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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2000;20:982.)
© 2000 American Heart Association, Inc.

Vascular Biology

Regulation of Lysyl Oxidase by Interferon- in Rat Aortic Smooth Muscle Cells

Yun Ling Song; John W. Ford; David Gordon; Charles J. Shanley

From the Departments of Surgery and Pathology, University of Michigan Medical School, Ann Arbor.

Correspondence to Charles J. Shanley, MD, Michigan Heart and Vascular Institute, PO Box 974, Ann Arbor, MI 48106.

	Abstract

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Abstract—Lysyl oxidase is an essential catalyst for the cross-linking of extracellular collagen and elastin. Abnormalities in lysyl oxidase activity may contribute to the pathogenesis of arterial diseases characterized by abnormal matrix remodeling. This study tested the hypothesis that interferon (IFN)-, a proinflammatory cytokine present in aortic aneurysm and arteriosclerotic plaque rupture, downregulates lysyl oxidase gene expression in rat aortic smooth muscle cells. Steady-state lysyl oxidase mRNA levels decreased in a concentration- and time-dependent manner to 30% of control levels after 24 hours of treatment with IFN-. Cell layer lysyl oxidase activity decreased in parallel with the observed changes in steady-state mRNA. Nuclear runoff studies suggested that transcriptional regulation was responsible for at least 40% of the observed downregulation. mRNA decay studies suggested that IFN- also decreased lysyl oxidase mRNA half-life from 9 to 6 hours. Downregulation of lysyl oxidase by IFN- did not appear to require new protein synthesis. This study documents that IFN- downregulates lysyl oxidase gene expression in rat aortic smooth muscle cells by transcriptional and posttranscriptional mechanisms. If similar regulation occurs in vivo, it is possible that IFN-–mediated changes in lysyl oxidase may contribute to arterial diseases characterized by abnormal extracellular matrix.

Key Words: interferon- • lysyl oxidase • smooth muscle cells

	Introduction

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Arterial aneurysm and arteriosclerotic plaque rupture are characterized by inflammatory cell infiltration and abnormal extracellular matrix remodeling.^{1 2 3} The basic mechanisms by which inflammatory cells regulate remodeling of the extracellular matrix are the subject of intense investigation.⁴ Cytokines, produced by a variety of cell types, contribute to the synthesis and degradation of extracellular matrix proteins. Interferon (IFN)-, a cytokine produced primarily by macrophages and T lymphocytes, inhibits collagen accumulation and fibronectin synthesis.⁵ Interestingly, T lymphocytes and IFN- are increased not only in arterial aneurysms but also in arteriosclerotic plaque rupture.^{2 6} Thus, it is possible that local production of IFN- exerts an important control mechanism for extracellular matrix remodeling in these disease processes. Unfortunately, the mechanisms by which IFN- regulates this remodeling remain poorly understood.

Lysyl oxidase (EC 1.4.3.13) is a copper-dependent metalloenzyme that is secreted by vascular smooth muscle cells and fibroblasts and that catalyzes a key step in the posttranslational cross-linking of collagen and elastin in the extracellular matrix.^{7 8 9} Reduced lysyl oxidase activity is associated with reduced cross-linking of nascent collagen and elastin.¹⁰ In humans, reduced lysyl oxidase activity is associated with increased skin laxity and joint hyperextensibility.^{13 14 15} In experimental animals, reduced lysyl oxidase activity is associated with aortic aneurysm development, and increased lysyl oxidase activity has been implicated in the pathogenesis of atherosclerosis.^{14 16} Furthermore, diminished cross-linking appears to increase the susceptibility of collagen and elastin to degradation by metalloproteinases.¹⁷ A better knowledge of factors that regulate lysyl oxidase expression may provide new insight into the pathogenesis of arterial diseases characterized by abnormal extracellular matrix remodeling.

Recent studies have demonstrated that certain cytokines regulate lysyl oxidase gene expression in vascular smooth muscle cells. Recent data from our laboratory have documented that the profibrotic cytokine, transforming growth factor-ß1, increases not only steady-state lysyl oxidase mRNA but also lysyl oxidase enzyme activity in cultured vascular smooth muscle cells.¹⁸ These findings have been confirmed and extended by other investigators.⁹ On the other hand, basic fibroblast growth factor downregulates lysyl oxidase gene expression.⁸ Thus, it appears that transcriptional and posttranscriptional mechanisms are involved in the positive and negative regulation of lysyl oxidase gene expression.^{8 9} Interestingly, an IFN-sensitive motif has been recently identified in the promoter of the lysyl oxidase gene.¹⁹ Thus, it seems logical to assume that lysyl oxidase may be an IFN-inducible gene; nevertheless, the available literature does not reference any studies dealing specifically with the role of IFN- in the regulation of lysyl oxidase gene expression.

The purpose of the present study was to test the hypothesis that IFN- downregulates lysyl oxidase gene expression in cultured rat vascular smooth muscle cells. Our results suggest that IFN- decreases steady-state lysyl oxidase mRNA levels in a time- and concentration-dependent manner. Furthermore, our data suggest that transcriptional and posttranscriptional mechanisms are involved. Taken together with the observation that local production of IFN- is increased in aortic aneurysms and arteriosclerotic plaque rupture, our data support the tenet that IFN-–dependent effects on lysyl oxidase gene expression may contribute to abnormal matrix remodeling in certain arterial diseases.

	Methods

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Materials
Recombinant human IFN- was from Collaborative Research. DMEM, FBS, trypsin-EDTA solution, and TRIzol reagent were from GIBCO-BRL. 5,6-Dichloro-1ß-D-ribofuranosylbenzimidazole (DRB), cycloheximide, and ß-aminopropionitrile were from Sigma Chemical Co. The Reverse Transcription System and Riboprobe Combination System were from Promega. The Genius System, a luminescence detection kit, was from Boehringer-Mannheim. [L-4,5-³H]Lysine was from Amersham. The pTrcHis2A vector and Xpress system were from Invitrogen. All chemicals were reagent grade.

Cell Culture
Rat aortic smooth muscle cells were isolated as previously described¹⁸ Cells were plated at 1.2x10⁶ cells per 75-cm² plastic tissue culture flask and maintained at 37°C in a humidified atmosphere of 5% CO₂ in air. Growth medium consisted of DMEM supplemented with 10% FCS, 0.68 mmol/L L-glutamine, 100 U/mL penicillin, and 100 µg/mL streptomycin. Medium was changed every 3 days. To determine the effect of IFN- on lysyl oxidase gene expression, 80% confluent cultures in passages 2 to 5 were washed in PBS and incubated for 24 hours in serum-free medium consisting of DMEM supplemented with 0.2% BSA and antibiotics. Quiescent cells were subsequently incubated in the presence or absence of log concentrations (0.01, 0.1, and 10 ng/mL) of IFN- for time periods of 0, 2, 4, 6, 8, 12, 24, and 48 hours.

RNA Extraction and Cloning of Lysyl Oxidase cDNA
Total RNA was extracted by using TRIzol reagent according to the method of Chomczynski and Sacchi²⁰ and further purified by digestion with RNase-free DNase for 60 minutes at 37°C. For cDNA synthesis, an aliquot containing 15 µg of purified RNA was added to 50 µL of an aqueous solution containing 300 U SuperScript II (Gibco) reverse transcriptase, 60 U RNasin (Promega), 25 µg random hexanucleotide primers, 5 µL of 10x reverse transcription buffer, and 1.25 mmol/L dNTP. The synthetic reaction was carried out for 60 minutes at 42°C. Polymerase chain reaction (PCR) was used to amplify lysyl oxidase cDNA. Oligonucleotide primers for rat lysyl oxidase were designed according to published sequences (sense 5'-CCG-GAT-CCA-TGC-GTT-TCG-GCT-GGA-CCG-TGC-TC-3', antisense 5'-CCG-TGA-GCT-TCT-TTC-TAA-TAC-GGT-GAA-AT-3').²¹ A typical PCR cycle consisted of denaturing for 1 minute at 94°C, annealing for 1 minute at 57°C, and extension for 3 minutes at 72°C. Forty cycles were carried out in the presence of 2 U Taq DNA polymerase. Restriction endonuclease cut sites were incorporated at the 5' end of the primers to facilitate subcloning. The PCR product was separated by electrophoresis and isolated from the agarose gel by use of a Geneclean II kit. The purified PCR product was digested with appropriate restriction enzymes and subcloned into the pGEM-3 plasmid vector. The sequence specificity of the cloned cDNA was further assessed with an automatic DNA sequencer (LKB) by the dideoxy chain termination method.

Northern Analysis
Purified total RNA (15 µg per lane) was separated by electrophoresis in 1.2% agarose-formaldehyde gels. The gel-separated RNA was transferred onto nylon membranes and immobilized by UV cross-linking. The membranes were prehybridized for 5 hours at 65°C in a solution containing 50% formamide, 5x SSC, 2% blocking reagent, 0.1% N-lauroylsarcosine, 50 mmol/L sodium phosphate (pH 7.0), and 7% SDS, followed by hybridization for 16 to 18 hours at 65°C in the same solution containing digoxigenin (DIG)-conjugated antisense riboprobes for lysyl oxidase. Riboprobes were synthesized in an in vitro transcription reaction by using the Riboprobe in vitro transcription system and DIG-labeled UTP. A 400-bp EcoRI-SalI cDNA segment from the cloned rat lysyl oxidase cDNA was used as a template for synthesis of the lysyl oxidase riboprobe. After hybridization, the mRNA signals were detected by use of a DIG luminescence detection kit. In brief, the membrane was washed sequentially in SSC and blocking buffer; incubated in a solution containing 75 mU/mL of anti–DIG-alkaline phosphatase complex for 30 minutes, washed with detection solution (0.1 mol/L Tris-HCl, 0.1 mol/L NaCl, 50 mmol/L MgCl₂, and 1% CSPD), and exposed at room temperature to x-ray film for 5 to 10 minutes. Signal intensity was measured with use of a scanning densitometer and NIH Image Analysis software. For normalization, the membranes were stripped and rehybridized with probes for 18S rRNA or GAPDH.

Synthesis of ³H-Labeled Recombinant Human Tropoelastin Substrate
A plasmid containing human tropoelastin cDNA (rTE) was kindly provided by Dr Joel Rosenbloom (University of Pennsylvania, Philadelphia). A 2.2-kb EcoRI/HindIII rTE fragment was subcloned into a pTrcHis2A expression plasmid containing a trc promoter and encoding a C-terminal 6-residue polyhistidine tail in the expressed protein. This polyhistidine tail functions as a metal binding site in the expressed protein, which markedly facilitates affinity purification of the recombinant protein. In brief, the rTE-containing pTrcHis2A plasmid was transformed into competent Escherichia coli by use of calcium phosphate precipitation and cultured overnight in LB medium. Subsequently, a 20 mL aliquot of this culture was transferred into 1 L of LB medium containing 50 µg/mL ampicillin and incubated with shaking at 37°C to an optical density (600 nm) of 0.6. The resultant bacterial pellet was collected, washed, and resuspended in 1 L lysine-free RPMI medium for 10 minutes. Protein synthesis was induced by adding isopropyl ß-D-thiogalactopyranoside to a final concentration of 1 mmol/L for 1 hour, and the bacteria were incubated for another 3 hours in the presence of 1.0 mCi/L of [L-4,5-³H]lysine. The bacterial pellet was collected by centrifugation, resuspended in 200 mL of binding buffer (20 mmol/L phosphate and 500 mmol/L NaCl, pH 7.8), treated with lysozyme (100 µg/mL) at 0°C for 20 minutes, and sonicated with three 10-second bursts on ice. The lysate was then rapidly frozen in liquid N₂ and thawed at 37°C. This process was repeated 4 times. The lysate was spun in a centrifuge at 3000g for 15 minutes to remove insoluble debris, and the lysate was passed through an 0.8-µm syringe filter. The lysate was further affinity-purified by using the Xpress protein purification system. After affinity purification, the recombinant protein was further purified and concentrated by using 10 000–molecular weight cutoff microconcentrating filters. The ³H-labeled rTE protein was confirmed by Western blot with the use of not only an anti-elastin antibody but also an anti-histidine antibody (data not shown). The ³H-labeled rTE substrate was stored at -70°C for later activity assay.

Lysyl Oxidase Activity Assay
Lysyl oxidase activity was measured by a modification of a previously described bioassay that used recombinant human [³H]tropoelastin as the substrate.^{22 23} Enzyme activity was measured in the conditioned culture media and in the insoluble cell layer. To measure lysyl oxidase activity in conditioned culture medium, 0.5 mL of conditioned medium was added to a solution containing 0.1 mol/L sodium borate, 0.15 mol/L sodium chloride, and 125 000 cpm [³H]rTE substrate in a final reaction volume of 1 mL. This reaction mixture was incubated for 2 hours at 37°C in the presence or absence of 50 µmol/L ß-aminopropionitrile. The reaction was stopped on ice, and the mixture was spun in a centrifuge at 15 000g for 15 minutes at 4°C to remove the insoluble substrate. A 700 µL aliquot of the supernatant was transferred to 10 000–molecular weight cutoff microconcentrating filters and spun at 5000g for 1 hour at 2°C. Enzyme activity was measured in 400 µL aliquots by liquid scintillation spectrometry, defined as the difference in counts per minute released from paired reaction mixtures carried out in the presence or absence of ß-aminopropionitrile and normalized to cell number. Triplicate assays were performed on triplicate cultures for each experimental condition. Lysyl oxidase activity was measured in the insoluble cell layer. Other urea-soluble protein was extracted in a buffer solution containing 0.02 mol/L boric acid, 4 mol/L urea, 0.15 mol/L NaCl (pH 8.0), 0.001 mol/L PMSF, and 10 µL of aprotinin per 10 mL of extraction buffer. The extraction mixture was scraped with a rubber policeman and triturated, and the solution was spun at 15 000g for 15 minutes at 4°C. The supernatant was dialyzed overnight against a solution containing 0.02 mol/L boric acid (pH 8.0), and 500 µL aliquots of the concentrated extract were assayed as described for the conditioned medium.

Nuclear Runoff Transcription Assay
Newly synthesized lysyl oxidase mRNA was assayed by using a modified nuclear runoff transcription assay.²⁴ In brief, cell nuclei isolated after treatment with NP-40 lysis buffer (10 mmol/L Tris-HCl [pH 7.4], 10 mmol/L NaCl, 3 mmol/L MgCl, and 0.5% [vol/vol] NP-40) to remove the cytoplasm. The isolated nuclei were resuspended in 90 µL nuclear storage buffer (50 mmol/L Tris-HCl [pH 8.3], 40% [vol/wt] glycerol, 0.1 mmol/L dithiothreitol, and 0.1 mmol/L EDTA) and stored at -70°C or processed directly to in vitro transcription. For in vitro transcription, 100 µL of 2x reaction buffer (10 mmol/L Tris-HCl [pH 7.5], 5 mmol/L MgCl₂, 0.3 mol/L KCl, 5 mmol/L dithiothreitol, and 1 mmol/L each of ATP, GTP, and CTP) and 3 µL DIG-labeled UTP were added to the resuspended nuclei; the reaction mixture was incubated at room temperature for 15 minutes. Nuclear DNA was digested by adding 1 µL of 20 000 U/mL RNase-free DNase and incubated at room temperature for 5 minutes. The nuclear RNA was then isolated by phenol chloroform extraction and ethanol precipitation. The DIG-labeled nuclear RNA pellet was dissolved in Tris-EDTA buffer and transferred via slot blot to nitrocellulose membranes on which lysyl oxidase cDNA had been previously immobilized by UV cross-linking. The hybridization signal was detected by using a DIG luminescence detection kit and normalized to GAPDH signal.

Statistical Analysis
Data were expressed as mean±SE and analyzed by 1-way ANOVA. A value of P<0.05 was considered statistically significant.

	Results

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Concentration-Dependent Regulation of Lysyl Oxidase mRNA by IFN-
Northern analysis was used to determine the effect of IFN- on steady-state lysyl oxidase mRNA levels in cultured rat aortic smooth muscle cells. Quiescent cells were cultured for 24 hours in the presence or absence of log concentrations of IFN-, and total RNA was isolated. IFN- decreased steady-state lysyl oxidase mRNA in a concentration-dependent manner (Figure 1). Steady-state lysyl oxidase mRNA levels decreased significantly to 73%, 50%, and 30% of control levels in the presence of 0.1, 1, and 10 ng/mL IFN-, respectively. Conversely, IFN- treatment had no effect on cell number over 48 hours (data not shown). These results suggest that the regulation of lysyl oxidase mRNA expression in cultured rat aortic smooth muscle cells by IFN- is concentration dependent.

View larger version (32K): [in this window] [in a new window]   Figure 1. Concentration-dependent regulation of steady-state lysyl oxidase mRNA levels by IFN-. Subconfluent rat aortic smooth muscle cell cultures were grown for 24 hours in serum-free DMEM containing 0.2% BSA. Quiescent cultures were subsequently treated for an additional 24 hours in the presence or absence of recombinant human IFN-. A, Autoradiogram of Northern blot of total RNA from a representative experiment. Lysyl oxidase mRNA transcript signals were quantified by laser densitometry and normalized to 18S rRNA signal. Lanes are as follows: lane 1, control vehicle; lane 2, 0.01 ng/mL IFN-; lane 3, 0.10 ng/mL IFN-; lane 4, 1.0 ng/mL IFN-; and lane 5, 10.0 ng/mL IFN-. B, Pooled data from 3 independent experiments. Data are mean±SE of 3 independent experiments. *P<0.05; **P<0.01.

Time-Dependent Regulation of Lysyl Oxidase mRNA by IFN-
To determine whether the observed IFN-–mediated changes in steady-state lysyl oxidase mRNA levels were time dependent, cells were treated for various time periods in the presence or absence of 1.0 ng/mL IFN-. Steady-state lysyl oxidase mRNA decreased to 75%, 50%, and 35% of control levels after treatment with 1 ng/mL IFN- for 6, 24, and 48 hours, respectively (Figure 2). These results suggest that the concentration-dependent changes in lysyl oxidase mRNA levels in response to IFN- are also time dependent.

View larger version (39K): [in this window] [in a new window]   Figure 2. Time-dependent regulation of steady-state lysyl oxidase mRNA levels by IFN-. Subconfluent rat aortic smooth muscle cell cultures were grown for 24 hours in serum-free DMEM containing 0.2% BSA. Quiescent cultures were subsequently treated in the presence or absence of 1.0 ng/mL recombinant human IFN- for up to 48 hours. A, Autoradiogram of a representative Northern blot of total RNA from rat aortic smooth muscle cells treated in the presence of either control vehicle (lanes 1, 3, and 5) or in the presence of 1.0 ng/mL IFN- (lanes 2, 4, and 6) for 6, 24, or 48 hours, respectively. Lysyl oxidase mRNA transcript signals were quantified by scanning laser densitometry and normalized to 18S rRNA signal. B, Pooled data from 3 independent experiments. Data are mean±SE of 3 independent experiments. *P<0.05; **P<0.01.

Transcriptional Regulation of Lysyl Oxidase by IFN-
A modified nuclear runoff transcription assay was used to determine whether IFN-–mediated changes in steady-state lysyl oxidase mRNA were due to alterations in the rate of transcription. Nuclear mRNA was isolated from cultured rat aortic smooth muscle cells after treatment in the presence or absence of 1 ng/mL IFN- for various time periods. IFN- reduced lysyl oxidase mRNA transcription to 91%, 75%, and 65% of control levels at 6, 24, and 48 hours, respectively (Figure 3). These results suggest that changes in steady-state lysyl oxidase mRNA in response to IFN- were mediated, in part, by decreased mRNA transcription. Furthermore, these results suggest that transcriptional effects of IFN- are responsible for 40% of the observed downregulation.

View larger version (13K): [in this window] [in a new window]   Figure 3. Effect of IFN- on transcription rate of lysyl oxidase mRNA. Subconfluent rat aortic smooth muscle cell cultures were treated in the presence or absence of 1 ng/mL IFN- for 6, 24, and 48 hours, respectively. Nuclei were isolated, and in vitro transcription was allowed to resume in the presence of DIG-labeled UTP. Equal amounts of DIG-labeled in vitro–transcribed cRNA probes from each group were hybridized to 1 µg denatured rat lysyl oxidase or GAPDH cDNA that had been immobilized on nitrocellulose filters. Data are mean±SE for 3 independent experiments. *P<0.05; **P<0.01.

Effect of IFN- on Lysyl Oxidase mRNA Decay Rates
The decay rate of lysyl oxidase mRNA in the presence of IFN- was examined to determine whether IFN- alters the posttranscriptional stability of lysyl oxidase mRNA. Cells were first treated in the presence or absence of 1.0 ng/mL IFN- for 24 hours, and then DRB (a selective inhibitor of RNA polymerase II) was added to inhibit new mRNA synthesis. The DRB-treated cells were harvested at 2, 4, 6, 8, 10, and 12 hours, and RNA was isolated. Northern analysis was used to determine relative lysyl oxidase mRNA levels. The half-time of lysyl oxidase mRNA decay for control cells in the absence of IFN- was 9 hours; conversely, the half-time of lysyl oxidase mRNA decay in the presence of IFN- decreased by 33% to 6 hours (Figure 4). Thus, these results suggest that posttranscriptional effects of IFN- on lysyl oxidase mRNA decay rates accounted for 50% of the observed downregulation in steady-state lysyl oxidase mRNA levels.

View larger version (38K): [in this window] [in a new window]   Figure 4. Effect of IFN- on steady-state mRNA decay rates. Subconfluent rat aortic smooth muscle cultures were treated in the presence or absence of 1.0 ng/mL IFN- for 24 hours and subsequently for up to 12 hours in the presence of 20 ng/mL DRB to inhibit transcription. Total RNA was isolated at the indicated time points, and Northern analysis was performed. A, Representative Northern blot from cultures in the presence of IFN-. B, Representative Northern blot from IFN-–treated cultures. For panels A and B, lanes are as follows: lane 1, 0 hours; lane 2, 2 hours; lane 3, 4 hours; lane 4, 6 hours; lane 5, 8 hours; lane 6, 10 hours; and lane 7, 12 hours after addition of DRB. C, Pooled data for 3 independent experiments. Error bars are the ranges of log of percent mRNA remaining from the densitometric scanning. All scanning data from panels A and B were subjected to linear analysis of the semilog plots of the percentage of mRNA remaining vs time; lysyl oxidase mRNA decayed with a half-life of 9 and 6 hours in DRB-control () and in DRB-IFN-–treated () cells, respectively. Parallel cultures were treated in the presence or absence of 1.0 ng/mL IFN- for 24, 36, and 48 hours but not DRB to establish IFN-–dependent changes in steady-state mRNA (data not shown).

Effect of Inhibiting New Protein Synthesis on Lysyl Oxidase mRNA
Synthesis of labile proteins can influence the stability of newly transcribed mRNA and thus may influence IFN-mediated effects on lysyl oxidase gene expression. Inhibition of new protein synthesis may cause large increases in steady-state mRNA (superinduction) secondary to the loss of labile proteins, which serve to destabilize mRNA, or to protection of the mRNA by bound ribosomes. Cycloheximide, an inhibitor of new protein synthesis, was used to explore the effect of inhibiting protein synthesis on IFN-–mediated changes in steady-state lysyl oxidase mRNA levels. In the absence of IFN-, cycloheximide increased steady-state lysyl oxidase mRNA levels by 2.5 fold above control levels (Figure 5). Similarly, in the presence of IFN-, cycloheximide increased lysyl oxidase mRNA 2.5 fold above control levels. Thus, it appears that cycloheximide influences steady-state levels of lysyl oxidase mRNA to a similar degree in the presence or absence of IFN-. Thus, IFN-–mediated changes in lysyl oxidase mRNA may not require new protein synthesis.

View larger version (11K): [in this window] [in a new window]   Figure 5. Effect of cycloheximide (CHX) on steady-state lysyl oxidase mRNA. Rat aortic smooth muscle cells were treated in the presence or absence of 1 ng/mL IFN- for 12 hours and subsequently treated in the presence or absence of 5 µg/mL CHX for 8 hours. Total RNA was isolated, and Northern analysis was performed. Data are derived from a representative of 3 independent experiments with similar results. *P<0.05; **P<0.01.

Effect of IFN- on Lysyl Oxidase Enzyme Activity
A modification of a previously described tritium-release bioassay was used to determine the effect of IFN- on lysyl oxidase activity in cultured rat aortic smooth muscle cells. Cells were cultured in the presence or absence of 1 ng/mL IFN- for various time periods, and enzyme activity was determined against a ³H-labeled rTE substrate for cell layer extracts or conditioned cell culture media. IFN- significantly decreased lysyl oxidase enzyme activity in the insoluble cell layer fraction to 80%, 51%, and 34% of control levels at 6, 24, and 48 hours, respectively (Table). These results suggest that IFN- downregulates not only lysyl oxidase mRNA but also functional enzyme activity levels. Furthermore, changes in enzyme activity appeared to parallel changes in mRNA levels, providing suggestive evidence that the observed changes in enzyme activity were at least partly due to the observed changes in mRNA levels.

View this table: [in this window] [in a new window]   Table 1. Effect of IFN- on Lysyl Oxidase Activity and Steady-State mRNA Levels

	Discussion

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Coronary artery disease, cerebrovascular occlusive disease, and aortic aneurysms are leading causes of death in the United States.^{25 26 26A} Despite the apparent dissimilarity between occlusive and aneurysmal diseases, both are characterized by extensive remodeling of the extracellular matrix in the vascular wall. A clear understanding of the mechanisms by which this remodeling occurs has potential implications for the development of novel pharmacological and genetic approaches to these vascular diseases.

Lysyl oxidase is a critical enzyme for normal collagen and elastin cross-linking and may be relevant in the pathogenesis of arterial occlusive and aneurysmal diseases. Regulation of lysyl oxidase production and activity plays a key role in maintaining the structural integrity of the extracellular matrix. Furthermore, reductions in lysyl oxidase activity have been associated with human diseases characterized by abnormal matrix remodeling.^{13 14 15} Unfortunately, comparatively little is known about the factors that regulate lysyl oxidase production in the blood vessel wall.

It appears that IFN- may play a key role in the regulation of collagen metabolism in the extracellular matrix. For example, IFN- is known to be an important negative regulator of collagen biosynthesis. In human lung fibroblasts, IFN- inhibits not only 1(I) and 1(III) procollagen mRNA expression but also the accumulation of newly synthesized collagen protein.¹⁴ In the present study, IFN- downregulated lysyl oxidase gene expression in cultured vascular smooth muscle cells. Thus, IFN- appears to regulate not only procollagen gene expression but also a key enzyme involved in the posttranslational processing of this structural protein. It is possible that regional differences in the expression of IFN- profoundly affect the synthesis, maturation, and stability of vascular wall collagen and elastin.

In addition to the role played by IFN- in biosynthesis of collagen and its maturation, IFN- may also regulate degradation of collagen and elastin in the vascular wall. Recent studies suggest that IFN- upregulates urokinase plasminogen activator gene expression in macrophages.²⁷ Given the observation that plasmin is a potent activator of matrix metalloproteinases and that metalloproteinases are known to degrade many extracellular matrix components, including collagen and elastin, it is likely that IFN- enhances extracellular matrix degradation in vivo.¹⁷ Furthermore, IFN- is known to directly stimulate the expression of matrix metalloproteinases in certain mesenchymal cells.²⁹ Finally, diminished collagen cross-linking secondary to a reduction in lysyl oxidase activity appears to increase the susceptibility of collagen to degradation by metalloproteinases.¹⁷ Thus, it is reasonable to postulate that IFN- may play a central role in the regulation of extracellular matrix synthesis, maturation, and degradation in the vascular wall.

Additional evidence supporting the hypothesis that IFN- regulates matrix metabolism in certain vascular disorders comes from the observation that IFN- production is increased in arteriosclerotic plaque rupture and aneurysm formation.^{2 16 30} Thus, it is possible that IFN-–dependent changes in lysyl oxidase activity reduce the mechanical integrity of the arterial wall, not only by reducing collagen accumulation and stabilization but also by possibly enhancing the degradation of these proteins by metalloproteinases.^{17 31}

With respect to the intracellular mechanisms by which IFN- controls lysyl oxidase gene expression, the results of the present study suggest that regulation occurs at transcriptional and posttranscriptional levels. Transcriptional regulation of lysyl oxidase mRNA is consistent with recent data regarding the murine lysyl oxidase promoter.^{32 33} Results from experiments using lysyl oxidase promoter–luciferase reporter constructs suggest that positive and negative regulatory motifs are associated with the lysyl oxidase promoter.³² Binding of nuclear regulatory factors in regions -784 to -970 of the mouse lysyl oxidase gene results in a 50% inhibition of the promoter.

Although the nature of these nuclear regulatory factor are not yet fully characterized, it is interesting to note that lysyl oxidase has recently been identified as an interferon-inducible gene.¹⁹ An interferon-sensitive transcriptional activator, interferon regulatory factor (IRF)-1, is known to bind to region the IRF-binding consensus sequence, -886 to -988, of the murine lysyl oxidase promoter and to stimulate transcription. However, an antagonistic transcriptional repressor, IRF-2, which shares a high degree of homology with IRF-1, can also bind to the IRF-binding consensus sequence in DNA.³⁴ It is reasonable to speculate that IRF-1 and IRF-2 can bind to the lysyl oxidase promoter in vascular smooth muscle cells.

Furthermore, it is noteworthy that lysyl oxidase and collagen 1(I) promoters share many similar features.³⁵ For example, the consensus binding motif for the CCAAT-binding factors, C-Krox A and C-Krox B, and SP1-like factors exist in both promoters.³² Given the fact that IFN- is a potent negative regulator of procollagen mRNA transcription³⁶ and that the promoters of these genes possess many similar characteristics, it is reasonable to conclude that the same or related mechanisms are involved in the synchronous regulation of these genes by IFN- in vivo.

Posttranscriptional regulation is an additional important mechanism by which the expression of extracellular matrix genes are regulated in vivo. Clearly, posttranscriptional regulation of lysyl oxidase mRNA stability by cytokines influences lysyl oxidase gene expression. For example, transforming growth factor-ß1 is known to increase the lysyl oxidase mRNA half-life in smooth muscle cells, whereas basic fibroblast growth factor decreases the lysyl oxidase mRNA half-life in osteoblastic MC3T3-E1 cells.^{8 9} In the present study, IFN- decreased the lysyl oxidase mRNA half-life in rat aortic smooth muscle cells. The mechanisms and signaling pathways responsible for these observations are unclear. However, participation of IFN-inducible labile proteins in this process is not supported by the results of the present study. Nevertheless, activation of stable protein factors may play a role in this process.^{37 38} Further studies are clearly needed to characterize the complex intracellular signaling pathways mediating the observed downregulation of lysyl oxidase gene expression by IFN-.

This study documents that IFN- downregulates lysyl oxidase mRNA expression and enzyme activity in cultured rat aortic smooth muscle cells. These effects appear to occur by transcriptional and posttranscriptional mechanisms. In light of the observation that IFN- downregulates procollagen mRNA and protein synthesis, the results of the present study suggest an additional posttranslational mechanism by which IFN- is likely to reduce the mechanical integrity and stability of the arterial wall. In this regard, IFN-–mediated reductions in the local activity of lysyl oxidase within the vascular wall may not only reduce the mechanical strength of newly synthesized collagen and elastin, but they are also likely to increase the susceptibility of these proteins to degradation by matrix metalloproteinases. Given the association between IFN- expression and arterial diseases characterized by abnormal matrix remodeling, the results of the present study suggest that IFN-–mediated changes in lysyl oxidase expression may play a critical role in the pathogenesis of arterial aneurysms and arteriosclerotic occlusive disease, including plaque rupture.

Received August 16, 1999; accepted November 19, 1999.

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