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Published as doi: 10.1096/fj.05-4554fje.
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(The FASEB Journal. 2006;20:997-999.)
© 2006 FASEB

Transcriptional up-regulation of MMP12 and MMP13 by asbestos occurs via a PKC{delta}-dependent pathway in murine lung

Arti Shukla*, Trisha F. Barrett*, Keiichi I. Nakayama{dagger}, Kieko Nakayama{dagger}, Brooke T. Mossman* and Karen M. Lounsbury*,1

* Departments of Pathology and Pharmacology, University of Vermont, USA, Burlington, Vermont, USA; and

{dagger} Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan

1Correspondence: Department of Pharmacology, University of Vermont College of Medicine, 89 Beaumont Ave. Burlington, VT 05405, USA. E-mail: karen.lounsbury{at}uvm.edu

ABSTRACT

Asbestos is a known inflammatory, carcinogenic, and fibrotic agent, but the mechanisms leading to asbestos-induced lung diseases are unclear. Using a murine inhalation model of fibrogenesis, we show that asbestos causes significant increases in mRNA levels of lung matrix metalloproteinases (MMPs 12 and 13) and tissue inhibitor of metalloproteinases (TIMP1), as well as increased activities of MMP 2, 9, and 12 in bronchoalveolar lavage fluids (BALF). Asbestos-exposed PKC{delta} knockout (PKC{delta}–/–) mice exhibited decreased expression of lung MMP12 and MMP13 compared with asbestos-exposed wild-type mice. Studies using small molecule inhibitors in murine alveolar epithelial type II cells (C10) and primary lung fibroblasts confirmed that asbestos transcriptionally up-regulates MMPs via an EGFR (or other growth factor receptors)/PI3K/PKC{delta}/ERK1/2 pathway. Moreover, use of a broad-spectrum MMP inhibitor showed that MMPs play an important role in further enhancing asbestos-induced signaling events by activating EGFR. These data reveal a potentially important link between asbestos signaling and integrity of the extracellular matrix (ECM) that likely contributes to asbestos-induced lung remodeling and diseases.—Shukla, A., Barrett, T. F., Nakayama, K. I., Nakayama, K., Mossman, B. T., Lounsbury, K. M. Transcriptional up-regulation of MMP12 and 13 by asbestos occurs via a PKC{delta}-dependent pathway in murine lung.


Key Words: TIMP • fibrosis • lung cancer • lung epithelium

THE DEVELOPMENT OF cancers (lung cancer, mesothelioma) and pulmonary fibrosis (asbestosis) is associated with the inhalation of asbestos fibers [reviewed in (1 , 2) ]. Although these diseases have been studied intensely by basic and clinical research scientists, little is known about the crucial cellular mechanisms that initiate and drive the processes of carcinogenesis and fibrogenesis. A multiplicity of interactions between effector cells of the immune system (alveolar macrophages, neutrophils, lymphocytes) and target cells, including bronchiolar and alveolar epithelial cells and fibroblasts, may govern the pathogenesis and progression of these diseases. We have shown previously that oxidative stress by asbestos fibers is linked causally to inflammation and the development of pulmonary fibrosis (3) , as well as airway epithelial cell injury and proliferation (4) .

Matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) appear to be critical in the development and maintenance of lung architecture and function, their dysregulation resulting in lung damage, and remodeling (5 6 7) . Abnormal ECM deposition is observed in the lungs of patients with idiopathic pulmonary fibrosis (IPF), due in part to an imbalance between MMPs and TIMPs (8 , 9) . Gene expression analysis also reveals that matrilysin (MMP7) may be a key regulator of pulmonary fibrosis in mice and humans (10) .

In the pathogenesis of lung cancers and fibrosis, MMPs may be critical, not only in the breakdown and remodeling of lung tissues but also in the release and/or activation of profibrotic growth factors such as insulin growth factors (IGFs), transforming growth factor-beta (TGF-ß), and tumor necrosis factor-alpha (TNF-{alpha}) (11 12 13) .

We have previously shown a role for PKC{delta} signaling in the regulation of proliferation and apoptosis of lung epithelial cells exposed to asbestos (14 , 15) . In the present investigation, using a mouse inhalation model of fibrogenesis (16) and isolated lung epithelial cells (C10 line) and fibroblasts, we show for the first time that asbestos causes up-regulation of MMPs 12 and 13 via a PKC{delta}-dependent pathway. In vitro studies also implicate interactions between phosphatidylinositol 3 kinase (PI3K)-PKC{delta}-extracellular signal-regulated kinase (ERK1/2) pathways in transcriptional up-regulation of MMP12 and MMP13. Increases in expression of MMP12 and MMP13 in lung tissues of normal asbestos-exposed mice were attenuated in asbestos-exposed PKC{delta} knockout [PKC{delta} (–/–)] mice, confirming a regulatory role of PKC{delta} in MMP transcription. Finally, we show that MMPs up-regulated by asbestos stimulate asbestos-induced signaling pathways via activation of the EGFR in epithelial cells.

MATERIALS AND METHODS

Inhalation experiments
Animal experiments were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals (publication 85–23, 1985) following protocols approved by the University of Vermont Institutional Animal Care and Use Committee. C57Bl/6 mice (8 to 12 wk of age) were exposed to ambient air or the NIEHS reference sample of chrysotile asbestos (7 mg/m3 air; 6 h/day; 5 d/week for 3, 9, or 40 d) as described previously (16) . Briefly, mice were euthanized with an intraperitoneal (i.p.) injection of pentobarbital (Abbott Laboratories, Abbot Park, IL), chest cavities were opened, and lungs were cannulated via the trachea with polyethylene tubing. Lungs were then lavaged 1x with sterile Ca2+- and Mg2+-free PBS at a vol of 1 ml. The vol of retrieved PBS in bronchoalveolar lavage fluid (BALF) was also recorded. One lung lobe was excised following lavage and stored in RNA-later (Ambion, Austin, TX) for RNA analysis using ribonuclease protection assays rNase protection assay (RPA) and Affymetrix microarray analyses (Affymetrix Inc., Santa Clara, CA). BALF was centrifuged at 600 g to obtain a cell-free supernatant for MMP activity assays using gelatin zymography (see below).

PKC{delta} (–/–) mice
A breeding pair of PKC{delta} (±) mice (17) , originally bred into the C57Bl/6 background, was a kind gift from Dr. K. I. Nakayama. These mice were subsequently maintained in the UVM facility and bred into the C57Bl/6 background 4–6x before use with normal wildtype (WT) [PKC{delta} (+/+)] littermates in inhalation experiments. Tail DNA was evaluated using the polymerase chain reaction (PCR), and primers for PKC{delta} were obtained from MWG Biotech. Inc. (High Point, NC). Lung tissue from PKC{delta} (–/–) mice was examined by Western blot analyses using antibodies for PKC{delta} and other isoforms ({alpha}, {zeta}, {theta}) of PKC (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) to confirm that only PKC{delta} protein is absent while other isoforms of PKC are present.

Cell cultures and exposures to agents
A contact inhibited, nontransformed murine alveolar epithelial type II cell line (C10) (18) was propagated in CMRL-1066 medium containing penicillin (100 U/ml), streptomycin (100 µg/ml), L-glutamine (2 mM), and 10% FBS (Life Technologies, Inc., Grand Island, NY). For all experiments, cells were grown to confluence, complete medium was removed, and medium containing 0.5% FBS or no serum was added 24 h before exposure to agents. Primary mouse lung fibroblasts were isolated from 10-wk-old C57Bl/6 mice using an enzyme digestion method (collagenase-trypsin-DNase) and propagated in Dulbecco’s modified Eagle medium (DMEM, GIBCO Life Technologies, Inc., Grand Island, NY) with 10% FBS, penicillin, streptomycin, and L-glutamine at concentrations above. Antibodies for phosphorylated form (p-ERK44/42) and total ERK1/2 (ERK44/42) and EGFR were from Cell Signaling (Beverly, MA). Inhibitors of PI3K (LY249002, 10 and 20 µM), ERK1/2 (U1026, 10 µM), PKC{delta} [Rottlerin, 5 µM (19) ], general PKCs (Bisinolymaleimide I, 5 µM), EGFR phosphorylation (AG1478, 10 and 20 µM) and broad-spectrum MMP inhibitor (GM6001, 10 µM) were obtained from Calbiochem (La Jolla, CA) and used at nontoxic and selective concentrations in vitro as reported previously (15 , 20 21 22 23) . These inhibitors were added 1 h prior to addition of asbestos. Actinomycin D (inhibitor for RNA synthesis, 50–500 ng/ml), gelatin, and Briz 35 were purchased from Sigma Chemical Co. (St. Louis, MO). Standard gelatinase mix was obtained from Chemicon International (Chemicon International, Temecula, CA).

For in vitro studies, crocidolite asbestos fibers (Na2(Fe3+)2(Fe2+)3(OH)2[Si8O22]) (NIEHS reference sample) were suspended in Hank’s balanced salt solution (HBSS) (Life Technologies, Inc. Grand Island, NY) at 1 mg/ml, sonicated, and then triturated 10x through a 22-gauge needle to obtain a homogenous suspension before addition directly to medium at noncytolytic concentrations of 5 µg/cm2 surface area of culture dish. Because available samples of NIEHS reference samples of crocidolite are limited and insufficient in quantities required for inhalation experiments, NIEHS reference standards of chrysotile asbestos (Mg3[Si2O5](OH)4) were used in animal studies. Long fibers (≥5 µm) of both crocidolite and chrysotile asbestos are fibrogenic and carcinogenic and cause oxidant generation from cells via frustrated phagocytosis or iron catalyzed reactions (i.e., crocidolite) (4) . NIEHS crocidolite and chrysotile reference samples of asbestos have been characterized previously for their chemical and physical properties (24) .

Microarray analysis for MMP mRNA levels in lungs
Total RNA was isolated from lung tissues as described above using TriZol reagent (Invitrogen, Life Technologies, CA) and submitted to the Vermont Cancer Center Microarray Facility for target preparation using standard Affymetrix protocols. Briefly, 3 µg of total RNA from each sample was reverse-transcribed using oligo-dT primer coupled to a T7 RNA polymerase binding sequence. Following double-stranded cDNA preparation, biotinylated-cRNA was synthesized using T7 polymerase and hybridized to Affymetrix murine genome U74Av2 oligonucleotide arrays for 16 h. The arrays were first incubated with a streptavidin-conjugated to phycoerythrin, followed by sequential incubation with biotin-coupled polyclonal antistreptavidin antibody (Ab) and strepavidin-phycoerythrin as an amplification step. After washing and laser scanning (Hewlett-Packard GeneArray Scanner, Agilent Technologies, Inc.), data were collected and analyzed by using GeneSifter software (GeneSifter VizX Labs, Seattle, WA). Results were confirmed by RPA using a larger number of animals per group (n=6).

Ribonuclease protection assays (RPA)
Total RNA was prepared from cells and murine lung tissue after 9 and 40 d of exposure to asbestos as described by Shukla et al. (25) . Steady-state mRNA levels of MMP3, MMP7 MMP8, MMP9, MMP12, MMP13, TIMP1, TIMP2, TIMP3, and the ribosomal probe L32 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) were examined with a RiboQuant multiprobe RPA system and the mMMP2 multiprobe template set (Pharmingen, San Diego, CA) according to the manufacturer’s protocol. Autoradiograms were quantitated with a Bio-Rad (Richmond, CA) phosphoimager. Results were normalized to expression of the housekeeping gene, L32.

TaqMan [Quantitative reverse transcriptase PCR (RT-PCR)]
Total RNA was extracted from cells as described for the RPA and then further purified using an RNA cleaning kit (Qiagen Inc., Valencia, CA) according to the manufacturer’s instructions. Samples were also treated with RNase-free DNase I to remove contaminating genomic DNA. The RNA was then used to generate cDNA with the Reverse Transcription System (Promega, Madison, WI), according to the manufacturer’s instruction. The Perkin-Elmer AB1 7700 prism Sequence Detection System (Applied Biosystems, Foster City, CA) was used to determine relative levels of expression of MMP12. All values were normalized to the expression of HPRT (TaqMan primers and probes for MMP12 and HPRT were purchased from Applied Biosystems).

Zymography on BALF samples from mice
BALF from sham and asbestos-exposed mice after 9 or 40 d of asbestos inhalation was collected for determination of MMP activity. The BALF was centrifuged at 600 X g for 5 min, and the supernatant was recovered and frozen at –70°C. Cell-free BALF (75 µl) was subjected to SDS-PAGE under nonreducing conditions. MMP activity was determined by in gel zymography with gelatin (Type A from Porcine skin, Sigma) as a substrate. Samples were loaded under nonreducing conditions onto 4% stacking/10% separating SDS-polyacrylamide gels with 1 mg/ml gelatin polymerized in the separating gel before electrophoresis. After separation, gels were washed 3x in 2.5% Triton X-100 for 20 min with gentle shaking. All gels were incubated for 48 h at 37°C in substrate buffer (50 mM Tris.HCl, pH 7.6, 5 mM CaCl2, 0.05% Brij 35, and 0.02% NaN3), stained in Coomassie blue R-250 in 7% acetic acid and 40% methanol, and then destained in 7% acetic acid and 40% methanol. Clear, digested regions represented MMP activity and were identified based on MW markers (MMP12) as described by Shipley et al. (26) and/or standard gelatinases (MMP 2 and 9, Chemicon Int., CA). MMP identity was confirmed by an additional 30 min incubation of selected gels with the metal chelators EDTA (10 mM).

Western blot analysis for extracellular signal-regulated kinases (ERK1/2 and p-ERK1/2)
Cells grown in culture dishes were washed three times with ice-cold PBS and collected in lysis buffer (20 mM Tris, pH 7.6; 1% Triton-X100; 137 mM NaCl; 2 mM EDTA; 1 mM Na3VO4; 1 mM DTT; 1 mM phenylmethylsulfonyl fluoride; 10 µg/ml leupeptin; and 10 µg/ml aprotinin) before incubation on ice for 30 min. Cells were then sonicated (3 bursts of 5 s each) and centrifuged at 14,000 rpm for 15 min at 4°C. Supernatants were collected, and protein concentrations were determined using the Bradford assay (Bio-Rad). Cell lysates (40 µg) were resolved by SDS-PAGE and transferred to nitrocellulose membranes according to standard procedures. Equal loading of protein was verified by Ponceau stain (Sigma). Membranes were washed in TBS, blocked for 30 min with TBS containing 5% nonfat milk, then incubated with primary antibodies at 1:1000 (p-ERK1/2, ERK1/2) dilution in TBS containing 1% BSA (0.01% Azide) overnight at 4°C. Membranes were then washed twice with TBS alone and twice with PBS containing 0.1% Tween 20 before incubation with horseradish peroxidase-conjugated secondary Ab (1:5000 in PBS containing 0.1% Tween 20 and 5% nonfat milk) for 1 h at room temperature. Membranes were washed once with PBS containing 0.1% Tween 20 and 3 times with PBS before Ab binding was visualized by enhanced chemiluminescence (Amersham Pharmacia Biotech, Piscataway, NJ) according to the manufacturer’s protocol.

Kinase activity assay for epidermal growth factor receptor (EGFR)
EGFR kinase activity was determined with an immunoprecipitation kinase assay as follows. Soluble protein was prepared as described elsewhere (15) ; the protein (300 µg) then was immunoprecipitated for 2 h at 4°C with an anti-EGFR Ab (1:100, Cell Signaling); and the antigen-antibody complexes were collected by incubation with agarose protein A (Life Technologies, Inc.) for 1 h at 4°C. Then, pellets were washed three times with lysis buffer and three times with kinase buffer (20 mM HEPES, pH 7.4; 10 mM MnCl2; 10 mM MgCl2; 1 mM DTT; 100 µM Na3VO4; and 10 µM ATP) before resuspension in a reaction buffer containing 25 µl kinase buffer, myelin basic protein (MBP, 5 µg), and 5 µCi of [{gamma}-32P]ATP (New England Nuclear, Life Science Products, Inc., Boston, MA). All of the samples were incubated for 20 min at 30°C. Reactions were terminated by addition of 2x SDS sample buffer and boiled, and the reaction products were resolved on a 15% SDS-polyacrylamide gel. The extent of myelin basic protein phophorylation was determined by autoradiography.

Statistical analyses
In all experiments, duplicate or triplicate determinations per group per time point were performed. Experiments were repeated 3x or more. Results were evaluated by one-way ANOVA with the Student-Newman-Keuls procedure for adjustment of multiple pair-wise comparisons between treatment groups. Differences of P ≤ 0.05 were considered statistically significant.

RESULTS

Asbestos inhalation causes up-regulation of MMP12, MMP13, and TIMP1 mRNA levels in lung: MMP12 and MMP13 are inhibited in PKC{delta} (–/–) mice
Microarray analysis of lung tissue
Oligonucleotide microarray analysis (Affymetrix) on RNA prepared from whole lungs of mice (n=3/group) was used to verify transcriptional changes in MMPs in asbestos-exposed lungs. As shown in Fig. 1 A, asbestos exposure resulted in significant increases in MMP12 mRNA levels in the lungs of WT mice at 3, 9, and 40 d. Significant attenuation of asbestos-induced up-regulation of MMP12 mRNA levels occurred in PKC{delta} (–/–) mice (Fig. 1B , (3 d). Gene profiling also revealed an elevation of MMP13 levels after asbestos inhalation for 9 d that was reduced in PKC{delta} knockout mice (Fig. 1C ). The complete results of microarray analyses of sham and asbestos-exposed WT mice have been recently published (27) .


Figure 1
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  		Figure 1. Microarray analysis showing that asbestos inhalation causes increases in lung mRNA levels of MMP12 and 13, which are inhibited in PKC{delta} (–/–) mice. PKC{delta} (–/–) mice and WT littermates bred into the C57Bl/6 background were exposed to asbestos (7 mg/m3, 6 h/day, 5 d/wk) for 3, 9, or 40 d. RNA was prepared from the lung, purified, and subjected to microarray analysis using an U74Av2 oligonucleotide chip. Data were analyzed using the GeneSifter program. A) Time-dependent effects of asbestos inhalation on MMP12. B) Effect of asbestos inhalation (3 d) on MMP12 levels in PKC{delta} (–/–) mice. C) Effect of asbestos inhalation (9 d) on MMP13 levels in WT and PKC{delta} (–/–) mice. *P ≤ 0.05 as compared with respective control group, #P ≤ 0.05 compared with asbestos-exposed group. (n=3 per group).

Ribonuclease protection assays
The microarray data were confirmed by ribonuclease protection assays using a larger number of animals. After 9 or 40 d of asbestos inhalation, lungs from WT and PKC{delta} (–/–) mice (n=6/group/time period) were analyzed for steady-state mRNA levels of MMPs and TIMPs by ribonuclease protection assays. Increased levels of MMP12, MMP13, and TIMP1 were observed in lungs of WT mice exposed to asbestos for 9 d (Fig. 2 A) and MMP12 levels remained elevated up to 40 d of asbestos exposure (data not shown), whereas levels of MMP3, 7, 8, 9, and TIMP2, 3 were similar to those of sham mice. As shown in Fig 2A , (a representative RPA showing two animals/group), PKC{delta} (–/–) mice showed attenuation of asbestos-induced steady-state mRNA levels of MMP12 and MMP13 as compared to WT animals. Fig. 2B and C represents the quantitation of 9 d autoradiograms for MMP12 and MMP13, respectively, with each group having 6 animals.


Figure 2
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  		Figure 2. Asbestos inhalation causes increases in steady-state mRNA levels of lung MMP12, 13, and TIMP1 in wild-type mice; MMP12 and 13 are inhibited in PKC{delta} (–/–) mice. C57Bl/6 and PKC{delta} (–/–) mice (n=6 per group) were exposed to chrysotile asbestos (7 mg/m3, 6 h/day, 5 d/wk) for 9 d. Lung RNA was prepared and analyzed by ribonuclease protection assay using an mMMP2 template. A) A representative autoradiogram using 2 mice per group (out of 24 mice run on the same gel). Quantitation of autoradiograms using 6 mice per group for MMP12 (B) and for MMP13 (C). *P ≤ 0.05 compared with untreated control.

MMP activity in BALF from WT and PKC{delta} (–/–) mice
BALF was analyzed for MMP activity using gelatin zymography. As shown in Fig. 3 A, proMMP9, proMMP2 (identified based on standard gelatinases) and MMP12 activities [as identified based on MW (fully processed 22 kDa) as described by Shipley et al. (26) ] were increased in BALF from 9 d asbestos-exposed animals. After 40 d of asbestos inhalation, MMP2 and MMP9 activity levels returned to normal control levels; however, MMP12 activity remained elevated in BALF (Fig. 3B ). Gelatin zymography on BALF from both WT (Fig. 3A ) and PKC{delta} (–/–) asbestos-exposed mice showed increased MMP2, MMP9, and MMP12 activities (Fig. 3C ). The discrepancy between lack of inhibition in MMP12 activity vs. decreased MMP12 transcript levels in PKC{delta} (–/–) mice might be attributed to the altered inflammatory response shown by these animals in response to asbestos (28) . High levels of MMP12 activity observed in PKC{delta} (–/–) sham animals could possibly be related to inflammatory patches we see in the lungs of these mice.


Figure 3
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  		Figure 3. Gelatin zymogram showing increased MMP2, 9, and 12 activities in BALF after asbestos inhalation. PKC{delta} (–/–) mice and WT littermates were exposed to asbestos (7 mg/m3, 6 h/day, 5 d/wk) for 9 (A) or 40 (B). BAL fluid was collected, and gelatin zymography was run under nonreducing conditions. Clear digested areas were identified based on molecular wt markers (MMP12) and positive controls (MMP2 and 9). C) Zymogram from BALF of PKC{delta} (–/–) mice after 9 d of asbestos exposure.

Asbestos increases steady-state mRNA levels of MMP12, MMP13, and TIMP1 in a time-dependent manner in lung epithelial cells and fibroblasts
In vitro studies were performed to determine the mechanisms of MMP up-regulation and relevant signaling pathways in lung epithelial cells and fibroblasts, target cell types of asbestos-induced lung cancers, and fibrosis, respectively. Exposure of C10 epithelial cells to asbestos (5 µg/cm2) for 4, 8, and 24 h caused time-dependent increases in steady-state mRNA levels of MMP13 and TIMP1 as determined by RPA (Fig. 4 A, B). Primary lung fibroblasts also showed time-dependent increases in steady-state mRNA levels of MMP12, MMP13, and TIMP1 after exposure to asbestos. MMP12 was abundant in fibroblasts but was undetectable in epithelial cells. The induction of MMP12 by asbestos in fibroblasts as detected by RPA was confirmed using quantitative RT-PCR (TaqMan) (Fig. 4C ).


Figure 4
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  		Figure 4. Asbestos exposure causes time-dependent increases in MMPs and TIMP steady-state mRNA levels in lung epithelial cells and fibroblasts. Lung epithelial type II cells (C10) or primary lung fibroblasts were exposed to asbestos (5 µg/cm2) for different time periods (4 to 24 h). RNA was prepared and analyzed by ribonuclease protection assay using a mMMP2 template. Autoradiograms were developed (A) and selected genes were quantitated using phospoimaging (B). Results are represented as a ratio to the housekeeping gene, L32. *P ≤ 0.05 in comparison to respective untreated control. C) Fibroblasts were exposed to asbestos for 24 or 48 h, RNA was prepared and analyzed by quantitative real-time PCR (TaqMan) for MMP12 levels. *P ≤ 0.05 in comparison to untreated controls.

Asbestos affects transcription of MMP13 and TIMP1 in lung epithelial cells
To show that increased steady-state mRNA levels of MMP13 and TIMP1 by asbestos were not due to stabilization of mRNA, C10 cells were treated with actinomycin D (a transcription inhibitor) at different concentrations (50, 100, 200, 500 ng/ml) for 30 min prior to exposure to asbestos. Pretreatment with actinomycin D completely blocked asbestos-induced increases in MMP13 and TIMP1 mRNA (Fig. 5 ) as determined by RPA, indicating transcriptional up-regulation of MMP13 and TIMP1 by asbestos.


Figure 5
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  		Figure 5. Asbestos transcriptionally up-regulates MMP13 and TIMP1. Lung epithelial type II cells (C10) were pretreated with various concentrations of actinomycin D (50–500 ng/ml) for 30 min before exposing them to asbestos (5 µg/cm2) for 24 h. RNA was prepared and analyzed by ribonuclease protection assays using a mMMP2 template. Quantitation of autoradiograms was performed using a phosphoimager. *P ≤ 0.05 as compared to untreated control.

Multiple cell signaling pathways are involved in regulation of asbestos-induced MMPs
Asbestos exerts its effects on lung epithelial cell proliferation via EGFR dependent and independent pathways leading to ERK1/2 and ERK5 activation and activating protein (AP)-1 transactivation (29) . To reveal the signaling pathways involved in regulation of MMP and TIMP transcription by asbestos, C10 epithelial cells (MMP13 and TIMP1) or fibroblasts (MMP12) were exposed to different small molecule kinase inhibitors before addition of asbestos for 24 h and then analyzed by RPA. As shown in Fig. 6 A, pretreatment of cells with an ERK1/2 inhibitor (U1026, 10 µM) decreased asbestos-associated increases in MMP12, MMP13, and TIMP1 mRNA. Whereas the PI3K inhibitor (LY294002 at 10 and 20 µM) inhibited both MMP12 and MMP13 transcription by asbestos, the EGFR phosphorylation inhibitor (AG1478 10 and 20 µM) inhibited MMP13 but not MMP12 mRNA expression by asbestos (Fig. 6B ). The PKC{delta}-specific inhibitor rottlerin at 5 µM blocked asbestos-induced transcription of both MMP12 and MMP13; however, a general PKC inhibitor (Bis at 5 µM) inhibited asbestos-induced up-regulation of MMP12 but had no effect on steady-state mRNA levels of MMP13 (Fig. 6C ), indicating different pathways of regulation in different cell types. Constitutive levels of MMP12 mRNA were also inhibited by rottlerin and Bis.


Figure 6
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  		Figure 6. Asbestos-induced MMP12 and 13 are regulated via an EGFR(or other growth factor)/PI3K/PKC{delta}/ERK1/2 pathway. Lung epithelial type II cells (C10) and primary lung fibroblasts were pretreated with either (A) a mitogen-activated protein kinase (ERK1/2) inhibitor (U1026 10 µM for 1 h), (B) a phosphatidylinositol 3-kinase (PI3K) inhibitor (LY 294002, 10 or 20 µM for 1 h) or an EGF receptor inhibitor (AG1478, 10 or 20 µM for 1 h) or (C) a protein kinase C general inhibitor (Bis 5 µM for 1 h) or the PKC{delta} specific inhibitor (rottlerin 5 µM for 1 h), before exposing them to asbestos (5 µg/cm2) for 24 h. RNA was prepared and analyzed by a ribonuclease protection assay. Quantitation of autoradiograms was performed using a phosphoimager. *P ≤ 0.05 as compared with respective untreated control, #P ≤ 0.05 as compared to respective asbestos-exposed group. All MMP12 studies were performed in fibroblasts whereas MMP13 and TIMP 1 studies were performed in lung epithelial type II cells (C10).

Asbestos-induced up-regulation of MMPs can further enhance signaling pathways via EGFR activation
Using a broad-spectrum inhibitor of MMPs (GM6001, 10 µM), we show that inhibition of MMPs inhibits asbestos-induced EGFR activation (Fig. 7 A) and ERK1/2 phosphorylation (Fig. 7B, C ) in C10 lung epithelial cells, indicating an important role of MMPs in initiation of asbestos-induced cell signaling. Addition of GM6001 alone to cells had no effects on EGFR or ERK1/2 phosphorylation.


Figure 7
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  		Figure 7. MMPs regulate asbestos-induced signaling pathways via EGFR activation in epithelial cells. Lung epithelial type II cells (C10) were pretreated with a broad-spectrum MMP inhibitor (GM6001, 10 µM) for 1 h before exposure with asbestos for 8 h. Western blot for ERK1/2 and EGFR kinase activity assays were performed as described in Materials and Methods. A) Kinase activity assay using MBP as a substrate showing inhibition of asbestos-induced EGFR activation by MMP inhibitor GM6001 (MBP = myelin basic protein). B) Western blot showing inhibition of asbestos-induced ERK1/2 (p-p44/p-p42) phosphorylation by GM6001. C) Quantitation of the Western blot in (B). *P ≤ 0.05 as compared to untreated control, #P ≤ 0.05 as compared with asbestos exposed group.

DISCUSSION

Asbestos fibers cause pulmonary fibrosis and lung cancers (2) , diseases involving epithelial cell-fibroblast interactions and resulting in lung remodeling. MMPs are a family of secreted or transmembrane zinc-dependent endopeptidases that can degrade ECM and basement membrane components and may be important in re-epithelization and remodeling of damaged lungs. In addition to enhancing ECM turnover and tissue remodeling, MMPs may also have profound effects on the release of pro-fibrotic growth factors and cytokines (11 12 13) .

Recent data implicate MMP7 or matrilysin as a key regulator of pulmonary fibrosis in mice and humans, and matrilysin knockout mice are resistant to pulmonary fibrosis (10) . Histological examination of normal lungs and lungs from patients with interstitial lung disease also implicate changes in distribution and amounts of MMPs and TIMPs (30) . Isolated alveolar macrophages obtained from untreated patients with idiopathic pulmonary fibrosis show marked increases in MMP9 secretion compared with macrophages collected from normal individuals (31) . In addition, a synthetic inhibitor of MMP, Batimastat, reduces bleomycin-induced lung fibrosis (32) . In studies here, we show that exposure to asbestos cause significant increases in MMP12, MMP13, and TIMP1 expression levels, which are attenuated in lungs of PKC{delta} (–/–) mice. These changes may be explained by our observations that PKC{delta} (–/–) mice exhibit altered inflammatory profiles and less pulmonary fibrosis in response to asbestos in comparison to WT littermates (28) . The observed increase in MMP12 activity in PKC{delta} (–/–) sham mice as compared to WT sham animals is hard to explain but might be related to altered immune responses and the presence of patches of inflammatory cells in lungs of PKC{delta} (–/–) mice.

Our results suggest that asbestos can induce ECM remodeling affecting both matrix deposition and degradation. For example, MMP13 (collagenase 3) is an epithelial matrix metalloproteinase that degrades mainly fibrillar collagens and gelatinases A and B (MMP2 and MMP9), which degrade type IV basement membrane collagen (33) . TIMP1 is a multifunctional molecule that inhibits matrix metalloproteinase activity and promotes the proliferation of receptive cells. Increased activity of MMP13 in BALF was not observed despite several-fold increases in mRNA levels of MMP13 in lung. This observation may reflect increased TIMP1 levels in lung that inhibited MMP13 activity. Increased expression of MMP13 and TIMP1 was also reported by Ortiz et al. (34) in a murine model of silicosis.

Here we show that inhalation of asbestos causes increases in mRNA levels and activity of MMP12. Macrophage metalloelastase (MME) or MMP12 can hydrolyze a broad spectrum of substrates (35 , 36) . Although most of the available literature indicates that the macrophage is the main cell type making MMP12, here we show for the first time that primary lung fibroblasts also express MMP12. MMP12 expression in this cell type was confirmed by two different techniques, RPA and quantitative RT-PCR (TaqMan), which revealed similar results (Fig. 4) . In support of our observation that cells other than macrophages can also express MMP12, a recent report shows the induction of MMP12 gene expression in airway-like epithelial cell by cigarette smoke (37) . Use of MMP12 knockout mice in our inhalation model may shed light on the importance of this protein in development of asbestos-induced fibrosis.

Asbestos inhalation also results in increased MMP2 and MMP9 activities in BALF, although no effect on transcript levels of these two MMPs was observed. These increases in activity could reflect contributions of increased inflammatory cells in BALF, which are features of this animal model (16) .

MMP production and activity are highly regulated at different levels. In general, basal transcription in normal adult tissues is low, but MMPs are up-regulated by a variety of factors at the transcriptional, postranscriptional, and postranslational levels as well as by the interaction of secreted enzymes with TIMPs (7 , 38) . Our studies using epithelial cells and fibroblasts with small molecule inhibitors show that asbestos-induced increases in MMP12, MMP13, and TIMP1 mRNA levels are ERK1/2 dependent. These results are consistent with many studies showing that MMPs (MMP1, MMP3, MMP7, MMP9, MMP10, MMP12, and MMP13) are regulated by extracellular stimuli, which activate activator protein-1 (AP-1) [reviewed in (39 , 40) ]. Previous work from our laboratory has shown that asbestos activates AP-1 dependent gene transcription via the ERK1/2 pathway, whereas c-Jun NH2-terminal kinase and p38 pathways are not activated by asbestos (41) . Experiments here reveal that asbestos-induced MMP12 and MMP13 expression are also regulated by EGF receptor (EGFR), phosphatidylinositol 3-kinase (PI3K), and PKC{delta}, results consistent with other reports implicating these pathways in MMP regulation by other agents (42 , 43) . For example, EGFR-mediated signaling promotes MMP9 activation by enhancing PI3K-dependent cell surface association of the receptor (44) . Our studies indicate nonsignificant effects of an EGFR inhibitor on asbestos-induced MMP12 mRNA levels in fibroblasts; however, asbestos-induced MMP13 mRNA levels in epithelial cells were significantly inhibited by higher concentrations (20 µM) of an EGFR inhibitor (Fig. 6B ). These findings indicate that asbestos-induced responses in epithelial cells occur via EGFR activation, whereas in fibroblasts other growth factor(s) may be responsible.

The MMPs play a critical role in the processing of EGF and EGF-like ligand precursors, thereby contributing to the EGFR signal transactivation (45 46 47) . In our study, blocking of MMP activation with a broad-spectrum small molecule inhibitor GM6001 in epithelial cells inhibited asbestos-induced EGFR activation and ERK1/2 phosphorylation, an EGFR-dependent event in cells after exposure to asbestos or cigarette smoke (23 , 48) . This finding demonstrates that once MMPs are activated, they can further enhance asbestos-induced signaling pathways via EGFR activation.

We note that the results in our study have focused largely on the expression of MMP and TIMP transcripts, which may or may not always equate with protein expression. Although it is desirable to determine protein expression of MMPs or TIMPs as well, the reagents (e.g., antibodies) to detect most nongelatinase MMPs in mice, including MMP12 and MMP13, are rudimentary and the specificity suspect. Future studies to better define MMP protein expression and secretion will extend our current data.

Putting the current findings in the context of previous findings by our research group and others (49 50 51) , we propose a new hypothetical model wherein asbestos transcriptionally up-regulates MMP12 and MMP13 in a growth factor/PI3K/PKC{delta}/ERK1/2 dependent manner (Fig. 8 ). We also predict that MMPs activated by asbestos have the potential to further regulate asbestos-induced signaling pathways via activating EGFR in epithelial cells. The interplay between asbestos-induced MMPs and TIMPs may be crucial in the development of asbestos-induced lung diseases, and relevant signaling pathways may be targets for intervention and therapy. Though this study indicates that MMPs can regulate asbestos-induced signaling pathways, the precise role of individual MMPs in asbestos-induced lung pathologies will be explored in the future by using MMP12 and MMP13 knockout models.


Figure 8
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  		Figure 8. Hypothetical schema showing regulation of MMPs by asbestos. Asbestos exposure leads to up-regulation of MMPs via growth factor receptors, utilizing the ERK1/2, PKC{delta}, and PI3K pathways. Up-regulation and activation of MMPs may promote EGF shedding resulting in further EGFR activation and phosphorylation of ERK1/2 in epithelial cells. Inhibition of MMPs by the broad-spectrum inhibitor GM6001 prevents asbestos-induced EGFR activation and ERK1/2 phosphorylation in epithelial cells.

ACKNOWLEDGMENTS

This work was supported by National Institutes of Health Grant PO1HL67004. Dr. Pamela Vacek (Department of Medical Biostatistics, University of Vermont, Burlington, VT) assisted in the statistical analyses. We also wish to acknowledge Scott Tighe and Timothy Hunter from the Vermont Cancer Center DNA Analysis Facility at the University of Vermont for performing oligonucleotide microarrays with the provided RNA samples. Oligonucleotide microarrays and real-time quantitative polymerase chain reactions were performed in the VT Cancer Center DNA Analysis Facility and were supported in part by grant P30CA22435 from the NCI. The views expressed are those of author and do not represent the views of the NCI.

Received for publication September 2, 2005. Accepted for publication December 29, 2005.

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A. Shukla, K. M. Lounsbury, T. F. Barrett, J. Gell, M. Rincon, K. J. Butnor, D. J. Taatjes, G. S. Davis, P. Vacek, K. I. Nakayama, et al.
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