Expression of matrix metalloproteinases, tissue inhibitors of metalloproteinases, and extracellular matrix mRNA following exposure to mineral fibers and cigarette smoke in vivo.

To determine the effect of mineral fibers and cigarette smoke on remodeling of lung tissues, we examined matrix metalloproteinase-1 (MMP-1), MMP-2, tissue inhibitors of metalloproteinase-1 (TIMP-1), TIMP-2, and types I and IV collagen mRNA levels from rat lungs exposed to mineral fibers and/or cigarette smoke in vivo. Male Wistar rats (10 weeks of age) were given a single intratracheal instillation of 2 mg of chrysotile or alumina silicate ceramic fibers (RCF). Animals were then exposed to cigarette smoke (side stream) 5 days per week for 4 weeks. Transcriptional levels of mRNA extracted from the lungs were assessed by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR). Exposure to cigarette smoke induced increases in MMP-1 and TIMP-1 mRNA levels and decreased TIMP-2 and type I collagen mRNA levels in lung. Chrysotile or RCF stimulated the expression of MMP-1 mRNA in the lung. The mineral fibers and cigarette smoke had more than additive effects on the expression of MMP-2 and TIMP-1 in the lung. These data suggest that the imbalance of the expression of MMPs, TIMPs, and extracellular matrix may be associated with the remodeling of lung tissues induced by mineral fibers and/or cigarette smoke. ImagesFigure 2.Figure 6. AFigure 6. BFigure 6. CFigure 6. DFigure 6. EFigure 6. F


Introduction
Occupational and environmental inhalation of asbestos and various types of man-made mineral fibers is thought to cause remodeling of lung tissues that can result in fibrosis (1)(2). Furthermore, some reports show that combined exposure to mineral fibers and cigarette smoke may result in remodeling of lung tissues (3). Extracellular matrix (ECM) degradation presumably contributes to the initial phase of tissue remodeling inherent to the physiological processes of morphogenesis, angiogenesis, inflammation, and wound healing (4). Matrix metalloproteinases (MMPs) are a family of zinc and calciumdependent endopeptidases that play a key role in ECM remodeling in the lung (5). MMPs have the combined ability to degrade the various components of connective tissue matrices specifically directed to ECM components (4,5). However, the activities of MMPs are controlled at several levels including their interactions with specific inhibitors, e.g., the tissue inhibitors of This paper is based on a presentation-at The Sixth International Meeting on the Toxicology of Natural and Man-Made Fibrous and Non-Fibrous Particles held 15-18 September 1996  yasuom@med.uoeh-u.ac.jp Abbreviations used: ECM, extracellular matrix; MMP, matrix metalloproteinase; NIH, National Institutes of Health; NS, normal saline; PCR, polymerase chain reaction; RCF, refractory ceramic fiber; RT-PCR, reverse transcription-polymerase chain reaction; SD, geometric mean diameter; TIMP, tissue inhibitor of metalloproteinase. metalloproteinases (TIMPs) (5). MMP-1 is an interstitial collagenase that specifically degrades connective tissue fibrillar collagen (type I collagen), and MMP-2 is a gelatinase that degrades type IV globular basement membrane collagen (4). The representative specific inhibitors MMP-1 and MMP-2 are TIMP-1 and TIMP-2, respectively (5). An imbalance of MMPs and TIMPs results in metalloproteinase activation, and relatively higher levels of MMPs than TIMPs may stimulate the degradation of collagen in the interstitial space and beneath epithelial and endothelial cells, leading to acute injury (6).
To determine the effect of the mineral fibers and cigarette smoke on remodeling of rat lung tissues, we examined MMP-1, MMP-2, TIMP-1, TIMP-2, and types I and IV collagen mRNA levels from rat lungs exposed to mineral fibers and cigarette smoke in vivo, using reverse transcription-polymerase chain reaction (RT-PCR).

Fibers
The fibers used in this study were chrysotile (Union Internationale Contre le Cancer) and alumina silicate ceramic fibers (RCF) (Japan). For chrysotile the geometric mean diameter (SD), geometric mean length, and number/mass were 0.085 (1.4) pm, 0.7 (1.9) pm and 4.0x 104/pg, respectively. For RCF the SD and geometric mean length were 1.2 (1.7) pm and 9.6 (1.9) pm, respectively. In our previous report, the detailed physical and chemical characteristics of these fibers were investigated (7).

EFxperimental Design
Male Wistar Rats (10 weeks of age) were used in this study, and thirty rats were divided into six groups (saline, chrysotile, RCF, cigarette smoke, chrysotile + cigarette smoke, RCF + cigarette smoke). Saline or chrysotile suspension or ceramic suspension (2 mg/0.2 ml) was administered to rats intratracheally. Rats were housed in an exposure chamber (8), and 20 cigarettes were smoked for 4 hr/day, 5 days/week for 4 weeks. The concentration of cigarette smoke in the chamber was 10 mg/m3, as measured gravimetrically from glass fiber samples. The chamber volume was 100 liters and the flow rate in the chamber was 50 liters/min. Environmental Health Perspectives * Vol 105, Supplement 5 * September 1997 Preparation of RNA, cDNA Synthesis, and Polymerase Chain Reaction RNA was extracted from the lung using guanidinium thiocyanate-phenol-chloroform (9). Total RNA (0.5 pg) was used for the synthesis of single-strand cDNA using Moloney murine leukemia virus-derived reverse transcriptase as described by Wang and Mark (10). The amplification was performed with a Thermocycler (Astech, Japan) under the following conditions: 940C for denaturation for 45 sec, 60°C for annealing for 45 sec, and 720C for extension for 2 min for MMPs, TIMPs, collagen, and  (10). PCR products were resolved by gel electrophoresis and visualized by ethidium bromide staining. The gels were photographed with Polaroid Type 665 positive/negative film (Polaroid Corporation, Cambridge, MA) over UV light at the same exposure and developing time. The bands of the positive film were scanned and the density of each PCR product was measured using National Institutes of Health (NIH) image 1.55 software (written by W. Rasband at NIH, Bethesda, MD). To quantify the transcriptional level of mRNA, the data were normalized to represent equivalent RNA loading based on the density of Pactin at the appropriate cycle of a given gene product (10). to 0-actin the group exposed to chrysotile+cigarette saline con-smoke was maximally expressed relative to all groups. Elevations in MMP-2 mRNA A were not observed in lungs exposed to mineral fibers or cigarette smoke. However, the level of MMP-2 mRNA was elevated significantly in rat lungs exposed to chrysotile+cigarette smoke and RCF+ cigarette smoke (Figure 2). Expression ofTIMP-1 and TIMP-2 mRNA in Lungs Transcripts for TIMP-1 in the control-or mineral fiber-treated lung were not detected ( Figure 3). However, gene expression of TIMP-1 in groups exposed to cigarette smoke and/or mineral fibers was observed. Combined effects of chrysotile and cigarette smoke on message level of TIMP-1 in rat lungs were observed.
Although mineral fibers did not change TIMP-2 mRNA levels in the lung, cigarette smoke markedly decreased levels of TIMP-2 mRNA in the lung. Combined effects of the two agents were not observed ( Figure 4).

Expression ofTypes I and IV Collagen mRNA in Lung
In rats exposed to mineral fibers, mRNA levels for type I collagen in the lung were not increased. Cigarette smoke decreased in type I collagen mRNA levels in rat lungs ( Figure 5). Message for type IV collagen in the lung was not detected in any of the groups except in a positive control, although the cycle number for PCR in these experiment was up to 40. Results are expressed as the ratio of MMP-2 to P-actin (mean ±SEM). *p<0.05 in comparison with saline control rats. **p<0.0l in comparison with saline control rats. M, DNA marker (ox 174 Hae Ill).

Pathological Findings
Environmental Health Perspectives -Vol 105, Supplement 5 * September 1997 MMP-2, lung m-NS I groups exposed to chrysotile and chrysotile + cigarette smoke ( Figure 6). In the other four groups, definite pathological changes were not observed.  (11) showed that exposure to facilitate inflammatory cell migration and induce ECM solubilization, subsequently extending the area of connective tissue destruction (6). In this sense, MMP-1 in the lung may contribute to the remodeling of lung tissues exposed to chrysotile and/or cigarette smoke. An additive, combined effect of two agents on the gene expression of TIMP-1 in the lung was observed in our study. This phenomenon might represent a compensatory mechanism against increases in MMP-1; it has been reported that TIMP suppressed the aberrant activation of MMP (17), and TIMPs and MMPs are coexpressed in many cell types in response to a variety of inducing stimuli (18). MMP-2, TIMP-2, and Type IV Coligen Mineral Fibers and Cigarette Smoke. In our study, single exposure to mineral fibers or cigarette smoke alone did not cause an increase in levels of MMP-2 mRNA in the lung. D'Ortho et al. (19) demonstrated an increase in the gelatinase and stromelysin activities in the bronchoalveolar lavage fluid in the course of acute lung injury in rats exposed to ozone. However, MMP-2, unlike other MMPs, was not affected by proinflammatory cytokines or PMA, which suggests a specific regulation of MMP-2 (20). The gene of TIMP-2 was expressed in the saline-exposed lung, whereas exposure to mineral fibers did not induce message levels of TIMP-2. TIMP-2 is expressed in many normal tissues; mRNA is expressed in most mesenchymal-derived tissues (21,22). A series of ras-transformed derivatives of C3H1OT1/2 fibroblasts showed great variability in TIMP-1 expression, whereas TIMP-2 expression was insensitive to transformation (22). However, we found that TIMP-2 mRNA levels in the lung were markedly decreased by cigarette smoke, which might result in the elevation of gelatinolytic activities. Skup and Ponton (23) reported that increased metalloproteinase activity in different pathologies could be due to decreased levels of the specific TIMP. In our experiments, the message for type IV collagen in the lung was not detected in all groups. The primer for type IV collagen was based on the sequence of the mouse. Using the same primer, Kaneto et al. (24) reported that the level of type IV collagen mRNA measured by RT-PCR was increased in rat kidney with interstitial fibrosis but undetectable in control kidney. Hence, our failure to detect type IV collagen mRNA was not related to primer selection. Combined Exposure to Mineral Fibers and Cigarette Smoke. Combined exposure to mineral fibers and cigarette smoke markedly induced an increase in levels of MMP-2 mRNA in the lungs. Therefore, increases in MMP-2 induced by the two agents suggest a possible elevation of gelatinolytic activity that could degrade basement membrane. Nery et al. (25) reported additive effects of smoking and exposure to silica dust in increasing epithelial permeability in patients with silicosis who smoke.

Conclusion
In summary, we examined gene expression of MMPs, TIMPs, and collagen in the rat lungs exposed to mineral fibers and/or cigarette smoke. Mineral fibers and cigarette smoke had combined effects on the expression of MMP-2 and TIMP-1 in the lungs. These data suggest that MMPs and TIMPs may be associated with remodeling of lung tissues induced by mineral fibers and cigarette smoke.