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Originally published In Press as doi:10.1074/jbc.M404882200 on May 24, 2004

J. Biol. Chem., Vol. 279, Issue 31, 32897-32903, July 30, 2004
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Intron Retention Generates a Novel Id3 Isoform That Inhibits Vascular Lesion Formation*

Scott T. Forrest{ddagger}, Kurt G. Barringhaus{ddagger}, Demetra Perlegas{ddagger}, Marie-Louise Hammarskjold§, and Coleen A. McNamara{ddagger}||

From the {ddagger}Cardiovascular Division, Department of Internal Medicine, and the Cardiovascular Research Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908 and §Department of Microbiology and the Myles H. Thaler Center, University of Virginia Health Sciences Center, Charlottesville, Virginia 22908

Received for publication, May 3, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The expression of intron-containing messages has been shown to occur in a variety of diseases including lactic acidosis, Cowden Syndrome, and several cancers. However, it is unknown whether these intron-containing messages result in protein production in vivo. Indeed, intron-containing RNAs are typically retained in the nucleus, targeted for degradation, or are repressed translationally. Here, we show that during vascular lesion formation in rats, an alternative isoform of the helix-loop-helix transcription factor Id3 (Id3a) generated by intron retention is abundantly expressed. We demonstrate that Id3 is expressed early in lesion formation when the proliferative index of the neointima is highest and that Id3 promotes smooth muscle cell (SMC) proliferation and S-phase entry and inhibits transcription of the cell-cycle inhibitor p21Cip1. Using an Id3a-specific antibody developed by our laboratory, we show that Id3a protein is induced during vascular lesion formation and that Id3a expression peaks late when the proliferative index is low or declining and extensive apoptosis is observed. Furthermore, Id3a fails to promote SMC growth and S-phase entry or to inhibit p21Cip1 promoter transactivation. In contrast, Id3a stimulates SMC apoptosis and inhibits endogenous Id3 production. Adenoviral delivery of Id3a inhibited lesion formation in balloon-injured rat carotid arteries in vivo. These data describe a novel feedback loop whereby intron retention generates an Id3 isoform that acts to limit SMC growth during vascular lesion formation, providing the first evidence that regulated intron retention can modulate a pathologic process in vivo.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Modulation of vascular smooth muscle cell (SMC)1 growth has emerged as a promising strategy for treating vascular proliferative disorders such as restenosis, vein graft failure, and transplant arteriopathy (1, 2). During vascular lesion formation, normally quiescent SMCs in the vessel wall re-enter the cell cycle, proliferate, and secrete matrix (3). In addition to growth and proliferation, SMC in developing lesions demonstrate altered morphology and apoptotic activity (4). Apoptosis may function to limit SMC growth and lesion size in vivo and has been associated with changes in the stability of atherosclerotic plaques (5, 6). Thus, understanding the mechanisms that regulate SMC growth and apoptosis is of key importance.

The Id class of helix-loop-helix proteins have been linked to cell-cycle control in a variety of cell types (7, 8). The Id family of proteins consists of four members, Id1-4, whose functions do not appear to be completely redundant (7, 9, 10). Id proteins act as dominant-negative transcription factors and appear to promote cellular proliferation by blocking basic helix-loop-helix-mediated transactivation of genes involved in cell-cycle control such as the cyclin-dependent kinase inhibitor p21Cip1 (11, 12). Recently, Id3 has emerged as a key regulator of SMC proliferation. Id3 expression is induced in cultured SMC in response to angiotensin II and reactive oxygen species, and Id3 message expression is increased in vivo in SMC in response to vascular injury (13, 14).

The Id3 gene encodes two distinct isoforms generated as a result of alternative splicing. Both messages of the rat gene termed Id3 and Id3a are expressed in vivo in SMC within vascular lesions. Id3a message, generated by retention of intron 1 in Id3 pre-mRNA, is normally absent from the vessel wall yet is abundantly expressed at late time points following vascular injury in rats. In addition, the message of the Id3a human homologue is expressed in advanced atherosclerotic plaques. Interestingly, Id3a inhibits growth and stimulates apoptosis in cultured SMC (13).

Intron-containing message expression has been associated previously with disease states such as breast and colon cancer (15, 16). However, intron-containing messages are frequently retained in the nucleus or targeted for degradation, preventing protein expression. In this study we provide the first evidence of intron retention giving rise to a distinct protein with important functional consequences in a disease model. Here we describe a novel feedback loop whereby Id3-induced SMC growth is limited by induction of Id3a via intron retention and show that Id3a expression following vascular injury inhibits lesion formation in vivo.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Cell Culture—Primary rat aortic SMCs were obtained from adult male Sprague-Dawley rats by enzymatic digestion and grown in Dulbecco's modified Eagle's medium/F-12 (1:1) (Invitrogen) with 20% fetal bovine serum (HyClone, Logan, UT). Following the establishment of a cultured line, cells were cultured in 10% fetal bovine serum. Id3-/- and wild-type littermate control mouse aortic SMCs were prepared as described above.

Adenoviral Expression—The construction of Ad-Id3 and Ad-Id3a has been described previously (11). Ad-GFP was purchased from the University of Iowa. Cultured rat aortic SMCs were infected with 25 multiplicities of infection of virus in serum-free medium. After 4 h, virus was removed and cells were cultured in medium containing 10% fetal bovine serum.

Transient Transfection—Transfections were performed using Fu-GENE 6 transfection reagent (Roche Applied Science) according to the manufacturer's instructions.

Cell Number Analysis—Cultured rat aortic SMCs were plated in 96-well plates at a density of 1,000 cells/well and infected with either Ad-GFP or Ad-Id3. Sorted SMCs were transfected with 0.5 µg of pAd-Lox GFP and 0.5 µg of empty pAdLox or pAdLox Id3. 24 h later, cells were harvested by scraping, and GFP-positive cells were sorted into 96-well plates at a density of 500 cell/well using a FACSVantage SE Turbo sorter (BD Biosciences). At the indicated time points following infection or seeding, cells were counted using a colorimetric cell number assay (Celltitre, Promega).

S-phase Analysis—Cultured rat aortic SMCs were plated in 96-well plates at a density of 1,000 cells/well and infected with Ad-GFP, Ad-Id3, or Ad-Id3a. At the indicated time points, BrdUrd incorporation was measured according to the manufacturer's specifications (cell proliferation enzyme-linked immunosorbent assay BrdUrd, Roche Applied Science) and quantitated colorimetrically by measuring Abs370.

Promoter Reporter Assays—Rat aortic SMCs were transfected with 0.9 µg of expression plasmids together with 0.1 µg of p21-luciferase or Id3-pGL3 reporter plasmids. 48 h following transfection, cells were harvested in luciferase lysis buffer, incubated with luciferase substrate (Promega), and measured for luciferase activity.

Analysis of Apoptotic Activity—Cultured rat aortic SMCs infected with Ad-GFP, Ad-Id3, or Ad-Id3a and cytoplasmic histone-bound DNA fragments were measured using the cell death detection enzyme-linked immunosorbent assay kit (Roche Applied Science).

Western Blotting—Lysates were collected in mPER lysis buffer (Pierce). Samples were electrophoresed with 4-20% SDS gradient gels (NuPAGE, Invitrogen) and transferred to polyvinylidene difluoride membrane (Sigma). Western blotting was performed with antibodies to Id3 (0.5 µg/ml, C-20, Santa Cruz Biotechnology), p21 (1.0 µg/ml, BD Biosciences), {alpha}-tubulin (0.5 µg/ml, Sigma), or Id3a (1.0 µg/ml) followed by a 1:2,000 dilution of horseradish peroxidase-linked secondary antibody (Santa Cruz Biotechnology).

Rat Balloon Endothelial Denudation and Gene Delivery—The rat carotid endothelial denudation model was performed using 350 g of male Sprague-Dawley rats (Harlan Laboratories, San Diego, CA) as described elsewhere (16). Left common carotid arteries were blunt-dissected at the bifurcation, and internal and external carotids were ligated. A 1-cm tip percutaneous transluminal coronary angioplasty dilation catheter (Boston Scientific) was inserted through the external carotid into the common carotid, and endothelial denudation was performed by inflation of the balloon and three passages down the common carotid. For vessels treated with gene delivery, the common carotid artery was ligated ~1 cm distal to the bifurcation and a polyethylene catheter, inserted through the external carotid, was used to deliver 100 µl of adenovirus (1 x 1010 plaque-forming unit). After a 20-min incubation, vessels were flushed with phosphate-buffered saline and blood flow was restored through the carotid artery.

Vessel Harvesting and Immunohistochemistry—Rats were given an overdose of intraperitoneal ketamine/xlyazine, and animals were pressure-perfused with 4% paraformaldehyde in phosphate-buffered saline. Injured arteries were removed and postfixed in 4% paraformaldehyde overnight at 4 °C, dehydrated in a graded alcohol series, and paraffin-embedded for thin sectioning. 5-µm arterial sections were stained using the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, UT) according to the manufacturer's instructions using a 1:100 dilution anti-Id3 (Santa Cruz Biotechnology) or mouse monoclonal anti-Id3a antibody. Slides then were incubated for 30 min in 0.3% hydrogen peroxidase substrate (Sigma) for 2 min, counterstained with hematoxylin, and mounted with Vectashield mounting medium (Vector Laboratories).

Analysis of Vessel Morphometry—Five cross-sections from six (Ad-{beta}-galactosidase) or seven (Ad-Id3a) balloon-injured in vivo fixed rat arteries were stained with hematoxylin and eosin (Sigma). Cross-sections were imaged using an Olympus BH 2-RFCA microscope with an on-line CCD camera (Olympus DP-70). The circumference of the lumen, internal elastic lamina (IEL), and external elastic lamina (EEL) was measured and used to calculate neointima and medial area (Image-Pro Plus, Media Cybernetics, Carlsbad, CA). Statistical analysis was performed using a Mann-Whitney U test due to the small sample size. A p < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Id3a Is Generated by Intron Retention—Alternative splicing of Id3 pre-mRNA resulted in messages encoding two distinct protein isoforms with different C termini (Fig. 1a). Id3 message is generated by the removal of both introns in the pre-mRNA. Id3a message is the result of inclusion of intron 1, a 115-bp sequence encoding a unique 29-amino acid C terminus.



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  		FIG. 1.
Id3 pre-mRNA is spliced to Id3 in cultured SMC. a, Id3 and Id3a contain different C-terminal peptide sequences. The unique C terminus of Id3a is generated by inclusion of intron 1. b, expression constructs analyzed. pAdLox Id3 contains Id3 cDNA lacking both introns. pAdLox Id3a was generated by point mutation of both the 5'-splice site and a pseudo-splice site intron 1 and deletion of the 3'-splice site. pAdLox Id3 FL encodes a 1.5-kb section of the Id3 pre-mRNA, which includes all three exons and both introns. c, SMCs were transiently transfected with empty vector (pAdLox empty), pAdLox Id3, pAdLox Id3a, or pAdLox Id3 FL, and lysates were analyzed by Western blotting.

 
To examine the processing of Id3 pre-mRNA, we used three expression plasmid constructs for transient transfection of cultured SMCs: 1) a plasmid encoding full-length Id3 pre-mRNA containing intron 1 (pAdLox Id3 FL); 2) a plasmid encoding Id3 cDNA lacking intron 1 (pAdLox Id3); and 3) a plasmid encoding an Id3 construct that contains intron 1 but does not allow for removal of the intron (pAdLox Id3a) (Fig. 1b). This non-spliceable construct contains two point mutations (330G-> A and 409G-> A) disturbing both the 5'-splice site and a pseudo-splice site within intron 1 and a deletion removing the 3'-splice site (Fig. 1b). We transiently transfected SMC with empty vector, pAdLox Id3, pAdLox Id3-FL, and pAdLox Id3a and analyzed lysates by Western blotting. Because no commercial antibody to Id3a exists, we developed an Id3a-specific mouse monoclonal antibody directed against the predicted unique C terminus of the Id3a protein. The expression of full-length Id3 pre-mRNA in SMC resulted in the expression of the Id3 but not Id3a protein (Fig. 1c). This finding suggests that in cultured proliferating SMC, intron 1 is removed from Id3 pre-mRNA to yield the Id3 protein. Disturbing the splice sites in Id3 pre-mRNA resulted in the inclusion of intron 1 and thus the production of Id3a, providing evidence that intron retention is the mechanism determining which message (and thus protein) is produced.

Id3 and Id3a Proteins Are Expressed Differentially following Balloon Injury—We previously have used in situ hybridization to show that Id3a message is induced following vascular injury (13). However, intron-containing messages are retained frequently in the nucleus or targeted for degradation, preventing protein expression. To determine whether the expression of the intron 1-containing Id3a message results in the expression of a unique protein in vivo, we analyzed the Id3 and Id3a protein expression following vascular injury.

To determine the time course of Id3 and Id3a expression in vivo following balloon injury, we used the rat carotid injury model (17) and harvested vessel lysates at various time points following injury for Western blot analysis (Fig. 2, a and b). Id3 protein expression present at low levels in uninjured control vessels was increased by 3 days post-injury, peaked at 7 days, and returned to base-line levels by 28 days. In contrast, Id3a protein not detected in uninjured arteries was induced following injury, peaked at 14 days post-injury, and remained high through 28 days.



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  		FIG. 2.
Id3 and Id3a have different patterns of expression following vascular injury. a, Western blot analysis of homogenates from injured rat carotid arteries collected at various time points following balloon injury. b, densitometric analysis of Id3 ({blacksquare}) and Id3a ()Western blot results normalized to {alpha}-tubulin immunoreactivity. c, the Id3a mouse monoclonal antibody is specific for Id3a. HEK cells were transiently transfected with an Id3 expression plasmid (pAdLox Id3) or an Id3a expression plasmid (pAdLox Id3a), and lysates were analyzed by Western blotting using the antibodies indicated. d-g, immunohistochemistry using anti-Id3 (d and e) or anti-Id3a antibody (f and g) was performed on rat carotid arteries 14 days following balloon injury. Magnification is x100 (e and g) and x40 (d and f). Arrows denote the internal elastic lamina.

 
To confirm that our Id3a antibody does not cross-react with Id3 protein, we transfected HEK cells with Id3 or Id3a expression constructs (pAdLox Id3 and pAdLox Id3a) and examined lysates by Western blotting with our monoclonal Id3a antibody or a commercially available Id3 antibody (Fig. 2c). Our Id3a antibody reacted with ectopically expressed Id3a protein but showed no immunoreactivity with Id3.

To examine the expression pattern of Id3 and Id3a in response to injury, we balloon-injured rat carotid arteries and fixed injured arteries 14 days later for immunohistochemistry. Id3 protein expression was observed in the media and in the developing neointima in injured vessels (Fig. 2, d and e). Id3a is abundantly expressed in the neointima of injured vessels but was absent from the media (Fig. 2, f and g).

Id3 Enhances SMC Proliferation—To determine the time course of Id3-induced effects on SMC proliferation, we infected cells with an adenoviral construct encoding either Id3 cDNA (lacking intron 1, Ad-Id3) or GFP (Ad-GFP) and assayed for the cell number. Ad-Id3 infection resulted in a significant increase in cell number at 12 and 24 h post-infection (p < 0.03) with the effect at 24 h being marked (Fig. 3a). In agreement with this finding, cultured SMC from Id3-/- mice demonstrated significantly reduced proliferation in the presence of serum (Fig. 3b). Ad-Id3-infected SMC displayed increased S-phase entry as determined by BrdUrd incorporation 4 and 16 h (p < 0.03) post-infection (Fig. 3c). Consistent with this finding, Id3-/- SMC entered S-phase at a reduced rate (p < 0.003) when compared with wild-type controls (Fig. 3d).



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  		FIG. 3.
Id3 stimulates SMC proliferation. a, Id3 increases SMC number. Rat SMCs were infected with Ad-Id3 or Ad-GFP and assayed for cell number at the time points indicated. b, mouse aortic SMC from Id3-/- ({blacksquare}) or wild-type ({blacktriangleup}) mice were plated in 96-well plates and assayed for cell number at various time points after plating. c, Id3 increases S-phase entry. SMCs were infected with Ad-Id3 () or Ad-GFP ({blacksquare}) and assayed for BrdUrd incorporation at the time points indicated. d, mouse aortic SMC from Id3-/- or wild-type mice were assayed for BrdUrd incorporation. Id3-/- SMC demonstrated an ~50% reduction in S-phase entry when compared with wild-type controls. e, Id3 inhibits p21 promoter activation. Rat SMCs were transiently co-transfected with p21-luciferase reporter plasmid (p21-Luc) and pAdLox empty vector (base line), or pAdLox Id3 and lysates were assayed for luciferase activity. f, mouse aortic SMC from Id3-/- or wild-type mice were transiently transfected with p21-Luc, and lysates were assayed for luciferase activity. Inset, Western blot analysis of p21 protein levels in lysates from Id3-/- or wild-type SMC.

 
Recent data provide evidence that all of the functions of the Id proteins are not redundant (7, 9, 10). Id3 has been implicated in the regulation of p21Cip1 protein expression; however, the effects of Id3 on p21Cip1 promoter activity are unknown (11, 14). In promoter-reporter experiments, transient transfection of SMC with pAdLox Id3 was capable of dramatically inhibiting endogenous promoter activation of the cell-cycle inhibitor p21Cip1 (Fig. 3e, p < 0.003). Further, Id3-/- SMC displayed a significant increase in p21Cip1 promoter activation (p < 0.0006) and p21Cip1 protein levels relative to wild-type control SMC (Fig. 3f).

The Effects of Id3a on SMC Growth and Viability—Previous studies have demonstrated that the E-protein E47 effectively activates p21Cip1 transcription, resulting in inhibition of cellular growth (11). Accordingly, we evaluated the effect of Id3 and Id3a on E-protein-mediated p21Cip1 transcription in SMC using a p21Cip1 promoter-reporter construct. Co-transfection of SMC with pcDNA-E47 and pAdLox Id3 but not pAdLox Id3a reduces p21Cip1-luciferase activity (p < 0.01) to levels comparable to base line, demonstrating that Id3a lacks the ability of Id3 to block E47-mediated p21Cip1 transactivation. These observations are not attributable to differences in Id3 and Id3a expression, because both pAdLox Id3 and pAdLox Id3a transfection results in similar protein levels as detected by Western blot using an antibody to their common N terminus (Fig. 4a, inset). Consistent with these data and in contrast to Ad-Id3, infection of SMC with an Id3a-expressing adenovirus, Ad-Id3a, did not result in a significant difference in S-phase entry (Fig. 4b).



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  		FIG. 4.
The effects of Id3a on SMC growth and viability. a, Id3a is not effective at inhibiting E47-mediated p21 transcription. Rat SMCs were transiently co-transfected with p21-Luc together with pCDNA-E47 and pAdLox empty vector (base line), pAdLox Id3, or pAdLox Id3a. Lysates were assayed for luciferase activity. Inset, Id3 and Id3a show similar levels of overexpression. Rat SMCs were transiently transfected with pAdLox Id3, pAdLox Id3a, and empty pAdLox vector control plasmids, lysed, and subjected to Western blotting using an antibody against the N-terminal portion of Id3/Id3a. b, Id3a does not promote S-phase entry. Rat SMCs were infected with Ad-Id3a () or Ad-GFP ({blacksquare}) and assayed for BrdUrd incorporation at the time points indicated. c, Id3a expression inhibits SMC proliferation. SMCs were co-transfected with pAdLox GFP and either pAdLox Id3a ({blacksquare}) or pAdLox empty plasmid ({blacktriangleup}). Following transfection, GFP-positive cells were sorted into 96-well plates for cell number assays, which were performed at various time points following seeding. d, Id3a stimulates SMC apoptosis. SMCs were infected with Ad-Id3a () or Ad-GFP ({blacksquare}). At various time points following infection, cytoplasmic lysates were collected and apoptosis was quantitated by an enzyme-linked immunosorbent assay-based method measuring cytoplasmic histone-bound DNA. e, Id3a expression decreases endogenous Id3 protein levels. SMCs were infected with 25, 100, or 200 multiplicities of infection of Ad-Id3a or Ad-GFP control virus, and lysates were analyzed by Western blotting. f, Id3a inhibits Id3 transcription. SMCs were transiently transfected with Id3-pGL3 reporter plasmid together with pAdLox empty plasmid, pAdLox Id3, or pAdLox Id3a.

 
Advanced vascular lesions demonstrate extensive apoptosis, a low proliferative index, and robust Id3a expression. Accordingly, we assayed the effects of Id3a on SMC proliferation and apoptosis. Results confirm that, relative to control plasmid, pAdLox Id3a expression inhibits SMC proliferation (Fig. 4c, p < 0.01). This finding correlates with the ability of Ad-Id3a to promote apoptosis in SMC 48 h following infection (Fig. 4d, p < 0.003).

It is intriguing to postulate that Id3a acts as a negative feedback molecule to limit Id3-induced growth. Therefore, we examined the effect of Ad-Id3a on Id3 protein levels and found that Ad-Id3a infection results in a dose-dependent decrease in endogenous Id3 levels in SMC (Fig. 4e). Because Id3 acts as a transcription regulator, we hypothesized that Id3a inhibits Id3 expression at the level of transcription. When co-transfected with an Id3 luciferase promoter-reporter construct, pAdLox Id3a resulted in an ~50% reduction in luciferase activity relative to empty vector (p < 0.001), whereas pAdLox Id3 had no significant effect on Id3 transcription (Fig. 4f).

Overexpression of Id3a in Injured Vessels Inhibits Neointimal Formation—Given the ability of Id3a to inhibit SMC growth and promote SMC apoptosis and that Id3a expression in vivo peaks as neointimal SMC proliferation declines, Id3a may represent an endogenous mechanism whereby neointimal formation is limited. Therefore, we hypothesized that early Id3a overexpression may inhibit vascular lesion formation in response to injury. To examine the effects of Id3a gene expression on lesion formation, we infected rat carotid arteries with Ad-Id3a or Ad-{beta}-galactosidase control virus immediately following balloon injury. 28 days following injury, arteries were fixed and embedded and vessel morphometry was analyzed by hematoxylin and eosin staining. Representative sections are shown (Fig. 5a and b). We assayed the effect of Id3a gene expression on neointimal formation by quantitating the intimal:medial ratio of injured vessels. Ad-Id3a delivery resulted in a 55% reduction in intimal:medial ratio when compared with Ad-{beta}-galactosidase control virus (Fig. 5c; p < 0.05). These results indicate that Id3a expression in vivo reduces vascular lesion formation in response to injury.



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  		FIG. 5.
Ad-Id3a delivery inhibits vascular lesion formation. a and b, representative cross-sections from carotid arteries of rats treated with either Ad-{beta}-galactosidase (a) or Ad-Id3a (b) following balloon injury. 28 days following injury, vessels were harvested, embedded, and hematoxinand eosin-stained. c, cross-sectional areas of the intima and media were calculated. The lesion sized is expressed as the ratio of neointima:media. d, proposed schematic of SMC growth regulation by Id3 and Id3a. Id3 up-regulated in vivo following vascular lesion or in vitro via ectopic expression acts to promote SMC growth via an inhibition of p21 transcription, thus increasing S-phase entry. Retention of intron 1 in Id3 pre-mRNA results in Id3a production, which inhibits SMC growth and stimulates apoptosis. Id3a participates in a negative feedback loop that functions to down-regulate Id3 expression.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Regulation of protein expression by alternative splicing is emerging as a potentially important means by which vascular lesion formation is controlled. SMC migration and secretion of extracellular matrix play significant roles in lesion formation. Interestingly, the expression of fibronectin and tenascin-C, both extracellular matrix proteins, is regulated by alternative splicing following vascular injury in rats (19, 20). Further, it has been shown that the expression of RA301/Tra2{beta}, an RNA-binding protein and putative splicing factor, is strongly up-regulated following balloon injury in rats (21). Therefore, it is intriguing to postulate that alternative splicing plays a central role in regulating SMC migration, proliferation, and extracellular matrix secretion by altering the expression of genes such as Id3, tenascin-C, and fibronectin.

Here we describe, to our knowledge, the first example in an in vivo disease model of alternative protein expression from an intron-containing message. The expression of intron-containing messages has been described in cancer cells, such as Her-2 and CD44 messages in breast and colon cancer (15, 16). Retention of intron 8 in the Her-2 transcript results in a sequence coding for herstatin, a Her-2 isoform containing a unique C terminus. Recombinant herstatin protein is capable of antagonizing Her-2-mediated growth in breast cancer cells, and herstatin message is down-regulated in carcinoma cells, providing a potential link between herstatin production and growth inhibition in breast cancer, yet it remains unclear whether herstatin protein is produced in vivo (15, 22, 23). Protein expression from intron-containing message has been described in the literature (24, 25) for several protein isoforms including periaxin and sodium channel subunits. However, the in vivo consequences of these events are not clear.

During early stages of vascular lesion formation, the expression of the fully spliced Id3 protein is increased when the proliferative index of the neointima is high, suggesting that Id3 acts to promote SMC growth in response to vascular injury (26, 27). Consistent with this hypothesis, ectopic Id3 expression promoted SMC proliferation and S-phase entry, whereas Id3-/- SMC displayed reduced proliferation and S-phase entry. It has been shown previously that other Id proteins promote cellular growth by negatively regulating transcription of cellcycle inhibitors such as p21Cip1 (11, 12). Indeed, we show that Id3 inhibited endogenous and E47-driven p21Cip1 transcription in SMC and that Id3-/- SMC had markedly increased p21Cip1 transcription and protein levels, providing a mechanism for Id3-induced SMC proliferation.

Retention of intron 1 in the Id3 message yields a distinct protein isoform termed Id3a, which encodes an alternative C terminus. Id3a protein expression is not observed in vivo in quiescent vessels but is induced following vascular injury. Unlike Id3, peak Id3a expression corresponds to time points late in lesion formation when the proliferative index of the neointima is declining or low and extensive apoptosis is observed (18, 27). Thus, we hypothesize that Id3a functions as part of a negative feedback loop to limit pathologic SMC proliferation (Fig. 5d). This is in agreement with our cell culture data, indicating that Id3a expression causes a decrease in SMC proliferation and the onset of apoptosis as well as inhibition of Id3 expression. The ability of Id3a to inhibit neointimal formation in vivo when expressed immediately following vascular injury further supports this hypothesis.

Our results provide the first evidence that regulated intron retention is a key step in controlling SMC growth and viability and vascular lesion formation. The mechanisms that regulate the splicing and translation of Id3a and other intron-containing messages may provide novel and important insights into the molecular mechanisms that regulate cellular growth in response to vascular injury and other proliferative disorders.


   FOOTNOTES
 
* This work was supported by National Institutes of Health Grants T32HL007284 (to S. T. F.), T32HLOO7355 (to K. G. B.), RO1 HL062522 and PO1 HL55798 (to C. A. M.), and AI34721, AI054335 [GenBank] , and CA097095 [GenBank] (to M.-L. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Supported by the Charles H. Ross Jr. Endowment at the University of Virginia. Back

|| To whom correspondence should be addressed. Tel.: 434-982-3366; Fax: 434-924-2828; E-mail: cam8c{at}virginia.edu.

1 The abbreviations used are: SMC, smooth muscle cell; Ad, adenovirus; BrdUrd, bromodeoxyuridine; Her, herstatin. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Yuan Zhuang (Duke University) for the generous gift of Id3-/- mice.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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