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(The FASEB Journal. 1999;13:1423-1434.)
© 1999 FASEB

Nitric oxide mediates LC-3-dependent regulation of fibronectin in ductus arteriosus intimal cushion formation

CATHERINE A. E. MASON*, PETER CHANG*, CAROLINE FALLERY* and MARLENE RABINOVITCH*,,{dagger},1

* Division of Cardiovascular Research, Research Institute, The Hospital for Sick Children,
{dagger} Departments of Pediatrics, Pathology and Medicine, University of Toronto, Toronto, Ontario, Canada M5G 1X8

1Correspondence: Division of Cardiovascular Research, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8. E-mail: MR{at}sickkids.on.ca


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Ductus arteriosus intimal cushion formation is characterized by fibronectin-dependent smooth muscle cell (SMC) migration. Enhanced fibronectin synthesis in ductus SMC is regulated by the interaction of LC-3, a microtubule-associated protein, with an AU-rich element (ARE) in the 3'-untranslated region of fibronectin mRNA, facilitating its recruitment to polyribosomes for translation. Since nitric oxide (NO) is implicated in posttranscriptional gene regulation and is produced in the ductus, we investigated its mechanistic role in LC-3-mediated fibronectin synthesis. NO production was sevenfold higher in ductus vs. aortic SMC (P<0.005) associated with increased neuronal NO synthase (nNOS) expression. The NOS inhibitor L-NMMA decreased fibronectin synthesis by ~45–50% (P<0.05), whereas the NO donor, SNAP, increased ductus fibronectin synthesis ~onefold (P<0.05); neither agent altered fibronectin mRNA levels. Immunoblotting revealed that SNAP increased and L-NMMA reduced a membrane-associated phosphorylated form of LC-3. RNA gel mobility shift assays confirmed that NO enhanced LC-3 binding to the fibronectin mRNA ARE. Our studies indicate a tissue-specific program in the ductus arteriosus whereby elevated nNOS expression and NO production regulate the posttranscriptional increase in fibronectin synthesis required for SMC motility.—Mason, C. A. E., Chang, P., Fallery, C., Rabinovitch, M. Nitric oxide mediates LC-3-dependent regulation of fibronectin in ductus arteriosus intimal cushion formation.


Key Words: extracellular matrix • cell migration • microtubule • atherosclerosis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
CELLULAR FIBRONECTIN IS an extracellular matrix glycoprotein secreted in situ by several cell types, including vascular smooth muscle cells (1) . It plays a central role in regulating morphogenesis and functional maturation in developing tissues through its effects on cell adhesion, differentiation, and migration (1 , 2) . In the ductus arteriosus (DA)2 (2) beginning at 100 days of a 145 day gestation in fetal lambs (3 , 4) , fibronectin-dependent migration of smooth muscle cells into the subendothelium (5) results in the formation of intimal cushions, which protrude into the vessel lumen and ultimately are required for closure of the ductus when it constricts in the postnatal period (6) .

We previously demonstrated that elevated fibronectin synthesis in smooth muscle cells of the neointima-forming DA, relative to that in smooth muscle cells of the adjacent aorta (Ao), is regulated at the level of mRNA translation (7) . An adenosine-uracil rich element (ARE) in the 3'-untranslated region (3'-UTR) of fibronectin mRNA confers increased translational efficiency in the DA smooth muscle cells. A ~15 kDa protein that binds to the ARE of fibronectin mRNA in RNA gel mobility shift assays was purified and identified as light chain 3 (LC-3) of microtubule-associated protein complexes 1A and 1B (8) . Ductus arteriosus smooth muscle cells express increased levels of LC-3 and enhanced LC-3/fibronectin mRNA complex formation relative to Ao smooth muscle cells. Transfection of Ao smooth muscle cells with LC-3 enhances fibronectin mRNA translation (8) . The mechanism is related to the ability of LC-3 to facilitate recruitment of fibronectin mRNA into polyribosomes, a process requiring intact microtubules (9) .

Nitric oxide (NO) has been implicated as both a positive (10 11 12 13) and negative (14 , 15) modulator of migration for smooth muscle and endothelial cells. It is also a signaling molecule that regulates gene expression at transcriptional (16 , 17) and posttranscriptional levels (18 19 20) . Nitric oxide can orchestrate posttranscriptional gene regulation by modulating interactions between RNA binding proteins and regulatory elements in the untranslated regions of mRNA, thereby altering mRNA stability and translational efficiency (18 , 19) . However, studies have largely been limited to analysis of regulatory factors binding to the iron response elements in mRNAs encoding proteins involved in iron metabolism (18 , 19) .

Nitric oxide is produced in the developing DA, where it can act to decrease vascular tone (21) . However, its physiological role as a vasodilator in the DA has been shown to be less important than that of prostaglandin E2 (PGE2) (22 , 23) . We therefore examined whether NO plays a role in remodeling of the developing DA, through modulating the posttranscriptional up-regulation of fibronectin synthesis required for the migration of smooth muscle cells into the subendothelium during the formation of intimal cushions (24) .

In this report we demonstrate significantly increased NO production in primary cultured DA compared with Ao smooth muscle cells. We relate this to enhanced expression of neuronal NO synthase (nNOS) in the intact DA as well as in cultured smooth muscle cells harvested at 100 days gestation, a time point coincident with the onset of intimal cushion formation. We also show that NO enhances fibronectin synthesis without modulating mRNA levels in DA smooth muscle cells. This NO-dependent posttranscriptional mechanism of elevated fibronectin synthesis in DA smooth muscle cells involves increased expression, and binding to the fibronectin mRNA ARE, of a membrane-associated phosphorylated form of LC-3.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture
Smooth muscle cells from the DA and Ao were isolated for primary cultures and characterized by smooth muscle {alpha}-actin immunostaining as described previously (25 , 26) . The DA and aortic arch, harvested from fetal lambs at 100 days gestation, were opened, rinsed in Dulbecco's phosphate-buffered saline (PBS) containing 3% antibiotics/antimycotics (Gibco BRL, Burlington, Ontario), and the adventitia was removed. Endothelial cells were removed with a No. 11 scalpel blade. The media was cut into 1 mm square pieces, which were allowed to adhere to 100 mm culture dishes before the addition of Medium 199 (OCI, Toronto, Ontario) containing 1% antibiotics/antimycotics and 10% fetal calf serum (Intergen, Purchase, N.Y.). Smooth muscle cells propagated from these explants were used after two passages.

Measurement of NO
Nitric oxide production was assessed in culture media from DA and Ao smooth muscle cells by measuring the accumulation of nitrite, a stable end product of NO. Nitrite concentration in cell culture media was determined over a 24 h incubation using spectrophotometry after a Griess reaction as described previously (27) . Experimental samples of culture medium (500 µl) and fresh culture medium containing standard levels of sodium nitrite were reacted with 500 µl 0.1% naphthylethylenediamine, 1% sulfanilamide, and 5% phosphoric acid for 10 min at room temperature and analyzed by spectrophotometry at 540 nm. A standard curve was constructed and used to determine nitrite concentration in the culture medium from DA and Ao smooth muscle cells, which was then normalized to cell number.

Western immunoblots
Ductus and Ao smooth muscle cells cultured from 100 day gestation fetal lambs were harvested at semi-confluency by scraping into PBS and pelleted at 1000 x g in a TJ-6 centrifuge (Beckman, Mississauga, Ontario) for 10 min. Cell pellets were then dissolved in 1% sodium dodecyl sulfate (SDS), 10 mM TRIS-HCl pH 7.4. Intact DAs and Ao's from fetal lambs at 100 days gestation were frozen in liquid nitrogen, ground into a fine powder with a mortar and pestle, and dissolved in 1% SDS, 10 mM TRIS-HCl pH 7.4. Protein concentrations were measured by standard Bradford assay (Bio-Rad, Hercules, Calif.), followed by spectrophotometry at 595 nm. Protein extracts from cultured DA or Ao smooth muscle cells or intact vessels in Laemmli sample buffer (5% ß-mercaptoethanol, 2% SDS, 10% glycerol, 62.5 mM TRIS-HCl pH 6.8) were separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes.

Membranes were blocked for 1 h in 5% non-fat dry milk/TRIS-buffered saline (TBS) containing 0.1% Tween-20 for immunoblotting of endothelial constitutive NO synthase (ecNOS) and nNOS, 0.5% for LC-3 and tubulin or 1% for inducible NOS (iNOS). Blots were then probed for 1 h at room temperature with polyclonal antibodies to amino acids 1095–1289 of human nNOS (1:500) (Transduction Laboratories, Lexington, Ky.), polyclonal antibodies to the NH2 terminus of murine iNOS (1:200) (Santa Cruz Biotechnology, Santa Cruz, Calif.), a monoclonal antibody to amino acids 1030–1209 of human ecNOS (1:2500) (Transduction Laboratories), polyclonal antibodies to LC-3 (1:1000) (kindly supplied by Dr. J. Hammarback, Department of Neurobiology and Anatomy, Bowman Gray School of Medicine, Winston-Salem, N.C.), or a monoclonal antibody to tubulin (1:2000) (Sigma, Mississauga, Ontario). Blots were washed three times with TBS containing 0.1%, 0.5% or 1% Tween-20 as indicated for the blocking buffer, followed by incubation with peroxidase-conjugated goat anti-rabbit immunoglobulin (IgG) or goat anti-mouse IgG (both 1:3000) (Sigma). The blots were washed as described above three times before developing using enhanced chemiluminescence Western blotting detection reagents (Amersham, Buckinghamshire, England).

Modulation of NO in cultured smooth muscle cells
The influence of NO on fibronectin synthesis, steady-state fibronectin mRNA levels, and LC-3 expression in cultured smooth muscle cells was examined by modulating cellular levels of NO using diphenyleneiodonium (DPI) (2 µM) (Sigma), a nonspecific inhibitor of flavonoid containing enzymes including NO synthases as well as NADPH oxidases (28) , L-NG-monomethylarginine (L-NMMA) (250 µM) (Sigma), a competitive inhibitor of NOS, or S-nitroso-N-acetylpenicillamine (SNAP) (100 µM) (BIOMOL Research Laboratories, Plymouth Meeting, Pa.), an NO donor. In the experiments where DPI or L-NMMA were used to inhibit NO production, cells were preincubated for 1 h in serum-free and cysteine/methionine-free medium 199 containing 1% bovine serum albumin (BSA) alone or in the presence of DPI or L-NMMA, followed by a 4 h incubation in the presence of fresh media containing the inhibitors. When SNAP was used to examine the effect of NO addition, cells were first preincubated in serum free media for 1 h and then SNAP was added for 4 h.

Measurement of fibronectin synthesis
Semi-confluent DA and Ao smooth muscle cells plated in 35 mm dishes at passage 2 were incubated in 2 ml serum-free and cysteine/methionine-free medium 199 containing 1% BSA for 1 h. This medium was replaced and 10 µCi/ml of [35S]-methionine (Amersham) was added for 4 h. Triplicate assessments of total protein synthesis were obtained from 50 µl aliquots of culture medium precipitated in 2% BSA/25% trichloroacetic acid and analyzed by liquid scintillation spectrometry. To measure fibronectin synthesis, 1 ml aliquots of culture medium were incubated with 100 µl gelatin 4B-Sepharose (Pharmacia Biotech, Uppsala, Sweden) beads overnight at 4°C. The beads were washed three times in PBS and bound fibronectin was eluted from the beads into 100 µl Laemmli sample buffer (5% ß-mercaptoethanol, 2% SDS, 10% glycerol, 62.5 mM TRIS-HCl pH 6.8) by boiling for 5 min. The samples were then separated on 6% SDS-polyacrylamide gels. Gels were fixed in 5% acetic acid/10% methanol for 30 min and soaked in Amplify (Amersham) for 15 min before drying under vacuum at 80°C on a Model 443 slab dryer (Bio-Rad). Dried gels were exposed to Kodak X-OMAT AR film for 2 to 4 days and a 220 kDa band corresponding to fibronectin was excised from the gels and counted by liquid scintillation spectrometry. Identification of this 220 kDa band as fibronectin was confirmed in our laboratory by Western blotting (3) . Fibronectin synthesis was standardized to total protein synthesis.

Northern blots
Total RNA was isolated from cultured DA smooth muscle cells using TRIZOL reagent (Life Technologies, Grand Island, N.Y.). Cells were lysed in TRIZOL (2 ml/100 mm dish) by repeated pipetting. The cell lysate was extracted with chloroform; RNA was precipitated with isopropyl alcohol and dissolved in 0.5% SDS in 0.1% diethylpyrocarbonate-treated water. RNA samples were separated in a 1% agarose gel containing 6% formaldehyde and transferred to a nitrocellulose membrane. Membranes were probed with a [32P]-dCTP (Amersham) -labeled human fibronectin cDNA probe (1.4 kB) (Gibco BRL) and a control human GAPDH cDNA probe (1.2 kB) (ATCC, Rockville, Md.).

Immunofluorescence staining of LC-3
For staining of LC-3, cultured DA smooth muscle cells on glass coverslips were fixed in 100% methanol at -20°C for 3 min and allowed to air dry. After rehydration in PBS, cells were probed with polyclonal antibodies to LC-3 (1:1000) in 0.1% BSA/PBS for 30 min, washed 3 times with 0.1% BSA/PBS, and incubated with fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson ImmunoResearch Laboratories, West Grove, Pa.; 1:100), followed by three washes in 0.1% BSA/PBS.

DA smooth muscle cell fractionation
DA smooth muscle cells from semi-confluent cultures in 100 mm culture dishes were resuspended in two volumes of hypotonic buffer (0.1 mM EDTA, 25 mM TRIS-HCl pH 7.9) and lysed by three cycles of freeze-thaw. Cytosolic extracts were isolated by centrifugation for 1 h at 16,000 x g as described previously (29) . Pellets were either solubilized in Laemmli sample buffer directly for SDS-PAGE or digested with RNase-free DNase I (Pharmacia Biotech) for 30 min at 37°C for RNA gel mobility shift assays. Protein concentration was determined using a standard Bradford protein assay kit (Bio-Rad), followed by spectrophotometry at 595 nm.

Phosphatase treatment of cell extracts containing LC-3
Cytosolic and membrane extracts (20 µg) isolated from cultured DA smooth muscle cells were incubated with potato acid phosphatase (0.24 units) (Calbiochem, Hornby, Ontario) at 30°C for 1 h as described previously (30 , 31) and analyzed by SDS-PAGE, followed by Western immunoblotting for LC-3 expression.

RNA gel mobility shift assays
Cytosolic extracts and 1 M KCl extracts of membranes in lysed cell pellets were dialyzed against RNA binding buffer (5 mM MgCl2, 100 mM KCl, 10% glycerol, 15 mM HEPES pH 7.9 in diethylpyrocarbonate-treated water). As described previously (8) , for each assay, 20 or 30 µg of protein extracts from DA smooth muscle cells were incubated with 105 cpm of [32P]-labeled mRNA oligonucleotide probe containing the wild-type fibronectin ARE (underlined), 5'-ACCUGUUAUUUAUCAAUU-3', or a mutated probe (mutation underlined) 5'-ACCUGGGAGGGAGCAAUU-3' (synthesized by Biotechnology Center, University of Calgary, Calgary, Alberta) in RNA binding buffer containing 2 µg Escherichia coli transfer RNA (Sigma) in a total volume of 20 µl for 30 min at 30°C. The samples were then separated on a 6% native polyacrylamide gel in 0.25 X TRIS-borate-EDTA (TBE) buffer (90 mM TRIS, 90 mM boric acid, 2 mM EDTA). For UV cross-linking assays, protein-ARE binding was carried out as described above, followed by cross-linking with 254 nm UV radiation (120 mJ/cm2) in a UV 1800 Stratalinker (Stratagene, La Jolla, Calif.) and analysis by SDS-PAGE.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
NO production is increased in DA compared with Ao smooth muscle cells
To examine whether elevated NO levels might regulate increased fibronectin synthesis in the neointima forming DA, NO production was first compared in primary cultured smooth muscle cells from the 100 day gestation fetal lamb DA and adjacent Ao. Levels of nitrites, the stable end products of NO, were sevenfold higher in culture medium from DA compared with Ao smooth muscle cells (P<0.005) (Fig. 1 ).



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  		Figure 1. NO production is greater in DA than in Ao smooth muscle cells. Graph representing nitrite accumulation (µM/105 cells) in culture media from DA and Ao smooth muscle cells. NO production as indicated by nitrite accumulation was sevenfold higher in DA compared to Ao smooth muscle cells (*P<0.005). Bars represent mean values ± SE from three separate experiments.

Western immunoblotting indicates increased nNOS and ecNOS in 100 day DA
To determine the source of elevated NO in DA smooth muscle cells, we compared expression of the three characterized NO-generating enzymes—neuronal NOS, endothelial constitutive NOS, and inducible NOS—by Western immunoblotting and densitometric analysis in DA and Ao smooth muscle cells (Fig. 2 ) and whole vessel homogenates (Fig. 3 ) from 100 day gestation fetal lambs. Expression of nNOS was increased in cultured DA relative to Ao smooth muscle cells (P<0.05), whereas ecNOS and iNOS expression were similar in the two cell types (Fig. 2) . Although iNOS appeared slightly decreased in DA relative to Ao smooth muscle cells, a statistically significant difference was not detected (Fig. 2) . Western immunoblots of whole vessel homogenates from 100 day gestation fetal lambs confirmed a onefold increase in nNOS expression in the DA compared with Ao, but also demonstrated a onefold increase in ecNOS expression (P<0.05 for both) (Fig. 3) . There was no appreciable difference in iNOS expression between the DA and Ao. By immunohistochemical analysis (data not shown), we demonstrated that the intensity of nNOS, ecNOS and fibronectin staining was increased in the DA at 100 days gestation when compared with the Ao at 100 days and with the DA and Ao at term.



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  		Figure 2. Expression of nNOS, ecNOS, and iNOS in cultured DA compared to Ao smooth muscle cells. Top: Representative Western blots of nNOS, ecNOS, and iNOS expression in positive controls [rat pituitary lysate (5 µg), human endothelial cell lysate (4 µg), or lipopolysaccharide and IFN-{gamma}-stimulated human macrophage lysate (5 µg) respectively] and cultured DA compared to Ao smooth muscle cells (20 µg/lane). Bottom: Densitometric analysis of nNOS, ecNOS, and iNOS expression levels in DA smooth muscle cells relative to Ao smooth muscle cell control levels. Expression of nNOS is increased by ~onefold in DA relative to Ao smooth muscle cells, whereas no significant differences in ecNOS or iNOS levels were detected. Bars represent mean band density of three separate experiments ± SD (*P<0.05).



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  		Figure 3. Expression of nNOS, ecNOS, and iNOS in DA and Ao tissue from 100 day gestation fetal lambs. Western immunoblot and densitometric analysis of nNOS, ecNOS and iNOS expression in three fetal lamb DAs and Ao's. Densitometric analyses revealed ~2.5-fold increased nNOS expression and ~2-fold increased ecNOS expression in the DA compared to the Ao, but no difference in iNOS expression. Bars represent mean band density from the three different animals ± SE (*P<0.05).

NO regulation of fibronectin synthesis
Having correlated elevated DA smooth muscle cell NO production with increased nNOS expression in cultured smooth muscle cells and with both increased nNOS and ecNOS expression in intact vessels at the critical 100 day gestational time point, where we have previously demonstrated elevated fibronectin synthesis (3) , we next addressed whether NO regulates fibronectin. We compared the influence of the organic NO donor, SNAP, and inhibitors of NOS activity, DPI and L-NMMA, on fibronectin synthesis in DA and Ao smooth muscle cells (Fig. 4 ). Treatment of DA smooth muscle cells with SNAP (100 µM) for 4 h caused a greater than onefold increase in fibronectin synthesis above basal levels (P<0.05). Furthermore, incubation with both the nonselective NOS inhibitor DPI (2 µM) and the specific NOS inhibitor L-NMMA (250 mM) caused a ~50% decrease in DA smooth muscle cell fibronectin synthesis relative to levels in control, untreated DA smooth muscle cells (P<0.05) (Fig. 4) . Whereas fibronectin synthesis in Ao smooth muscle cells was significantly increased by SNAP to the level of fibronectin synthesis observed in DA smooth muscle cells (P<0.05), the already lower basal levels of fibronectin synthesis in Ao cells were not significantly affected by treatment with DPI or L-NMMA (Fig. 4) . Thus enhanced fibronectin synthesis in DA smooth muscle cells is regulated by increased NO, while basal levels of fibronectin synthesis in Ao smooth muscle cells are not regulated by NO.



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  		Figure 4. NO regulates enhanced fibronectin synthesis in DA smooth muscle cells, but not basal levels in Ao smooth muscle cells. Top: Graph comparing fibronectin synthesis in untreated DA smooth muscle cells (CON) with that after 4 h treatment with the NO donor, SNAP, the NO synthase inhibitor, L-NMMA, or the general flavonoid enzyme inhibitor DPI. Bottom: Graph comparing fibronectin synthesis by untreated Ao smooth muscle cells (CON) to that after 4 h treatment with the NO donor SNAP (100 µM), the NO synthase inhibitor, L-NMMA (250 µM), or the general flavonoid enzyme inhibitor DPI (2 µM). Bars represent the mean ± SE of three separate experiments. SNAP significantly increases (*P<0.05) fibronectin synthesis in DA smooth muscle cells, and both L-NMMA and DPI significantly ({dagger}P<0.05) inhibit enhanced DA smooth muscle cell fibronectin synthesis. SNAP significantly increases (*P<0.05) fibronectin synthesis in Ao smooth muscle cells to a level equal to that found in DA smooth muscle cells. However, in contrast to their effects on DA smooth muscle cells, L-NMMA and DPI do not affect basal levels of fibronectin synthesis in Ao cells.

Northern blot analyses demonstrated no effect of SNAP or L-NMMA on steady-state levels of fibronectin mRNA in DA smooth muscle cells with respect to GAPDH mRNA levels or 18s and 28s ribosomal RNA (Fig. 5 ). This suggested that NO might regulate the previously described posttranscriptional mechanism involving enhanced production and binding of LC-3 to the ARE in the fibronectin mRNA 3'-UTR.



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  		Figure 5. Steady-state levels of fibronectin mRNA are not regulated by NO in DA smooth muscle cells. Top: Northern blot analysis of total RNA extracts from untreated DA smooth muscle cells (CON) and cells treated with SNAP and L-NMMA probed with [32P] dCTP-labeled human fibronectin cDNA and human GAPDH cDNA. Ethidium bromide stained 18S and 28S ribosomal RNA from each sample demonstrate equal loading of the lanes. Bottom: Densitometric analysis of fibronectin mRNA levels illustrates no significant difference in fibronectin mRNA levels relative to 28S ribosomal RNA after SNAP or L-NMMA treatment.

NO regulation of LC-3 by immunohistochemistry and Western immunoblotting
Immunofluorescence microscopy and Western immunoblot analysis of DA smooth muscle cells treated with SNAP and L-NMMA were used to examine whether LC-3 is regulated by NO. LC-3, a component of microtubule-associated protein complexes 1A and 1B, binds to tubulin, and has been colocalized with microtubule-associated protein 1B (MAP 1B) and microtubules in cultured rat neurons (32 , 33) . In methanol-fixed DA smooth muscle cells, immunofluorescence microscopy revealed a punctate `beaded' pattern of LC-3 staining (Fig. 6 A), which codistributed with microtubules (data not shown), as well as diffuse perinuclear staining, which could reflect polyribosome-associated LC-3 at the rough endoplasmic reticulum (Fig. 6A ). Increasing NO levels with SNAP enhanced the intensity of the microtubule-associated as well as the diffuse perinuclear LC-3 staining (Fig. 6B ). Decreasing NO levels with L-NMMA also caused an increase in microtubule-associated LC-3 staining; however, the perinuclear staining appeared to be reduced (Fig. 6C ). Furthermore, cells treated with SNAP appeared more elongated than control cells, suggesting an exaggerated motile phenotype (Fig. 6B ), whereas cells treated with L-NMMA appeared stellate, consistent with a nonmotile phenotype (Fig. 6C ).



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  		Figure 6. Immmunofluorescence labeling of LC-3 in distinct subcellular locations in DA smooth muscle cells (A) after treatment with SNAP (B) or L-NMMA (C). LC-3 labeling is found in concise beads along microtubules (arrows) and in a diffuse perinuclear staining pattern (arrowheads). SNAP (B) increases the intensity of perinuclear LC-3 staining and causes the cells to take on an elongate phenotype. Both SNAP (B) and L-NMMA (C) cause an increase in the intensity of beaded staining along microtubules (high magnification insets).

To verify the effect of NO on LC-3 expression and localization, we compared tubulin-containing cytosolic fractions with cellular membrane fractions containing rough endoplasmic reticulum, by densitometric analysis of LC-3 Western immunoblots (Fig. 7 A, B). In the membrane fraction, the majority of LC-3 was of a slightly lower molecular weight form than in the cytosolic fraction (Fig. 7A ). Treatment with SNAP caused an increase and L-NMMA a decrease in this form of LC-3 (Fig. 7A, B ), correlating with changes in fibronectin synthesis (Fig. 4) and intensity of LC-3 perinuclear immunostaining (Fig. 6) . Like other RNA binding proteins (29) , LC-3 could also be removed from cellular membranes in high-salt (1 M KCl) (data not shown).



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  		Figure 7. Identification of cytosolic tubulin-associated and membrane-associated forms of LC-3 that are differentially regulated by NO. A) Representative Western blot of tubulin and LC-3 levels and Coomassie blue stain of total protein in cytosolic (left) and cell membrane (right) protein extracts (20 µg loaded in each lane) from control DA smooth muscle cells (CON) and cells treated with SNAP (S) and L-NMMA (L) for 4 h in culture. B) Densitometric analysis of LC-3 expression in DA smooth muscle cells cytosolic and membrane extracts separately and expressed as a total (C). Total LC-3 levels were significantly increased by SNAP (*P<0.05) but not affected by L-NMMA. Bars represent mean ± SE of three experiments. D) Representative Western blot of three separate experiments demonstrating treatment of membrane-associated LC-3 with potato acid phosphatase (PAP) results in a shift in apparent molecular weight of the lower band to the higher molecular weight band (arrowheads indicate the two LC-3 bands).

A slightly higher molecular weight form of LC-3 was observed in cytosolic extracts, where it codistributed with tubulin and exhibited increased expression after either SNAP or L-NMMA treatment (Fig. 7A, B ), correlating with intensity of microtubule-associated LC-3 immunostaining (Fig. 6) .

Densitometric analysis of LC-3 bands from both membrane and cytosolic fractions after SNAP or L-NMMA treatment demonstrated that addition of excess NO to DA smooth muscle cells by SNAP significantly increased total expression of LC-3 (sum of both bands) within 4 h (P<0.05), whereas inhibition of endogenous NO production by L-NMMA did not affect overall LC-3 expression over 4 h (Fig. 7C ). Treatment with L-NMMA appeared instead to cause a shift in LC-3 localization away from the membrane-associated pool and into the cytosolic pool (Fig. 7A, B ).

We next investigated whether the two forms of LC-3 might represent different phosphorylation states, as this might influence their localization and function. Many phosphorylated proteins exhibit either upward or downward shifts in apparent molecular weight on SDS-PAGE relative to their unphosphorylated form (34 35 36) . We therefore treated DA smooth muscle cell cytosolic and membrane extracts with potato acid phosphatase, as described previously (31) , and assessed changes in LC-3 molecular weights by Western immunoblotting (Fig. 7D ). When membrane extracts containing both forms of LC-3 were treated with potato acid phosphatase, the lower molecular weight band disappeared and the higher molecular weight band was increased in intensity, whereas no effect on molecular weight of the cytosolic form of LC-3 was observed. These results suggested that the lower molecular weight form of LC-3 is a phosphorylated form of the higher molecular weight band (Fig. 7D ).

NO modulation of LC-3 binding to fibronectin ARE
To further confirm that the phosphorylated and membrane-associated form of LC-3 exhibits fibronectin mRNA binding affinity, we carried out RNA gel mobility shift assays using an 18-mer oligonucleotide containing the wild-type ARE or an ARE-mutated 18-mer oligonucleotide and cytosolic and membrane fractions from DA smooth muscle cells treated with SNAP or L-NMMA. As previously demonstrated (8) , retarded LC-3-RNA binding complexes were not detected on the gels when the mutated ARE oligonucleotide probe was used or when the [32P]-labeled ARE was competed out with unlabeled ARE in excess (500:1) (data not shown).

After a short exposure, strong ARE binding complex formation could be detected in the membrane fractions, likely reflecting the phosphorylated form of LC-3 (Fig. 8 A), whereas much less binding activity was observed using the cytosolic fractions. In both cases however, there appeared to be an increase in binding activity with SNAP and a decrease with L-NMMA (Fig. 8A ). In the cytosolic fraction, the decrease in LC-3-RNA complex formation with L-NMMA seemed to be discrepant with the increase in the level of LC-3 detected by Western immunoblotting. This could be explained if LC-3-RNA complex formation in the cytosol is related to only a small amount of the phosphorylated form of LC-3 not detected by Western immunoblot. To support this, UV cross-linking assays using membrane as well as cytosolic fractions resolved a similar ~22 kDa binding complex, reflecting binding of a ~15 kDa protein (the molecular weight of LC-3) to the ~7 kDa fibronectin ARE. This complex was barely detectable in the cytosol compared with the membrane fraction, but was increased by SNAP and decreased after L-NMMA (Fig. 8B ).



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  		Figure 8. RNA gel mobility shift and UV cross-linking assays show NO regulation of DA smooth muscle cell protein binding to the fibronectin ARE. A) Representative autoradiograph demonstrating results of an RNA gel mobility shift assay show that binding of complexes of membrane-associated proteins (20 µg) with the fibronectin ARE is enhanced compared to binding of cytosolic proteins (30 µg). Binding of the middle complex from cytosolic fractions and of the complex from membrane extracts is increased by SNAP (S) and decreased by L-NMMA (L). B) Representative UV cross-linking assay showing a ~22 kDa complex (arrowhead) formed by membrane (memb) and cytosolic proteins from DA smooth muscle cells with the ~7 kDa fibronectin ARE probe, indicating a ~15 kDa molecular mass of the bound protein. Binding of this ~15 kDa protein from DA smooth muscle cell cytosolic extracts (C) was enhanced by treatment with SNAP (S) and decreased by L-NMMA (L).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In this paper we have investigated the developmental program determining fibronectin up-regulation in the DA at the onset of intimal cushion formation. We showed that elevated NO production is likely the result of increased nNOS and ecNOS expression in the intact DA, and results in increased phosphorylation and binding of LC-3 to the ARE of the fibronectin mRNA and enhanced DA smooth muscle cell fibronectin synthesis.

Since NO production was previously demonstrated in the endothelial-denuded fetal DA (21) , it was presumably derived from smooth muscle cells, but it was not examined in comparison to the Ao. Our demonstration of a sevenfold enhanced release of NO from fetal DA compared with Ao smooth muscle cells suggests a specific role for NO in DA physiology or development. Endothelial-derived NO is a key regulator of smooth muscle tone in the systemic and pulmonary vasculatures (37 , 38) . However, the physiological role of NO in maintaining DA vessel patency in fetal life has been shown to be less important, as PGE2 appears to be the main vasodilator (21 22 23 , 39 , 40) . Thus, the role of increased NO levels in the DA remained to be determined.

Of the three characterized NOS isoforms, neuronal NOS was first detected in neurons (41) , but it is also expressed in other cell types, including cardiac (42) and skeletal muscle (43 , 44) and intimal cells of atherosclerotic lesions (45) . Endothelial constitutive NOS was first identified in vascular endothelial cells. It regulates vessel tone and blood pressure as well as inhibition of platelet and polymorphonuclear granulocyte adhesion to the endothelium (46) . A third isoform, iNOS, is inducible by a variety of proinflammatory agents and cytokines, and has been implicated in vascular inflammatory responses and associated pathological neointimal formation in atherosclerosis and postcardiac transplant coronary arteriopathy (47 48 49 50) .

Our report is the first demonstrating expression of nNOS in the normal fetal lamb DA and aorta. There appears to be a developmental program regulating increased expression of ecNOS and nNOS isoforms at 100 days gestation that results in posttranscriptional up-regulation of fibronectin synthesis and the onset of intimal cushion formation (3) . Clyman et al. (51) recently demonstrated ecNOS expression in the fetal lamb DA at 100 days gestation but did not detect nNOS expression. The difference may be related to our use of a different antibody previously shown to label nNOS in sections of sheep pineal gland (52) and rat pituitary gland (Fig. 2) .

Our demonstration that endogenous NO regulates fibronectin synthesis in smooth muscle cells of the 100 day gestation DA, which have a migratory phenotype, but does not influence basal levels in the relatively quiescent Ao smooth muscle cells supports the proposed role of increased NO levels in regulating the formation of intimal cushions in the DA. However, the finding that addition of an exogenous NO donor (SNAP) to Ao smooth muscle cells increases fibronectin synthesis points to a potential role for NO in pathological neointimal formation of the systemic vasculature.

Our experiments indicating no change in steady-state levels of fibronectin mRNA after treatment with SNAP or L-NMMA are in keeping with our earlier work, which showed that despite the relative increase in fibronectin synthesis, levels of fibronectin mRNA are similar in DA and Ao smooth muscle cells. NO has previously been shown to modulate posttranscriptional regulation of genes involved in iron metabolism by inducing binding of iron regulatory factors to iron response elements in the untranslated regions of ferritin and the transferrin receptor mRNAs (18 , 19) . Erythropoietin mRNA stability appears to be regulated by reactive oxygen intermediates in a similar fashion, since its stabilization by complexing of erythropoietin RNA binding protein with an element in its 3'-UTR is induced by hypoxia (53 , 54) . Vascular endothelial-derived growth factor (VEGF) is increased in hypoxia in response to superoxide and hydrogen peroxide, largely due to increased VEGF mRNA stability, mediated by enhanced binding of proteins to an ARE in the 3'-UTR of VEGF mRNA (55 56 57) . However, this is the first report demonstrating NO modulation of RNA binding protein interaction with an ARE. Since AREs are present in many cytokines and proto-oncogenes, these data provide insights into potential regulatory mechanisms in inflammation and proliferation.

We previously showed that enhanced smooth muscle cell fibronectin synthesis involves increased mRNA translation through recruitment of fibronectin mRNA to the polyribosomes as a result of binding of the microtubule-associated protein LC-3 to the ARE of fibronectin mRNA (8 , 9) . In this report we demonstrate that NO regulates phosphorylation of LC-3 associated with increased binding efficiency to the fibronectin ARE and localization to the membrane fraction of DA smooth muscle cells that contains rough endoplasmic reticulum. Our finding that membrane-associated LC-3 was increased by SNAP and decreased by L-NMMA suggests that it is the form involved in facilitating fibronectin mRNA translation on polyribosomes at the rough endoplasmic reticulum. This is in keeping with other studies of RNA binding protein function. The mRNAs encoding cyclin A and the small unit of ribonucleotide reductase are quiescent in oocytes until after fertilization, when their translation is activated, a phenomenon associated with phosphorylation of an 82 kDa RNA binding protein that binds to these mRNAs (58 , 59) . We speculate that NO-mediated phosphorylation of LC-3 may allow this protein to facilitate fibronectin mRNA access to the rough endoplasmic reticulum where it is translated. This could explain our observation that total LC-3 is unchanged in DA smooth muscle cells treated with L-NMMA. Instead, there appears to be a shift in LC-3 away from the membrane-associated phosphorylated pool and translational machinery and into the tubulin-associated cytosolic pool.

Our finding that dephosphorylated LC-3 localizes to a cytosolic fraction, where it is associated with tubulin, suggests that phosphorylation of LC-3 removes it from microtubules. Such a scenario has previously been described for the microtubule-associated protein E-MAP-115, a protein involved in microtubule dynamics (60) . Cell cycle-dependent hyperphosphorylation of E-MAP-115 results in its unstable binding to microtubules, which appears to be a prerequisite for increasing the dynamic properties of microtubules required during mitosis (60) . Conversely, phosphorylation of some RNA binding proteins is associated with modification of their ribonucleoprotein particles such that mRNA recruitment into polyribosomes is facilitated (59 , 61) . Therefore, an examination of the specific sites of NO-dependent LC-3 phosphorylation and how this determines LC-3 function in fibronectin translational efficiency and microtubule dynamics is warranted.

The mitogen-activated protein (MAP) kinase pathway has previously been implicated in smooth muscle cell migration (62 63 64) . Furthermore, reactive oxygen intermediates and NO are known to activate specific MAP kinases in vascular cells (65 66 67) . LC-3 contains four potential sites for serine/threonine phosphorylation by a proline-directed MAP kinase. Our current studies suggest that the regulation of fibronectin synthesis in DA smooth muscle cells by NO may be mediated by a MAP kinase pathway, since the MAP kinase kinase inhibitor PD98059 inhibits fibronectin synthesis in untreated DA smooth muscle cells as well as cells treated with the NO donor SNAP. This supports a role for the MAP kinase pathway in NO regulation of fibronectin synthesis; however, the effect of the MAP kinase inhibitor on LC-3 phosphorylation and localization within the cell remains to be examined. Our ongoing studies are aimed at examining the role of MAP kinase in regulating LC-3 phosphorylation and function in smooth muscle cell fibronectin synthesis and migration through the use of MAP kinase inhibitor studies and site-directed mutagenesis of the four potential MAP kinase sites within LC-3.

LC-3 facilitation of fibronectin mRNA translation has also been shown to require intact microtubules (9) . While Han et al. (68) demonstrated that a testis-brain RNA binding protein binds translationally repressed mRNAs to microtubules, we have no evidence for this function with respect to LC-3 and fibronectin mRNA. On the other hand, Tau mRNA has been proposed to use microtubule tracks for its intracellular transport and hence its polarized expression (69) , and it is conceivable that fibronectin mRNA translation may depend on microtubule-based transport. This could be the form of LC-3 described by Mann and Hammarback (32 , 33) , which is thought to play a role in microtubule dynamics. Expression of this form might therefore be enhanced by either increased or decreased NO levels, both of which appear to signal changes in cell shape.

The microtubule-associated form of LC-3 may also play a role in microtubule dynamics required for changes in cell shape associated with acquiring a motile or nonmotile phenotype. MAP 1B, of which LC-3 is a component, is expressed at high levels in growing axons of neurons in the developing and adult nervous systems, and has been proposed to play a specific role in cytoskeleton remodeling in neurons (70 71 72) . Since both SNAP and L-NMMA cause increases in the level of the microtubule-associated nonphosphorylated form of LC-3, this form could conceivably be involved in microtubule-based shape changes in DA smooth muscle cells regulated by NO.

Our data suggest a mechanistic role for NO in fibronectin-dependent smooth muscle cell migration into the intima. Though NO can protect against vascular disease (73) , there is increasing evidence implicating a pathogenic role for NO when associated with high levels of superoxide (O2.-) and generation of peroxynitrite (ONOO-) resulting from inflammatory processes (47 , 74 , 75) . We have not determined whether NO itself, or ONOO- as the result of enhanced NO production in the DA, is the cause of increased fibronectin synthesis. In the pathophysiology of occlusive neointimal formation induced by an inflammatory stimulus, e.g., restenosis and postcardiac transplant coronary arteriopathy, induction of fibronectin synthesis represents one of the earliest changes (76 77 78) and may be mediated by NO (our unpublished data).

Our results not only support other evidence indicating the importance of NO in vascular neointimal formation, but also provide novel insights into the mechanism involved by elucidating the role of NO in posttranscriptional up-regulation of fibronectin, an extracellular matrix glycoprotein key to vascular smooth muscle migration. Furthermore, our demonstration that NO can regulate fibronectin is exciting, because this mechanism may be relevant to inflammatory responses in the vessel wall and other tissues in which induction of NO and extracellular matrix deposition are observed.


   ACKNOWLEDGMENTS
 
We thank Dr. James Hammarback, Department of Neurobiology and Anatomy, Bowman Gray School of Medicine, Winston-Salem, N.C., for supplying us with polyclonal antibodies to LC-3. We are grateful to Joan Jowlabar for secretarial assistance in preparing the manuscript and to Claire Coulber for technical assistance.


   FOOTNOTES
 
2 Abbreviations: ARE, adenosine-uracil rich element; Ao, aorta; BSA, bovine serum albumin; DA, ductus arteriosus; DPI, diphenylene-iodonium; ecNOS, endothelial constitutive nitric oxide synthase; Ig, immunoglobulin; iNOS, inducible nitric oxide synthase; LC-3, light chain 3; L-NMMA, L-NG-monomethylarginine; MAP, mitogen-activated protein; MAP 1B, microtubule-associated protein 1B; nNOS, neuronal nitric oxide synthase; NO, nitric oxide; ONOO-, peroxynitrite; PBS, phosphate-buffered saline; PGE2, prostaglandin E2; SD, standard deviation; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; SE, standard error of the mean; SNAP, S-nitroso-N-acetylpenicillamine; TBS, TRIS-buffered saline; 3'-UTR, 3'-untranslated region; VEGF, vascular endothelial-derived growth factor.

Received for publication January 4, 1999. Revision received March 9, 1999.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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