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Asbestos Induces Nitric Oxide Synthesis in Mesothelioma Cells via

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					Asbestos Induces Nitric Oxide Synthesis in
Mesothelioma Cells via Rho Signaling Inhibition
Chiara Riganti, Sara Orecchia, Francesca Silvagno, Gianpiero Pescarmona, Pier Giacomo Betta, Elena Gazzano,
Elisabetta Aldieri, Dario Ghigo, and Amalia Bosia

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


We have observed that in three human malignant mesothelioma cell
lines, crocidolite asbestos induced the activation of the transcription
                                                                                        CLINICAL RELEVANCE
factor NF- B and the synthesis of nitric oxide (NO) by inhibiting the
RhoA signaling pathway. The incubation with crocidolite decreased                       In human malignant mesothelioma cells, crocidolite asbes-
the level of GTP-bound RhoA and the activity of Rho-dependent                           tos induces the activation of NF- B and the synthesis of
kinase, and induced the activation of Akt/PKB and IkB kinase,                           nitric oxide by impairing the prenylation and subsequent
leading to the nuclear translocation of NF- B. The effects of crocido-                  activation of RhoA.
lite 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 coen-
                                                                                      eration, differentiation, and apoptosis (4). NO is synthesized by
zyme A (HMGCoA) reductase. Furthermore, crocidolite, similarly to
mevastatin, inhibited the synthesis of cholesterol and ubiquinone
                                                                                      three NO synthase (NOS; EC 1.14.13.39) isoforms, which cata-
and the prenylation of RhoA: these effects were prevented in the                      lyze the conversion of L-arginine to L-citrulline and NO with a
presence of mevalonic acid. This suggests that crocidolite fibers                     1:1 stoichiometry (5). Various stimuli, such as bacterial lipopoly-
might inhibit the synthesis of isoprenoid molecules at the level                      saccharide, inflammatory cytokines, and oxidative stress, can
of the HMGCoA reductase reaction or of an upstream step, thus                         stimulate the expression of the inducible NOS isoform (iNOS,
impairing the prenylation and subsequent activation of RhoA. Akt                      NOS II) via the activation of NF- B (6). In addition, asbestos
can stimulate NO synthesis via a double mechanism: it can activate                    fibers have been shown to induce both NF- B activation and
the inducible NO synthase via the NF- B pathway and the endothe-                      NO synthesis in alveolar macrophages, lung epithelial cells (7,
lial NO synthase via a direct phosphorylation. Our results suggest                    8), and, more recently, in mesothelial cells (9).
that crocidolite increases the NO levels in mesothelioma cells by                         NF- B activity is controlled by members of the IkB family
modulating both NO synthase isoforms.                                                 which bind directly to NF- B dimers in the cytoplasm, preventing
                                                                                      the nuclear localization of the transcription factor, which is re-
Keywords: crocidolite; mesothelioma; nitric oxide; RhoA; NF- B
                                                                                      quired for DNA binding (3). Many agents activate NF- B via
Human malignant mesothelioma (HMM) is a rare but aggressive                           serine phosphorylation, ubiquitination and proteasomal degra-
tumor that originates from mesothelial cells and exhibits a strong                    dation of I B : in this way, NF- B is free to translocate to the
correlation with exposure to asbestos fibers, such as crocidolite,                     nucleus and modulate the expression of many genes, including
amosite, and chrysotile (1, 2). Key changes in the development                        iNOS (6).
of the disease are the loss of the normal restraints on prolifera-                        An increased iNOS activity has been induced in different
tion and the acquisition of resistance to apoptosis: asbestos,                        cellular models by inhibitors of RhoA prenylation, such as statins
which is a complete carcinogen for mesothelial cells, plays a                         (10–13), and by direct inhibitors of RhoA GTPase activity, such
complex role in altering both proliferation and apoptosis (2).                        as the toxin B from Clostridium difficile (10, 13). A link between
    NF- B is a redox-sensitive transcription factor comprised of                      inhibition of the small G proteins Rho and NF- B activation
protein dimers, including the transcription-activating hetero-                        has been already suggested (13).
dimer consisting of p50 and p65 subunits, that regulates expres-                          Starting from these observations, our study has been aimed
sion of genes intrinsic to inflammation, cell proliferation, and                       to investigate in HMM cells the role played by the RhoA sig-
apoptosis (3). One of the effects induced by NF- B activation                         naling pathway in crocidolite-induced NO synthesis and cell
is the increased synthesis of nitric oxide (NO), a highly reactive                    proliferation.
molecule involved in different cellular functions, including prolif-
                                                                                      MATERIALS AND METHODS
                                                                                      Materials
(Received in original form January 9, 2006 and in final form November 29, 2006 )      Fetal bovine serum (FBS) and Ham’s F-12 nutrient mixture medium
                                                                                      were supplied by BioWhittaker (Verviers, Belgium); plasticware for
This work has been supported with grants from Fondazione Internazionale
                                                                                      cell culture was from Falcon (Becton Dickinson, Bedford, MA); the
Ricerche Medicina Sperimentale (FIRMS), Compagnia di SanPaolo, Regione
Piemonte (Ricerca Sanitaria Finalizzata CIPE A201, 2004/2005), and Ministero          cationic exchange resin Dowex AG50WX-8, N-(1-naphthylethylenedia-
dell’Universita e della Ricerca. E.G. is a recipient of a post-doctoral fellowship
              `                                                                       mine) dihydrochloride and sulfanilamide were from Aldrich (Milan,
funded by Regione Piemonte.                                                           Italy); L-[2,3,4,5-3H]arginine monohydrochloride (62 Ci/mmol) was ob-
Correspondence and requests for reprints should be addressed to Dario Ghigo,
                                                                                      tained from Amersham International (Bucks, UK); Y27632 was from
Dipartimento di Genetica, Biologia e Biochimica (Sezione di Biochimica), Via          Calbiochem (La Jolla, CA). Electrophoresis reagents were obtained
Santena, 5/bis, 10126 Torino, Italy. E-mail: dario.ghigo@unito.it                     from Bio-Rad Laboratories (Hercules, CA), and the protein content
Am J Respir Cell Mol Biol Vol 36. pp 746–756, 2007
                                                                                      of cell monolayers and cell lysates was assessed with the BCA kit from
Originally Published in Press as DOI: 10.1165/rcmb.2006-0011OC on February 22, 2007   Pierce (Rockford, IL). When not otherwise specified, other reagents
Internet address: www.atsjournals.org                                                 were purchased from Sigma Chemical Co (St. Louis, MO).
Riganti, Orecchia, Silvagno, et al.: Asbestos Inhibits Rho Signaling                                                                           747

Cells                                                                       2 g/ml leupeptin, and 0.1% NP-40, pH 7.6). This suspension was
                                                                            incubated for 10 min on ice with occasional vortexing, and centrifuged
HMM cell lines MM98, OC99, and GF99 were obtained from the pleural
                                                                            for 30 s at 13,000    g to pellet nuclei, which were rinsed with 0.2 ml
effusions of three patients with histologically confirmed malignant me-
                                                                            of wash buffer B (2 M KCl, 25 mM Hepes, 0.1 mM EDTA, 1 mM
sothelioma; the mesothelial origin of the isolated cells was confirmed
                                                                            PMSF, 1 mM DTT, 10 g/ml aprotinin, and 2 g/ml leupeptin, pH 7.6)
by positive immunostaining as previously described (14). Cells were
                                                                            and incubated at 4 C for 20 min. Then an equal volume of buffer C
cultured in Ham’s F-12 medium supplemented with 10% FBS, 1%
penicillin/streptomycin, and 1% L-glutamine, and were maintained in         (25 mM Hepes, 0.1 mM EDTA, and 20% glycerol, pH 7.6) was added,
a humidified atmosphere at 37 C and 5% CO2. The N11 mouse glial              the mix was centrifuged for 15 min at 20,000 g, and the supernatant
cell line was a gift from Dr. Marco Righi (CNR Institute of Neurosci-       stored at 80 C until used for Western blotting or electrophoretic
ence, Section of Cellular and Molecular Pharmacology, Milan, Italy).        mobility shift assay (EMSA).
At the end of each incubation period the experiments were performed
on adherent cells, after having removed the incubation medium together      EMSA
with detached cells. When measurements were performed on extracellu-        The probe containing the NF- B oligonucleotide consensus sequence
lar medium, the medium was centrifuged previously at 12,000 g for           was labeled with [ -32P]ATP (Amersham International) (3,000 Ci/mmol,
15 min to pellet cellular debris. During the 24-h incubation period         250 Ci), using T4 polynucleotide kinase (Roche, Basel, Switzerland).
preceding experiments aimed to investigate cell protein phosphoryla-        The sequence of oligonucleotide was (binding site underlined): 5 -AGT
tion (I B , I       , eNOS), cells were maintained with the above-          TGAGGGGACTTTCCCAGG-3 (Promega Corporation, Madison,
mentioned culture medium containing a low level of FBS (2% instead          WI). Ten g of the nuclear extracts, obtained as described above, were
of 10%), to minimize the known effects of serum on the baseline pattern     incubated for 20 min with 20,000 cpm of 32P-labeled double-stranded
of protein phosphorylation. MM98, OC99, and GF99 cells showed the           oligonucleotide at 4 C in a reaction mixture containing: 2 l of 10 g/ml
same behavior under each experimental condition: for sake of simplicity     BSA, 2 l of buffer D (100 mM KCl, 20 mM Hepes, 0.5 mM EDTA,
we chose to report in Results the data obtained in MM98 cells as            2 mM DTT, 0.1 mM PMSF, 20% glycerol, and 0.25% NP-40, pH 7.6),
representative of the other two cell lines.                                 4 l of buffer E (300 mM KCl, 100 mM Hepes, 10 mM DTT, 100 M
                                                                            PMSF, and 20% Ficoll, pH 7.6) and 2 g of poly(dI-dC) (Roche). The
Asbestos Fibers                                                             final volume of the mix was brought to 25 l with water. In supershift
UICC (Union International Contre le Cancer) crocidolite fibers were          assay, nuclear extracts were pre-incubated for 30 min at room tempera-
                                                            ¨
sonicated (Labsonic sonicator, 100 W, 10 s; Labsonic, Gottingen,            ture with 2 l of anti-p50 antibody (PC136; Calbiochem) or anti-p65
Germany) before incubation with cell cultures, in order to dissociate       antibody (PC138; Calbiochem); then the reaction mixture containing
fiber bundles and allow their better suspension and diffusion in the         the 32P-labeled double-stranded oligonucleotide was added and samples
culture medium.                                                             were treated as described previously. The DNA–protein complex was
                                                                            separated on a not-denaturating 4% polyacrilamide gel in TBE buffer
Propidium Iodide Exclusion Assay                                            (pH 8.0). After electrophoresis, the gel was dried and autoradiographed
After incubation under different experimental conditions in 24-well         by exposure to X-ray film for 48 h.
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/         Western Blot Analysis
NaOH, pH 7.5, 140 mM NaCl, 25 mM CaCl2) containing 2.5 M propid-            Cells were directly solubilized in the lysis buffer (25 mM Hepes,
ium iodide (PI). Then cells were washed three times with fresh PBS          135 mM NaCl, 1% NP40, 5 mM EDTA, 1 mM EGTA, 1 mM ZnCl2,
and rinsed with 1 ml of binding buffer. An aliquot of cells suspension      50 mM NaF, 10% glycerol), supplemented with protease inhibitor cock-
was sonicated and used to determine intracellular protein content. Fluo-    tail set III (100 mM AEBSF, 80 M aprotinin, 5 mM bestatin, 1.5 mM
rescence of each sample was recorded using a Perkin-Elmer LS-5 spec-        E-64, 2 mM leupeptin, and 1 mM pepstatin; Calbiochem), 2 mM PMSF,
trofluorimeter (Shelton, CT). Excitation and emission wavelengths were       and 1 mM sodium orthovanadate. Whole cell extracts (or nuclear ex-
536 and 617 nm, respectively. A blank was prepared testing the cells        tracts, for p50 and p65 detection) containing 30 g of proteins were
in the absence of PI in each set of experiments, and its fluorescence        separated by SDS-PAGE, transferred to PVDF membrane sheets
was subtracted from that measured in the samples.                           (Immobilon-P; Millipore, Bedford, MA), and probed with the following
                                                                            antibodies: anti-I B (from rabbit, diluted 1:500 in PBS-BSA 1%; Santa
Lactate Dehydrogenase Activity                                              Cruz Biotechnology, Santa Cruz, CA), anti-phospho(Ser 32)-I B
After incubation under different experimental conditions, the extracel-     (from mouse, diluted 1:250 in PBS-BSA 1%; Santa Cruz Biotechnol-
lular medium was centrifuged at 12,000 g for 15 min to pellet cellular      ogy), anti-I B kinase(IKK) (from rabbit, diluted 1:500 in PBS-BSA
debris, whereas cells were washed with fresh medium, detached with          1%, Santa Cruz Biotechnology), anti-phospho(Ser 180)-IKK (from
trypsin/EDTA, washed with PBS, re-suspended at 1         105 cells/ml in    rabbit, diluted 1:250 in PBS-BSA 1%; Cell Signaling Technology Inc.,
0.2 ml of 82.3 mM triethanolamine phosphate hydrochloride (TRAP,            Beverly, MA), anti-Rho kinase (Rock) 1 (from rabbit, diluted 1:500 in
pH 7.6), and sonicated on ice with two 10-s bursts. Lactate dehydroge-      PBS-BSA 1%; Santa Cruz Biotechnology), anti-Rock2 (from rabbit,
nase (LDH) activity was measured in the extracellular medium and in         diluted 1:500 in PBS-BSA 1%; Santa Cruz Biotechnology), anti-eNOS
the cell lysate, as previously described (15). One hundred microliters      (from mouse, diluted 1:500 in PBS-BSA 1%; Transduction Labora-
of supernatant from extracellular medium or 10 l of cell lysate were        tories, Lexington, KY), anti-phospho-(Ser 1177) eNOS (from mouse,
incubated at 37 C with 82.3 mM TRAP (pH 7.6) and 5 mM NADH                  diluted 1:500 in PBS-BSA 1%; Cell Signaling Technology Inc.), anti-
(final volume: 1 ml). The reaction was started by adding 20 mM pyruvic       p50 (from mouse, diluted 1:250 in PBS-BSA 1%; Santa Cruz Biotech-
acid and was followed for 10 min, measuring absorbance at 340 nm            nology), anti-p65 (from rabbit, diluted 1:500 in PBS-BSA 1%; Santa
with a Lambda 3 spectrophotometer (Perkin-Elmer). The reaction ki-          Cruz Biotechnology), and anti-glyceraldehyde-3-phosphate dehydroge-
netics were linear throughout the time of measurement. Both intracellu-     nase (anti-GAPDH, from rabbit, diluted 1:500 in PBS-BSA 1%; Santa
lar and extracellular enzyme activity were expressed as mol NADH            Cruz Biotechnology). Expression of GAPDH, the product of an
oxidized/min/dish, then extracellular LDH activity was calculated as        housekeeping gene, was used as a control of equal loading. After a
percentage of the total LDH activity in the dish.                           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,
Preparation of Nuclear Extracts                                             or anti-goat IgG (Amersham International, diluted 1:1,000 in PBS-
Cells were plated in 100-mm-diameter dishes at confluence, and all           Tween with Blocker Not-Fat Dry Milk 5% [Bio-Rad]). The membrane
procedures for nuclear protein extraction were performed at 4 C using       was washed again with PBS-Tween and proteins were detected by
ice-cold reagents, as described (16). Cells were mechanically scraped       enhanced chemiluminescence (Immun-Star; Bio-Rad). To assess the
in PBS, washed, and resuspended (1      107 cells/0.5 ml) in lysis buffer   presence of the Proliferating Nuclear Cellular Antigen (PCNA), lysates
A (15 mM KCl, 10 mM Hepes, 2 mM MgCl2, 0.1 mM EDTA, 1 mM                    were directly resolved on a 12% SDS-PAGE, transferred to PVDF
phenylmethylsulfonylfluoride [PMSF], 1 mM DTT, 10 g/ml aprotinin,            membrane sheets and probed overnight with an anti-PCNA antibody
748                                                   AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 36                      2007

(from mouse, 1:500 in PBS-BSA 1%; Santa Cruz Biotechnology). The           was incubated for 45 min at 4 C with agarose-glutathione beads coupled
membrane was then washed and treated as described above.                   to a fusion protein containing glutathione S-transferase (GST) and the
                                                                           Rho-binding domain of the Rho effector protein rhotekin (Upstate,
IKK Activity Assay                                                         Charlottesville, VA). The beads were then washed three times in MLB
IKK activity was measured as previously described (17). Cells were         buffer and harvested by addition of Laemmli buffer 2 . The RhoA
washed with ice-cold PBS and solubilized in 0.5 ml of lysis buffer         activity was analyzed, resolving the samples by 12% SDS-PAGE and
(50 mM Hepes, 150 mM NaCl, 2 mM MgCl2, 1 mM EDTA, 0.1% NP40,               Western blotting using anti-RhoA antibody to detect GTP-bound acti-
100 M NaF, 10 mM sodium orthovanadate, 10 g/ml leupeptin,                  vated RhoA. RhoA-GTP/total RhoA ratio was taken as an index of
10 g/ml pepstatin, 10 g/ml aprotinin, 1 mM PMSF, and 250 M                 the active fraction of RhoA.
DTT). Samples were centrifuged at 13,000         g for 15 min and the
supernatant was used for the assay and cell protein quantification. To      Rock Assay
purify the IKK complex, equal amounts of the whole lysate (0.5 mg          Rock activity was measured using the CycLex Rho-Kinase Assay Kit
cell proteins/test) were immunoprecipitated with an anti-IKK antibody      (CycLex Co.), a single site binding immunoassay. Cells were cultured
(from rabbit, diluted 1:200 in PBS-BSA 1%; Santa Cruz Biotechnology)       in 35-mm-diameter Petri dishes, washed with ice-cold PBS and lysed
for 90 min at 4 C. Samples were centrifuged (13,000       g for 15 min)    in 0.2 ml lysis buffer (50 mM Tris-HCl, pH 8.0, 0.1% Triton X-100,
and washed three times with kinase buffer (20 mM Hepes, 20 mM              1mM EDTA, 1mM EGTA, 2 mM NaF, 2 mM sodium orthovanadate,
  -glycerolphosphate, 1 mM MnCl2, 5 mM MgCl2, 2 mM NaF, and                0.5 g/ml leupeptin, 1 g/ml pepstatin, 0.2 mM PMSF, and 10 mM
250 M DTT). A quantity of 0.1 g of the immunoprecipitated proteins           -mercaptoethanol). Samples were sonicated on crushed ice with two
was incubated with 1 mM ATP in the presence of the proteasome              10-s bursts and centrifuged at 13,000 g for 5 min at 4 C. Supernatants
inhibitor MG132 (10 M); to provide the reaction mix with an excess         were treated following manufacturer’s instructions, and protein content
of I B protein (the substrate of IKK), 30 g of total cellular lysate       was measured. Briefly, samples were diluted 1:4 in the kinase buffer
of SV40-positive HMM cells, obtained under not-denaturing conditions,      provided with the kit, containing 20 mM ATP, and incubated fo
were added. SV40-positive HMM cells were chosen because they were          60 min at 30 C in 96-well plates, pre-coated with the recombinant
previously shown to exhibit a very high basal amount of I B (9).           C-terminus of the myosin binding subunit (MBS) of myosin phospha-
MG132 was added in the reaction mixture in order to avoid, during          tase. Wells were washed five times with 2% Tween-20, and 100 l of
the assay, the degradation of phospho-IkB protein by the proteasome        the HRP-conjugated anti phospho(Thr 696)-MBS antibody were added.
eventually still present in the cell lysate (used as a provider of the     After a 60-min incubation at room temperature, samples were washed
substrate). Reaction was carried over at 30 C for 30 min and stopped       again, and 100 l of the chromogenic substrate tetra-methylbenzidine
with 30 l of Laemmli buffer. Finally, samples were subjected to electro-   were added. After a 15-min incubation at room temperature, the reac-
phoresis in a 12% SDS-PAGE, transferred to PVDF membrane sheets,           tion was stopped with 100 l of 0.5 N H2SO4 and absorbance was
and probed with an anti-I B antibody (from rabbit, diluted 1:250 in        read at 450 nm, using a Packard EL340 microplate reader (Bio-Tek
PBS-BSA 1%; Santa Cruz Biotechnology) and an anti-phospho(Ser              Instruments). For each set of experiments, a titration curve was pre-
32)-IkB antibody (from mouse, diluted 1:250 in PBS-BSA 1%; Santa
                                                                           pared, using serial dilution of recombinant Rho-Kinase II (MBL Inc.,
Cruz Biotechnology), respectively.
                                                                           Woburn, MA) in kinase buffer. Data were expressed as mU absorbance/
                                                                           mg cell proteins.
Akt Activity Assay
Akt activity was measured using the CycLex Akt/PKB Kinase Assay/           RT-PCR
Inhibitor Screening Kit (CycLex Co., Nagano, Japan). Cells were cul-
                                                                           Total RNA was obtained by the guanidinium thiocyanate-phenol-
tured in 35-mm-diameter Petri dishes, washed with ice-cold PBS, and
                                                                           chloroform method (19). A quantity of 30 ng of total RNA was reversely
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 orthovan-                transcribed into cDNA with the Superscript II One-Step RT-PCR Sys-
adate, 0.5 g/ml leupeptin, 1 g/ml pepstatin, 0.5 mM PMSF, and              tem with Platinum Taq DNA Polymerase (cycling conditions: 1 cycle
10 mM -mercaptoethanol). Samples were sonicated on crushed ice             50 C for 30 min, 1 cycle 94 C for 2 min). cDNA products were deter-
with two 10-s bursts and centrifuged at 13,000     g for 30 min at 4 C.    mined by PCR amplification, carried out in a total volume of 50 l,
Supernatants were treated following manufacturer’s instructions, and       according to the manufacturer’s recommendations. The RT-PCR effi-
protein content was measured. Briefly, 15 l of samples were diluted         ciency was controlled by amplifying a -actin fragment, used as an
in 85 l of the kinase buffer provided with the kit, containing 125 M       housekeeping gene. Primers for iNOS (0.3 M) were: 5 -TCCGAGG
ATP, and incubated for 60 min at 30 C in 96-well plates, pre-coated        CAAACAGCACATTCA-3 , 5 -GGGTTGGGGGTGTGGTGATGT-3
with the Akt substrate AkTide-2T, which is efficiently phosphorylated       (462 bp); primers for -actin (0.5 M) were: 5 -GGTCATCTTCTCG
by Akt at a serine residue. Wells were washed five times with 2%            CGGTTGGCCTTGGGGT-3 , 5 -CCCCAGGCACCAGGGCGTGAT-3
Tween-20, and 100 l of horseradish peroxidase (HRP)-conjugated             (230 bp). PCR amplification for iNOS was: 1 cycle of denaturation at
anti-phospho-AkTide-2T monoclonal antibody were added. After a             95 C for 2 min, 30 cycles of denaturation at 95 C for 30 s, annealing
60-min incubation at room temperature, samples were washed again,          at 55 C for 1 min, elongation at 72 C for 30 s, and 1 cycle of extension
and 100 l of the chromogenic substrate tetra-methylbenzidine were          at 72 C for 10 min; for -actin: 1 cycle of denaturation at 94 C for
added. After a 15-min incubation at room temperature, the reaction was     3 min, 35 cycles of denaturation at 94 C for 1 min, annealing at 58 C
stopped with 100 l of 0.5 N H2SO4 and absorbance was read at 450 nm,       for 1 min, elongation at 72 C for 1 min, and 1 cycle of extension at
using a Packard EL340 microplate reader (Bio-Tek Instruments, Winooski,    72 C for 7 min. Samples were electrophoresed in 1.5% agarose gels
VT). For each set of experiments, a titration curve was prepared, using    containing ethidium bromide in Tris-acetate/EDTA buffer to visualize
serial dilutions of recombinant Akt (CycLex Co.) in kinase buffer. Akt     the PCR products.
activity was expressed as mU absorbance/mg cell proteins.
                                                                           Nitrite Production
RhoA-GTP Pull-Down                                                         Confluent cell monolayers in 35-mm-diameter Petri dishes were incu-
Biochemical assay for activity of RhoA was performed as described          bated in fresh medium for 24 h under the experimental conditions
(18). Cells were lysed in MLB buffer (125 mM Tris-HCl, pH 7.4,             indicated in Results. Then nitrite production was measured by adding
750 mM NaCl, 1% NP40, 10% glycerol, 50 mM MgCl2, 5 mM EDTA,                0.15 ml of cell culture medium (centrifuged previously at 12,000      g
25 mM NaF, 1 mM sodium orthovanadate, 10 g/ml leupeptin, 10 g/ml           for 15 min to pellet cellular debris) to 0.15 ml of Griess reagent (20)
pepstatin, 10 g/ml aprotinin, and 1 mM PMSF) and centrifuged at            in a 96-well plate, and, after a 10 min incubation at 37 C in the dark,
13,000 g for 10 min at 4 C. An aliquot of supernatant was taken out        absorbance was measured at 540 nm with a Packard EL340 microplate
for determination of protein content and analysis of total amount of       reader (Bio-Tek Instruments). A blank was prepared for each experi-
RhoA. Another aliquot of the same lysate was directly probed with an       mental condition in the absence of cells, and its absorbance was sub-
anti-RhoA antibody (1:250, in PBS-BSA 1%; Santa Cruz Biotechnol-           tracted from that measured in the samples. Nitrite concentration was
ogy), to measure total RhoA protein. A further 30 g of the supernatant     expressed as nmol nitrite/mg cell proteins.
Riganti, Orecchia, Silvagno, et al.: Asbestos Inhibits Rho Signaling                                                                           749

Measurement of NOS Activity                                                ice-cold PBS, detached by trypsin/EDTA, and resuspended in 200 l
                                                                           of PBS. A 50- l aliquot was used for protein quantification, while the
Cells grown at confluence on 35-mm-diameter Petri dishes, after incuba-
                                                                           remaining part was transferred in poliethylene vials and the radioactiv-
tion under the experimental conditions described in Results, were          ity was measured by liquid scintillation. [3H]thymidine incorporated in
detached by trypsin/EDTA, washed with PBS, resuspended in 0.3 ml           each sample was expressed as pmol/mg cell proteins.
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             Statistical Analysis
two 10-s bursts. In each assay tube, the following reagents were added
to 100 l of lysate at the following final concentrations: 2 mM NADPH,       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
2.5 Ci L-[3H]arginine ( 0.4 M), 100 M tetrahydrobiopterin,
                                                                           considered significant.
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              RESULTS
applied to 2 ml columns of Dowex AG50WX-8 (Na form) and eluted             Crocidolite Asbestos Elicits in HMM Cells a Dose- and Time-
with 4 ml of water. The radioactivity corresponding to [3H]citrulline
                                                                           Dependent Increase of Both NO Synthesis and LDH Leakage,
content in 6.1 ml eluate was measured by liquid scintillation counting.
Citrulline synthesis was expressed as pmol citrulline/min/mg cell          Which Are Inhibited by Coincubation with Mevalonic Acid
proteins.                                                                  We investigated the effects of different concentrations (1, 5,
                                                                           25 g/cm2) of crocidolite asbestos fibers on HMM cells viability
Measurement of Isoprenoid Molecules Synthesis                              after a 24- to 48-h incubation (Figure 1). The incorporation of
The synthesis of isoprenoid molecules was checked as intracellular         PI, a marker of decreased cell viability (23), increased as a function
accumulation of cholesterol and ubiquinone, and measured as pre-           of time and concentration. We investigated further the effects
viously described (21). Cells were incubated for 24 h with 10 Ci/ml        of the incubation of HMM cells with different concentrations
of [3H]acetate (3600 mCi/mmol; Amersham International) or 10 Ci/ml         (1, 5, 25 g/cm2) of crocidolite asbestos fibers, under different
of [14C]mevalonic acid (67 mCi/mmol; Amersham International), then         incubation times (6, 24, 48 h), on the extracellular accumulation
washed twice with PBS and resuspended in 200 l of PBS. A 50- l             of nitrite (a stable derivative of NO synthesis), and on the release
aliquot was used for protein quantification, while the remaining part       of LDH in the extracellular medium (a sensitive index of cytotox-
was transferred in glass microcentrifuge tubes. A quantity of 1.5 ml of    icity) (24) (Figure 2A). Since after 6 h the NO synthesis and
a 1:2 methanol/hexane solution was added, and cellular suspensions
                                                                           cytotoxic effect were negligible, whereas after 48 h the cytotoxic
were vortexed for 1 h, then centrifuged at 2,000      g for 5 min. The
                                                                           effect was too marked, we decided to perform the subsequent
upper phase was transferred in a new set of glass microcentrifuge tubes,
while the lower phase was resuspended in 1 ml of hexane, vortexed
                                                                           experiments using a 24-h incubation time and a 25 g/cm2 con-
overnight, and centrifuged at 2,000 g for 5 min: the new upper phase       centration of fibers, which appeared to be the minimal dose
was added to the previously isolated phase. After a 24-h evaporation,      among our conditions able to induce significant accumulation
samples were dissolved in 100 l of chloroform and resolved by thin         of nitrite. Under these experimental conditions, we incubated
layer chromatography on silica gel, using 1:1 diethyl ether/exane as       the cells in the absence or presence of variable concentrations
mobile phase. Standard solutions of 10 l of cholesterol (2 g/ml) and       (50, 100, 200 M) of mevalonic acid (Figure 2B). A quantity of
ubiquinone (2 g/ml) were employed. After the separation, the gel was       100 M mevalonic acid completely reverted both nitrite increase
exposed to iodine gas for 2 h and the spots corresponding to cholesterol   and LDH leakage induced by crocidolite.
and ubiquinone were isolated. The radioactivity of each spot was mea-
sured by liquid scintillation counting and expressed as cpm/mg cellular    Crocidolite Asbestos and Inhibitors of RhoA and
proteins.                                                                  Rocks Activate NF- B in HMM Cells
                                                                           After a 24-h incubation, crocidolite, Y27632 (an inhibitor of the
Assessment of RhoA Prenylation
                                                                           Rocks) and toxin B (an inhibitor of RhoA GTPase activity)
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 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 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 condensa-
tion was discarded. The protein content of total lysate, aqueous phase,
and detergent phase was determined by Bradford test (Bio-Rad), and
6 g 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          Figure 1. Effect of crocidolite on PI exclusion in HMM cells. HMM
anti-GAPDH antibody (Santa Cruz Biotechnology) was used to verify          (MM98) cells were incubated for 24 and 48 h in the absence (CTRL)
the partitioning of soluble GAPDH in the aqueous phase.                    or presence of crocidolite (CRO, 1, 5, 25 g/cm2). Camptothecin (CAM,
                                                                           10 nM for 24 h), a genotoxic and proapoptotic compound in mesotheli-
[3H]thymidine Incorporation Assay                                          oma cells, was used as a positive control. After these incubation times,
Cells were grown in 35-mm-diameter Petri dishes and incubated for 24 h     cells were checked for their content of PI, as described in MATERIALS AND
under the experimental conditions described in Results, in a culture       METHODS. Measurements were performed in duplicate and data are
medium containing 1 Ci/ml [3H]thymidine (62 Ci/mmol; Amersham              presented as means SE (n 6). *P 0.05, **P 0.001 versus CTRL;
International). At the end of the incubation, cells were washed with        P 0.05 versus CRO 25 g/cm2 24 h.
750                                               AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 36                      2007




                                                                              Figure 2. Effect of crocidolite and mevalonic acid on NO syn-
                                                                              thesis 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 g/cm2) (A ), and for
                                                                              24 h in the absence (CTRL) or presence of crocidolite (CRO,
                                                                              25 g/cm2) and/or mevalonic acid (MA, 50, 100, 200 M)
                                                                              (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 MATERI-
                                                                              ALS AND METHODS. Each measurement was performed in dupli-
                                                                              cate. 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.




strongly activated NF- B in HMM cells. The NF- B nuclear             lysate) to phosphorylate its substrate I B : asbestos fibers, as
translocation was demonstrated by EMSA (Figure 3A) and by            well as Y27632 and toxin B, induced IKK activity in HMM cells
Western blotting performed on nuclear extracts using anti-p50        (Figure 4A). Mevalonic acid, per se devoid of any effect on IKK
and anti-p65 subunits antibodies (Figure 3B). A similar nuclear      activity, completely prevented the crocidolite-induced increase
translocation was observed in mouse N11 glial cells (used as         of IKK activity (Figure 4A). Activation of IKK implies its
a positive control) incubated with bacterial lipopolysaccharide      phosphorylation on serine and threonine (3): asbestos fibers
(LPS), a well-known NF- B activator and iNOS inducer (Figures        significantly increased the intracellular amount of phospho(Ser
3A and 3B). Cells were also incubated with mevalonic acid, the       180)-IKK , without changing the total levels of IKK (Figure
product of 3-hydroxy-3-methylglutaryl coenzyme A (HMGCoA)            4B). Y27632 and toxin B also elicited an increment of phospho
reductase: mevalonic acid alone did not induce nuclear transloca-    (Ser 180)-IKK . Mevalonic acid, both alone and incubated together
tion of the p50/p65 heterodimer, but it completely abolished         with crocidolite, prevented IKK phosphorylation (Figure 4B).
the effect of crocidolite (Figures 3A and 3B). Under the same        LPS induced both IKK activity and phosphorylation in N11 cells
experimental conditions, the NF- B inhibitor I B , which was         (Figure 4).
abundant in the cytosol of resting HMM cells, disappeared in             Akt/PKB activates the IKK complex in some experimental
cells incubated with crocidolite, Y27632, and toxin B (Figure 3B).   models (25): after the incubation of HMM cells with crocidolite
The appearance of phosphorylated I B followed a complemen-           asbestos, the Akt activity in the cell lysate increased (Figure 5),
tary pattern (Figure 3B). Again, mevalonic acid completely re-       an effect which was completely prevented when cells were incu-
verted the effect of asbestos fibers on I B (Figure 3B). In N11       bated with crocidolite and mevalonic acid together (Figure 5).
glial cells LPS induced a clear disappearance of the cytosolic       A significant increase of intracellular Akt activity was elicited also
level of I B and the appearance of phospho-I B (Figure 3B).          by Y27632 and toxin B in HMM cells and by LPS in N11 cells
                                                                     (Figure 5).
Crocidolite Asbestos and Inhibitors of RhoA and of Rocks Elicit          Since RhoA can bind GTP only when prenylated, GTP-bound
IKK Phosphorylation, Akt Activation, and RhoA/Rock                   RhoA can be measured as an index of RhoA prenylation and
Signaling Inhibition in HMM Cells                                    activation (26). In HMM cells crocidolite fibers markedly low-
IKK is responsible for I B phosphorylation and degradation           ered the level of GTP-bound RhoA, without changing the ex-
(3). IKK activity in HMM cells was assayed as the ability of IKK     pression of total RhoA: this effect was prevented when crocido-
complex (immunoprecipitated from equal amounts of whole cell         lite was incubated together with mevalonic acid (Figure 6A). As
Riganti, Orecchia, Silvagno, et al.: Asbestos Inhibits Rho Signaling                                                                         751




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 g/cm2, CRO), in the absence or presence of mevalonic acid (MA, 100 M), Y27632 (Y276, 0.2 M),
or toxin B (TOX, 0.1 ng/ml). N11 cells, incubated with bacterial lipopolysaccharide (LPS, 20 g/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 g/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 I B , phospho-I B , 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 I B followed the same pattern in the other two cell lines (OC99, GF99).


expected, Y27632 did not modify the amount of GTP-bound                    Crocidolite Increases the Expression of NOS II, the Activity
RhoA or total RhoA protein, while toxin B reduced the level                of NOS III, and the Synthesis of NO via NF- B and
of GTP-bound RhoA (Figure 6A). After a 24-h incubation,                    Akt Activation in HMM Cells
crocidolite asbestos, Y27632, and toxin B lowered the activity
                                                                           After a 24-h incubation, crocidolite, Y27632, and toxin B induced
of Rock in HMM cells, and mevalonic acid reverted the effect
                                                                           the expression of iNOS mRNA in HMM cells: the effect of
of crocidolite (Figure 6B). Neither crocidolite, Y27632, nor toxin         crocidolite was blocked by the co-incubation with mevalonic
B changed the expression of Rock1 and Rock2 proteins (data                 acid (Figure 8A). None of these agents modified the transcript
not shown).                                                                of -actin, chosen as an housekeeping gene (Figure 8A).
Crocidolite Inhibits the Synthesis of Isoprenoid Molecules and                In a number of cellular models Akt phosphorylates the consti-
the Prenylation of RhoA in HMM Cells                                       tutive endothelial NOS (eNOS, NOS III) on serine 1177, causing
                                                                           a further enzyme activation (28). In untreated HMM cells most
The ability of crocidolite to inhibit the synthesis of isoprenoid          eNOS was in the nonphosphorylated form, and crocidolite in-
molecules was checked by investigating the incorporation of 3H             duced a marked increase of phospho-eNOS (Figure 8B). This
and 14C in cholesterol and ubiquinone in HMM cells incubated               effect was prevented by mevalonic acid.
with [3H]acetate or [14C]mevalonic acid. In the presence of either            These effects on NOS expression and phosphorylation were
crocidolite or the HMGCoA reductase inhibitor mevastatin, the              accompanied by changes of NOS activity. After a 24-h incuba-
intracellular conversion of [3H]acetate into both cholesterol and          tion, crocidolite, Y27632, and toxin B induced a significant aug-
ubiquinone was significantly decreased (Figure 7A). Contrarily,             mentation of extracellular nitrite (an NO stable derivative in
the conversion of [14C]mevalonic acid to these two molecules               oxygenated cell systems) and intracellular NOS activity in HMM
was not affected, whereas in the same experimental conditions              cultures (Figure 9A). Mevalonic acid significantly prevented
the squalene synthase inhibitor squalestatin (27) was fully effec-         such effects (Figure 9A). Acting as NO scavengers (due to their
tive in inhibiting cholesterol synthesis (favoring, as expected, a         content of oxyhemoglobin), red blood cells lowered the nitrite
greater conversion of mevalonic acid to the isoprenoid chain of            concentration without modifying the NOS activity, as expected
ubiquinone) (Figure 7B). We also observed that crocidolite, as             (Figure 9A).
well as mevastatin, inhibited the prenylation of RhoA, evaluated
by investigating the relative amount of prenylated membrane-               Crocidolite Induces Both Proliferation and Death of HMM
associated and nonprenylated cytosolic RhoA in HMM cells                   Cells in an NO-Dependent Manner
(Figure 7C). These effects were prevented by the presence of               After a 24-h incubation, the expression of PCNA, an antigen
mevalonic acid (Figure 7C).                                                that accumulates in the cells during the early S phase (29), was
752                                                   AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 36                      2007




                                                                           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 g/
                                                                           cm2), in absence or presence of mevalonic acid (MA, 100 M), Y27632
                                                                           (Y276, 0.2 M), or toxin B (TOX, 0.1 ng/ml). Cell lysates were analysed
                                                                           for Akt activity as described in MATERIALS AND METHODS. N11 cells, incu-
                                                                           bated with bacterial lipopolysaccharide (LPS, 20 g/ml), were used as
Figure 4. Effect of crocidolite, mevalonic acid, and inhibitors of RhoA/   a positive control; in the absence of LPS the pattern was superimposable
Rho kinase on Ikk activation. HMM (MM98) cells were incubated for          to that of HMM CTRL (not shown). Each measurement was performed
24 h in fresh medium only (CTRL) or with crocidolite fibers (CRO,          in duplicate. The experiments were repeated with the other two cell
25 g/cm2), in the absence or presence of mevalonic acid (MA, 100           lines (OC99, GF99), giving superimposable results (data not shown).
  M), Y27632 (Y276, 0.2 M), or toxin B (TOX, 0.1 ng/ml). (A ) IKK          Data are presented as means SEM (n            6). *P 0.05, **P 0.001
activity was assayed as the ability of the immunoprecipitated IKK com-     versus CTRL; P       0.001 versus CRO.
plex to phosphorylate I B , and its outcome was checked using an
anti-phospho(Ser 32)-I B and an anti-I B antibody (as described in
MATERIALS AND METHODS). MG132 (10 M) was added in the reaction
mixture in order to avoid, during the assay, the degradation of phospho-
                                                                           also been implicated (30). The molecular basis of crocidolite-
I B protein by the proteasome eventually still present in the SV40-
positive HMM cell lysate (used as a provider of the substrate).            associated carcinogenesis is still under investigation. Asbestos
(B ) Detection of Ikk phosphorylation. Whole cell lysates were analysed    can stimulate in a variety of cell types the expression of genes
by Western blotting using the following antibodies: anti-phospho(-         critical to cellular injury, proliferation, and inflammation, via
Ser180)-I     , anti-I     , anti-GAPDH (as described in MATERIALS AND     the activation of transcription factors, such as activated protein-1
METHODS). N11 cells, incubated with LPS ( , 20 g/ml), were used as         and NF- B (31). The strong oxidative stress evoked by asbestos
a positive control; in the absence of LPS the pattern was superimposable   is likely to be responsible for NF- B activation (31), but other
to that of HMM CTRL (not shown). Each panel is representative of two       mechanisms may be involved. NF- B activation is known to be
experiments with similar results. The experiments shown in B were          promoted by different serine/threonine kinases, such as mitogen-
repeated in duplicate with the other two cell lines (OC99, GF99), giving   activated protein kinases and Akt (3).
superimposable results (data not shown).                                       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
clearly increased in HMM cells by crocidolite fibers, Y27632,               RhoA signaling pathway. RhoA is involved in different cellular
and toxin B (Figure 9B). This effect was prevented by mevalonic            crucial events, such as proliferation, tumorigenesis, and tumor
acid and packed red blood cells, with a pattern similar to that            invasion (32). It usually cycles between a nonprenylated, inactive
observed in nitrite measurements (Figure 9A). Since PCNA                   form and a prenylated, GTP-bound active form (26): if active,
may increase also as a consequence of DNA damage, we also                  RhoA can interact with several downstream effectors, such as the
measured the cell incorporation of [3H]thymidine under the same            two serine-threonine Rho-dependent kinases Rock1 and Rock2
experimental conditions: again, crocidolite, Y27632, and toxin B           (33). The effect of RhoA modulation on NF- B activation may
increased the DNA synthesis (Figure 10). In parallel, crocidolite,         change depending on the cellular model. We observed that in
Y27632, and toxin B increased also the leakage of LDH (a                   HMM cells crocidolite fibers promoted NF- B translocation into
sensitive marker of cell damage), thus suggesting that these               the nucleus, elicited the phosphorylation of I B and lowered
agents can exert both mitogenic and cytotoxic effects (Figure
                                                                           the intracellular content of I B protein. A similar response
10). Mevalonic acid and packed erythrocytes prevented these
                                                                           pattern was observed when cells were incubated with both toxin
effects: this phenomenon was less evident after 48 h (not shown),
                                                                           B from Clostridium difficile, which inhibits the GTPase activity
probably because of mevalonic acid consumption and oxidation
                                                                           of RhoA and other small G proteins (34), and Y27632, a selective
of red blood cell hemoglobin to methemoglobin (unable to scav-
                                                                           Rock inhibitor (35). Mevalonic acid, the product of HMGCoA
enge NO).
                                                                           reductase, being a substrate for the synthesis of isoprenoid mole-
                                                                           cules may favor the prenylation and activation of RhoA. In our
DISCUSSION
                                                                           experimental conditions mevalonic acid, which per se did not
Crocidolite asbestos is considered to be the major causative factor        change the nuclear content of NF- B, completely prevented
in the development of HMM, although other co-carcinogens, such             both the NF- B activation and the phosphorylation and decrease
as simian virus 40 (SV40) infection and genetic factors, have              of I B induced by crocidolite.
Riganti, Orecchia, Silvagno, et al.: Asbestos Inhibits Rho Signaling                                                                            753




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 g/cm2),
mevalonic acid (MA, 100 M), Y27632 (Y276, 0.2 M), 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).   Figure 7. Effect of crocidolite and mevastatin on the synthesis of iso-
                                                                           prenoid 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 follow-
    The IKK complex phosphorylates I B favoring its proteaso-              ing compounds, alone or in different combinations: crocidolite fibers
mal degradation and allowing NF- B to translocate to the nu-               (CRO, 25 g/cm2), mevastatin (MVS, 100 M), and squalestatin (SQ,
cleus. IKK is fully active only when phosphorylated, mainly on             1 M). Subsequently, the cells were lysed and checked for incorporation
serine 176 and 180 of IKK (3). In nonstimulated HMM cells,                 of 3H (A ) or 14C (B ) into cholesterol and ubiquinone (see MATERIALS AND
IKK was weakly phosphorylated and its activity (measured as                METHODS). Data are presented as means          SE (n   6). *P    0.05, **P
the ability of immunoprecipitated intracellular IKK complex to                0.001 versus CTRL. (C ) After the incubation, cells were lysed and
phosphorylate I B ) was not detectable, while both crocidolite             checked for the expression of total, prenylated, and nonprenylated
and inhibitors of RhoA and Rock increased both IKK activity                RhoA and of GAPDH (see MATERIALS AND METHODS). The figure is represen-
and the amount of phospho-IKK . Mevalonic acid completely                  tative of two experiments with similar results. The experiments show
blocked the effect of crocidolite.                                         in A and B were repeated in duplicate with the other two cell lines
                                                                           (OC99, GF99), giving superimposable results (data not shown).
    The mechanisms of IKK phosphorylation have been exten-
sively 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 trans-                In other experimental models, a relationship between IKK /
location, cellular survival, and/or inhibition of apoptosis (36).          I B /NF- B pathway and RhoA and Rock signaling has already
Interestingly, crocidolite has been shown to activate Akt (by              been proposed (12, 13). Exposure of HMM cells to crocidolite
inducing its phosphorylation on serine 473) in a rat pleural meso-         fibers induced a significant decrease of GTP-bound (active)
thelial cell line (37). After a 24-h incubation of HMM cells with          RhoA and of Rock activity, a result similar to that observed
crocidolite, Akt activity was significantly increased, an effect pre-       after the incubation with toxin B, a specific RhoA inhibitor (34).
vented by the presence of mevalonic acid. Moreover, Y27632 and             Again, mevalonic acid prevented these crocidolite effects.
toxin B, although at a lower extent, induced a significant increase            To our knowledge, this is the first evidence that crocidolite
of the Akt activity as well. So far, the crocidolite-dependent activa-     inhibits the RhoA/Rock pathway. Furthermore, our data suggest
tion of Akt is likely to be linked to the inhibition of RhoA and           that the asbestos elicits the activation of Akt, IKK, and NF- B
Rock.                                                                      by inducing the blockage of RhoA signaling: indeed, crocidolite,
754                                                        AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 36                     2007

                                                                                     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 phos-
                                                                                 phorylate 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 accu-
                                                                                 mulation. 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
Figure 8. (A ) Effect of crocidolite, mevalonic acid and inhibitors of           in mesothelial cells (2, 38) and lung epithelial cells (39). This
RhoA/Rho kinase on iNOS induction. HMM (MM98) cells were incu-                   phenomenon could be observed also in our cell cultures. Crocido-
bated in fresh medium only (CTRL) or with crocidolite fibers (CRO, 25            lite, as well as Y27632 and toxin B, increased the DNA synthesis,
  g/cm2), in the absence or presence of mevalonic acid (MA, 100 M),              but also the leakage of LDH (a sensitive marker of cell damage),
Y27632 (Y276, 0.2 M), and toxin B (TOX, 0.1 ng/ml). After 24 h,                  thus appearing able to exert both mitogenic and cytotoxic effects,
total RNA was extracted and subjected to RT-PCR for iNOS mRNA, as
                                                                                 which were prevented by mevalonic acid and packed erythrocytes.
described in MATERIALS AND METHODS. One lane was loaded with bidistilled
                                                                                 Interestingly, crocidolite, Y27632, and toxin B modified NOS activ-
water ( ) in place of cellular extracts. The figure is representative of
two experiments per cell line with similar results. (B ) Effect of crocidolite
                                                                                 ity and nitrite levels according to DNA synthesis and LDH release.
on eNOS phosphorylation. HMM (MM98, OC99, and GF99) cells were                   This suggests that NO could play a role both in the cytotoxic and
incubated 24 h in the absence (CTRL) or presence on crocidolite fibers           in the proliferative stimulus induced by crocidolite and by inhibitors
(CRO, 25 g/cm2), in the absence or presence of mevalonic acid (MA,               of RhoA signaling. Mevalonic acid, favoring the prenylation of
100 M), Y27632 (Y276, 0.2 M), and toxin B (TOX, 0.1 ng/ml).                      RhoA, is likely to inhibit cell toxicity and proliferation by decreas-
Cellular extracts were analyzed by Western blotting with an anti-eNOS            ing the synthesis of NO induced by crocidolite, Y27632, and toxin
antibody and an anti-phospho(Ser1177)eNOS antibody, as indicated                 B. To confirm this statement, the presence of packed erythrocytes,
in MATERIALS AND METHODS. The figure is representative of two experi-            used as scavengers of NO, clearly inhibited nitrite accumulation,
ments with similar results. The experiments were repeated with the               DNA synthesis, and LDH leakage under each experimental con-
other two cell lines (OC99, GF99), giving superimposable results (data           dition. This suggests that the prenylation status of RhoA may
not shown).                                                                      play a critical role in both mesothelioma apoptosis and prolifera-
                                                                                 tion. 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
like mevastatin, inhibits both the synthesis of isoprenoid mole-                 cell type and the NO concentration (4, 40); thus it may play a
cules (cholesterol, ubiquinone) and the prenylation of RhoA.                     role as both pro- or antitumoral agent (41).
These effects are prevented in the presence of mevalonic acid,                       During the preparation of this manuscript, a paper has been
which allows cells to synthesize isoprenoid groups and then to                   published (42) showing that in primary mesothelial cells TNF-
keep RhoA in the prenylated, active form. This observation                       induces NF- B activation and protects from crocidolite asbestos
suggests that crocidolite fibers might inhibit the synthesis of                   cytotoxicity. Since TNF- may induce Akt activation and NO
isoprenoid molecules at the level of the HMGCoA reductase                        synthesis, we investigated its effect on HMM cells: after a 24-h
reaction or of an upstream step, causing a depletion of mevalonic                incubation with 10 ng/ml TNF- , we did not observe any signifi-
                                                                                 cant increase of Akt or NOS activity (data not shown). More-
acid.
                                                                                 over, TNF- did not revert the activation of Akt and NOS and
    We have previously shown that in HMM cells crocidolite
                                                                                 the cytotoxic effect exerted by crocidolite in HMM (data not
increases iNOS expression and NO synthesis via activation of
                                                                                 shown). So far, our results would rule out a role for TNF- in
NF- B (9). Several authors have reported an increased iNOS                       asbestos-induced NO synthesis, cytotoxicity, and Akt activation
activity in cells incubated with different inhibitors of RhoA pro-               in HMM cells. The transformed malignant phenotype of HMM
teins, such as statins (10, 11) and inhibitors of geranylgeranyla-               cells may account for their different response to TNF- in com-
tion (13). Toxin B from C. difficile (10) and Y27632 (13) also                    parison with normal mesothelial cells (42).
elicited enhanced nitrite production in several cell lines. Our                      To verify whether mevalonic acid could influence the effects
data show that, like crocidolite, inhibitors of RhoA and Rock                    of asbestos by modifying directly the fiber reactivity, we have
may also increase iNOS mRNA and NO synthesis in HMM                              also incubated the crocidolite fibers for 24 h in culture medium
cells. More interestingly, overexpression and activation of iNOS                 without cells in the presence of mevalonic acid; the suspension
induced by crocidolite are likely to be mediated by inhibition                   was then centrifuged, and fibers were washed with fresh medium
of the RhoA prenylation, since these effects were prevented by                   and finally incubated with cell cultures for 24 h. In the same
the presence of mevalonic acid.                                                  experimental conditions previously shown in Results, such
Riganti, Orecchia, Silvagno, et al.: Asbestos Inhibits Rho Signaling                                                                        755




                                                                               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 g/cm2), mevalonic acid (MA, 100
                                                                                 M), Y27632 (Y276, 0.2 M), toxin B from C. difficile (TOX,
                                                                               0.1 ng/ml), and packed red blood cells (RBC, 10 l/ml). (A )
                                                                               NOS activity in cell lysates (open bars) and nitrite levels in the
                                                                               extracellular medium (hatched bars) was measured in tripli-
                                                                               cate, as described (see MATERIALS AND METHODS). Data are pre-
                                                                               sented 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 MATERI-
                                                                               ALS AND METHODS; the figure is representative of two experi-
                                                                               ments with similar results. The experiments were repeated with
                                                                               the other two cell lines (OC99, GF99), giving superimposable
                                                                               results (data not shown).




asbestos fibers pretreated with mevalonic acid elicited in HMM              In summary, our data suggest that crocidolite asbestos induces
cells the same effects of nontreated fibers, as far as nuclear          NF- B activation and stimulates the synthesis of NO by inhib-
translocation of NF- B, GTP binding of RhoA, and DNA syn-              iting the RhoA signaling pathway. The increased production of
thesis were concerned (data not shown). Furthermore, in order          NO may be implicated in the proliferative response of HMM
to check the putative antioxidant action of mevalonic acid, we         cells that survive after exposure to asbestos fibers. In our experi-
measured the accumulation of thiobarbituric acid–reactive sub-         mental conditions, mevalonic acid prevented any effect induced
stances (TBARS), which are known markers of lipid peroxida-            by crocidolite and by the HMGCoA reductase inhibitor mevas-
tion. As expected, a 24-h incubation of HMM cells with crocido-        tatin, under concentrations that per se did not exert toxic effects
lite induced a significant increase of TBARS accumulation versus        and did not inhibit Rho kinase signaling. The reverting effect
control (n 6, P 0.02; data not shown): the coincubation of             of mevalonic acid suggests that crocidolite impairs the prenyla-
cells with fibers and mevalonic acid obtained the same effect of        tion of RhoA, which is necessary to the activation of this small
crocidolite alone, thus suggesting that MA does not exert per          GTPAse. Although many steps of this pathway still remain un-
se an antioxidant effect and that the correction elicited by MA on     clear, we think that our results may open a new line of research
crocidolite-dependent NF- B activation and RhoA GTP binding            in the investigation of crocidolite effects on human mesothelial
inhibition is not mediated by such a mechanism.                        cells and in the pathogenesis of malignant mesothelioma.




                                                                                        Figure 10. Effects of crocidolite and signaling inhibi-
                                                                                        tors on cell proliferation (measured as [3H]thymidine
                                                                                        incorporation) and on the release of LDH in the extra-
                                                                                        cellular medium. HMM (MM98) cells were grown for
                                                                                        24 h in the absence (CTRL) or presence of the follow-
                                                                                        ing components, alone or in different combinations:
                                                                                        crocidolite fibers (CRO, 25 g/cm2), mevalonic acid
                                                                                        (MA, 100 M), Y27632 (Y276, 0.2 M), toxin B from
                                                                                        C. difficile (TOX, 0.1 ng/ml), and packed red blood
                                                                                        cells (RBC, 10 l/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 de-
                                                                                        scribed (see MATERIALS AND METHODS). Data are pre-
                                                                                        sented 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 re-
                                                                                        peated with the other two cell lines (OC99, GF99),
                                                                                        giving superimposable results (data not shown).
756                                                          AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 36                                2007

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