for humans. Although elevated mutant frequencies were detected, these responses were not clearly associated with NNK exposure, so that overall, the in vivo Pig-a assays were negative. Thus, while NNK induces mutations in the in vitro Pig-a assay, the in vivo Pig-a assay has limited ability to detect NNK mutagenicity under conditions relevant to NNK exposure in smokers.

Evaluating Mode of Action of Acrolein Toxicity in an In Vitro Human Airway Tissue Model.

Acrolein is a reactive unsaturated aldehyde and is found at high concentrations in both mainstream and side-stream tobacco smoke. Exposure to acrolein via cigarette smoking has been associated with acute lung injury, chronic obstructive pulmonary diseases (COPDs), and asthma. In this study, we developed an in vitro treatment strategy that resembles the inhalation exposure to acrolein experienced by smokers and systematically examined the adverse respiratory effects induced by the noncytotoxic doses of acrolein in a human airway epithelial tissue model. A single 10-min exposure to buffered saline containing acrolein significantly induced oxidative stress and inflammatory responses, with changes in protein oxidation and GSH depletion occurring immediately after the treatment whereas responses in inflammation requiring a manifestation time of at least 24 h. Repeated exposure to acrolein for 10 consecutive days resulted in structural and functional changes that recapitulate the pathological lesions of COPD, including alterations in the beating frequency and structures of ciliated cells, inhibition of mucin expression and secretion apparatus, and development of squamous differentiation. Although some of the early responses caused by acrolein exposure were reversible after a 10-day recovery, perturbations in the functions and structures of the air-liquid-interface (ALI) cultures, such as mucin production, cilia structures, and morphological changes, failed to fully recover over the observation period. Taken together, these findings are consistent with its mode of action that oxidative stress and inflammation have fundamental roles in acrolein-induced tissue remodeling. Furthermore, these data demonstrate the usefulness of analytical methods and testing strategy for recapitulating the key events in acrolein toxicity using an in vitro model.

Establishing a novel Pig-a gene mutation assay in L5178YTk+/- mouse lymphoma cells.

The X-linked Pig-a gene encodes an enzyme required for the biosynthesis of glycosyl phosphatidylinositol (GPI) anchors. Pig-a mutant cells fail to synthesize GPI and to express GPI-anchored protein markers (e.g., CD90) on their surface. Marker deficiency serves as a phenotypic indicator of Pig-a mutation in various in vivo assays. Here, we describe an in vitro Pig-a mutation assay in L5178YTk+/- mouse lymphoma cells, in which mutant-phenotype cells are measured by flow cytometry using a fluorescent anti-CD90 antibody. Increased frequencies of CD90-deficient mutants were detected in cells treated with benzo[a]pyrene (B[a]P), N-ethyl-N-nitrosourea (ENU), ethyl methanesulphonate, and 7,12-dimethylbenz[a]anthracene, with near maximum mutant frequencies measured eight days after treatment. The CD90 deficiency in mutant cells quantified by flow cytometry was shown to be due to loss of GPI anchors in a limiting-dilution cloning assay using proaerolysin selection. Individual CD90-deficient cells from cultures treated with ENU, B[a]P, and vehicle were sorted and clonally expanded for molecular analysis of their Pig-a gene. Pig-a mutations with agent-specific signatures were found in nearly all clones that developed from sorted CD90-deficient cells. These results indicate that a Pig-a mutation assay can be successfully conducted in L5178YTk+/- cells. The assay may be useful for mutagenicity screening of environmental agents as well as for testing hypotheses in vitro before committing to in vivo Pig-a assays. Environ. Mol. Mutagen. 59:4-17, 2018. Published 2017. This article is a US Government work and is in the public domain in the USA.

Proposed Mode of Action for Acrolein Respiratory Toxicity Associated with Inhaled Tobacco Smoke.

This article presents a mode of action (MOA) analysis that identifies key mechanisms in the respiratory toxicity of inhaled acrolein and proposes key acrolein-related toxic events resulting from the inhalation of tobacco smoke. Smoking causes chronic obstructive pulmonary disorder (COPD) and acrolein has been previously linked to the majority of smoking-induced noncancer respiratory toxicity. In contrast to previous MOA analyses for acrolein, this MOA focuses on the toxicity of acrolein in the lower respiratory system, reflecting the exposure that smokers experience upon tobacco smoke inhalation. The key mechanisms of acrolein toxicity identified in this proposed MOA include (1) acrolein chemical reactivity with proteins and other macromolecules of cells lining the respiratory tract, (2) cellular oxidative stress, including compromise of the important anti-oxidant glutathione, (3) chronic inflammation, (4) necrotic cell death leading to a feedback loop where necrosis-induced inflammation leads to more necrosis and oxidative damage and vice versa, (5) tissue remodeling and destruction, and (6) loss of lung elasticity and enlarged lung airspaces. From these mechanisms, the proposed MOA analysis identifies the key cellular processes in acrolein respiratory toxicity that consistently occur with the development of COPD: inflammation and necrosis in the middle and lower regions of the respiratory tract. Moreover, the acrolein exposures that occur as a result of smoking are well above exposures that induce both inflammation and necrosis in laboratory animals, highlighting the importance of the role of acrolein in smoking-related respiratory disease.

Sphingosine-1-phosphate stimulates contraction of human airway smooth muscle cells.

The bioactive sphingolipid sphingosine-1-phosphate (S1P) that is increased in airways of asthmatic subjects markedly induced contraction of human airway smooth muscle (HASM) cells embedded in collagen matrices in a Gi-independent manner. Dihydro-S1P, which binds to S1P receptors, also stimulated contractility. S1P induced formation of stress fibers, contraction of individual HASM cells, and stimulated myosin light chain phosphorylation, which was inhibited by the Rho-associated kinase inhibitor Y-27632. S1P-stimulated HASM cell contractility was independent of the ERK1/2 and PKC signaling pathways, important regulators of airway smooth muscle contraction. However, removal of extracellular calcium completely blocked S1P-mediated contraction and Y-27632 reduced it. S1P also induced calcium mobilization that was not desensitized by repeated additions. Pretreatment with thapsigargin to deplete InsP3-sensitive calcium stores partially blocked increases in [Ca2+]i induced by S1P, yet did not inhibit S1P-stimulated contraction. In sharp contrast, the L-type calcium channel blocker verapamil markedly decreased S1P-induced HASM cell contraction, supporting a role for calcium influx from extracellular sources. Collectively, our results suggest that S1P may regulate HASM contractility, important in the pathobiology of asthma.

The roles of sphingosine-1-phosphate in asthma.

Asthma is a complex condition in which exposure to environmental antigens induces inflammatory reactions in the airway characterized by activation of mast cells and eosinophils. Mast cells are known to be the main effector cells in eliciting IgE-mediated allergic response. These cells secrete various substances that perpetuate inflammation and provoke airway smooth muscle (ASM) contraction. A newly recognized addition to the repertoire of FcepsilonRI-mediated signaling events is the activation of sphingosine kinase leading to the generation of the potent sphingolipid mediator, sphingosine-1-phosphate (S1P) from sphingosine. S1P secretion by the lung significantly increases after challenge with an allergen, adding this sphingolipid metabolite to the variety of mediators that are released during an allergic reaction [FASEB J. 15 (2001) 1212]. Indeed, similar to previous reports, we found that FcepsilonRI cross-linking not only increased cellular levels of S1P, it also markedly enhanced its secretion from rat basophilic leukemia RBL-2H3 cells. Moreover, S1P induced degranulation of RBL and bone marrow derived mast cells (BMMCs) cells as determined by hexosaminidase release. Treatment of BMMCs with the sphingosine kinase inhibitors, DL-threo-dihydrosphingosine and dimethylsphingosine, reduced IgE/Ag stimulated histamine release. RT-PCR analysis demonstrated that these mast cells express S1P receptors EDG-1 and EDG-5 but not EDG-3, EDG-6 or EDG-8 transcripts. Further studies are needed to determine whether IgE triggering results in transactivation of EDG-1 or EDG-5 present on mast cells and whether this is a critical event for mast cell activation.

P-REX2, a novel PI-3-kinase sensitive Rac exchange factor.

We have identified an activator of Rac, P-REX2, that is structurally related to the exchange factor PtdIns(3,4,5)-dependent Rac exchanger (P-REX1), but exhibits distinct tissue-specific expression. P-REX2 is spliced into two RNA species, approximately 3.5 and approximately 10 kb in size. The cDNA corresponding to the smaller transcript encodes a protein that exhibits strong similarity with P-REX1 within its N-terminal domains, but differs in the C-terminal region. P-REX2 promoted increased levels of GTP-bound Rac that could be further stimulated by enhancing PI-3K activity. Thus, P-REX2 may serve as a novel link between Rac activation and the PI-3 kinase pathway.

Persistent activation of Rac1 in squamous carcinomas of the head and neck: evidence for an EGFR/Vav2 signaling axis involved in cell invasion.

The poor prognosis associated with head and neck squamous cell carcinoma (HNSCC) is primarily due to both local invasion and the regional and/or distant metastatic spread. Recent findings have provided evidence that the acquisition of a motile and invasive phenotype by cancer cells involves the dysregulated function of key intracellular molecular mechanisms together with aberrant signaling events initiated by the surrounding microenvironment. These intrinsic and extrinsic biochemical pathways in turn often converge to stimulate the activity of members of the Rho family of Ras-related guanosine triphosphate (GTP)-binding proteins, including RhoA, Rac and Cdc42, which control the organization of the actin cytoskeleton thereby regulating cell adhesion, polarity and motility. In this study, we examined the status of activation of these GTPases in a representative collection of HNSCC cell lines. Surprisingly, we found that most HNSCC cells exhibit remarkably high levels of GTP-bound Rac1. Further analysis revealed that the activation of Rac1 in these HNSCC cells could be due to two independent signaling events, an epidermal growth factor receptor (EGFR)-based autocrine loop that leads to the activation of the Rac1 exchange factor Vav2 and an EGFR/Vav2-independent pathway that arises as a consequence of the oncogenic mutation of the H-ras proto-oncogene. Indeed, we provide evidence that the EGFR/Vav2/Rac1 axis is a crucial pathway for the acquisition of motile and invasive properties of most HNSCC cells. These findings shed light onto the molecular mechanisms involved in HNSCC cell invasion, and may reveal new therapeutic opportunities to halt the metastatic spread of these aggressive malignancies.

P-Rex1 links mammalian target of rapamycin signaling to Rac activation and cell migration.

Polarized cell migration results from the transduction of extra-cellular cues promoting the activation of Rho GTPases with the intervention of multidomain proteins, including guanine exchange factors. P-Rex1 and P-Rex2 are Rac GEFs connecting Gbetagamma and phosphatidylinositol 3-kinase signaling to Rac activation. Their complex architecture suggests their regulation by protein-protein interactions. Novel mechanisms of activation of Rho GTPases are associated with mammalian target of rapamycin (mTOR), a serine/threonine kinase known as a central regulator of cell growth and proliferation. Recently, two independent multiprotein complexes containing mTOR have been described. mTORC1 links to the classical rapamycin-sensitive pathways relevant for protein synthesis; mTORC2 links to the activation of Rho GTPases and cytoskeletal events via undefined mechanisms. Here we demonstrate that P-Rex1 and P-Rex2 establish, through their tandem DEP domains, interactions with mTOR, suggesting their potential as effectors in the signaling of mTOR to Rac activation and cell migration. This possibility was consistent with the effect of dominant-negative constructs and short hairpin RNA-mediated knockdown of P-Rex1, which decreased mTOR-dependent leucine-induced activation of Rac and cell migration. Rapamycin, a widely used inhibitor of mTOR signaling, did not inhibit Rac activity and cell migration induced by leucine, indicating that P-Rex1, which we found associated to both mTOR complexes, is only active when in the mTORC2 complex. mTORC2 has been described as the catalytic complex that phosphorylates AKT/PKB at Ser-473 and elicits activation of Rho GTPases and cytoskeletal reorganization. Thus, P-Rex1 links mTOR signaling to Rac activation and cell migration.

Rac inhibits thrombin-induced Rho activation: evidence of a Pak-dependent GTPase crosstalk.

The strict spatio-temporal control of Rho GTPases is critical for many cellular functions, including cell motility, contractility, and growth. In this regard, the prototypical Rho family GTPases, Rho, Rac, and Cdc42 regulate the activity of each other by a still poorly understood mechanism. Indeed, we found that constitutively active forms of Rac inhibit stress fiber formation and Rho stimulation by thrombin. Surprisingly, a mutant of Rac that is unable to activate Pak1 failed to inhibit thrombin signaling to Rho. To explore the underlying mechanism, we investigated whether Pak1 could regulate guanine nucleotide exchange factors (GEFs) for Rho. We found that Pak1 associates with P115-RhoGEF but not with PDZ-RhoGEF or LARG, and knock down experiments revealed that P115-RhoGEF plays a major role in signaling from thrombin receptors to Rho in HEK293T cells. Pak1 binds the DH-PH domain of P115-RhoGEF, thus suggesting a mechanism by which Rac stimulation of Pak1 may disrupt receptor-dependent Rho signaling. In agreement, expression of a dominant-negative Pak-Inhibitory Domain potentiated the activation of Rho by thrombin, and prevented the inhibition of Rho by Rac. These findings indicate that Rac interferes with receptor-dependent Rho stimulation through Pak1, thus providing a mechanism for cross-talk between these two small-GTPases.

Sphingosine kinase type 1 induces G12/13-mediated stress fiber formation, yet promotes growth and survival independent of G protein-coupled receptors.

Sphingosine 1-phosphate (S1P) is the ligand for a family of specific G protein-coupled receptors (GPCRs) that regulate a wide variety of important cellular functions, including growth, survival, cytoskeletal rearrangements, and cell motility. However, whether it also has an intracellular function is still a matter of great debate. Overexpression of sphingosine kinase type 1, which generated S1P, induced extensive stress fibers and impaired formation of the Src-focal adhesion kinase signaling complex, with consequent aberrant focal adhesion turnover, leading to inhibition of cell locomotion. We have dissected biological responses dependent on intracellular S1P from those that are receptor-mediated by specifically blocking signaling of Galphaq, Galphai, Galpha12/13, and Gbetagamma subunits, the G proteins that S1P receptors (S1PRs) couple to and signal through. We found that intracellular S1P signaled "inside out" through its cell-surface receptors linked to G12/13-mediated stress fiber formation, important for cell motility. Remarkably, cell growth stimulation and suppression of apoptosis by endogenous S1P were independent of GPCRs and inside-out signaling. Using fibroblasts from embryonic mice devoid of functional S1PRs, we also demonstrated that, in contrast to exogenous S1P, intracellular S1P formed by overexpression of sphingosine kinase type 1 promoted growth and survival independent of its GPCRs. Hence, exogenous and intracellularly generated S1Ps affect cell growth and survival by divergent pathways. Our results demonstrate a receptor-independent intracellular function of S1P, reminiscent of its action in yeast cells that lack S1PRs.