Published as doi: 10.1096/fj.05-4554fje.
(The FASEB Journal. 2006;20:997-999.)
© 2006 FASEB
Transcriptional up-regulation of MMPs 12 and 13 by asbestos occurs via a PKC
-dependent pathway in murine lung
Arti Shukla*,
Trisha F. Barrett*,
Keiichi I. Nakayama
,
Keiko Nakayama,
Brooke T. Mossman* and
Karen M. Lounsbury*,1
* Departments of Pathology and Pharmacology, University of Vermont, Burlington, Vermont, USA; and
Department of Molecular Genetics, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan
1Correspondence: Department of Pharmacology, University of Vermont, 89, Beaumont Ave., Burlington, VT 05405, USA. E-mail: karen.lounsbury{at}uvm.edu
SPECIFIC AIMS
Matrix metalloproteases (MMPs) play an important role in lung remodeling in response to various environmental agents; however, their role in asbestos-induced lung diseases remains unexplored. In this study we tested the hypothesis that asbestos causes transcriptional up-regulation of various MMPs via a PKC
-dependent pathway both in vitro and in vivo. Moreover, we hypothesized that asbestos-induced MMPs were regulated by multiple signaling pathways and governed extracellular signal-regulated kinase (ERK1/2) phosphorylation via epidermal growth factor receptor (EGFR) activation in epithelial cells.
PRINCIPAL FINDINGS
1. Asbestos exposure causes up-regulation of MMP mRNA levels in lungs that are reduced in PKC (–/–) mice
Oligonucleotide microarray analysis (Affymetrix) on RNA prepared from whole lungs of C57Bl/6 wild-type (WT) [PKC (+/+)] and PKC (–/–) mice after asbestos inhalation (
7 mg/m3 air, 6 h/day, 5 d/week for 3, 9, or 40 d) was used to identify transcriptional changes in MMPs. Asbestos exposure resulted in significant increases in MMP12 mRNA levels in the lungs of WT mice at 3, 9, and 40 d (Fig 1
A). Asbestos-induced up-regulation of MMP12 mRNA levels was significantly attenuated in PKC (–/–) mice (Fig 1B
). Gene profiling also revealed an elevation of MMP13 levels after asbestos inhalation for 9 d that was reduced in PKC (–/–) mice (Fig 1C
). Microarray results were confirmed and expanded using ribonuclease protection assays [rNase protection assay (RPA)] (n=6 mice/group/time point). Increased steady-state mRNA levels of MMP12, MMP13, and TIMP1 were observed in lungs of WT mice exposed to asbestos, whereas other MMPs (MMP3, 7, 8, and 9) and TIMP2 and 3 did not change. As observed in microarrays, PKC (–/–) mice showed reduced steady-state mRNA levels of MMP12 and MMP13 compared with WT animals. PKC (–/–) did not inhibit asbestos-induced TIMP1 mRNA levels.
2. 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 epithelial cells and fibroblasts, the known target cell types of asbestos-induced lung cancers and fibrosis, respectively. Murine alveolar epithelial type II cells (C10) exposed to asbestos (5 µg/cm2) for 4, 8, and 24 h showed time-dependent increases in steady-state mRNA levels of MMP13 and TIMP1 as determined by RPA. 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 as confirmed by quantitative reverse-transcriptase polymerase chain reaction (TaqMan).
To show that increased steady-state mRNA levels of MMPs by asbestos were not due to stabilization of mRNA, epithelial cells were treated with actinomycin D 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, indicating transcriptional up-regulation of MMP13 and TIMP1 by asbestos.
3. Multiple cell signaling pathways are involved in regulation of asbestos-induced MMP12, MMP13, and TIMP1
To reveal the signaling pathways involved in regulation of MMPs and TIMP1 transcription by asbestos, C10 cells (MMP13 and TIMP1) or primary lung fibroblasts (MMP12) were exposed to different small molecule kinase inhibitors before addition of asbestos for 24 h. As shown in Fig 2
A, pretreatment of cells with an ERK1/2 inhibitor (U1026, 10 µM) decreased asbestos-associated increases in MMP12, MMP13, and TIMP1. Whereas the PI3K inhibitor LY294002 (10 and 20 µM) inhibited transcription of both MMP12 and MMP13 by asbestos, the EGFR phosphorylation inhibitor AG1478 (10 and 20 µM) inhibited MMP13 expression significantly but MMP12 expression only slightly (Fig 2B
). The PKC-specific inhibitor rottlerin (5 µM) blocked asbestos-induced transcription of both MMP12 and MMP13; however, a general PKC inhibitor, bisindolymaleimide I (Bis, 5 µM), inhibited asbestos-induced up-regulation of MMP12 but had no effect on steady-state mRNA levels of MMP13 (Fig 2C
).
4. MMPs regulate asbestos-induced EGFR activation and ERK1/2 phosphorylation
To directly show the consequences of MMPs up-regulated by asbestos, C10 cells were pretreated with the broad range MMP inhibitor GM6001 (10 µM), 1 h prior to addition of asbestos fibers for 8 h. GM6001 significantly inhibited both asbestos-induced ERK1/2 phosphorylation and EGFR activation. These events may be mediated by shedding of EGF by MMPs (a known phenomenon), which can then activate EGFR and down-stream ERK1/2 signaling. These studies show that asbestos can up-regulate MMPs via multiple signaling pathways and that MMPs can further activate asbestos-induced responses via subsequent activation of EGFR (Fig. 3
) in epithelial cells.
CONCLUSIONS AND SIGNIFICANCE
Asbestos fibers cause pulmonary fibrosis and lung cancers, diseases that involve epithelial cell-fibroblast interactions and result in lung remodeling. MMPs are a family of secreted or transmembrane zinc-dependent endopeptidases that can degrade extracellular matrix (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.
We show that asbestos exposure causes increased MMP12, MMP13, and TIMP1 transcription both in vitro (lung epithelial cells and fibroblasts) and after inhalation of fibers. Using PKC (–/–) mice, we also show that PKC plays an important role in transcriptional up-regulation of asbestos-induced MMP12 and MMP13. Further studies using cell cultures and small molecule inhibitors showed that asbestos-induced MMP up-regulation is dependent on signaling by growth factor receptors as well as the PI3K, PKC and ERK1/2 pathways.
MMPs up-regulated by asbestos can further activate asbestos-induced signaling pathways in epithelial cells. Inhibition of MMPs inhibited EGFR activation and ERK1/2 phosphorylation by asbestos, suggesting a role for MMPs in previously characterized asbestos-induced signaling pathways. In support of our findings several studies have shown that various MMPs play a critical role in shedding of EGF and activation of the EGFR.
In conclusion, our study is the first demonstration that asbestos can transcriptionally up-regulate MMP12 and MMP13 in a growth factor receptor/PI3K/PKC/ERK1/2-dependent manner (Fig. 3)
. Once up-regulated, MMPs can further activate asbestos-induced signaling pathways via EGFR activation. The interplay between asbestos-induced MMPs and TIMPs may be crucial in the development of asbestos-induced lung diseases, and relevant signaling pathways or MMPs may be targeted for intervention and therapy.
FOOTNOTES
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Copyright © 2006 by The Federation of American Societies for Experimental Biology.
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