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American Journal of Respiratory Cell and Molecular Biology. Vol. 28, pp. 504-511, 2003
© 2003 American Thoracic Society
DOI: 10.1165/rcmb.2002-0124OC

Tissue Inhibitor of Metalloproteinase-3 Downregulation in Lymphangioleiomyomatosis

Potential Consequence of Abnormal Serum Response Factor Expression

Xiaoning Zhe, Yan Yang, Sandhya Jakkaraju and Lucia Schuger

Department of Pathology, Wayne State University, School of Medicine, Detroit, Michigan

Address correspondence to: Lucia Schuger, M.D., Department of Pathology, Wayne State University, 540 E. Canfield St., Rm. 9248, Detroit, MI 48201. E-mail: lschuger{at}med.wayne.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Pulmonary lymphangioleiomyomatosis (LAM) is characterized by abnormal smooth muscle–like cell proliferation leading to tissue destruction and cyst formation. We demonstrate that serum response factor (SRF), a critical smooth muscle transcription factor, is overexpressed in LAM cells. To determine whether abnormal SRF levels might have a pathogenic role in LAM, we transfected SRF into mouse lung fibroblasts and performed a cDNA array analysis. High SRF level upregulated the expression of matrix metalloproteinase (MMP)-2 and MMP-14, two MMPs previously shown to be increased in LAM. In addition, SRF down-regulated tissue inhibitor of metalloproteinase (TIMP)-3, one of their inhibitors. TIMP-3 inhibition was further confirmed by reverse transcriptase/polymerase chain reaction, immunoblotting, and immunostaining of human lung fibroblasts transfected with SRF fused to DsRed2 (a red variant of green fluorescent protein). To determine the in vivo significance of our findings, we immunostained 12 LAM cases for TIMP-3. In eight of them, TIMP-3 was ubiquitously present in normal lung parenchyma, but it was absent in LAM lesions. In the remaining cases, including two out of five normal control lungs, the antibody immunoreacted exclusively with elastin, probably due to suboptimal tissue processing. Because timp-3–null mice develop spontaneous emphysema, our findings suggest that SRF-mediated TIMP-3 inhibition might contribute to the tissue damage seen in LAM.

Abbreviations: extracellular matrix, ECM • lymphangioleiomyomatosis, LAM • matrix metalloproteinases, MMPs • reverse transcriptase/polymerase chain reaction, RT-PCR • serum response factor, SRF • smooth muscle, SM • tissue inhibitor of metalloproteinases, TIMPs • tuberous sclerosis complex, TSC


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Lymphangioleiomyomatosis (LAM) is a rare systemic disorder affecting primarily childbearing-age women (1, 2). The disease is characterized by an abnormal proliferation of smooth muscle (SM)-like cells (LAM cells) in the pulmonary interstitium, as well as by the formation of thin-walled cysts and a high incidence of angiomyolipomas (3, 4). Morphologically, the abnormally proliferating LAM cells resemble immature SM cells and express SM proteins such as SM {alpha}-actin and desmin. In addition, some LAM cells express gp100, a melanocytic marker that is recognized by antibody HMB-45 (5). In many cases LAM cells are also immunoreactive for estrogen and/or progesterone receptors, but the role of these receptors in the pathogenesis of the disease remains unclear (6, 7). Although LAM cells lack significant cellular atypia, mitotic activity, or metastatic potential, over time these cells proliferate to obstruct and destroy the lung parenchyma, leading to progressive loss of pulmonary function and eventually death. LAM occurs almost exclusively in women, with the mean age of disease onset in the thirties (8). The most common presenting symptoms are dyspnea and pneumothorax (1, 8).

LAM occurs either as an isolated disorder or in association with tuberous sclerosis complex (TSC) (9). TSC is an autosomally dominant inherited disorder caused by a mutation in a tumor suppressor gene, referred to as tsc. The latter has two genetic loci, tsc1 and tsc2, and only tsc2 mutations have been found in association with LAM (10). The clinical manifestations of TSC include mental retardation, seizures, and hamartomatous tumors in brain, heart, kidney, lung, and skin. The critical role of TSC2 is strongly supported by the findings of Carsillo and coworkers (11), who demonstrated mutations in the tsc2 gene in LAM cells from sporadic LAM cases. The nature of the relationship between LAM and TSC, however, remains unknown. Recently Noonan and colleagues have shown that alteration of CaM signaling through TSC2 may be involved in the pathogenesis of LAM (12). Other investigators have shown that abnormal expression of cytokines such as insulin-like growth factor system and transforming growth factor-ß1 are also involved in the atypical phenotype of LAM cells (13, 14). Because the etiology of LAM is unclear, the therapeutic approaches have been limited and include the use of progesterone, tamoxifen, and lung transplant for patients with advanced disease (15).

We and others have found that SRF plays a critical role in SM developing myoblasts, in which it shows a high level of expression compared with adult SM (16, 17). Undifferentiated SM cell precursors express a dominant-negative truncated isoform of SRF, referred to as SRF{Delta}5. However, this isoform rapidly disappears upon myoblast differentiation (17), and it is not present in adult visceral SM (16, 17) or LAM-affected lungs (Zhe and Schuger, unpublished observations). Because LAM cells morphologically resemble immature SM myoblasts, we determined their SRF expression levels. Our studies demonstrated that LAM cells express high levels of SRF, supporting the concept that they might represent abnormal immature SM cells. SRF is a member of the MADs box family of transcription factors, and plays a critical role in the regulation of myogenesis (18). SRF binds to the CArG box or CArG box-like motif, an essential cis-element present in the muscle-specific proteins, such as SM {alpha}-actin, and stimulates their transcription (16, 17, 19, 20).

To determine whether high SRF levels may have a deleterious biological effect, we transiently transfected SRF into mouse and human lung fibroblasts and assessed the impact on gene expression. Among the genes modulated by SRF, we found upregulation of mmp-2 and mmp-14 expression, which have already been shown increased in LAM (21, 22), whereas it suppressed the expression of timp-3, one of their inhibitors. In comparison, SR{Delta}F5 transfection had no effect. Because, to the best of our knowledge, there are no publications on TIMP-3 in LAM, we studied the presence of TIMP-3 in the latter. Immunohistochemical examination confirmed significant decrease or absence of TIMP-3 in most LAM lesions. Because high MMP activity, as well as absence of TIMP-3, have been linked to lung damage (23, 24), our studies suggest that abnormal SRF levels may contribute to the pathogenesis of LAM through the promotion of an ECM enzymatic imbalance which favors ECM degradation and tissue destruction.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cells
Human lung fibroblast cell lines WI-38 and IMR-90 were purchased from American Type Culture Collection (Manassas, VA). Primary cultures of lung fibroblasts were obtained from adult CD-1 (ICR) BR mice (Charles River, Wilmington, MA), or from normal human lungs (through our Tissue Bank). The lungs were minced in 0.1% collagenase I (Sigma, St. Louis, MO) and incubated at 37°C for 60 min to obtain single cell suspensions. Fibroblasts were then isolated by differential plating as previously described (25). All cells were cultured in complete medium composed of Dulbecco's modified Eagle's medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Invitrogen), 1.0 mM of sodium pyruvate, 0.1 mM of nonessential amino acids, 1.5 g/liter of sodium bicarbonate, 100 U/ml of penicillin, 100 µg/ml of streptomycin, and 0.25 µg/ml of fungizone (all from Sigma).

Construction of SRF-Expressing Plasmids
SRF and SRF{Delta}5 cDNAs containing the full-length coding sequences, previously cloned into pGEM-T easy vector (Promega, Madison, WI) (26), were retrieved by EcoRI and ligated into EcoRI-digested pcDNA3 expression vector (Invitrogen). The orientation of the clones was determined by restriction digestion with Pst I and the sequences were confirmed by nucleotide sequencing. These constructs were used for transfection into mouse lung fibroblasts. To clone SRF cDNA into the pDsRed2-N1 vector (Clontech, Palo Alto, CA) by in-frame fusion to the N-terminus of DsRed2 protein (a red variant of green fluorescent protein), a set of primers were designed to generate the orientated SRF insert with the unique restriction sites Xho I and EcoR I: 5' forward primer: 5'-gccctcgagcactgagcgccatgtt-3' and 3' reverse primer: 5'-ggaattctttcactcttggtgctgtgggtg-3'. The PCR product was retrieved from the agarose gel by using the QIAEX II Gel Extraction Kit (Qiagen Inc., Valencia, CA). After this recombination, the stop codon of SRF cDNA was eliminated and the open reading frame of downstream DsRed2 protein was left unchanged. The correct orientation and in-frame fusion were confirmed by sequencing before transfection.

Transient Transfections
Cells were grown in 6-well plates to 70% confluence and transfected using lipofectamine plus reagent (Invitrogen) following the manufacturer's instructions. The recombinant plasmids and null vector (used as a control) were mixed with the lipofectamine reagent in a 1:3.5 wt/vol proportion, and the cells were transfected for 3 h in OPTI-MEM I medium (Invitrogen). After 3 h of incubation at 37°C, the transfection medium was replaced with complete Dulbecco's modified Eagle's medium and the cultures were incubated for 18–24 additional hours. The transfection efficiency for the SRF-DsRed2 plasmid construct varied from a minimum of 15% to a maximum of 60%, as indicated by percentage of cells showing red fluorescence. The SRF- and SRF{Delta}5-pcDNA3 plasmid constructs did not allow for determination of transfection efficiency because they expressed untagged protein; we assumed, however, that it was similar or slightly higher than that of the SRF-DsRed2 plasmid, because the untagged plasmids are smaller in size.

cDNA Array Analysis
This was done using the Atlas cDNA Expression Array System from Clontech and following the manufacturer's instructions. Briefly, 5 µg of DNase I (Invitrogen)-treated total RNA was isolated from SRF-, SRF{Delta}5-, and null vector–transfected mouse lung fibroblasts using TRIzol (Invitrogen). Total RNA was incubated with random primers provided with the cDNA array kit at 50°C. Reaction buffer, dNTPs, 35 µCi {alpha}-32P-dATP (Amersham Life Sciences, Arlington Heights, IL), and MMLV reverse transcriptase (Invitrogen) were then added and incubated for 25 min to allow cDNA extension. Reactions were terminated and purified by column chromatography. An aliquot of the eluted cDNAs was used to determine {alpha}-32P incorporation by scintillation counting. Equal counts of labeled probes from control and SRF transfected cell samples (> 100,000 counts/min) were added, respectively, onto nylon membranes containing over 500 dotted cDNAs together with several housekeeping cDNAs as positive controls (Cat. #7741–1; Clontech). The arrays were exposed to X-ray film (BioMax MR; Kodak, Rochester, NY) for 18–72 h in the presence of an intensifying screen at -70°C. Exposure time was adjusted for each array until the signals for the housekeeping genes were the same on both. The autoradiographs were scanned with a MultiImager-Max system (Bio-Rad Laboratories, Hercules, CA) and analyzed using Quantity One software (Bio-Rad) by constructing a grid with a window for each gene.

Immunoblot analysis
Cells were lysed and immunoblots were performed as previously described (27, 28). Goat polyclonal antibody to TIMP-3 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) was used at a concentration of 1 µg/ml. Rabbit polyclonal antibody that recognizes both SRF and SRF{Delta}5 (Santa Cruz Biotechnology Inc.) (17, 19) was used at a 1:200 dilution. Primary antibodies were detected with horseradish peroxidase–conjugated secondary antibody diluted 1:3,000 (Bio-Rad). The bands were visualized by chemiluminescence using a commercial kit (Amersham) according to the manufacturer's instructions. Semiquantitative densitometric analysis of the bands was accomplished using Quantity One software (Bio-Rad) following subtraction of background density. Results were calculated as fold changes from control values.

Reverse Transcriptase/Polymerase Chain Reaction Analysis
RNA was isolated from the human lung fibroblasts with TRIzol reagent (Invitrogen) following the manufacturer's instructions. The following primers were used for PCR: TIMP-1, 5' forward primer; 5'-aattccgacctcgtcatcag-3' and 3' reverse primer, 5'-tgcagttttccagcaatgag-3'; TIMP-2, 5' forward primer; 5'-gatgcacatcaccctctgtg-3' and 3' reverse primer, 5'-gtcgagaaactcctgcttgg-3'; TIMP-3, 5' forward primer 5'-ctgacaggtcgcgtctatga-3' and 3' reverse primer, 5'-ggcgtagtgtttggactggt-3'; TIMP-4, 5' forward primer 5'-cagaccctgctgacactgaa-3' and 3' reverse primer, 5'-agactttccctctgcaccaa-3'; GAPDH, 5' forward primer 5'-acccagaagactgtggatgg-3' and 3' reverse primer, 5'-gggtcttactccttggaggc-3'. Reverse transcriptase/polymerase chain reaction (RT-PCR) was performed with the GeneAmp RNA PCR kit (PerkinElmer, Foster City, CA) following the manufacturer's instructions. All amplifications shown here represent the product of 22 cycles. GAPDH primer set was used to produce a 464 base-pair amplicon as an internal control. Under the conditions used in these studies, plateau of internal control and most amplicons was reached at 30 cycles.

Immunohistochemistry
Formalin-fixed, paraffin-embedded tissues from 12 LAM cases were obtained from the NHLBI LAM Registry in compliance with the required regulations and consents. All the tissues were obtained during open lung biopsy, and all were diagnostic for LAM. Five normal lung controls were selected from our pathology archival material. Samples were then used to determine the expressions of SRF and TIMP-3 by immunohistochemical analysis. Serial 5-µm-thick sections from each of the cases were immunostained with antibodies against SRF, TIMP-3, SM {alpha}-actin (Boehringer Mannheim Biochemicals Inc., Indianapolis, IN), and HMB-45 (Dako Corp., Carpinteria, CA). Antibodies were all used at a concentration of 4 µg/ml. Staining was completed using a commercial peroxidase–antiperoxidase kit following the manufacturer's instructions (ABC kit from Vector, Burlington, CA) as previously described (27, 29). Human lung fibroblasts transfected with SRF-DsRed2 fusion plasmid were fixed for 5 min in absolute ethanol, immunostained for TIMP-3, and then exposed to a 1:60 dilution of mouse anti-goat fluorescein isothiocyanate secondary antibody (Sigma). Controls for these experiments included omission of first antibody, substitution with pre-immune mouse or goat IgG (Sigma), and pre-adsorption of anti–TIMP-3 antibody with 40 µg/ml of recombinant TIMP-3 (Novagen, Madison, WI) for TIMP-3 immunostaining.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Immunohistochemical Studies Revealed High SRF Level in LAM Lesions
Immunohistochemical analysis of LAM cases and normal lung controls showed low SRF levels in most lung cells, including bronchial and vascular SM (Figure 1, uppermost panels, arrows). In contrast, LAM cells presented strong SRF immunoreactivity (Figure 1, middle and lower panels). Although LAM cells are histologically distinguishable due to their immature/fetal appearance, the lesions were immunostained for SM {alpha}-actin and HMB-45 to confirm presence of LAM markers (small panels below each SRF-immunostained LAM lesion). These were counterstained blue with hematoxylin (Sigma). Two patterns of SRF localization were noticed, and these changed from one lesion to another within the same LAM case. In one type of pattern, SRF was detected within the nucleus of multiple LAM cells, but not in all of them (Figure 1, middle panels; arrowheads point to the lesion, arrow in inset points to SRF-positive nucleus). In such cases, SRF localization was unrelated to HMB-45 expression (not shown). The other type of SRF distribution was diffuse in the nucleus and the cytoplasm, with slight predominance in the nucleus (Figure 1, lower panels; arrowheads point to the lesion, arrows in inset point to SRF-positive nucleus and cytoplasm). In such lesions, all the cells showed SRF upregulation. The two patterns of SRF distribution were found in patients with sporadic LAM and with TSC inherited disease.



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  		Figure 1. Increased immunodetection of SRF in LAM lesions. Immunostaining of lung samples with anti-SRF antibodies shows very low SRF levels in bronchial and vascular SM (upper two panels, arrows). The same applies to the rest of the cells in the normal lung. LAM lesions, on the contrary show strong immunoreactivity for SRF (middle and lowest composed panels, arrowheads). Notice that in some LAM lesions, SRF concentrates in the nucleus (middle panels, with inset showing a higher magnification), whereas in others it localizes to the nucleus and the cytoplasm (lowest panels, with inset showing a higher magnification). Consecutive sections of each respective lesion were immunostained for SM {alpha}-actin and HMB45 (and counterstained with hematoxylin) to confirm that these are indeed LAM cells. Magnification bar: 10 µm in insets and HMB45 pictures, 60 µm in the rest.

 
High SRF Level Stimulates Expression of ECM Degrading Enzymes While it Downregulates TIMP-3 in Lung Fibroblasts
cDNA array analysis was done to identify potential effects of high SRF levels that might explain the tissue damage occurring in LAM. It should be stressed that all our studies involved transient transfections. Therefore, only a percentage of cells incorporated the plasmid construct, resulting in less pronounced differences in gene expression than those one should see in stable transfections. Western blotting confirmed upregulation of SRF production in cells transiently transfected with the SRF cDNA plasmid construct, whereas cells transfected with empty vector showed no significant change compared with untransfected cells (Figure 2A). Approximately 80% of the 588 mouse genes represented on the cDNA array membrane showed visible signals. The average intensity of all the nine housekeeping genes on the array was used to normalize the results. Densitometric analysis of the autoradiographs showed that upon SRF upregulation, ~ 12% of the genes were increased over 2-fold, whereas 4% were decreased. Among the genes upregulated by SRF were several ECM-degrading enzymes, including mmp-2 and mmp-14, and among the genes downregulated by SRF was timp-3 (Figure 2B). SRF-induced changes in mRNA levels for ECM proteases and inhibitors are summarized in Table 1. SRF{Delta}5 did not affect the proteolytic profile tested here (not shown). Bcl-2 and estrogen receptor, two proteins characteristically upregulated in LAM (7, 15, 30), were also found to be upregulated by SRF, and to a lesser extent by SRF{Delta}5 in our cDNA array screening (not shown).



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  		Figure 2. Transient transfection of SRF into mouse lung fibroblasts downregulates TIMP-3 and upregulates MMP-2 and MMP-14 expression. (A) Western blot demonstrating an increase in SRF in mouse lung fibroblasts transfected with a SRF-expressing plasmid, (SRF, middle lane), compared with cells transfected with empty vector (EV, left lane), or untransfected cells (UT, right lane). Lower row shows Ponceau S staining of the same membrane to demonstrate equal protein loading and transfer among lanes. (B) Portions of cDNA expression array demonstrating equal density for two controls (GAPDH and ornithine decarboxylase or ODC), decrease in TIMP-3, and upregulation of MMP-2 and MMP-14 in cells respectively transfected with empty vector (EV) and SRF expression construct. Dots represent duplicates of same cDNA. Notice that these are the results of transient transfections, therefore only part of the cells incorporated the SRF plasmid construct, blunting the differences between SRF overexpressing cells and controls.

 

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  		TABLE 1 SRF-induced changes in the expression of ECM-degrading enzymes and their inhibitors

 
Because timp-3 null mice develop lung damage (24), we decided to focus on this MMP inhibitor. To further confirm the effect of SRF over TIMP-3, we performed additional studies on human lung fibroblasts, including all other known TIMP family members, TIMP-1 through -4. Three types of human lung fibroblasts were used in these studies, WI-38, IMR-90, and normal lung fibroblasts in primary culture, and all yielded similar results. Therefore, we selected results from normal lung fibroblasts to prepare Figure 3. Semiquantitative RT-PCR showed no significant changes in the levels of TIMP-1 and TIMP-2 message, whereas TIMP-3 was downregulated, as in the cDNA array analysis (Figure 3A). We were unable to amplify TIMP-4 mRNA, and this observation was consistent with a previous study showing no TIMP-4 expression in normal lungs (31) (Figure 3A). Immunoblots confirmed TIMP-3 downregulation at the protein level in human lung fibroblasts transiently transfected with SRF, but not in those transfected with vector alone or SRF{Delta}5, used as controls (Figure 3B). All the experiments were repeated at least three times with each of the three human cell cultures to assure that the results were highly consistent.



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  		Figure 3. Transient transfection of SRF into human lung fibroblasts downregulates TIMP-3 expression. (A) RT-PCR showing TIMP-3 mRNA downregulation in SRF-transfected human lung fibroblasts compared with those transfected with empty vector or untransfected cells. GAPDH represents an internal control to demonstrate equal amplification. TIMP-1 and TIMP-2 show no significant change upon SRF upregulation, whereas TIMP-4 is not detected. (B) Immunoblots demonstrating TIMP-3 downregulation in SRF-transfected human lung fibroblasts comparing to SRF{Delta}5 and empty vector (EV) transfection and untransfected cells (UT). Third row showing Ponceau S staining of the same membrane used to detect TIMP-3 demonstrating similar protein loading and transfer among lanes.

 
To determine the effect of SRF on a per-cell basis, we transiently transfected human lung fibroblasts with SRF-DsRed2 and immunostained them for TIMPs 1, 2 and 3. SRF-transfected cells showed a significant downregulation in TIMP-3 immunoreactivity, whereas untransfected cells within the same culture were positive for the latter (Figure 4, three upper left panels). Corresponding Normaski microscopy was added to show all the cells in the field (Figure 4, three upper right panels). SRF-transfected cells showed no change in TIMP-1 and TIMP-2 expression (Figure 4, right and left lowest panels).



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  		Figure 4. Immunostaining of human lung fibroblasts shows TIMP-3 downregulation upon transient transfection with a SRF-DsRed2 plasmid construct. TIMP-3 was immunodetected using a fluorescein isothiocyanate–conjugated secondary antibody (green fluorescence). Cells expressing SRF-DsRed2 fusion protein are easily identified by their red-orange fluorescent nucleus. SRF-transfected cells show either traces or absence of TIMP-3 in their cytoplasm, whereas untransfected cells in the same field are positive for TIMP-3 (three left upper panels). Normaski image showing the profile of all cells in the field is presented in corresponding right panels. Immunostaining with anti–TIMP-1 and anti–TIMP-2 antibodies shows no changes in these two TIMPs after SRF-DsRed2 fusion plasmid transfection (lowest two panels). Magnification bar: 20 µm.

 
Immunohistochemical Studies Revealed TIMP-3 Downregulation in LAM Lesions
Immunostaining of 12 formalin-fixed, paraffin-embedded LAM tissue samples from 12 different patients demonstrated TIMP-3 absence in LAM cells in eight cases (Figure 5, upper and middle left panels and small panel in right upper corner). Notice the strong red-orange immunoreactivity for TIMP-3 in endothelial (black arrowheads) and alveolar epithelial cells (black arrows) compared with the bluish LAM cells (hematoxylin counterstaining, black asterisk). Serial sections corresponding to same areas have been immunostained for SM {alpha}-actin, to confirm that these are LAM cells (Figure 5, upper and middle right panels). In these eight cases, as well as in three out of five control lungs, TIMP-3 immunoreactivity was detected in most normal cells, including bronchial and vascular SM (white arrows), bronchial and alveolar epithelium (black arrows), endothelium (black arrowheads), fibroblasts (white arrowheads), and areas of collagen deposition (white asterisk). In the remaining four LAM cases and two controls, the antibody immunoreacted only with elastic tissue (not shown). When anti–TIMP-3 antibody was preadsorbed with recombinant TIMP-3, immunostaining, whether specific or nonspecific, disappeared in all cases. We interpreted the disappearance of crossreactivity as an indication that the antibody crossreacts with elastin through its active site. Therefore, anti–TIMP-3/TIMP-3 complexes were unable to bind either TIMP-3 or elastin (not shown).



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  		Figure 5. Decreased immunodetection of SRF in LAM lesions. Immunostaining using anti–TIMP-3 antibodies demonstrates absence or only traces of TIMP-3 in LAM lesions, which appear bluish due to hematoxylin counterstaining (upper and middle left panels and small panel in right lower corner of the composite, black asterisk). Consecutive serial sections immunostained for SM {alpha}-actin are presented for each of the same areas (upper and middle right panels). TIMP-3 immunoreactivity (red-orange) was detected in most normal cells, including bronchial and vascular SM (white arrows), alveolar and bronchial epithelium (black arrows), endothelium (black arrowheads), fibroblasts (white arrowheads), and areas of collagen deposition (white asterisk). Notice presence of TIMP-3–negative LAM cells in the same field as TIMP-3–positive cells (normal endothelial and epithelial cells) in the upper and middle left panels and in small panel at right lower corner. Magnification bar: 50 µm in lowest right four panels and 200 µm in the rest.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
LAM is a rare, fatal disease characterized by abnormal proliferation of SM-like cells in the lung, leading to progressive parenchymal destruction and eventually respiratory insufficiency (32). The mechanisms whereby the lung is damaged during the course of this disease are not fully understood. However, there is evidence suggesting increased ECM degradation as part of the pathophysiology of LAM (21, 22, 32). The molecular pathways that modulate LAM cells' ability to degrade ECM are currently unknown. In this regard, the studies presented here are the first to demonstrate that LAM cells have abnormally high levels of SRF and that this might contribute to enhanced ECM proteolysis. SRF is a critical muscle-related transcription factor highly expressed in developing SM cells (16), but with low expression in their mature counterparts (17, 19). In addition to being present in embryonic SM cells, SRF was present in the nucleus and cytoplasm of LAM cells (33).

To determine whether high SRF levels might contribute to the progression of LAM, we overexpressed it in lung fibroblasts and searched for genes whose alteration by SRF could account for the lung damage seen in this disease. We considered the possibility of using lung undifferentiated embryonic mesenchymal cells, because LAM might originate from an undifferentiated mesenchymal cell precursor. However, we ruled out this possibility based on the fact that embryonic mesenchymal cells undergo spontaneous SM myogenesis upon spreading in culture (17, 27, 28). Moreover, the identity of the LAM cell precursor is currently unknown. Like LAM cells, fibroblasts are of mesenchymal nature and express SM {alpha}-actin under several conditions (34). However, unlike LAM cells, fibroblasts have significantly lower levels of SRF, facilitating the study of SRF upregulation.

We found that SRF stimulated MMP-2 and MMP-14 (MT1-MMP), whereas it downregulated one of their inhibitors, TIMP-3. These two MMPs have been previously reported to be upregulated in LAM lesions (21, 22). Hayashi and coworkers studied the expression of MMP-1, -2, -3, and -9, and TIMP-1 and -2, and found that MMP-9 and particularly MMP-2 levels were increased in LAM lesions, whereas their inhibitors TIMP-1 and TIMP-2 were expressed at levels similar to those detected in normal lung parenchyma (21). MMPs are a family of zinc endoproteinases capable of degrading essentially all the ECM components (35). Therefore, Hayashi and coworkers (21) proposed that MMP-2 and MMP-9 upregulation might contribute to the connective tissue destruction and cyst formation occurring in LAM. MMP-14, the other metalloproteinase upregulated by SRF, was also reported to be increased in LAM cells, particularly in those cells that also expressed the cell proliferation marker PCNA (22). Because MMP-14 is a well-established activator of MMP-2 (36–38), Matsui and colleagues proposed that MMP-14 may contribute to pulmonary damage by acting upon MMP-2 (22) and that its presence is associated with a higher proliferation rate, another critical factor that should increase lung damage by LAM cells. Our data now suggest that MMP-2 and MMP-14 upregulation, as well as the increment in other ECM degrading enzymes, may be a consequence of SRF overexpression; thus, high SRF level may be an important factor in LAM cell-mediated tissue destruction.

In accordance to the overall data pointing to SRF as a promoter of ECM proteolysis, we found that TIMP-3 expression was inhibited by SRF. To the best of our knowledge, TIMP-3 was never studied in the context of LAM; therefore, we determined whether TIMP-3 was also downregulated in LAM lesions. In correlation to what we found in vitro, immunohistochemical studies demonstrated significant decrease to near absence of TIMP-3 in the lesions of eight out of twelve patients with LAM, whereas in each of these cases, TIMP-3 was ubiquitously present in the normal lung parenchyma. Unexpectedly, in the other four cases as well as in two out of five normal lung controls, the immunostaining pattern given by the anti–TIMP-3 antibody was completely different, decorating only elastic fibers. The reason for such differences in results is currently unclear. However, a likely possibility could be spurious cross-reactivity with elastin resulting from suboptimal histologic tissue processing. Although not reported in the literature, cross-reactivity of antibodies with elastin is not an uncommon artifact that can be seen after delayed fixation, prolonged exposure to formalin, or other tissue processing variables.

Although timp-1 and timp-2 deletions are known to cause no lung abnormalities (24), the lack of TIMP-3 may have particular significance in the progression of LAM, because timp-3–null mice are the only ones having a destructive lung phenotype, which leads to progressive emphysema and shorter life span (24). The protective effects of TIMP-3 may be related to its unique biological characteristics. First, TIMP-3 is the only nonsoluble member of the TIMP family (39, 40) and therefore binds to the ECM immediately adjacent to the cells, allowing for a more effective inhibition of pericellular MMPs. Furthermore, unlike other TIMPs, TIMP-3 is capable of inhibiting not only MMPs but also members of the adamalysin family, namely the ADAMs (a disintegrin and metalloproteinase) and the ADAMTSs (ADAM with thrombospondin-like repeat) (35). These two families of cell surface or secreted proteolytic enzymes have been shown to play a role in diseases of the lung and other organs (41–43).

The mechanism whereby SRF downregulates TIMP-3 is currently unknown, and overall the control of TIMP-3 gene expression has not been a focus of systematic study. Kang and colleagues have shown that gastric cancer cell lines lacking TIMP-3 expression have aberrant hypermethylation near the transcription-start site of the timp-3 gene, and that its expression can be reactivated by compounds that prevent methylation (44). This mechanism of timp-3 silencing has been demonstrated in several other circumstances (45, 46). Further studies are required to determine whether timp-3 DNA hypermethylation is playing a role in LAM. Another possibility is that LAM cells produce an autocrine/paracrine factor inhibitor of TIMP-3. This may explain why in some LAM lesions, in which SRF is expressed only in a portion of the cells, the decrease in TIMP-3 is generalized. It should be noticed that neither timp-3 nor mmp-2 or mmp-14 have CarG-elements in their 5'-flanking sequences; therefore, it is unlikely that SRF would exert its pro-proteolytic effect by direct binding to these genes.

In addition to increased MMP-2 and MMP-14 and decreased TIMP-3, our studies showed imbalances in other main ECM proteinases and corresponding inhibitors, all of them indicative of SRF-mediated increased proteolytic status. These findings seem to suggest that SRF acts broadly in the regulation of ECM remodeling process by altering different categories of proteinases and proteinase inhibitors. However, whether all or some of them are altered in LAM remains to be elucidated. Understanding the factors that modulate LAM cell ability to degrade ECM should provide a new basis for understanding and hopefully treating this disease.


   Acknowledgments
 
This work has been supported by NHLBI grants HL-48730 and HL-67100, a grant from the Children's Research Center of Michigan (to L.S.), and a Pilot Project Award from The LAM Foundation (to X.Z.). The authors are thankful to the patients with LAM for having donated their tissues for this study, and for the tremendous help of Mrs. Sue Byrnes, director of The LAM Foundation.

Received in original form July 19, 2002

Received in final form November 11, 2002


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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