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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Murine exposure to silica is associated with enhanced tumor necrosis factor 
(TNF-) expression and matrix deposition. The regulation of TNF is mediated through TNF receptor (TNFR) activation of
transcription factors. In the present work we have studied the importance of the individual TNFR in silica-induced lung inflammation and matrix deposition in mice. We studied RNA expression of TNF, 1(I) collagen, interstitial collagenase (MMP-13), and its inhibitor (TIMP-1) in the lungs of silica-treated mice. Furthermore, we correlated MMP-13/TIMP-1 RNA abundance with activation of the transcription factors AP-1 and NF-
B in the lungs of C57BL/6 mice, and
of mice deficient in one of the two types of TNFR (p55
/ or
p75/), exposed to silica (0.2 g/kg) or saline by intratracheal instillation. Animals were killed 28 d after exposure and lung hydroxyproline (HP), TNF, 1(I) collagen, MMP-13, and TIMP-1 RNA abundance was measured. AP-1 and NF-B activation was studied by
gel-shift assays. Compared with C57BL/6 mice, p55/ and p75/
mice significantly (*p < 0.05) decreased lung HP accumulation in response to silica. All murine strains enhanced TNF and 1(I) collagen
mRNA in response to silica. Enhanced (p < 0.05) MMP-13 RNA expression was also observed in all murine strains in response to silica.
Enhanced (p < 0.05) TIMP-1 RNA expression was observed in
C57BL/6 mice, but not in p55/ or p75/ mice, in response to silica. NF-B activation was observed in all murine strains, whereas
AP-1 activation was observed only in C57BL/6 mice after silica treatment. These data suggest that TNFR deletion modifies MMP-13/
TIMP-1 expression in favor of matrix degradation.
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INTRODUCTION |
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Murine exposure to silica results in the development of lung
inflammation and accumulation of extracellular matrix with
characteristics similar to the fibroproliferative changes observed in humans after occupational exposure (1, 2). The
mechanisms responsible for the development of silica-induced
fibrotic lung injury are not fully understood but may involve
the upregulated expression of cytokines in the lung (1, 2). Tumor necrosis factor (TNF) has been shown to play an important role in the pathogenesis of silicosis (1, 2). Mice exposed to
silica overexpress lung TNF mRNA and show increased TNF
production in the lung (3, 4). This enhanced TNF expression
precedes the onset of fibroblast proliferation and subsequent
collagen deposition in the lung (3, 4). Neutralization of TNF
with anti-TNF antibodies or the administration of soluble
TNF receptors can prevent or diminish the development of silica-induced lung fibrosis in mice (3, 5).
The mechanisms by which TNF promotes the accumulation
of collagen in the lung of silica-treated mice are not completely understood and a controversial body of information
regarding the effects of TNF on collagen expression is available (6, 7). In vitro data suggest that TNF regulates collagen
transcription, and TNF has been shown to induce collagen
mRNA expression in cultured human fibroblasts (6, 7). However, the effects of TNF on collagen transcription may be mediated indirectly by upregulating the expression of cytokines
such as transforming growth factor 
(8).
Although these findings support a profibrotic role for TNF, data also suggest that TNF is capable of inducing matrix remodeling by enhancing the expression of metalloproteinases (9). Silica-exposed rats demonstrate enhanced lung expression of collagenase 3 (MMP-13), a matrix metalloproteinase that degrades mainly fibrillar collagens, and gelatinases A and B (MMP-2 and MMP-9), which are known to degrade type IV basement membrane collagen (10). Therefore, it is possible that TNF can induce extracellular matrix remodeling affecting both matrix deposition and degradation in response to silica.
TNF transduces its biologic activities by binding to two receptors of 55 and 75 kDa (11, 12). The trimeric occupation of the
TNF receptor by the ligand results in the recruitment of receptor-specific proteins that activate kinases and promote the activation
of transcription factors, such as NF-B and AP-1, that will induce
transcription of TNF-sensitive genes (11, 12). The importance of
the TNF receptors in the pathogenesis of silica-induced lung fibrosis is demonstrated by the fact that mice in which both of
these receptors have been deleted are protected from the inflammatory and fibrotic effects of silica (13). The contribution of the individual TNF receptors during silicosis is not well understood.
To study the role of TNF and its receptors in the pathogenesis of lung fibrosis, we evaluated the effect that deletion of either TNF receptor (p55 or p75) has on 1(I) collagen, interstitial collagenase (MMP-13), and tissue inhibitor of metalloproteinases 1 (TIMP-1) mRNA expression after silica exposure in mice. We also characterized the activation of the NF-B
and AP-1 transcription factors in the lungs of wild-type (silica-sensitive C57BL/6) and TNF receptor-deficient mice, and correlated this activation with changes in MMP-13 and TIMP-1
mRNA expression in murine lung.
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INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Chemicals
Silica particles were crystalline (-quartz) and had an average particle
size of 1 µm. These particles were filtered and sterilized at 200° C as
previously described (13). Silica suspension was made by adding sterile
0.9% NaCl and sonicating immediately before intratracheal instillation.
Animals
Specific pathogen-free female C57BL/6 mice (Charles River Laboratories, Kingston, NY) weighing 20 to 25 g (6-10 wk old) were housed in
pathogen-free cabinets and provided with food, and water ad libitum.
Mice deficient in either p55 (p55/) or p75 (p75/) TNF receptors
were generated at Immunex (Seattle, WA) on a C57BL/6 background
and have been previously described (Peschon and coworkers [13-15]).
Silica Treatment
Animals were anesthetized with tribromoethanol and then exposed to
silica as previously described (13). Briefly, silica (0.2 g/kg; approximately 5 mg per animal) in 0.06 ml of 0.9% NaCl was slowly instilled
into the tracheal lumen. Control mice received the same volume of
sterile saline. Twenty-eight days after silica exposure animals were
anesthetized, the descending aorta was severed, and the left lungs were
removed and stored at 
80° C for RNA and hydroxyproline analysis.
Morphology
Right lungs were fixed in situ for 2 h by the intratracheal instillation of 10% neutral formalin at a constant pressure of 20 cm H2O. Lung tissues were then sectioned sagittally and embedded in paraffin. Sections 4 µm thick were stained with hematoxylin-eosin or Masson trichrome for light microscopic examination.
Lung Hydroxyproline Content
Lung hydroxyproline concentration was determined spectrophotometrically as previously described (13, 16, 17). Hydroxyproline content was computed as micrograms of hydroxyproline per whole left lung.
Northern Analysis
Total RNA was extracted from the lung by a cesium chloride method
and Northern analysis was performed as previously described (13, 18).
Murine TNF (pMuTNF) and 18S cDNAs were obtained from the
American Type Culture Collection (Rockville, MD) and have been
described elsewhere (17, 19). The murine 1(I) procollagen plasmid
(pMColla1-I) was graciously provided by E. Vuorio (Turku University, Turku, Finland) (20).
RT-PCR for MMP-13 and TIMP-1
One microgram of RNA was treated with 1 U of DNase (GIBCO-BRL, Rockville, MD). First-strand cDNA was synthesized by reverse transcriptase (RT). Primers used for polymerase chain reactions (PCRs) were custom synthesized (GIBCO-BRL), and are summarized below.
The set of primers used for the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) PCR was as follows: 5'-CCCCTTCATTGACCTCAACT-3' and 5'-TTGTCATGGATGACCTTGGC-3' with an amplified product of 395 bp. MMP-13 nucleotides 5'-CATCCATCCCGTGACCTTAT-3' and 5'-GCATGACTCTCACAATGCGA-3' were used as primers to amplify a 383-bp segment. For TIMP-1 amplification the primers 5'-GACCACCTTATACCAGCGTT-3' and 5'-GTCACTCTCCAGTTTGCAAG-3' were used for a fragment size of 333 bp. To quantify the housekeeping gene GAPDH, a competitor was constructed by cutting with NcoI an internal fragment of 155 bp from a GAPDH cDNA. The competitor (cGAPDH) sequence was obtained by PCR amplification of the modified plasmid, using the primers for GAPDH. The competitor size was 240 bp. Fourfold serial dilutions of the standard competitor (5, 20, 50, and 100 pg) were coamplified with a constant amount of cellular cDNA (1 pg/µl). Cycling conditions were as follows: 95° C/10 min for 1 cycle; 95° C/30 s, 58° C/30 s, and 72° C/ 120 s for 40 cycles; and a final incubation at 72° C for 7 min. Aliquots (5 µl) of the PCR product were resolved on a 1.5% agarose gel containing ethidium bromide. Scanning densitometry (Kodak digital analysis system 120; Eastman Kodak, Rochester, NY) quantitated band intensities. The logarithm of the GAPDH-to-cGAPDH ratio was plotted as a function of the logarithm of known cGAPDH amount. The point of equivalence represents the concentration of GAPDH in the cDNA sample. For collagenase amplification 5 pg of GAPDH was used in 35 cycles, and TIMP-1 was amplified with 3 pg of GAPDH in 40 cycles.
In Situ Hybridization for MMP-13 and TIMP-1
Riboprobes for in situ hybridization were generated as previously described (21). Rat collagenase cDNA was kindly provided by C. O. Quinn (St. Louis University, St. Louis, MO). Mouse TIMP-1 cDNA was provided by D. Edwards (University of East Anglia, Norwich, UK). In situ hybridization was performed on 4-µm sections as previously reported, using digoxigenin-labeled riboprobes (21, 22).
Electrophoretic Mobility Shift Assay
Nuclear extracts from the lungs of silica-treated mice were prepared as
previously described (23). For retardation assays, NF-B consensus oligonucleotide 5'-GGGGACTTTCCC-3' and AP-1 consensus oligonucleotide 5'-CGCTTGATGACTCAGCCGGAA-3' (Santa Cruz Biotechnology, Santa Cruz, CA) were end labeled with [
-32P]ATP and T4
polynucleotide kinase (GIBCO-BRL Life Technologies, Gaithersburg,
MD). Five micrograms of protein from the crude nuclear extract was
mixed with the labeled probe. DNA-protein complexes were separated
on a 6% polyacrylamide gel. Competition assays were performed with a
400-fold excess of unlabeled probe or NF-B or AP-1 mutant oligonucleotide (Santa Cruz Biotechnology). Supershifts were performed by
adding to the binding mixture antibodies to p50, p65, or c-Jun/AP-1
(Santa Cruz Biotechnology). Densitometric analysis of the gels was performed with the use of a Gel Doc 2000 (Bio-Rad, Hercules, CA).
Statistics
All values are expressed as means + SEM. Differences between murine strains were analyzed by analysis of variance (ANOVA) with the Fisher protected least-squares differences (PLSD) test for pairwise comparison (StatView 4; Abacus Concept, Berkeley, CA). A p value of < 0.05 was considered significant.
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RESULTS |
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INTRODUCTION
METHODS
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Silica-induced Lung Injury in Wild-type and Individual TNF Receptor-deficient Mice
Histologic evidence of lung injury was evaluated by light microscopy 28 d after the intratracheal exposure of mice to either silica or saline (Figure 1). Saline-exposed wild-type or
TNF receptor-deficient mice (either p55/ or p75/) demonstrated normal lung histology (not shown). In contrast, silica
exposure resulted in the development of peribronchiolar areas
of inflammation in the lungs of C57BL/6 mice (Figure 1A). These areas of inflammation formed well-defined nodules consisting of hystiocytes and lymphocyte aggregates that surrounded the terminal bronchioles and vessels (Figure 1B). In
contrast to C57BL/6 mice, p55 TNF receptor-deficient mice
reacted to silica exposure with an accumulation of acellular,
proteinaceous material that filled many alveolar spaces of the
lung (Figure 1C). This proteinaceous material stained positive
with periodic acid-Schiff (data not shown) and contained few
polymorphonuclear cells (Figure 1D). However, the granuloma-like lesions that were clearly identified in C57BL/6 mice
(Figure 1A) were largely absent in the lungs of p55/ mice
(Figure 1C). Well-organized nodules were observed in the lungs of p75 TNF receptor-deficient mice in response to silica exposure (Figure 1E). However, in contrast to the lesions observed in the lungs of C57BL/6 mice, these were predominantly located in perivascular areas of the lung (Figure 1F).
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Lung Collagen Content after Silica Exposure
Lung collagen content was assessed by measuring hydroxyproline from each murine strain 28 d after silica exposure. As
depicted in Figure 2, silica exposure significantly increased the
hydroxyproline content in the lungs of all groups. There was a
105 ± 3% increase in lung hydroxyproline in C57BL/6 mice
(201 ± 14 µg/left lung) compared with saline-treated mice (98 ± 8 µg/left lung) (p < 0.05). Compared with saline-treated animals (80 ± 8 µg/left lung in p55/ mice and 83 ± 5 µg/left
lung in p75/ mice, respectively) silica exposure also induced
significant increases in the lung hydroxyproline content of
TNF receptor-deficient mice (132 ± 5 µg/left lung in p55/
mice and 118 ± 11 µg/left lung in p75/ mice, respectively).
However, the increases in lung hydroxyproline content in p55-/-
and p75/ mice were significantly less (66 ± 2 and 42 ± 3%)
than in C57BL/6 mice (p < 0.05).
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Strains were significantly different
(p < 0.05) compared with silica-treated C57BL/6 mice.
1(I) Collagen and TNF mRNA Expression
after Silica Exposure
We studied the possibility that the decreased levels of collagen
accumulation in the lungs of TNF receptor-deficient mice were
the result of decreased gene transcription. Therefore, we studied, by Northern analysis, the expression of TNF and 1(I) procollagen mRNA in the lungs of C57BL/6 and TNF receptor-deficient mice 28 d after silica exposure. Silica exposure
resulted in an enhanced expression of TNF and 1(I) procollagen mRNA in the lungs of both C57BL/6 and individual
TNF receptor-deficient mice (data not shown).
Interstitial Collagenase 3 and TIMP-1 RNA Expression in Murine Lung after Silica Exposure
Lung expression of interstitial collagenase MMP-13 mRNA and
its inhibitor (TIMP-1) mRNA was studied by PCR 28 d after
silica exposure (Figure 3A). Figure 3B illustrates the densitometric analysis (measured in arbitrary units) of the expression
of interstitial collagenase mRNA in the lungs of C57BL/6 and
individual TNF receptor deficient mice exposed to silica. Compared with saline-treated mice (C57BL/6, 1.5 ± 0.95 AU × 104; p55/, 1.1 ± 0.9 AU × 104; p75/, 1.7 ± 0.9 AU × 104), silica exposure significantly (p < 0.05) increased the expression of interstitial collagenase in the lungs of all the murine strains (C57BL/6, 3.1 ± 0.4 AU × 104; p55/, 2.6 ± 0.7 AU × 104; p75/, 3.3 ± 0.7 AU × 104) (p < 0.05). However, no differences were found in MMP-13 expression between the wild-type and both strains of TNF receptor-deficient mouse.
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Figure 3C illustrates the expression of TIMP-1 mRNA in
the lungs of C57BL/6 and individual TNF receptor-deficient
mice exposed to silica. Compared with control mice (C57BL/6,
4.2 ± 0.3 AU × 104; p55/, 5 ± 1.1 AU × 104; p75/, 3.2 ± 0.6 AU × 104) silica exposure resulted in significant increases
(p < 0.05) in TIMP-1 mRNA expression in the lungs of C57BL/
6 mice (8.2 ± 1.6 AU × 104; p < 0.05), but not in the lungs of
p55/ (6.2 ± 1.2 AU × 104) or p75/ (4.5 ± 0.7 AU × 104) mice.
In Situ Hybridization for Interstitial Collagenase (MMP-13) and TIMP-1 in Murine Lungs
We studied the localization of MMP-13 and TIMP-1 mRNAs in the lungs of silica-treated C57BL/6 and individual TNF receptor-deficient, mice using digoxigenin-labeled riboprobes. Figure 4 illustrates the hybridization signal for MMP-13 mRNA in the lungs of saline- and silica-treated mice. Silica exposure resulted in overexpression of MMP-13 in the lungs of C57BL/6 mice (Figure 4A), and in both strains of TNF receptor-deficient mice (Figures 4B and 4C). Inside silicotic nodules, MMP-13 signal was primarily observed in the cytoplasm of macrophages (Figure 4E). In addition, alveolar epithelial cells and alveolar macrophages located in the neighborhood of the granulomatous lesions showed intense cytoplasmic labeling (Figures 4F and 4G). In saline-treated mice the expression of MMP-13 was limited to bronchiolar cells and scattered free alveolar macrophages (Figures 4H-4J). The specificity of the hybridization signal was demonstrated by the absence of signal with the use of a sense riboprobe (Figure 4D).
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Figure 5 depicts the localization, by in situ hybridization, of TIMP-1 mRNA in the lungs of C57BL/6 and individual TNF receptor-deficient mice. Silica exposure resulted in TIMP-1 mRNA expression in granuloma-like lesions in wild-type and both strains of TNF receptor-deficient mice (Figures 5A-5C). An intense staining was noticed in the silicotic nodules as well as in epithelial and inflammatory cells of the neighboring areas of C57BL/6 mouse lungs (Figure 5A). In saline-treated mice, expression of TIMP-1 mRNA was primarily observed in bronchiolar epithelial cells (Figures 5E-5G). Control experiments using sense riboprobe displayed no reactivity (Figure 5D).
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Transcription Factor Activation in Murine Lung after Silica Exposure
To further understand the mechanisms involved in the modification of silica-induced MMP-13 and TIMP-1 mRNA upregulation in TNF receptor-deficient mice, we studied the activation of the transcription factors NF-B (Figure 6) and AP-1
(Figure 7) in the lungs of C57BL/6 and individual TNF receptor-deficient mice. Silica exposure resulted in significantly enhanced (p < 0.05) NF-B activation in the lungs of C57BL/6
and in both p55 and p75 TNF receptor-deficient mice 28 d after exposure (Figures 6A and 6B, respectively). The observed
NF-B binding could be competed by a nonlabeled NF-B
oligonucleotide. The use of an antibody specific to the p50,
but not the p65, subunit of NF-B caused a shift of the NF-B
complexes. In contrast, silica exposure resulted in significant
(p < 0.05) AP-1 activation in the lungs of C57BL/6 mice. This
response was significantly decreased in the lungs of individual
TNF receptor deficient mice (Figures 7A and 7B). Use of antibodies against AP-1 depleted the corresponding AP-1 band, confirming AP-1-binding specificity.
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INTRODUCTION
METHODS
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DISCUSSION
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TNF and its receptors play a fundamental role in the pathogenesis of silicosis (3, 5). Silica induces oxidative stress in the
lung, with an enhanced production of reactive oxygen species that upregulates the TNF promoter (24, 25). Once produced, TNF mediates its biologic activities by binding to its two
known receptors (11, 12). The 55-kD receptor contains a cytoplasmic death domain that signals cytotoxic effects and triggers NF-B activation (26, 27). The 75-kD receptor similarly
mediates NF-B activation, triggers cell proliferation, and in
murine models of lung injury modulates TNF-mediated inflammation (11, 15, 26). The importance of the TNF receptors
in the pathogenesis of fibrogenic lung disease has been demonstrated in mice in which both of these receptors have been
deleted. These animals are protected from the inflammatory
and fibroproliferative effects of silica (13). However, the contribution of the individual receptors to the pathogenesis of the
disease process is poorly understood.
In the present work we found that deletion of either one of the TNF receptors (p55 or p75) modifies the nature of the inflammatory response observed in the lungs of wild-type (C57BL/6) mice. C57BL/6 mice are silica sensitive, demonstrating enhanced TNF expression in their lungs, and developing inflammation and fibrosis in response to silica (4, 13). The characteristic lesion observed in the lungs of silica-treated C57BL/6 mice is the development of well-formed nodules of peribronchiolar predominance (Figure 1B). In the present work it was found that deletion of either one of the TNF receptors (p55 or p75) resulted in histologic differences in the formation of these lesions. Thus, the characteristic nodular lesion was observed less frequently in p55 TNF receptor-deficient mice, and although p75 receptor-deficient animals did form granuloma-like nodules in response to silica, their location were predominantly perivascular (Figure 1D). These data suggest a possible role for the p55 TNF receptor in the formation of granulomas in response to silica. Similar results have been observed in the liver of BCG-challenged p55 TNF receptor-deficient mice. In these mice a decreased granulomatous response to BCG was demonstrated (28).
An important observation of the present work is that the
deletion of TNF receptors significantly decreased the accumulation of collagen in comparison with the lungs of C57BL/6 silica-treated mice. This result should be the consequence of
changes in collagen turnover either by decreased collagen production and/or increased collagen degradation. The effects of
TNF on collagen expression are complex and TNF has been
shown both to promote and inhibit collagen production in
vitro (6, 7). Alveolar macrophages, retrieved from the lungs of
asbestos-exposed patients, release TNF that promote upregulation of the collagen gene when added to human fibroblasts
(6, 7). In vivo studies have demonstrated that overexpression
of TNF in the lungs of rats (transfer of TNF cDNA via adenovirus infection) or mice (transgenic mice overexpressing TNF) is
followed by the development of inflammation and the accumulation of collagen in the lung (8, 29). However, these studies did not evaluate in a direct manner the impact of TNF on
collagen accumulation and, therefore, it is not clear whether
TNF overexpression enhanced lung collagen synthesis or decreased collagen degradation. Interestingly, Piguet and associates demonstrated that the administration of anti-TNF antibodies to normal adult mice decreased lung and bone collagen
content without altering collagen mRNA levels (30). In addition, these authors reported that treatment of mice with anti-TNF antibodies or soluble TNF receptors can prevent the deposition or promote the resorption of collagen in the lungs of
silica-treated mice (3, 5). Our finding of decreased lung collagen accumulation in response to silica in TNF receptor-deficient mice cannot be explained by changes in lung collagen
transcription, because no differences in 1(I) procollagen mRNA
were found. In contrast, our data strongly suggest that the observed reduction in collagen deposition in the lungs of individual TNF receptor-deficient mice could be better explained by
an enhanced degradation of lung collagen.
Matrix metalloproteinases (MMPs) are a family of secreted or transmembrane zinc-dependent endopeptidases that are capable of degrading virtually all extracellular matrix and basement membranes components. On the basis of their substrate affinity and structural domains, MMPs have been classified as collagenases, gelatinases, stromelysins, and membrane-type metalloproteinases (31). MMP production and activity are highly regulated at different levels. In general, basal transcription in normal adult tissues is low but MMPs are upregulated by a variety of factors at transcriptional, posttranscriptional, and posttranslational levels as well as by the interaction of the secreted enzymes with their tissue inhibitors (TIMPs) (33, 34).
Silica has been shown to upregulate MMP expression both in vitro and in vivo (9, 10). Pérez-Ramos and coworkers showed in a rat model of silicosis that early silicotic granulomas exhibited intense staining for collagenase 3 and gelatinases A and B, and for TIMP-1 and TIMP-2. However, in late, highly fibrotic nodules MMP signals were scarce. As TIMPs showed only a moderate reduction in late silicotic nodules, compared with MMPs, the findings suggested that an imbalance in the expression of MMPs and TIMPs might be implicated in extracellular matrix remodeling and basement membrane disruption during experimental lung silicosis (10). However, in this study, MMP and TIMP levels were not quantified.
In the present study, we found an enhanced gene expression of MMP-13, which degrades mainly fibrillar collagens, and its inhibitor TIMP-1 in the lungs of silica-sensitive C57BL/6 mice. Most importantly, TIMP-1, but not collagenase, mRNA expression was significantly lower in the lungs of both types of TNF receptor-deficient mice compared with wild-type mice. Because collagenase activity depends on a tight balance between the expression of the active enzyme and its inhibitors, these data suggest that the absence of either of the TNF receptors promotes matrix degradation by effectively decreasing the overexpression of TIMP-1.
TIMP-1 is a multifunctional molecule that inhibits matrix metalloproteinase activity and promotes the proliferation of receptive cells. Thus, any reduction in TIMP expression is likely to lead to a higher level of collagenase activity, thus facilitating collagen breakdown, but also may influence fibroblast proliferation. The mechanisms responsible for the regulation of MMPs and their inhibitors in silicosis are not well known. TNF induces collagenase production by fibroblasts and endothelial and leukemic cells (9, 35). This effect of TNF on MMP expression appears to be transduced, at least in human dermal fibroblasts, by the p55 TNF receptor (37, 38).
The TNF-mediated signal transduction pathways involved
in the regulation of MMPs and TIMPs in silicosis are unknown
at this time. Studies looking at the transcription level of the
MMPs and their inhibitors suggest that the predominant regulation of their respective promoters is mediated by AP-1 (39-
41). Most published data regarding TNF activation of AP-1 indicate that it is mediated via the p55 TNF receptor, whereas
NF-B activation can be signaled by either of the TNF receptors (26). Our results demonstrated that each type of TNF receptor is competent to induce overexpression of MMP-13 RNA
in mouse lung in response to silica exposure. We also found
that in response to silica exposure wild-type (C57BL/6) mice
enhanced the activation of both transcription factors, NF-B and AP-1. In contrast to C57BL/6 mice, TNF receptor-deficient animals demonstrated only enhanced activation of NF-B, and exhibited a greatly decreased activation of AP-1 in
their lungs in response to silica exposure. These data may suggest a role for NF-B in the regulation of MMP-13. Interestingly, in collagen-induced arthritis in mice, the expression of
both AP-1 and NF-B increased collagenase 3 gene expression, although a better correlation was observed with NF-B
activation (42).
Murine TIMP-1 gene is expressed by most cells at a low basal level, and during acute remodeling its transcription is activated by a variety of stimuli. TIMP-1 has also been shown to be upregulated by TNF. A nonconsensus AP-1-binding site (5'-TGAGTAA-3') that is conserved in mammalian TIMP-1 genes has been shown to be a critical element in basal and serum-stimulated transcription (43). In our findings, the decreased AP-1 activation observed in the lungs of individual TNF receptor-deficient mice correlates well with their decreased TIMP-1 lung RNA expression, suggesting that AP-1 may play a role in the regulation of TIMP-1 expression. However, these results might be the reflection of altered populations of inflammatory cells in the lungs from the different murine strains rather than specific changes in the level of activation of AP-1. Therefore, further analyses performed in specific lung cell populations in which the changes in both AP-1 activity and TIMP-1 transcription can be localized to the same cell type are necessary to establish this regulation. Further studies evaluating the transcription regulation of MMPs and their inhibitors in the lungs of TNF receptor-deficient mice are necessary to understand the importance of TNF receptors in lung injury.
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Footnotes |
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Correspondence and requests for reprints should be addressed to Luis A. Ortiz, M.D., Section of Pulmonary Diseases, Critical Care, and Environmental Medicine, Department of Medicine SL9, Tulane University Medical Center, New Orleans, LA 70112-2699. E-mail: lortiz{at}mailhost.tcs.tulane.edu
(Received in original form February 23, 2000 and in revised form July 26, 2000).
Acknowledgments: The authors thank Drs. J. Peschon, A. Holian, O. Quinn, and D. Edwards for their generous gift of TNF receptor-deficient mice, silica particles, MMP-13, and TIMP-1 cDNA, respectively, and Mrs. Mary Chelles and Boioang Tonthat, respectively, for technical assistance in the preparation of lung tissues and Northern analysis.
Supported by NIH grants HL 03569 (to L. A. Ortiz), HL 03374 (to J. A. Lasky), and ES 08663 (to M. Friedman).
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References |
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INTRODUCTION
METHODS
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DISCUSSION
REFERENCES
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