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Asbestos Induces Nitric Oxide Synthesis in Mesothelioma Cells via Rho Signaling Inhibition

Department of Genetics, Biology and Biochemistry, and Interdepartmental Center "G. Scansetti" for Studies on Asbestos and Other Toxic Particulates, Università di Torino; Research Center on Experimental Medicine (CeRMS), Torino; and Pathology Unit, Department of Oncology, Azienda Sanitaria Ospedaliera, Alessandria, Italy

Correspondence and requests for reprints should be addressed to Dario Ghigo, Dipartimento di Genetica, Biologia e Biochimica (Sezione di Biochimica), Via Santena, 5/bis, 10126 Torino, Italy. E-mail: dario.ghigo{at}unito.it

	Abstract

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
We have observed that in three human malignant mesothelioma cell lines, crocidolite asbestos induced the activation of the transcription factor NF-B and the synthesis of nitric oxide (NO) by inhibiting the RhoA signaling pathway. The incubation with crocidolite decreased the level of GTP-bound RhoA and the activity of Rho-dependent kinase, and induced the activation of Akt/PKB and IkB kinase, leading to the nuclear translocation of NF-B. The effects of crocidolite fibers on NF-B activation and NO synthesis were mimicked by Y27632 (an inhibitor of the Rho-dependent kinases) and toxin B (an inhibitor of RhoA GTPase activity), while they were reverted by mevalonic acid, the product of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA) reductase. Furthermore, crocidolite, similarly to mevastatin, inhibited the synthesis of cholesterol and ubiquinone and the prenylation of RhoA: these effects were prevented in the presence of mevalonic acid. This suggests that crocidolite fibers might inhibit the synthesis of isoprenoid molecules at the level of the HMGCoA reductase reaction or of an upstream step, thus impairing the prenylation and subsequent activation of RhoA. Akt can stimulate NO synthesis via a double mechanism: it can activate the inducible NO synthase via the NF-B pathway and the endothelial NO synthase via a direct phosphorylation. Our results suggest that crocidolite increases the NO levels in mesothelioma cells by modulating both NO synthase isoforms.

Key Words: crocidolite • mesothelioma • nitric oxide • RhoA • NF- • B

CLINICAL RELEVANCE Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References   In human malignant mesothelioma cells, crocidolite asbestos induces the activation of NF-B and the synthesis of nitric oxide by impairing the prenylation and subsequent activation of RhoA.

 
Human malignant mesothelioma (HMM) is a rare but aggressive tumor that originates from mesothelial cells and exhibits a strong correlation with exposure to asbestos fibers, such as crocidolite, amosite, and chrysotile (1, 2). Key changes in the development of the disease are the loss of the normal restraints on proliferation and the acquisition of resistance to apoptosis: asbestos, which is a complete carcinogen for mesothelial cells, plays a complex role in altering both proliferation and apoptosis (2).

NF-B is a redox-sensitive transcription factor comprised of protein dimers, including the transcription-activating heterodimer consisting of p50 and p65 subunits, that regulates expression of genes intrinsic to inflammation, cell proliferation, and apoptosis (3). One of the effects induced by NF-B activation is the increased synthesis of nitric oxide (NO), a highly reactive molecule involved in different cellular functions, including proliferation, differentiation, and apoptosis (4). NO is synthesized by three NO synthase (NOS; EC 1.14.13.39) isoforms, which catalyze the conversion of L-arginine to L-citrulline and NO with a 1:1 stoichiometry (5). Various stimuli, such as bacterial lipopolysaccharide, inflammatory cytokines, and oxidative stress, can stimulate the expression of the inducible NOS isoform (iNOS, NOS II) via the activation of NF-B (6). In addition, asbestos fibers have been shown to induce both NF-B activation and NO synthesis in alveolar macrophages, lung epithelial cells (7, 8), and, more recently, in mesothelial cells (9).

NF-B activity is controlled by members of the IkB family which bind directly to NF-B dimers in the cytoplasm, preventing the nuclear localization of the transcription factor, which is required for DNA binding (3). Many agents activate NF-B via serine phosphorylation, ubiquitination and proteasomal degradation of IB: in this way, NF-B is free to translocate to the nucleus and modulate the expression of many genes, including iNOS (6).

An increased iNOS activity has been induced in different cellular models by inhibitors of RhoA prenylation, such as statins (10–13), and by direct inhibitors of RhoA GTPase activity, such as the toxin B from Clostridium difficile (10, 13). A link between inhibition of the small G proteins Rho and NF-B activation has been already suggested (13).

Starting from these observations, our study has been aimed to investigate in HMM cells the role played by the RhoA signaling pathway in crocidolite-induced NO synthesis and cell proliferation.

	MATERIALS AND METHODS

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Materials
Fetal bovine serum (FBS) and Ham's F-12 nutrient mixture medium were supplied by BioWhittaker (Verviers, Belgium); plasticware for cell culture was from Falcon (Becton Dickinson, Bedford, MA); the cationic exchange resin Dowex AG50WX-8, N-(1-naphthylethylenediamine) dihydrochloride and sulfanilamide were from Aldrich (Milan, Italy); L-[2,3,4,5-³H]arginine monohydrochloride (62 Ci/mmol) was obtained from Amersham International (Bucks, UK); Y27632 was from Calbiochem (La Jolla, CA). Electrophoresis reagents were obtained from Bio-Rad Laboratories (Hercules, CA), and the protein content of cell monolayers and cell lysates was assessed with the BCA kit from Pierce (Rockford, IL). When not otherwise specified, other reagents were purchased from Sigma Chemical Co (St. Louis, MO).

Cells
HMM cell lines MM98, OC99, and GF99 were obtained from the pleural effusions of three patients with histologically confirmed malignant mesothelioma; the mesothelial origin of the isolated cells was confirmed by positive immunostaining as previously described (14). Cells were cultured in Ham's F-12 medium supplemented with 10% FBS, 1% penicillin/streptomycin, and 1% L-glutamine, and were maintained in a humidified atmosphere at 37°C and 5% CO₂. The N11 mouse glial cell line was a gift from Dr. Marco Righi (CNR Institute of Neuroscience, Section of Cellular and Molecular Pharmacology, Milan, Italy). At the end of each incubation period the experiments were performed on adherent cells, after having removed the incubation medium together with detached cells. When measurements were performed on extracellular medium, the medium was centrifuged previously at 12,000 x g for 15 min to pellet cellular debris. During the 24-h incubation period preceding experiments aimed to investigate cell protein phosphorylation (IB, I, eNOS), cells were maintained with the above-mentioned culture medium containing a low level of FBS (2% instead of 10%), to minimize the known effects of serum on the baseline pattern of protein phosphorylation. MM98, OC99, and GF99 cells showed the same behavior under each experimental condition: for sake of simplicity we chose to report in RESULTS the data obtained in MM98 cells as representative of the other two cell lines.

Asbestos Fibers
UICC (Union International Contre le Cancer) crocidolite fibers were sonicated (Labsonic sonicator, 100 W, 10 s; Labsonic, Göttingen, Germany) before incubation with cell cultures, in order to dissociate fiber bundles and allow their better suspension and diffusion in the culture medium.

Propidium Iodide Exclusion Assay
After incubation under different experimental conditions in 24-well plates, cells were washed twice with fresh PBS and incubated for 10 min at room temperature in 1 ml of binding buffer (100 mM Hepes/NaOH, pH 7.5, 140 mM NaCl, 25 mM CaCl₂) containing 2.5 µM propidium iodide (PI). Then cells were washed three times with fresh PBS and rinsed with 1 ml of binding buffer. An aliquot of cells suspension was sonicated and used to determine intracellular protein content. Fluorescence of each sample was recorded using a Perkin-Elmer LS-5 spectrofluorimeter (Shelton, CT). Excitation and emission wavelengths were 536 and 617 nm, respectively. A blank was prepared testing the cells in the absence of PI in each set of experiments, and its fluorescence was subtracted from that measured in the samples.

Lactate Dehydrogenase Activity
After incubation under different experimental conditions, the extracellular medium was centrifuged at 12,000 x g for 15 min to pellet cellular debris, whereas cells were washed with fresh medium, detached with trypsin/EDTA, washed with PBS, re-suspended at 1 x 10⁵ cells/ml in 0.2 ml of 82.3 mM triethanolamine phosphate hydrochloride (TRAP, pH 7.6), and sonicated on ice with two 10-s bursts. Lactate dehydrogenase (LDH) activity was measured in the extracellular medium and in the cell lysate, as previously described (15). One hundred microliters of supernatant from extracellular medium or 10 µl of cell lysate were incubated at 37°C with 82.3 mM TRAP (pH 7.6) and 5 mM NADH (final volume: 1 ml). The reaction was started by adding 20 mM pyruvic acid and was followed for 10 min, measuring absorbance at 340 nm with a Lambda 3 spectrophotometer (Perkin-Elmer). The reaction kinetics were linear throughout the time of measurement. Both intracellular and extracellular enzyme activity were expressed as µmol NADH oxidized/min/dish, then extracellular LDH activity was calculated as percentage of the total LDH activity in the dish.

Preparation of Nuclear Extracts
Cells were plated in 100-mm-diameter dishes at confluence, and all procedures for nuclear protein extraction were performed at 4°C using ice-cold reagents, as described (16). Cells were mechanically scraped in PBS, washed, and resuspended (1 x 10⁷ cells/0.5 ml) in lysis buffer A (15 mM KCl, 10 mM Hepes, 2 mM MgCl₂, 0.1 mM EDTA, 1 mM phenylmethylsulfonylfluoride [PMSF], 1 mM DTT, 10 µg/ml aprotinin, 2 µg/ml leupeptin, and 0.1% NP-40, pH 7.6). This suspension was incubated for 10 min on ice with occasional vortexing, and centrifuged for 30 s at 13,000 x g to pellet nuclei, which were rinsed with 0.2 ml of wash buffer B (2 M KCl, 25 mM Hepes, 0.1 mM EDTA, 1 mM PMSF, 1 mM DTT, 10 µg/ml aprotinin, and 2 µg/ml leupeptin, pH 7.6) and incubated at 4°C for 20 min. Then an equal volume of buffer C (25 mM Hepes, 0.1 mM EDTA, and 20% glycerol, pH 7.6) was added, the mix was centrifuged for 15 min at 20,000 x g, and the supernatant stored at –80°C until used for Western blotting or electrophoretic mobility shift assay (EMSA).

EMSA
The probe containing the NF-B oligonucleotide consensus sequence was labeled with [-³²P]ATP (Amersham International) (3,000 Ci/mmol, 250 µCi), using T4 polynucleotide kinase (Roche, Basel, Switzerland). The sequence of oligonucleotide was (binding site underlined): 5'-AGTTGAGGGGACTTTCCCAGG-3' (Promega Corporation, Madison, WI). Ten µg of the nuclear extracts, obtained as described above, were incubated for 20 min with 20,000 cpm of ³²P-labeled double-stranded oligonucleotide at 4°C in a reaction mixture containing: 2 µl of 10 µg/ml BSA, 2 µl of buffer D (100 mM KCl, 20 mM Hepes, 0.5 mM EDTA, 2 mM DTT, 0.1 mM PMSF, 20% glycerol, and 0.25% NP-40, pH 7.6), 4 µl of buffer E (300 mM KCl, 100 mM Hepes, 10 mM DTT, 100 µM PMSF, and 20% Ficoll, pH 7.6) and 2 µg of poly(dI-dC) (Roche). The final volume of the mix was brought to 25 µl with water. In supershift assay, nuclear extracts were pre-incubated for 30 min at room temperature with 2 µl of anti-p50 antibody (PC136; Calbiochem) or anti-p65 antibody (PC138; Calbiochem); then the reaction mixture containing the ³²P-labeled double-stranded oligonucleotide was added and samples were treated as described previously. The DNA–protein complex was separated on a not-denaturating 4% polyacrilamide gel in TBE buffer (pH 8.0). After electrophoresis, the gel was dried and autoradiographed by exposure to X-ray film for 48 h.

Western Blot Analysis
Cells were directly solubilized in the lysis buffer (25 mM Hepes, 135 mM NaCl, 1% NP40, 5 mM EDTA, 1 mM EGTA, 1 mM ZnCl₂, 50 mM NaF, 10% glycerol), supplemented with protease inhibitor cocktail set III (100 mM AEBSF, 80 µM aprotinin, 5 mM bestatin, 1.5 mM E-64, 2 mM leupeptin, and 1 mM pepstatin; Calbiochem), 2 mM PMSF, and 1 mM sodium orthovanadate. Whole cell extracts (or nuclear extracts, for p50 and p65 detection) containing 30 µg of proteins were separated by SDS-PAGE, transferred to PVDF membrane sheets (Immobilon-P; Millipore, Bedford, MA), and probed with the following antibodies: anti-IB (from rabbit, diluted 1:500 in PBS-BSA 1%; Santa Cruz Biotechnology, Santa Cruz, CA), anti-phospho(Ser 32)-IB (from mouse, diluted 1:250 in PBS-BSA 1%; Santa Cruz Biotechnology), anti-IB kinase(IKK) (from rabbit, diluted 1:500 in PBS-BSA 1%, Santa Cruz Biotechnology), anti-phospho(Ser 180)-IKK (from rabbit, diluted 1:250 in PBS-BSA 1%; Cell Signaling Technology Inc., Beverly, MA), anti-Rho kinase (Rock) 1 (from rabbit, diluted 1:500 in PBS-BSA 1%; Santa Cruz Biotechnology), anti-Rock2 (from rabbit, diluted 1:500 in PBS-BSA 1%; Santa Cruz Biotechnology), anti-eNOS (from mouse, diluted 1:500 in PBS-BSA 1%; Transduction Laboratories, Lexington, KY), anti-phospho-(Ser 1177) eNOS (from mouse, diluted 1:500 in PBS-BSA 1%; Cell Signaling Technology Inc.), anti-p50 (from mouse, diluted 1:250 in PBS-BSA 1%; Santa Cruz Biotechnology), anti-p65 (from rabbit, diluted 1:500 in PBS-BSA 1%; Santa Cruz Biotechnology), and anti-glyceraldehyde-3-phosphate dehydrogenase (anti-GAPDH, from rabbit, diluted 1:500 in PBS-BSA 1%; Santa Cruz Biotechnology). Expression of GAPDH, the product of an housekeeping gene, was used as a control of equal loading. After a 1-h incubation, the membrane was washed with PBS-Tween 0.1% and subjected for 1 h to a peroxidase-conjugated anti-rabbit, anti-mouse, or anti-goat IgG (Amersham International, diluted 1:1,000 in PBS-Tween with Blocker Not-Fat Dry Milk 5% [Bio-Rad]). The membrane was washed again with PBS-Tween and proteins were detected by enhanced chemiluminescence (Immun-Star; Bio-Rad). To assess the presence of the Proliferating Nuclear Cellular Antigen (PCNA), lysates were directly resolved on a 12% SDS-PAGE, transferred to PVDF membrane sheets and probed overnight with an anti-PCNA antibody (from mouse, 1:500 in PBS-BSA 1%; Santa Cruz Biotechnology). The membrane was then washed and treated as described above.

IKK Activity Assay
IKK activity was measured as previously described (17). Cells were washed with ice-cold PBS and solubilized in 0.5 ml of lysis buffer (50 mM Hepes, 150 mM NaCl, 2 mM MgCl₂, 1 mM EDTA, 0.1% NP40, 100 µM NaF, 10 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, 1 mM PMSF, and 250 µM DTT). Samples were centrifuged at 13,000 x g for 15 min and the supernatant was used for the assay and cell protein quantification. To purify the IKK complex, equal amounts of the whole lysate (0.5 mg cell proteins/test) were immunoprecipitated with an anti-IKK antibody (from rabbit, diluted 1:200 in PBS-BSA 1%; Santa Cruz Biotechnology) for 90 min at 4°C. Samples were centrifuged (13,000 x g for 15 min) and washed three times with kinase buffer (20 mM Hepes, 20 mM -glycerolphosphate, 1 mM MnCl₂, 5 mM MgCl₂, 2 mM NaF, and 250 µM DTT). A quantity of 0.1 µg of the immunoprecipitated proteins was incubated with 1 mM ATP in the presence of the proteasome inhibitor MG132 (10 µM); to provide the reaction mix with an excess of IB protein (the substrate of IKK), 30 µg of total cellular lysate of SV40-positive HMM cells, obtained under not-denaturing conditions, were added. SV40-positive HMM cells were chosen because they were previously shown to exhibit a very high basal amount of IB (9). MG132 was added in the reaction mixture in order to avoid, during the assay, the degradation of phospho-IkB protein by the proteasome eventually still present in the cell lysate (used as a provider of the substrate). Reaction was carried over at 30°C for 30 min and stopped with 30 µl of Laemmli buffer. Finally, samples were subjected to electrophoresis in a 12% SDS-PAGE, transferred to PVDF membrane sheets, and probed with an anti-IB antibody (from rabbit, diluted 1:250 in PBS-BSA 1%; Santa Cruz Biotechnology) and an anti-phospho(Ser 32)-IkB antibody (from mouse, diluted 1:250 in PBS-BSA 1%; Santa Cruz Biotechnology), respectively.

Akt Activity Assay
Akt activity was measured using the CycLex Akt/PKB Kinase Assay/Inhibitor Screening Kit (CycLex Co., Nagano, Japan). Cells were cultured in 35-mm-diameter Petri dishes, washed with ice-cold PBS, and lysed in 0.2 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 1mM EDTA, 1mM EGTA, 10 mM NaF, 2 mM sodium orthovanadate, 0.5 µg/ml leupeptin, 1 µg/ml pepstatin, 0.5 mM PMSF, and 10 mM -mercaptoethanol). Samples were sonicated on crushed ice with two 10-s bursts and centrifuged at 13,000 x g for 30 min at 4°C. Supernatants were treated following manufacturer's instructions, and protein content was measured. Briefly, 15 µl of samples were diluted in 85 µl of the kinase buffer provided with the kit, containing 125 µM ATP, and incubated for 60 min at 30°C in 96-well plates, pre-coated with the Akt substrate AkTide-2T, which is efficiently phosphorylated by Akt at a serine residue. Wells were washed five times with 2% Tween-20, and 100 µl of horseradish peroxidase (HRP)-conjugated anti-phospho-AkTide-2T monoclonal antibody were added. After a 60-min incubation at room temperature, samples were washed again, and 100 µl of the chromogenic substrate tetra-methylbenzidine were added. After a 15-min incubation at room temperature, the reaction was stopped with 100 µl of 0.5 N H₂SO₄ and absorbance was read at 450 nm, using a Packard EL340 microplate reader (Bio-Tek Instruments, Winooski, VT). For each set of experiments, a titration curve was prepared, using serial dilutions of recombinant Akt (CycLex Co.) in kinase buffer. Akt activity was expressed as mU absorbance/mg cell proteins.

RhoA-GTP Pull-Down
Biochemical assay for activity of RhoA was performed as described (18). Cells were lysed in MLB buffer (125 mM Tris-HCl, pH 7.4, 750 mM NaCl, 1% NP40, 10% glycerol, 50 mM MgCl₂, 5 mM EDTA, 25 mM NaF, 1 mM sodium orthovanadate, 10 µg/ml leupeptin, 10 µg/ml pepstatin, 10 µg/ml aprotinin, and 1 mM PMSF) and centrifuged at 13,000 x g for 10 min at 4°C. An aliquot of supernatant was taken out for determination of protein content and analysis of total amount of RhoA. Another aliquot of the same lysate was directly probed with an anti-RhoA antibody (1:250, in PBS-BSA 1%; Santa Cruz Biotechnology), to measure total RhoA protein. A further 30 µg of the supernatant was incubated for 45 min at 4°C with agarose-glutathione beads coupled to a fusion protein containing glutathione S-transferase (GST) and the Rho-binding domain of the Rho effector protein rhotekin (Upstate, Charlottesville, VA). The beads were then washed three times in MLB buffer and harvested by addition of Laemmli buffer 2x. The RhoA activity was analyzed, resolving the samples by 12% SDS-PAGE and Western blotting using anti-RhoA antibody to detect GTP-bound activated RhoA. RhoA-GTP/total RhoA ratio was taken as an index of the active fraction of RhoA.

Rock Assay
Rock activity was measured using the CycLex Rho-Kinase Assay Kit (CycLex Co.), a single site binding immunoassay. Cells were cultured in 35-mm-diameter Petri dishes, washed with ice-cold PBS and lysed in 0.2 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100, 1mM EDTA, 1mM EGTA, 2 mM NaF, 2 mM sodium orthovanadate, 0.5 µg/ml leupeptin, 1 µg/ml pepstatin, 0.2 mM PMSF, and 10 mM -mercaptoethanol). Samples were sonicated on crushed ice with two 10-s bursts and centrifuged at 13,000 x g for 5 min at 4°C. Supernatants were treated following manufacturer's instructions, and protein content was measured. Briefly, samples were diluted 1:4 in the kinase buffer provided with the kit, containing 20 mM ATP, and incubated fo 60 min at 30°C in 96-well plates, pre-coated with the recombinant C-terminus of the myosin binding subunit (MBS) of myosin phosphatase. Wells were washed five times with 2% Tween-20, and 100 µl of the HRP-conjugated anti phospho(Thr 696)-MBS antibody were added. After a 60-min incubation at room temperature, samples were washed again, and 100 µl of the chromogenic substrate tetra-methylbenzidine were added. After a 15-min incubation at room temperature, the reaction was stopped with 100 µl of 0.5 N H₂SO₄ and absorbance was read at 450 nm, using a Packard EL340 microplate reader (Bio-Tek Instruments). For each set of experiments, a titration curve was prepared, using serial dilution of recombinant Rho-Kinase II (MBL Inc., Woburn, MA) in kinase buffer. Data were expressed as mU absorbance/mg cell proteins.

RT-PCR
Total RNA was obtained by the guanidinium thiocyanate-phenol-chloroform method (19). A quantity of 30 ng of total RNA was reversely transcribed into cDNA with the Superscript II One-Step RT-PCR System with Platinum Taq DNA Polymerase (cycling conditions: 1 cycle 50°C for 30 min, 1 cycle 94°C for 2 min). cDNA products were determined by PCR amplification, carried out in a total volume of 50 µl, according to the manufacturer's recommendations. The RT-PCR efficiency was controlled by amplifying a -actin fragment, used as an housekeeping gene. Primers for iNOS (0.3 µM) were: 5'-TCCGAGGCAAACAGCACATTCA-3', 5'-GGGTTGGGGGTGTGGTGATGT-3' (462 bp); primers for -actin (0.5 µM) were: 5'-GGTCATCTTCTCGCGGTTGGCCTTGGGGT-3', 5'-CCCCAGGCACCAGGGCGTGAT-3' (230 bp). PCR amplification for iNOS was: 1 cycle of denaturation at 95°C for 2 min, 30 cycles of denaturation at 95°C for 30 s, annealing at 55°C for 1 min, elongation at 72°C for 30 s, and 1 cycle of extension at 72°C for 10 min; for -actin: 1 cycle of denaturation at 94°C for 3 min, 35 cycles of denaturation at 94°C for 1 min, annealing at 58°C for 1 min, elongation at 72°C for 1 min, and 1 cycle of extension at 72°C for 7 min. Samples were electrophoresed in 1.5% agarose gels containing ethidium bromide in Tris-acetate/EDTA buffer to visualize the PCR products.

Nitrite Production
Confluent cell monolayers in 35-mm-diameter Petri dishes were incubated in fresh medium for 24 h under the experimental conditions indicated in RESULTS. Then nitrite production was measured by adding 0.15 ml of cell culture medium (centrifuged previously at 12,000 x g for 15 min to pellet cellular debris) to 0.15 ml of Griess reagent (20) in a 96-well plate, and, after a 10 min incubation at 37°C in the dark, absorbance was measured at 540 nm with a Packard EL340 microplate reader (Bio-Tek Instruments). A blank was prepared for each experimental condition in the absence of cells, and its absorbance was subtracted from that measured in the samples. Nitrite concentration was expressed as nmol nitrite/mg cell proteins.

Measurement of NOS Activity
Cells grown at confluence on 35-mm-diameter Petri dishes, after incubation under the experimental conditions described in RESULTS, were detached by trypsin/EDTA, washed with PBS, resuspended in 0.3 ml of Hepes/EDTA/dithiotreitol (DTT) buffer (20 mM Hepes, 0.5 mM EDTA, 1 mM DTT, pH 7.2) and then sonicated on crushed ice with two 10-s bursts. In each assay tube, the following reagents were added to 100 µl of lysate at the following final concentrations: 2 mM NADPH, 2.5 µCi L-[³H]arginine (= 0.4 µM), 100 µM tetrahydrobiopterin, 1.5 mM CaCl₂ (20). After a 15-min incubation at 37°C, the reaction was stopped by adding 2 ml Hepes-Na/EDTA buffer (20 mM Hepes sodium salt, 2 mM EDTA, pH 6); the whole reaction mixture was applied to 2 ml columns of Dowex AG50WX-8 (Na⁺ form) and eluted with 4 ml of water. The radioactivity corresponding to [³H]citrulline content in 6.1 ml eluate was measured by liquid scintillation counting. Citrulline synthesis was expressed as pmol citrulline/min/mg cell proteins.

Measurement of Isoprenoid Molecules Synthesis
The synthesis of isoprenoid molecules was checked as intracellular accumulation of cholesterol and ubiquinone, and measured as previously described (21). Cells were incubated for 24 h with 10 µCi/ml of [³H]acetate (3600 mCi/mmol; Amersham International) or 10 µCi/ml of [¹⁴C]mevalonic acid (67 mCi/mmol; Amersham International), then washed twice with PBS and resuspended in 200 µl of PBS. A 50-µl aliquot was used for protein quantification, while the remaining part was transferred in glass microcentrifuge tubes. A quantity of 1.5 ml of a 1:2 methanol/hexane solution was added, and cellular suspensions were vortexed for 1 h, then centrifuged at 2,000 x g for 5 min. The upper phase was transferred in a new set of glass microcentrifuge tubes, while the lower phase was resuspended in 1 ml of hexane, vortexed overnight, and centrifuged at 2,000 x g for 5 min: the new upper phase was added to the previously isolated phase. After a 24-h evaporation, samples were dissolved in 100 µl of chloroform and resolved by thin layer chromatography on silica gel, using 1:1 diethyl ether/exane as mobile phase. Standard solutions of 10 µl of cholesterol (2 µg/ml) and ubiquinone (2 µg/ml) were employed. After the separation, the gel was exposed to iodine gas for 2 h and the spots corresponding to cholesterol and ubiquinone were isolated. The radioactivity of each spot was measured by liquid scintillation counting and expressed as cpm/mg cellular proteins.

Assessment of RhoA Prenylation
After 24 h of treatment cells were lysed with 2% ice cold Triton X-114 in Tris buffered saline pH 7.4 and phase-separated as previously described (22) with some modifications. Cells were harvested in lysis buffer (25 mM Tris-HCl, pH 7.4, 150 mM NaCl, 2% Triton X-114, 5 mM MgCl₂, 1 mM Na₂HPO₄, 1 mM sodium orthovanadate, 1 mM PMSF, protease inhibitor cocktail set III; Calbiochem) and incubated for 30 min at 4°C. After sonication, insoluble material was removed by centrifugation at 13,000 x g for 10 min at 4°C and the supernatant was phase-separated. Briefly, each sample was overlaid on a sucrose cushion and after warming at 37°C for 3 min the turbid solution was centrifuged at 300 x g for 5 min at room temperature to separate the hydrophobic and aqueous phase. Both phases were collected and the separation was repeated. Finally, the aqueous phase was rinsed with 2% Triton X-114 without sucrose cushion and the detergent phase of this last condensation was discarded. The protein content of total lysate, aqueous phase, and detergent phase was determined by Bradford test (Bio-Rad), and 6 µg aliquots were analyzed by SDS-PAGE. An anti-RhoA antibody (Santa Cruz Biotechnology) was used to evaluate RhoA distribution between aqueous phase (soluble unprenylated form of RhoA) and detergent phase (hydrophobic prenylated form of RhoA), whereas an anti-GAPDH antibody (Santa Cruz Biotechnology) was used to verify the partitioning of soluble GAPDH in the aqueous phase.

[³H]thymidine Incorporation Assay
Cells were grown in 35-mm-diameter Petri dishes and incubated for 24 h under the experimental conditions described in RESULTS, in a culture medium containing 1 µCi/ml [³H]thymidine (62 Ci/mmol; Amersham International). At the end of the incubation, cells were washed with ice-cold PBS, detached by trypsin/EDTA, and resuspended in 200 µl of PBS. A 50-µl aliquot was used for protein quantification, while the remaining part was transferred in poliethylene vials and the radioactivity was measured by liquid scintillation. [³H]thymidine incorporated in each sample was expressed as pmol/mg cell proteins.

Statistical Analysis
All data in text and figures are provided as means ± SE. The results were analysed by a one-way ANOVA and Tukey's test. P < 0.05 was considered significant.

	RESULTS

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

 
Crocidolite Asbestos Elicits in HMM Cells a Dose- and Time-Dependent Increase of Both NO Synthesis and LDH Leakage, Which Are Inhibited by Coincubation with Mevalonic Acid
We investigated the effects of different concentrations (1, 5, 25 µg/cm²) of crocidolite asbestos fibers on HMM cells viability after a 24- to 48-h incubation (Figure 1). The incorporation of PI, a marker of decreased cell viability (23), increased as a function of time and concentration. We investigated further the effects of the incubation of HMM cells with different concentrations (1, 5, 25 µg/cm²) of crocidolite asbestos fibers, under different incubation times (6, 24, 48 h), on the extracellular accumulation of nitrite (a stable derivative of NO synthesis), and on the release of LDH in the extracellular medium (a sensitive index of cytotoxicity) (24) (Figure 2A). Since after 6 h the NO synthesis and cytotoxic effect were negligible, whereas after 48 h the cytotoxic effect was too marked, we decided to perform the subsequent experiments using a 24-h incubation time and a 25 µg/cm² concentration of fibers, which appeared to be the minimal dose among our conditions able to induce significant accumulation of nitrite. Under these experimental conditions, we incubated the cells in the absence or presence of variable concentrations (50, 100, 200 µM) of mevalonic acid (Figure 2B). A quantity of 100 µM mevalonic acid completely reverted both nitrite increase and LDH leakage induced by crocidolite.

View larger version (10K): [in this window] [in a new window]   Figure 1. Effect of crocidolite on PI exclusion in HMM cells. HMM (MM98) cells were incubated for 24 and 48 h in the absence (CTRL) or presence of crocidolite (CRO, 1, 5, 25 µg/cm2). Camptothecin (CAM, 10 nM for 24 h), a genotoxic and proapoptotic compound in mesothelioma cells, was used as a positive control. After these incubation times, cells were checked for their content of PI, as described in MATERIALS AND METHODS. Measurements were performed in duplicate and data are presented as means ± SE (n = 6). *P < 0.05, **P < 0.001 versus CTRL; °P < 0.05 versus CRO 25 µg/cm2 24 h.

View larger version (22K): [in this window] [in a new window]   Figure 2. Effect of crocidolite and mevalonic acid on NO synthesis and LDH leakage in HMM cells. HMM (MM98) cells were incubated for 6, 24, 48 h in the absence (CTRL) or presence of crocidolite (CRO, 1, 5, 25 µg/cm2) (A), and for 24 h in the absence (CTRL) or presence of crocidolite (CRO, 25 µg/cm2) and/or mevalonic acid (MA, 50, 100, 200 µM) (B). After these incubation times, nitrite concentration in the extracellular medium and LDH activity in both extracellular medium and cell lysate were measured as described in MATERIALS AND METHODS. Each measurement was performed in duplicate. The experiments were repeated with the other two cell lines (OC99, GF99), giving superimposable results (data not shown). (A) Data are presented as means ± SEM (n = 6). *P < 0.05, **P < 0.001 versus CTRL. (B) Data are presented as means ± SEM (n = 5). *P < 0.05, ** p < 0.001 versus CTRL; °P < 0.05 versus CRO.

Crocidolite Asbestos and Inhibitors of RhoA and Rocks Activate NF-B in HMM Cells

View larger version (153K): [in this window] [in a new window]   Figure 3. Effect of crocidolite, mevalonic acid, and inhibitors of RhoA/Rock on the NF-B pathway. HMM (MM98) cells were incubated for 24 h without (CTRL) or with crocidolite fibers (25 µg/cm2, CRO), in the absence or presence of mevalonic acid (MA, 100 µM), Y27632 (Y276, 0.2 µM), or toxin B (TOX, 0.1 ng/ml). N11 cells, incubated with bacterial lipopolysaccharide (LPS, 20 µg/ml), were used as a positive control (+); in each experiment one lane was loaded with bidistilled water (–) in place of cellular extracts; in the absence of LPS the pattern was superimposable to that of HMM CTRL (not shown). (A) EMSA detection of NF-B nuclear translocation, as described in MATERIALS AND METHODS. In the lane marked with Anti p50 and Anti p65, a supershift assay was performed on HMM cells incubated with 25 µg/cm2 crocidolite fibers (see MATERIALS AND METHODS). EMSA was repeated also in the other two cell lines (OC99, GF99), giving superimposable results (data not shown). (B) The nuclear translocation of the two subunits of NF-B, p50 and p65, was detected with Western blotting performed on nuclear extracts as described in MATERIALS AND METHODS. The expression of IB, phospho-IB, and GAPDH was detected with Western blotting performed on whole cellular extracts (see MATERIALS AND METHODS for details). Detection of GAPDH, evidence of housekeeping gene expression, was used as a control of equal loading in this and the subsequent experiments. The level of IB followed the same pattern in the other two cell lines (OC99, GF99).

Crocidolite Asbestos and Inhibitors of RhoA and of Rocks Elicit IKK Phosphorylation, Akt Activation, and RhoA/Rock Signaling Inhibition in HMM Cells

View larger version (81K): [in this window] [in a new window]   Figure 4. Effect of crocidolite, mevalonic acid, and inhibitors of RhoA/Rho kinase on Ikk activation. HMM (MM98) cells were incubated for 24 h in fresh medium only (CTRL) or with crocidolite fibers (CRO, 25 µg/cm2), in the absence or presence of mevalonic acid (MA, 100 µM), Y27632 (Y276, 0.2 µM), or toxin B (TOX, 0.1 ng/ml). (A) IKK activity was assayed as the ability of the immunoprecipitated IKK complex to phosphorylate IB, and its outcome was checked using an anti-phospho(Ser 32)-IB and an anti-IB antibody (as described in MATERIALS AND METHODS). MG132 (10 µM) was added in the reaction mixture in order to avoid, during the assay, the degradation of phospho-IB protein by the proteasome eventually still present in the SV40-positive HMM cell lysate (used as a provider of the substrate). (B) Detection of Ikk phosphorylation. Whole cell lysates were analysed by Western blotting using the following antibodies: anti-phospho(Ser180)-I, anti-I, anti-GAPDH (as described in MATERIALS AND METHODS). N11 cells, incubated with LPS (+, 20 µg/ml), were used as a positive control; in the absence of LPS the pattern was superimposable to that of HMM CTRL (not shown). Each panel is representative of two experiments with similar results. The experiments shown in B were repeated in duplicate with the other two cell lines (OC99, GF99), giving superimposable results (data not shown).

View larger version (11K): [in this window] [in a new window]   Figure 5. Effect of crocidolite, mevalonic acid, and inhibitors of RhoA/Rho kinase on Akt activity. HMM (MM98) cells were incubated for 24 h in fresh medium only (CTRL) or with crocidolite fibers (CRO, 25 µg/cm2), in absence or presence of mevalonic acid (MA, 100 µM), Y27632 (Y276, 0.2 µM), or toxin B (TOX, 0.1 ng/ml). Cell lysates were analysed for Akt activity as described in MATERIALS AND METHODS. N11 cells, incubated with bacterial lipopolysaccharide (LPS, 20 µg/ml), were used as a positive control; in the absence of LPS the pattern was superimposable to that of HMM CTRL (not shown). Each measurement was performed in duplicate. The experiments were repeated with the other two cell lines (OC99, GF99), giving superimposable results (data not shown). Data are presented as means ± SEM (n = 6). *P < 0.05, **P < 0.001 versus CTRL; °P < 0.001 versus CRO.

View larger version (40K): [in this window] [in a new window]   Figure 6. Effect of crocidolite and mevalonic acid on GTP binding of RhoA and on Rho kinase activity. HMM (MM98) cells were cultured for 24 h in the absence (CTRL) or presence of the following compounds, alone or in different combinations: crocidolite fibers (CRO, 25 µg/cm2), mevalonic acid (MA, 100 µM), Y27632 (Y276, 0.2 µM), or toxin B from C. difficile (TOX, 0.1 ng/ml). Subsequently, the cells were lysed and checked for (A) expression of RhoA-GTP and total RhoA (see MATERIALS AND METHODS; panel is representative of two experiments with similar results), and (B) Rho kinase activity (see MATERIALS AND METHODS); data are presented as means ± SE (n = 6). *P < 0.001 versus CTRL; °P < 0.05 versus CRO. The experiments were repeated with the other two cell lines (OC99, GF99), giving superimposable results (data not shown).

View larger version (26K): [in this window] [in a new window]   Figure 7. Effect of crocidolite and mevastatin on the synthesis of isoprenoid molecules and on the prenylation and expression of RhoA. HMM (MM98) cells were cultured for 24 h with [3H]acetate (A) or [14C]mevalonic acid (B), in the absence (CTRL) or presence of the following compounds, alone or in different combinations: crocidolite fibers (CRO, 25 µg/cm2), mevastatin (MVS, 100 µM), and squalestatin (SQ, 1 µM). Subsequently, the cells were lysed and checked for incorporation of 3H (A) or 14C (B) into cholesterol and ubiquinone (see MATERIALS AND METHODS). Data are presented as means ± SE (n = 6). *P < 0.05, **P < 0.001 versus CTRL. (C) After the incubation, cells were lysed and checked for the expression of total, prenylated, and nonprenylated RhoA and of GAPDH (see MATERIALS AND METHODS). The figure is representative of two experiments with similar results. The experiments show in A and B were repeated in duplicate with the other two cell lines (OC99, GF99), giving superimposable results (data not shown).

Crocidolite Increases the Expression of NOS II, the Activity of NOS III, and the Synthesis of NO via NF-B and Akt Activation in HMM Cells

View larger version (63K): [in this window] [in a new window]   Figure 8. (A) Effect of crocidolite, mevalonic acid and inhibitors of RhoA/Rho kinase on iNOS induction. HMM (MM98) cells were incubated in fresh medium only (CTRL) or with crocidolite fibers (CRO, 25 µg/cm2), in the absence or presence of mevalonic acid (MA, 100 µM), Y27632 (Y276, 0.2 µM), and toxin B (TOX, 0.1 ng/ml). After 24 h, total RNA was extracted and subjected to RT-PCR for iNOS mRNA, as described in MATERIALS AND METHODS. One lane was loaded with bidistilled water (–) in place of cellular extracts. The figure is representative of two experiments per cell line with similar results. (B) Effect of crocidolite on eNOS phosphorylation. HMM (MM98, OC99, and GF99) cells were incubated 24 h in the absence (CTRL) or presence on crocidolite fibers (CRO, 25 µg/cm2), in the absence or presence of mevalonic acid (MA, 100 µM), Y27632 (Y276, 0.2 µM), and toxin B (TOX, 0.1 ng/ml). Cellular extracts were analyzed by Western blotting with an anti-eNOS antibody and an anti-phospho(Ser1177)eNOS antibody, as indicated in MATERIALS AND METHODS. The figure is representative of two experiments with similar results. The experiments were repeated with the other two cell lines (OC99, GF99), giving superimposable results (data not shown).

These effects on NOS expression and phosphorylation were accompanied by changes of NOS activity. After a 24-h incubation, crocidolite, Y27632, and toxin B induced a significant augmentation of extracellular nitrite (an NO stable derivative in oxygenated cell systems) and intracellular NOS activity in HMM cultures (Figure 9A). Mevalonic acid significantly prevented such effects (Figure 9A). Acting as NO scavengers (due to their content of oxyhemoglobin), red blood cells lowered the nitrite concentration without modifying the NOS activity, as expected (Figure 9A).

View larger version (49K): [in this window] [in a new window]   Figure 9. Effects of crocidolite and signaling inhibitors on NO synthesis and on the level of PCNA. HMM (MM98) cells were grown for 24 h in the absence (CTRL) or presence of the following components, alone or in different combinations: crocidolite fibers (CRO, 25 µg/cm2), mevalonic acid (MA, 100 µM), Y27632 (Y276, 0.2 µM), toxin B from C. difficile (TOX, 0.1 ng/ml), and packed red blood cells (RBC, 10 µl/ml). (A) NOS activity in cell lysates (open bars) and nitrite levels in the extracellular medium (hatched bars) was measured in triplicate, as described (see MATERIALS AND METHODS). Data are presented as means ± SE (n = 6). *P < 0.05, **P < 0.005 versus CTRL; °P < 0.05; °°P < 0.001 versus CRO or Y276 or TOX, respectively. (B) Whole extracts were subjected to Western blotting, using an anti-PCNA antibody, as described in MATERIALS AND METHODS; the figure is representative of two experiments with similar results. The experiments were repeated with the other two cell lines (OC99, GF99), giving superimposable results (data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 10. Effects of crocidolite and signaling inhibitors on cell proliferation (measured as [3H]thymidine incorporation) and on the release of LDH in the extracellular medium. HMM (MM98) cells were grown for 24 h in the absence (CTRL) or presence of the following components, alone or in different combinations: crocidolite fibers (CRO, 25 µg/cm2), mevalonic acid (MA, 100 µM), Y27632 (Y276, 0.2 µM), toxin B from C. difficile (TOX, 0.1 ng/ml), and packed red blood cells (RBC, 10 µl/ml). Cell proliferation, extrapolated from the [3H]thymidine incorporation assay (open bars), and LDH leakage in the extracellular medium (hatched bars) were measured in triplicate, as described (see MATERIALS AND METHODS). Data are presented as means ± SE (n = 3). *P < 0.05, **P < 0.005 versus CTRL; °P < 0.05, °°P < 0.005 versus CRO or Y276 or TOX, respectively. The experiments were repeated with the other two cell lines (OC99, GF99), giving superimposable results (data not shown).

	DISCUSSION

Top Abstract CLINICAL RELEVANCE MATERIALS AND METHODS RESULTS DISCUSSION References

We have previously observed that crocidolite can promote nuclear translocation of NF-B in HMM cells (9). The results of the present work show that the crocidolite-dependent nuclear translocation of NF-B is elicited through the inhibition of the RhoA signaling pathway. RhoA is involved in different cellular crucial events, such as proliferation, tumorigenesis, and tumor invasion (32). It usually cycles between a nonprenylated, inactive form and a prenylated, GTP-bound active form (26): if active, RhoA can interact with several downstream effectors, such as the two serine-threonine Rho-dependent kinases Rock1 and Rock2 (33). The effect of RhoA modulation on NF-B activation may change depending on the cellular model. We observed that in HMM cells crocidolite fibers promoted NF-B translocation into the nucleus, elicited the phosphorylation of IB and lowered the intracellular content of IB protein. A similar response pattern was observed when cells were incubated with both toxin B from Clostridium difficile, which inhibits the GTPase activity of RhoA and other small G proteins (34), and Y27632, a selective Rock inhibitor (35). Mevalonic acid, the product of HMGCoA reductase, being a substrate for the synthesis of isoprenoid molecules may favor the prenylation and activation of RhoA. In our experimental conditions mevalonic acid, which per se did not change the nuclear content of NF-B, completely prevented both the NF-B activation and the phosphorylation and decrease of IB induced by crocidolite.

The IKK complex phosphorylates IB favoring its proteasomal degradation and allowing NF-B to translocate to the nucleus. IKK is fully active only when phosphorylated, mainly on serine 176 and 180 of IKK (3). In nonstimulated HMM cells, IKK was weakly phosphorylated and its activity (measured as the ability of immunoprecipitated intracellular IKK complex to phosphorylate IB) was not detectable, while both crocidolite and inhibitors of RhoA and Rock increased both IKK activity and the amount of phospho-IKK. Mevalonic acid completely blocked the effect of crocidolite.

The mechanisms of IKK phosphorylation have been extensively investigated and many serine/threonine kinases seem to be involved (3). For instance, in some cell types IKK is a target of the kinase activity of Akt/PKB, which promotes NF-B translocation, cellular survival, and/or inhibition of apoptosis (36). Interestingly, crocidolite has been shown to activate Akt (by inducing its phosphorylation on serine 473) in a rat pleural mesothelial cell line (37). After a 24-h incubation of HMM cells with crocidolite, Akt activity was significantly increased, an effect prevented by the presence of mevalonic acid. Moreover, Y27632 and toxin B, although at a lower extent, induced a significant increase of the Akt activity as well. So far, the crocidolite-dependent activation of Akt is likely to be linked to the inhibition of RhoA and Rock.

In other experimental models, a relationship between IKK/IB/NF-B pathway and RhoA and Rock signaling has already been proposed (12, 13). Exposure of HMM cells to crocidolite fibers induced a significant decrease of GTP-bound (active) RhoA and of Rock activity, a result similar to that observed after the incubation with toxin B, a specific RhoA inhibitor (34). Again, mevalonic acid prevented these crocidolite effects.

To our knowledge, this is the first evidence that crocidolite inhibits the RhoA/Rock pathway. Furthermore, our data suggest that the asbestos elicits the activation of Akt, IKK, and NF-B by inducing the blockage of RhoA signaling: indeed, crocidolite, like mevastatin, inhibits both the synthesis of isoprenoid molecules (cholesterol, ubiquinone) and the prenylation of RhoA. These effects are prevented in the presence of mevalonic acid, which allows cells to synthesize isoprenoid groups and then to keep RhoA in the prenylated, active form. This observation suggests that crocidolite fibers might inhibit the synthesis of isoprenoid molecules at the level of the HMGCoA reductase reaction or of an upstream step, causing a depletion of mevalonic acid.

We have previously shown that in HMM cells crocidolite increases iNOS expression and NO synthesis via activation of NF-B (9). Several authors have reported an increased iNOS activity in cells incubated with different inhibitors of RhoA proteins, such as statins (10, 11) and inhibitors of geranylgeranylation (13). Toxin B from C. difficile (10) and Y27632 (13) also elicited enhanced nitrite production in several cell lines. Our data show that, like crocidolite, inhibitors of RhoA and Rock may also increase iNOS mRNA and NO synthesis in HMM cells. More interestingly, overexpression and activation of iNOS induced by crocidolite are likely to be mediated by inhibition of the RhoA prenylation, since these effects were prevented by the presence of mevalonic acid.

Until now, no data are available about the effect of crocidolite asbestos on the activity of the other NOS isoforms in human mesothelioma. Since we found that crocidolite and inhibitors of RhoA pathway activate Akt, we investigated whether they could exert some effect on eNOS, which is a well-known target of Akt (28). Actually, crocidolite induced phosphorylation of eNOS in HMM cells. It is already known that Akt can stimulate NO synthesis via a double mechanism: it can activate the NF-B/iNOS pathway by phosphorylating IKK (25), and it may phosphorylate the eNOS isoform, leading to its activation (28). Our results suggest that crocidolite increases the NO levels in HMM cells by modulating both iNOS and eNOS. Prenylated RhoA seems to play an important role in inducing this redundant signal, because in the presence of mevalonic acid crocidolite was unable to induce iNOS, phosphorylate eNOS, and increase nitrite accumulation. The role of RhoA and Rock in crocidolite-induced expression of iNOS RNA is inferred indirectly by the similar effect of pharmacologic inhibitors of Rho signaling pathway: this hypothesis has to be confirmed with the use of dominant-negative mutants of Rock.

A number of experimental evidences suggests that asbestos may activate simultaneously both proliferation and apoptosis in mesothelial cells (2, 38) and lung epithelial cells (39). This phenomenon could be observed also in our cell cultures. Crocidolite, as well as Y27632 and toxin B, increased the DNA synthesis, but also the leakage of LDH (a sensitive marker of cell damage), thus appearing able to exert both mitogenic and cytotoxic effects, which were prevented by mevalonic acid and packed erythrocytes. Interestingly, crocidolite, Y27632, and toxin B modified NOS activity and nitrite levels according to DNA synthesis and LDH release. This suggests that NO could play a role both in the cytotoxic and in the proliferative stimulus induced by crocidolite and by inhibitors of RhoA signaling. Mevalonic acid, favoring the prenylation of RhoA, is likely to inhibit cell toxicity and proliferation by decreasing the synthesis of NO induced by crocidolite, Y27632, and toxin B. To confirm this statement, the presence of packed erythrocytes, used as scavengers of NO, clearly inhibited nitrite accumulation, DNA synthesis, and LDH leakage under each experimental condition. This suggests that the prenylation status of RhoA may play a critical role in both mesothelioma apoptosis and proliferation. Although NO is more known to act as a pro-apoptotic and antiproliferative agent, it has been also observed to inhibit apoptosis and stimulate cellular proliferation, depending on the cell type and the NO concentration (4, 40); thus it may play a role as both pro- or antitumoral agent (41).

During the preparation of this manuscript, a paper has been published (42) showing that in primary mesothelial cells TNF- induces NF-B activation and protects from crocidolite asbestos cytotoxicity. Since TNF- may induce Akt activation and NO synthesis, we investigated its effect on HMM cells: after a 24-h incubation with 10 ng/ml TNF-, we did not observe any significant increase of Akt or NOS activity (data not shown). Moreover, TNF- did not revert the activation of Akt and NOS and the cytotoxic effect exerted by crocidolite in HMM (data not shown). So far, our results would rule out a role for TNF- in asbestos-induced NO synthesis, cytotoxicity, and Akt activation in HMM cells. The transformed malignant phenotype of HMM cells may account for their different response to TNF- in comparison with normal mesothelial cells (42).

To verify whether mevalonic acid could influence the effects of asbestos by modifying directly the fiber reactivity, we have also incubated the crocidolite fibers for 24 h in culture medium without cells in the presence of mevalonic acid; the suspension was then centrifuged, and fibers were washed with fresh medium and finally incubated with cell cultures for 24 h. In the same experimental conditions previously shown in RESULTS, such asbestos fibers pretreated with mevalonic acid elicited in HMM cells the same effects of nontreated fibers, as far as nuclear translocation of NF-B, GTP binding of RhoA, and DNA synthesis were concerned (data not shown). Furthermore, in order to check the putative antioxidant action of mevalonic acid, we measured the accumulation of thiobarbituric acid–reactive substances (TBARS), which are known markers of lipid peroxidation. As expected, a 24-h incubation of HMM cells with crocidolite induced a significant increase of TBARS accumulation versus control (n = 6, P < 0.02; data not shown): the coincubation of cells with fibers and mevalonic acid obtained the same effect of crocidolite alone, thus suggesting that MA does not exert per se an antioxidant effect and that the correction elicited by MA on crocidolite-dependent NF-B activation and RhoA GTP binding inhibition is not mediated by such a mechanism.

In summary, our data suggest that crocidolite asbestos induces NF-B activation and stimulates the synthesis of NO by inhibiting the RhoA signaling pathway. The increased production of NO may be implicated in the proliferative response of HMM cells that survive after exposure to asbestos fibers. In our experimental conditions, mevalonic acid prevented any effect induced by crocidolite and by the HMGCoA reductase inhibitor mevastatin, under concentrations that per se did not exert toxic effects and did not inhibit Rho kinase signaling. The reverting effect of mevalonic acid suggests that crocidolite impairs the prenylation of RhoA, which is necessary to the activation of this small GTPAse. Although many steps of this pathway still remain unclear, we think that our results may open a new line of research in the investigation of crocidolite effects on human mesothelial cells and in the pathogenesis of malignant mesothelioma.

	Footnotes

 
This work has been supported with grants from Fondazione Internazionale Ricerche Medicina Sperimentale (FIRMS), Compagnia di SanPaolo, Regione Piemonte (Ricerca Sanitaria Finalizzata CIPE A201, 2004/2005), and Ministero dell'Università e della Ricerca. E.G. is a recipient of a post-doctoral fellowship funded by Regione Piemonte.

Originally Published in Press as DOI: 10.1165/rcmb.2006-0011OC on February 22, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form January 9, 2006

Accepted in final form November 29, 2006

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