1 Instituto Nacional de Cancerología, Mexico DF 14000; 3 Instituto Nacional de Enfermedades Respiratorias, Mexico DF 14080; 4 Facultad de Ciencias, Universidad Nacional Autónoma de México, Mexico DF 04510, Mexico; and 2 University of Sheffields, Sheffields S10 2RX United Kingdom
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ABSTRACT |
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Tissue
inhibitors of metalloproteinases (TIMPs) play a key regulatory role in
extracellular matrix remodeling. By screening a lung library with a
human TIMP-2 cDNA probe, we have isolated the cDNA corresponding to
guinea pig TIMP-2. The 3.5-kb cDNA presents an open reading frame that
predicts a protein of 220 amino acids showing 97.2, 96.8, 97.2, and
77.3% overall identity with human, mouse, rat, and chicken TIMP-2,
respectively. Guinea pig TIMP-2 cDNA was expressed in CHO-K1 cells,
showing a protein with the expected molecular weight and activity.
Northern blot analysis revealed TIMP-2 expression in brain, kidney,
intestine, spleen, heart, and lung. Transforming growth factor-
downregulated TIMP-2 mRNA in guinea pig lung fibroblasts, whereas a
variety of other stimuli showed no effect. In normal and
hyperoxia-exposed lungs, TIMP-2 mRNA was mainly localized in alveolar
macrophages and epithelial cells. No quantitative differences were
found by Northern blot. These results confirm that TIMP-2 is highly
conserved in mammals and largely expressed in lungs.
tissue inhibitor of metalloproteinase-2; matrix metalloproteinase; hyperoxia
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INTRODUCTION |
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TISSUE INHIBITORS OF METALLOPROTEINASES (TIMPs) are a family of secreted proteins that regulate matrix metalloproteinase (MMP) activity. The TIMP family, currently consisting of four well-characterized members (TIMP-1, TIMP-2, TIMP-3, and TIMP-4), plays a major role in physiological events that involve tissue remodeling and repair and exhibits multifunctional roles including cell growth-stimulating activities and protection from apoptosis (2, 12, 27). The four known TIMPs share many properties but also have distinct activities, suggesting that they play different specific roles. TIMP-2 is a major constituent in the lung that is capable of inhibiting the activities of all known MMPs. Furthermore, it has been proposed to be an adapter molecule that allows proMMP-2 to become associated with the membrane type of MMP, forming progelatinase-TIMP-2-membrane-type MMP complexes that promote gelatinase A activation (26).
The guinea pig is a rodent that has been used as a model for several diseases including asthma, acute lung injury, osteoarthritis, and pulmonary emphysema (7, 8, 12, 16, 17, 20, 23). In most of these disorders, an imbalance in MMPs and/or TIMPs has been suggested, and, therefore, it is important to know the possible role of these molecules that are involved in the extracellular matrix remodeling in these pathological conditions.
Exposure to elevated concentrations of O2 causes extensive lung injury in all mammalian species studied to date (6). We have demonstrated the upregulation of gelatinases A and B in rat models of acute exposure to 100% O2 or subacute hyperoxia, suggesting that they might contribute to hyperoxic lung damage through the degradation of extracellular matrix components (19, 21). Concerning TIMPs, it has been reported that hyperoxia induces an increase in the mRNA encoding TIMP-1 in rats, rabbits, and mice (15, 19, 22), but studies with TIMP-2 are scanty.
TIMP-2 cDNAs have been cloned from rat, human, mouse, and, more recently, chicken (1, 3, 13, 24). In the present study, we describe the cloning of guinea pig TIMP-2 cDNA, the expression pattern in guinea pig tissues, its regulation in lung fibroblasts, and its expression and localization in a model of lung injury induced by 100% hyperoxia.
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MATERIALS AND METHODS |
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Isolation of guinea pig TIMP-2 cDNA. Total RNA from guinea pig adult lungs was isolated with TRIzol Reagent (GIBCO BRL, Life Technologies, Gaithersburg, MD). mRNA was purified with oligo(dT)-cellulose (USB, Cleveland, OH) and used to construct a directional library in Lambda Zap with a commercial kit (Stratagene, La Jolla, CA). Individual clones (1 × 106) were screened with human TIMP-2 cDNA (kindly donated by D. R. Edwards, University of East Anglia, Norwich, UK). Two clones were isolated and sequenced by the dideoxyribonucleotide chain-termination method with a Sequenase version 2.0 kit (USB). In any of the clones, a complete open reading frame was found. To isolate cDNA, 0.5 × 106 plaques from a newborn guinea pig lung library (28) were screened with the partial cDNA previously cloned. From this library, two clones were selected for further analysis and sequenced by an automated sequencer (ABI Prism model 310). The sequence of guinea pig TIMP-2 cDNA was deposited in GenBank (accession no. AF127803).
Guinea pig TIMP-2 protein expression. TIMP-2 cDNA was subcloned into the pBK-CMV eukaryotic expression vector (Stratagene). This construct was transfected into CHO-K1 cells with a cationic lipid {N-[1-(2,3-dioleoyl)propyl]-N,N,N-trimethylammonium methylsulfate (DOTAP), Boehringer Mannheim, Mannheim, Germany} as recommended by the manufacturer. The transfected cells were selected with G-418 antibiotic for 2 wk and then seeded in serum-free medium. The conditioned medium was collected and subjected to reverse zymography. Briefly, dialyzed samples (20 µl) were electrophoresed on a 12% SDS-PAGE with 1 mg/ml of gelatin (Sigma, St. Louis, MO) as the substrate and 100 ng/ml of purified gelatinases A and B (Chemicon International, Temecula, CA) as the source of substrate-degrading enzymes. After three rinses with 50 mM Tris · HCl, pH 7.5, and 2.5% Triton X-100, the gels were incubated for 15 h in 50 mM Tris · HCl and 10 mM CaCl2 at 37°C. Finally, the gels were fixed and stained with Coomassie blue.
Northern blot analysis. Total RNA was isolated from different
adult tissues of guinea pigs and from guinea pig lung fibroblasts (JH4
cell line, American Type Culture Collection). Lung fibroblasts were
grown in F-12K medium (GIBCO BRL, Life Technologies, Grand Island, NY)
supplemented with 8% fetal bovine serum under standard culture
conditions. At confluence, the cells were exposed to 10 ng/ml of either
epidermal growth factor, transforming growth factor (TGF)-,
interleukin-1
, or tumor necrosis factor (TNF)-
; 2 or 20 × 10
6 M phorbol 12-myristate 13-acetate; 1 × 10
7 M dexamethasone; 1 mM N-acetylcysteine;
and 0.4 mM H2O2. Ten micrograms of total RNA
derived from tissues or cells were electrophoresed, transferred to
nylon membranes, and hybridized with guinea pig TIMP-2 cDNA.
Experimental model of hyperoxia. Pathogen-free male Hartley guinea pigs weighing 400-450 g were exposed to humidified 100% O2 for 24 and 72 h maintained in a 68 × 99 × 83-cm forced-air environmental chamber. Food and water were available ad libitum. O2 concentration in the chamber was continuously monitored with Oxycheck Critikon (McNeil Laboratories, Irvine, CA). Three animals were killed at 24 and 72 h of hyperoxia. Control guinea pigs (n = 6) were kept under similar conditions in room air.
The animals were anesthetized, and the lungs were perfused with sterile saline solution through the left cardiac ventricle. The right lung was instilled with freshly prepared 4% paraformaldehyde for in situ hybridization studies, and the left lung was processed for RNA extraction and Northern blot as described in Northern blot analysis.
Separate exposures were performed for bronchoalveolar lavage and wet and dry lung weights. Lungs were lavaged by flushing twice with 10 ml of sterile saline solution at 37°C through a tracheal cannula. Seventy to eighty percent of the instilled volume was recovered without significant differences between hyperoxic and control guinea pigs. Total proteins were measured with the Bradford (5) method. Total cell counts were obtained on unfractionated bronchoalveolar lavage fluid (BALF) with a hemocytometer counting chamber. The BALF was then centrifuged at 400 g for 10 min at 4°C. The cells were fixed in 50% ethyl alcohol and 2% Carbowax (50% polyethylene glycol) and centrifuged at 400 g for 15 min. Several slides per sample were stained with hematoxylin and eosin and used for differential cell counting.
BALF and lung zymography. Lung samples (20 mg/ml) from control and hyperoxia-exposed animals were homogenized in 10 mM 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 20 mM HEPES, pH 7.5, and 150 M NaCl. After centrifugation, supernatant aliquots containing 5 µg of protein were used to analyze lung tissue gelatinase activity. For BALF zymograms, equal volumes of lavage fluid (10 µl) were mixed with Laemmli sample buffer containing 3% SDS. Gelatinase activity was detected on a gelatin substrate-SDS gel as previously described (20). Serum-free conditioned medium from human lung fibroblasts was used as gelatinase A marker, and serum-free conditioned medium from phorbol 12-myristate 13-acetate-stimulated U-2 OS cells was used as a marker of gelatinase B.
In situ hybridization. For in situ hybridization, a 484-bp fragment (nucleotides 376-850) derived from the opening reading frame of guinea pig TIMP-2 cDNA was used. The transcription of sense and antisense transcripts was performed with a labeling mixture containing digoxigenin-UTP (Boehringer Mannheim).
In situ hybridization was performed on 4-µm sections as previously described (20). Briefly, sections mounted on silanized slides were incubated in 0.001% proteinase K (Sigma) for 20 min at 37°C. After acetylation with acetic anhydride, the sections were prehybridized for 1 h at 45°C in a hybridization buffer. The sections were incubated with digoxigenin-labeled probes at 45°C overnight. Some sections were hybridized with digoxigenin-labeled sense RNA probe. The tissues were incubated with a polyclonal sheep anti-digoxigenin antibody coupled to alkaline phosphatase (Boehringer Mannheim) for 1 h at room temperature.
The color reaction was performed by incubation with Fast Red chromogen (Biomeda, Foster City, CA). Sections were lightly counterstained with hematoxylin.
Statistical analysis. For statistical comparison, two-tailed Student's t-test for unpaired observations was used. P < 0.05 was considered significant.
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RESULTS |
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Isolation and sequencing of cDNA clones encoding guinea pig
TIMP-2. With screening of two independent lung libraries with a
human TIMP-2 cDNA probe, four overlapping clones for guinea pig TIMP-2
were detected. Inserts were subcloned in the pBK-CMV eukaryotic
expression vector and sequenced. One insert was found to correspond to
a 3.5-kb cDNA, with an open reading frame encoding a putative protein
of 220 amino acids (Fig. 1). It presents
the unique regions characterizing TIMP-2 proteins (9), including the sequence DVGGKKEY (residues 103-110) and sequence
insertion NDIYGN (residues 59-64). The guinea pig TIMP-2 predicted
amino acid sequence revealed a high level of homology compared with other mammals that have been cloned to date (human, mouse, and rat
TIMP-2; overall identity 97.2, 96.8, and 97.2%, respectively). With
chicken TIMP-2, an overall identity of 77.3% was found (Fig. 2).
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Expression of TIMP-2 cDNA in CHO-K1 cells. CHO-K1 cells were
used to transiently overexpress guinea pig TIMP-2 cDNA. Although these
cells constitutively synthesize TIMP-2, the conditioned medium of cells
transfected with a plasmid containing TIMP-2 cDNA presented a threefold
increase in TIMP-2 activity compared with cells transfected only with
vector as revealed by reverse zymography (Fig.
3, top). Gelatin zymography showing
gelatinase B was performed in the same samples to evaluate similar
loading (Fig. 3, bottom).
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Expression of guinea pig TIMP-2 mRNA in tissues and lung
fibroblasts. Northern blot analysis revealed a relatively similar expression of TIMP-2 in the different tissues analyzed, with the exception of the liver where a faint signal was found (Fig.
4A). The size of the predominant
transcript detected was 3.8 kb, although when the films were
overexposed, a smaller transcript of 1.2 kb was also observed (data not
shown). Longer exposure of the blot and the use of specific 3'
probes revealed the presence of three transcripts as reported by
Hammani et al. (14) (data not shown).
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Guinea pig lung fibroblasts constitutively expressed TIMP-2 mRNA. When
the cells were exposed to TGF-, an ~50% downregulation was
noticed after normalization with glyceraldehyde-3-phosphate dehydrogenase (Fig. 4B). The decrease in TIMP-2 mRNA was
apparent as early as 18 h after exposure to TGF-
. TNF-
also
downregulated TIMP-2 expression, although when the same experiment was
performed in serum-free conditions, only an effect of TGF-
was
noticed (data not shown). In contrast, no effect on TIMP-2 expression was observed in the presence of other agents even when higher doses (50 ng/ml) were used.
Hyperoxia-induced lung injury. Animals were exposed to 100% O2 up to 3 days and were killed at 24 and 72 h. Wet-to-dry weight ratios were measured to estimate the degree of pulmonary edema. Although there was a trend toward higher values in the animals exposed to O2, no significant differences with the air control group were found (5.2 ± 0.2 vs. 5.46 ± 0.39 and 5.49 ± 0.23 at 24 and 72 h, respectively). BALF protein content revealed a significant increase in both O2-exposed groups (control, 103 ± 2 µg/ml; 24 h, 141 ± 4 µg/ml; 72 h, 194 ± 7.1 µg/ml; P < 0.01).
The number and types of cells obtained from BALFs are shown in Table
1. At 72 h of O2
exposure, a significant increase in total lung cells was observed
compared with that in the control lungs (P < 0.05).
Inflammatory cells in BALF from control guinea pigs consisted mostly of
macrophages. At 24 and 72 h of hyperoxia, a significant increase in the
percentage of eosinophils was observed (P < 0.03). This
increase was reflected in a decrease in the percentage of macrophages.
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Zymography of BALFs and lung tissue extracts. Gelatin
zymography of BALFs from control guinea pigs exhibited gelatinase A activity, and in some animals, gelatinase B was also observed. Compared
with BALF from control animals, BALF obtained at 72 h of hyperoxia
showed an increase in gelatinase A and B activity bands. At 24 h of
hyperoxia, some BALF values were similar to control values, whereas
others displayed a similar increase to that noticed at 72 h. A
representative zymogram is shown in Fig. 5A. In the case of lung tissue
extract supernatants, aliquots containing ~5 µg of protein (control
and experimental animals) were used. Gelatin zymography showed
increased gelatinases A and B at 72 h of O2 exposure (Fig.
5B). EDTA inhibited BALF and lung tissue gelatinolytic activity
bands (data not shown).
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Expression and localization of TIMP-2 in hyperoxia-induced lung
injury. We examined the expression of guinea pig TIMP-2 during hyperoxia-induced lung damage. To evaluate TIMP-2 expression, control
and hyperoxic lungs were processed for Northern blot analysis. Results
obtained from four different animals were semiquantified by
densitometry and normalized to 18S rRNA. There were no changes in the
TIMP-2 mRNA levels at 24 and 72 h of hyperoxia compared with that in
normal nonexposed control lungs (3.4 ± 2.2 and 10 ± 1.6% over
control value, respectively). A representative Northern blot is
illustrated in Fig. 6.
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The cellular source of TIMP-2 was evaluated by in situ hybridization
with digoxigenin-labeled antisense RNA probes. Lungs from control
guinea pigs revealed positive staining for TIMP-2 transcript in
alveolar epithelial cells, alveolar macrophages, and chondrocytes (Fig.
7, A and B). A similar
localization was observed in guinea pigs exposed for 24 h to a high
concentration of O2 (Fig. 7, D and E).
Although nonquantitative analysis was performed, positive staining was
more widely distributed, especially at 72 h of hyperoxia where alveolar
epithelial cells and free alveolar macrophages were noticed expressing
TIMP-2 mRNA (Fig. 7, F-H). Control tissues
evaluated with a sense riboprobe displayed no reactivity (Fig. 7,
C and I).
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DISCUSSION |
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In this study, we have isolated cDNA for guinea pig TIMP-2. The predicted amino acid sequence has a striking similarity to TIMP-2 from other species, conserving the general features of the TIMP family and the unique regions characterizing TIMP-2 proteins (9), including the sequence DVGGKKEY and the sequence insertion NDIYGN. Two of the clones isolated presented a large nontranslated 3'-end. This cDNA region, although previously not cloned from other species, coincides with the reported 3'-end of the human TIMP-2 gene (14), further supporting the presence of alternative polyadenylation sites for the different size transcripts.
The expression pattern of the tissues examined revealed a profile similar to that observed in other species, including low expression in the liver (18).
It has been reported that TIMP-2 is constitutively expressed and barely
regulated by several agents (4). TGF- has been shown to downregulate
TIMP-2 transcripts in several cell lines including human fetal and
adult lung fibroblasts and neoplastic cell lines (10, 25). Accordingly,
in this work, only TGF-
revealed a regulatory effect, inducing
TIMP-2 downregulation either in the presence of serum or in serum-free
conditions. TNF-
caused a slight decrease in TIMP-2 gene expression
in fibroblasts grown in the presence of serum. However, in the absence
of serum, no effect was noticed. A similar finding has been reported
for other genes including
1 type I collagen (11),
suggesting that the effect of TNF-
may be altered by one or more
serum factors.
The isolated cDNA for guinea pig TIMP-2 was used to examine the expression of this gene in lung injury induced by hyperoxia. Although it is difficult to identify unequivocally all the lung cell types at the level of light microscopy resolution, our results showed that free alveolar macrophages, alveolar epithelial cells, and chondrocytes expressed TIMP-2 mRNA in control lungs. Although by Northern blot analysis we were unable to demonstrate any change in the already high level of expression of TIMP-2 in control lungs, by in situ hybridization we observed a wider distribution of cells expressing TIMP-2 mRNA during hyperoxia. Free alveolar macrophages and putative alveolar epithelial cells were often positively stained. This pattern is similar to that observed for gelatinases in a rat hyperoxia model previously developed in our laboratory (21). Interestingly, an in situ zymography approach revealed in that model intense areas of gelatinolytic activity in the alveolar septa of hyperoxic lungs, suggesting that gelatinases were active in vivo. Similarly, in the model of hyperoxic injury in guinea pigs, an increase in gelatinase A and B activities was noticed in the lung tissue as well as in the BALF. It has been reported that TIMP-2 at low concentrations can enhance proMMP-2 activation and at high concentrations can inhibit MMP-2 activity (26). These findings let us speculate that the upregulation in the expression of TIMP-2 in some cells might not necessarily be related to the inhibition of MMPs but to the possible activation of gelatinase A.
In summary, we have isolated and cloned the TIMP-2 cDNA of the guinea pig and characterized the expression pattern in tissues and its regulation in lung fibroblasts. Additionally, we have evaluated its expression in an in vivo model of acute lung injury. Availability of guinea pig TIMP-2 cDNA will be a useful tool in the research of guinea pig lung disorders where an imbalance between MMPs and TIMPs is suspected.
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ACKNOWLEDGEMENTS |
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We thank Victor Ruiz and Javier Delgado for assistance with the experimental model.
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FOOTNOTES |
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This study was partially supported by Programa Universitario de Investigacion en Salud and Programa de Apoyo de Estudios de Posgrado Grant 002365 (Universidad Nacional Autónoma de México, Coyoacán, Mexico).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: A. Pardo, Facultad de Ciencias, UNAM, Apartado Postal 21-630, Coyoacán 04000, Mexico DF, Mexico (E-mail: aps{at}hp.fciencias.unam.mx).
Received 22 July 1999; accepted in final form 17 November 1999.
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