©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Structure and Expression of a Smooth Muscle Cell-specific Gene, SM22 (*)

Julian Solway , Jonathan Seltzer , Frederick F. Samaha , Steven Kim , Linda E. Alger , Qun Niu , Edward E. Morrisey , Hon S. Ip , Michael S. Parmacek (§)

From the (1) Department of Medicine, University of Chicago, Chicago, Illinois 60637

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

SM22 is expressed exclusively in smooth muscle-containing tissues of adult animals and is one of the earliest markers of differentiated smooth muscle cells (SMCs). To examine the molecular mechanisms that regulate SMC-specific gene expression, we have isolated and structurally characterized the murine SM22 gene. SM22 is a 6.2-kilobase single copy gene composed of five exons. SM22 mRNA is expressed at high levels in the aorta, uterus, lung, and intestine, and in primary cultures of rat aortic SMCs, and the SMC line, A7r5. In contrast to genes encoding SMC contractile proteins, SM22 gene expression is not decreased in proliferating SMCs. Transient transfection experiments demonstrated that 441 base pairs of SM22 5`-flanking sequence was necessary and sufficient to program high level transcription of a luciferase reporter gene in both primary rat aortic SMCs and A7r5 cells. DNA sequence analyses revealed that the 441-base pair promoter contains two CArG/SRF boxes, a CACC box, and one potential MEF-2 binding site, cis-acting elements which are each important regulators of striated muscle transcription. Taken together, these studies have identified the murine SM22 promoter as an excellent model system for studies of developmentally regulated, lineage-specific gene expression in SMCs.


INTRODUCTION

The phenotypic plasticity of smooth muscle cells (SMCs)() permits this muscle cell lineage to subserve diverse functions in multiple tissues including the arterial wall, uterus, respiratory, urinary and digestive tracts. In contrast to fast and slow skeletal muscle cells which fuse and terminally differentiate before expressing contractile protein isoforms, SMCs are capable of simultaneously proliferating and expressing a set of lineage-restricted proteins including myofibrillar isoforms, cell surface receptors, and SMC-restricted enzymes. Moreover, in response to specific physiological and pathophysiological stimuli, SMCs can modulate their phenotype by down-regulating a set of contractile protein genes, and in so doing, convert from the so called ``contractile phenotype'' to a dedifferentiated ``secretory phenotype'' (1, 2, 3, 4, 5, 6, 7, 8, 9, 10) . This phenotypic modulation has been implicated in the pathogenesis of a number of disease states including atherosclerosis and restenosis following coronary balloon angioplasty (11-14) and may also contribute to the airway remodeling seen in asthma (15) . One approach to understanding the molecular mechanisms that regulate SMC development and differentiation is to identify and characterize the cis-acting sequences and trans-acting factors that regulate SMC-specific, developmentally-regulated gene expression. This approach has provided a great deal of information concerning the molecular mechanisms that regulate skeletal muscle and cardiac muscle development (for review, see Refs. 16-19). In contrast, relatively little is understood about the molecular mechanisms that regulate SMC development due in part to the fact that few SMC-specific markers have been identified.

Previous studies have suggested that the SM22 gene product is expressed exclusively in smooth muscle-containing tissues of adult animals and is one of the earliest markers of differentiated smooth muscle cells (10, 20, 21, 22, 23) . SM22 is a 22-kDa protein with structural homology to the vertebrate thin filament myofibrillar regulatory protein calponin and the Drosophila muscle protein mp20 (24). Interestingly, Drosophila mp20 is expressed specifically in the synchronous muscles and is not found in the asynchronous oscillatory flight muscles. In addition, Drosophila mp20 has two potential calcium binding domains that are oriented in a helix-loop-helix or EF-hand conformation (25) . In contrast to calponin, which is associated physically with other SMC thin filament proteins including actin, caldesmon, and troponin T, both SM22 and Drosophila mp20 do not appear to be physically associated with the contractile apparatus (20) . In vitro analyses, including differential display performed using primary cultures of rat aortic SMCs, have revealed that SM22 is expressed at high levels in freshly dispersed SMCs, but is dramatically down-regulated in late passage cells (10) . Taken together, these data are consistent with the hypothesis that SM22, while not physically associated with the contractile apparatus, plays an important functional role in smooth muscle cells.

In the studies described in this report, we have used the murine SM22 gene as a model system to examine the molecular mechanisms that regulate SMC-specific gene expression. Specifically, we have isolated the murine SM22 cDNA and used it as a molecular probe to better define the tissue and cellular pattern of SM22 gene expression in vivo and in primary cultures of rat aortic SMCs during progression through the cell cycle. In addition, we have isolated and structurally characterized a murine SM22 genomic clone and performed a series of transient transfections using SM22/luciferase reporter plasmids in order to identify the functionally important cis-acting transcriptional regulatory elements that control SM22 gene expression in SMCs. These studies demonstrated that SM22 is expressed at high levels in all smooth muscle cell-containing tissues of the adult mouse, as well as in primary cultures of rat aortic SMCs and the smooth muscle cell line, A7r5. Transient transfection analyses revealed that a relatively small fragment in the 5`-flanking region of the SM22 gene (bp -441 to +41) is necessary and sufficient to program high level transcription of SM22/luciferase reporter constructs in primary rat aortic smooth muscle cells and in A7r5 cells, but is inactive in all non-smooth muscle cell lines analyzed. Taken together, these data suggest that the SM22 promoter may serve as an excellent model system to examine the molecular mechanisms that regulate SMC-specific gene expression.


EXPERIMENTAL PROCEDURES

Isolation of Murine SM22 cDNA Clones

The coding region of the murine SM22 cDNA was isolated by performing low stringency PCR using murine uterine RNA and synthetic 5` and 3` oligonucleotide PCR primers constructed from the previously published sequence of the rat SM22 cDNA (23) . The 5` PCR primer was constructed to be identical to the first 34-bp of the rat SM22 cDNA with the addition of a 5` EcoRI site (5`-ATCGAATTCCGCTACTCTCCTTCCAGCCCACAAACGACCAAGC-3`). The 3` primer was constructed to include the reverse complement of bp 759 to 782 of the rat SM22 cDNA with an additional 3` HindIII restriction site (5`-ATCAAGCTTGGTGGGAGCTGCCCATGTGCAGTC-3`). PCR reaction products were subcloned into EcoRI/HindIII-digested pGEM7Z (Promega, Madison, WI) as described previously (26) . The nucleotide sequence of the murine SM22 cDNA was confirmed by sequencing of the full-length murine SM22 genomic clone. MacVector DNA sequencing software (Kodak/IBI, Rochester, NY) was used for DNA sequence analyses.

To isolate the 3`-untranslated region of the SM22 cDNA, 5 10 recombinant clones from an oligo(dT)-primed gt11 C2C12 myotube cDNA library were screened with the P-labeled murine SM22 cDNA probe (bp 29-811) as described previously (27) . Twelve clones were purified to homogeneity and analyzed by Southern blot analyses as described (27) . Two independent clones, each of which contained a poly(A) tail, were subcloned into EcoRI-digested pGEM7Z and their nucleotide sequences determined. The nucleotide sequence of the 5`-untranslated region was determined from the sequence of the SM22 genomic clone. The 5`-untranslated region was localized on the genomic clone by Southern blot hybridizations, in addition to RNase protection and primer extension analyses as described below.

Isolation of Murine SM22 Genomic Clones

Approximately 1 10 recombinant phage from a murine 129SV Lambda FIX II genomic library (Stratagene, La Jolla, CA) were screened with the 783-bp murine SM22 cDNA probe (bp 29-811) labeled with [-P]dCTP, and three positive clones were purified to homogeneity as described previously (27) . One clone (SM22-3a) was found to include the entire coding region of the SM22 gene and 9-kb of 5`-flanking sequence and was used for all subsequent subcloning and sequencing experiments.

Southern Blot Analyses

High molecular weight DNA was prepared from the tails of strain 129SV mice as described previously (26). Southern blotting and hybridization to the radiolabeled 783-bp murine SM22 cDNA probe were performed as described previously (26) . Low stringency washing conditions were 2 SSC, 0.1% SDS at 50 °C. High stringency washing conditions were 0.1 SSC, 0.1% SDS at 68 °C.

Northern Blot Analyses

Tissues were isolated from 12-week-old 129SV mice (Jackson Laboratories) as described previously (26). Animals were housed and cared for according to NIH guidelines in the University of Chicago Laboratory Animal Medicine Veterinary Facility. RNA was prepared from organ samples and from cultures of primary rat aortic SMCs, the rat SMC line A7r5, and non-smooth muscle cell lines including murine NIH 3T3 cells, murine C3H10T1/2 cells, monkey COS-7 cells, murine C2C12 myoblasts and myotubes, human HepG2 cells, and murine EL-4 cells by the single step guanidinium isothiocyanate protocol described previously (28) . Northern blotting was performed using 10 µg of RNA/sample as described previously with the exception that 36 µg/ml ethidium bromide was added to the RNA resuspension buffer in order to permit quantitation of the 28 S and 18 S ribosomal RNA subunits in each lane. Probes included the 783-bp (bp 29-811) murine SM22 cDNA and the 754-bp (bp 659-1404) murine calponin cDNA probe.() Quantitative image analyses were performed using a Molecular Dynamics PhosphorImager (Sunnyvale, CA).

Primer Extension, 5`-RACE, and RNase Protection Analyses

A 25-mer oligonucleotide probe constructed to include the reverse complement of base pairs +80 to +104 of the SM22 cDNA (5`-TGCCGTAGGATGGACCCTTGTTGGC-3`) was 5` end-labeled with [-P]ATP and T4 polynucleotide kinase. 40 µg of mouse uterine RNA was hybridized to 2 10 dpm of labeled probe and primer extension reactions performed at 42, 50, and 56 °C as described previously (27) . 5`-RACE was performed using murine uterine RNA and a synthetic antisense cDNA probe corresponding to bp 234 to 258 of the murine SM22 cDNA according to the manufacturer's instructions (Perkin Elmer). RNase protection analyses were performed by subcloning the -441 to +41 murine SM22 genomic subfragment including a synthetic 3` HindIII linker into PstI/HindIII-digested pGEM4Z and performing in vitro transcription of the antisense strand of the genomic subfragment with T7 polymerase of the NcoI-linearized plasmid (NcoI cuts at bp -88 of the genomic clone) in order to obtain an antisense cRNA probe corresponding to bp -88 to +44. (Of note, the HindIII linker shares sequence identity with the SM22 cDNA resulting in a cRNA probe with sequence identity initiated at bp +44 (not +41) of the SM22 genomic clone.) The 142-bp probe was labeled with [-P]UTP and RNase protection analyses were performed using the RPAII kit (Ambion, Austin, TX) according to the manufacturer's instructions.

Cell Culture

The rat cell line A7r5 which was derived from embryonic thoracic aorta was grown in Dulbecco's modified essential media (Life Technologies, Inc.) supplemented with 10% fetal bovine serum (Life Technologies, Inc.) and 1% penicillin/streptomycin. The human hepatocellular carcinoma cell line HepG2 was grown in modified Eagle's medium supplemented with 10% fetal bovine serum and 0.1 mM minimal essential medium non-essential amino acids (Life Technologies, Inc.). Murine lymphoma-derived EL4 cells were grown in Dulbecco's modified Eagle's media supplemented with 10% horse serum (Life Technologies, Inc.). Murine NIH 3T3 cells, C3H10T1/2 cells, C2C12 myoblasts and myotubes were grown as described previously (29, 30). Primary cultures of rat aortic SMCs were isolated from 12-16-week-old Sprague-Dawley rats (Charles River Laboratories) using the method described previously (31) . Virtually all cells isolated using this method stain positive with anti-smooth muscle actin monoclonal antiserum. In all experiments, only early passage (passage 2 or 3) rat aortic SMCs were utilized. For the cell cycle analyses, SMCs from the third passage were placed in serum-free medium (50% Dulbecco's minimal essential medium, 50% Ham's F-12, L-glutamine (292 mg/ml), insulin (5 mg/ml), transferrin (5 mg/ml), and selenious acid (5 ng/ml)) for 72 h in order to synchronize the cells in G/G as described previously (31) . Following 72 h of serum starvation, cells were stimulated to proliferate by incubation in medium containing 45% Dulbecco's modified Eagle's medium, 45% Ham's F-12, and 10% fetal bovine serum.

Plasmids

The Rous sarcoma virus (RSV) long terminal repeat-driven luciferase reporter plasmid, pRSVL, and the pMSVgal reference plasmid have been described previously (27) . The promoterless pGL2-Basic plasmid (Promega, Madison, WI) served as the cloning backbone for all of the luciferase reporter plasmids described below. The p-5000/I1SM22luc plasmid contains 5-kb of SM22 5`-flanking sequence, the untranslated SM22 first exon, the SM22 first intron, and the first 12-bp of exon 2 of the SM22 gene subcloned 5` of the luciferase reporter gene. It was constructed by first subcloning the 8.5-kb BamHI/HindIII SM22 genomic subfragment (containing 5-kb of 5`-flanking sequence, exon 1, and 3.5 kb of intron 1) into BglII/HindIII digested pGL2-Basic vector. Next, a 488-bp PCR-generated HindIII-linked SM22 genomic subfragment including at its 5` end the SM22 intron 1 HindIII restriction site (see Fig. 5A) and running to bp +76 of the SM22 cDNA (which includes 12-bp of exon 2) was subcloned into the HindIII-digested vector and its correct orientation (5` to 3` relative to the luciferase reporter gene) confirmed by DNA sequence analysis. The p-5000SM22luc plasmid, containing 5-kb of SM22 5`-flanking sequence subcloned 5` of the luciferase reporter gene, was constructed by first subcloning the 2.2-kb BamHI/EcoRI SM22 genomic subfragment (corresponding to bp -5000 to -2800) into BamHI/EcoRI-digested pBluescript IIKS (Stratagene La Jolla, CA). Next, the 1251-bp EcoRI/NcoI SM22 genomic subfragment corresponding to bp -1339 to -89 and the 130-bp PCR-generated genomic subfragment containing bp -88 (including the NcoI site at its 5` end) to +41 (including a HindIII linker at its 3` end) was ligated into the EcoRI/HindIII-digested vector. Then, the 1.4-kb EcoRI SM22 genomic subfragment (corresponding to bp -2800 to -1340) was subcloned into the EcoRI-digested plasmid and its orientation confirmed by DNA sequence analysis. Finally, the resulting SM22 genomic subfragment corresponding to bp -5 kB to +41 was excised from the Bluescript phagemid with BamHI and HindIII and subcloned into BglII/HindIII-digested pGL2-Basic. The p-1339SM22luc plasmid containing the 1380-bp SM22 genomic subfragment (bp -1339 to +41) subcloned 5` of the luciferase reporter in the pGL2-Basic vector, was constructed using the 1251-bp EcoRI/NcoI SM22 genomic subfragment (bp -1338 to -89) and the 130-bp (bp -88 to +41) PCR-generated genomic subfragments described above. The p-441SM22luc plasmid contains the 482-bp (bp -441 to +41) PstI/HindIII SM22 genomic subfragment subcloned into BglII/HindIII-digested pGL2-Basic plasmid. The p-300SM22luc and p-162SM22luc luciferase reporter plasmids, respectively, contain the PCR-generated bp -300 to +41, and -162 to +41 SM22 genomic subfragments (including synthetic XhoI (5` end) and HindIII (3` end) linkers), subcloned into XhoI/HindIII-digested pGL2-Basic vector. All PCR-generated genomic subfragments were confirmed by dideoxy DNA sequence analysis.


Figure 5: Structure of the murine SM22 gene. A, a schematic representation and partial restriction endonuclease map of the murine SM22 gene. BamHI (B), EcoRI (R), XbaI (X), NcoI (N), and HindIII (H) restriction enzyme sites are shown. The transcriptional start site is indicated with an arrow. Exons are shown as shaded boxes.B, the nucleotide sequence of the murine SM22 gene. The nucleotide sequence of the exons (upper-case letters) and introns (lower-case letters), as well as 1340 bp of 5`-flanking sequence are shown. Nucleotides within the minimal functional SM22 promoter with sequence identity to previously characterized muscle regulatory cis-acting sequence elements are boxed (see text). The consensus splice donor-acceptor junctions (AG/GT) are underlined.



Transfections and Luciferase Assays

1 10 passage three primary rat aortic SMCs and A7r5 cells, respectively, were split and plated 24 h prior to transfection and transfected with 50 µg of Lipofectin reagent (Life Technologies), 15 µg of luciferase reporter plasmid, and 5 µg of the pMSVgal reference plasmid as described previously (27, 32) . 1 10 NIH 3T3 or COS-7 were transfected with 20 µg of Lipofectin reagent, 15 µg of the luciferase reporter plasmid, and 5 µg of the pMSVgal reference plasmid as described previously (32, 35) . 1 10 HepG2 cells were transfected using 360 µg of Lipofectamine reagent (Life Technologies), 26 µg of luciferase reporter plasmid, and 9 µg of the pMSVgal reference plasmid. Following transfection, cell lysates were prepared and luciferase and -galactosidase assays were performed as described previously (27) . All experiments were repeated at least three times to assure reproducibility and permit the calculation of standard errors. Luciferase activities (light units) were corrected for variations in transfection efficiencies as determined by assaying cell extracts for -galactosidase activities. Data are expressed as normalized light units ± S.E.


RESULTS

Isolation and Structural Characterization of the Murine SM22 cDNA

Murine SM22 cDNA clones were isolated using the polymerase chain reaction in conjunction with synthetic oligonucleotide primers derived from the previously published sequence of the rat SM22 cDNA (23) . A partial restriction endonuclease map and the nucleotide sequence of the full-length murine SM22 cDNA are shown in Fig. 1. The murine SM22 cDNA encodes a 201-amino acid polypeptide with a predicted molecular mass of 22.5 kDa. It is composed of a 76-bp 5`-untranslated region, a 603-bp open reading frame, and a 403-bp 3`-untranslated region. Of note, 23-bp 5` of the poly(A) tail there is an A/T-rich sequence (AATATA) which may function as the polyadenylation signal (Fig. 1B, boxed).


Figure 1: The primary structure of a full-length murine SM22 cDNA. A, schematic representation of a partial restriction endonuclease map of the SM22 cDNA. The size of the cDNA in bp is shown above the map. The protein coding region of the cDNA is shown as an open box; the 5`-untranslated region is shaded, and the 3`-untranslated region is hatched. MscI, NcoI, HincII (H2), PstI, and BstXI restriction enzyme sites are shown above the map. The size of the deduced protein in amino acids (aa) is shown below the map. B, the nucleotide and deduced amino acid sequence of the murine SM22 cDNA. The putative polyadenylation signal is boxed (see text). The two regions of the SM22 cDNA that share high level sequence identity with the Drosophila muscle protein mp20 are shaded. C, a Pustell protein matrix analysis of the mouse SM22 amino acid sequence (x axis) versus the Drosophila muscle protein mp20 amino acid sequence (y axis). MacVector Sequence Analysis Software (Kodak, IBI) was used to perform the Pustell protein matrix analysis, parameters were set to a window size of 6, min % score of 60, and hash value of 2. Regions of sequence identity are indicated by a line.



A comparison of the coding sequences of the murine and human SM22 cDNAs (10) demonstrated that the two sequences are 91 and 97% identical at the nucleotide and amino acid levels, respectively. In addition, a comparison of the coding sequences of the murine SM22 cDNA and the murine smooth muscle thin filament regulatory protein, calponin (33) , demonstrated that these two sequences are 23% identical and 32% conserved at the amino acid level. Interestingly, the protein sequence encoded by the murine SM22 cDNA exhibits partial sequence identity with the sequence of the Drosophila muscle protein mp20 (20) across the entire cDNA, suggesting that these two proteins may have evolved from a common ancestral gene (Fig. 1C). As shown in Fig. 1C, two domains were particularly well conserved between these proteins. One domain with 14/19 amino acid identity (corresponding to amino acids 104-122 of the murine SM22 protein, see Fig. 1B, shaded box) may represent a calcium binding domain oriented in an EF-hand conformation (25) . The second C-terminal conserved domain with 13/24 amino acid identity (corresponding to amino acids 158-181 of the murine SM22 protein) is a domain of unknown function (Fig. 1B, shaded box).

SM22 Is Encoded by a Single Copy Gene

The finding of a putative calcium binding domain oriented in an EF-hand conformation suggested that SM22 might be related to other members of the troponin C supergene family of intracellular calcium binding proteins including slow/cardiac troponin C, fast skeletal troponin C, calmodulin, myosin light chain, and parvalbumin (25) . In order to determine whether SM22 is encoded by a single copy gene in the murine genome and whether SM22 is related to other troponin C supergene family members, the murine SM22 cDNA was used to probe Southern blots containing murine genomic DNA under both high and low stringency conditions. As shown in Fig. 2, under high stringency conditions, the murine SM22 cDNA probe hybridized to one or two BamHI, EcoRI, HindIII, PstI, and XbaI bands, suggesting that SM22 is a single copy gene in the murine genome. Interestingly, no additional bands were demonstrated under low stringency conditions, suggesting that although the SM22 gene may have one EF-hand calcium binding domain, it is not closely related to other members of troponin C supergene family.


Figure 2: A Southern blot analysis of the murine SM22 gene. High molecular weight murine SV 129 DNA was digested with the restriction endonucleases BamHI, EcoRI, HindIII, PstI, and XbaI and hybridized to the radiolabeled SM22 cDNA probe under high and low (data not shown) stringency conditions. Size markers are shown in kb to the left of the blot.



Lineage-restricted Expression of the SM22 Gene

Previous studies have suggested that SM22 protein is expressed solely in smooth muscle-containing tissues of the adult and may be one of the earliest markers of the smooth muscle cell lineage (21-23). To determine the in vivo pattern of SM22 gene expression, the SM22 cDNA was hybridized to Northern blots containing RNAs prepared from 12-week-old murine tissues. As shown in Fig. 3A, the murine SM22 cDNA probe hybridized to one predominant mRNA species of approximately 1.2-kb. SM22 mRNA is expressed at high levels in the smooth muscle-containing tissues of aorta, small intestine, lung, spleen, and uterus. In addition, prolonged autoradiographic exposures revealed very low, but detectable, levels of SM22 mRNA in heart, kidney, skeletal muscle, and thymus (data not shown).


Figure 3: The in vivo tissue distribution and cellular-specificity of SM22 gene expression. A, the top panel shows a Northern blot analysis of RNA samples isolated form adult murine tissues hybridized to the radiolabeled SM22 cDNA probe. RNA size markers are shown in kb to the left of the blot. The SM22 cDNA probe hybridized to a single 1.2-kb species of mRNA which was present in smooth muscle-containing tissues (arrow). The bottom panel shows the ethidium stained formaldehyde-containing gel prior to membrane transfer of RNA. The locations of the 28 S and 18 S ribosomal RNA bands are indicated to the left of the gel. B, the top panel shows a Northern blot analysis of RNA samples isolated from primary rat aortic SMCs (VSMC), A7r5, NIH 3T3 (3T3), C3H10T1/2 (10T1/2), COS-7, C2C12 myoblast (C2 Blasts), C2C12 myotubes (C2 Tubes), HepG2, and EL-4 cells hybridized to the radiolabeled SM22 cDNA probe. The SM22 cDNA hybridized to a 1.2-kb species of mRNA (arrow) present in primary SMCs, A7r5 cells, C2C12 myoblasts and myotubes. The bottom panel shows the ethidium-stained formaldehyde-containing gel prior to transfer of RNA.



In order to determine the cell-specificity of SM22 gene expression, the SM22 cDNA probe was hybridized to Northern blots containing RNAs prepared from rat aortic vascular SMCs, the rat SMC line A7r5, murine NIH 3T3 and C3H10T1/2 fibroblasts, the SV40-transformed monkey kidney cell line COS-7, murine C2C12 myoblasts and myotubes, the human hepatocellular carcinoma cell line HepG2, and the murine lymphoid cell line EL4. As shown in Fig. 3B, high levels of SM22 mRNA were detected in primary rat aortic vascular SMCs and the smooth muscle cell line A7r5. Of note, detection of a second larger species of mRNA may represent either cross-hybridization of the SM22 probe to the murine calponin mRNA or to a rare SM22 transcript with an extended 5` untranslated region (70) . ()In addition, SM22 mRNA was expressed in both undifferentiated C2C12 myoblasts and terminally-differentiated C2C12 myotubes. Finally, a faint hybridization signal was detectable in NIH 3T3, C3H10T1/2, and HepG2 cells after a 3-day autoradiographic exposure (data not shown). Quantitative PhosphorImager analysis of these low level hybridization signals revealed that SM22 mRNA is expressed in these three non-myogenic cell lines at less than 1.5% the intensity of SM22 gene expression in A7r5 and primary SMCs. Thus, in addition to primary SMCs and SMC lines, SM22 mRNA is expressed in other embryonic skeletal muscle cell lineages such as C2C12 myoblasts and myotubes, but not in other non-myogenic cell lineages.

SM22 Is Expressed in Both Cell Cycle-arrested and Proliferating SMCs

Within the tunica media of the arterial wall the vast majority of vascular SMCs are maintained in a non-proliferating, quiescent state and express contractile proteins (2, 3, 4, 5, 9, 10, 34, 35) . However, in response to vascular injury, SMCs migrate from the tunica media to the intimal layer, proliferate, and assume a ``synthetic phenotype'' (11, 12, 13, 14, 35, 36, 37) . Previous studies have demonstrated that many genes encoding vascular SMC contractile proteins are down-regulated during this process (2, 3, 5, 38) . Thus, it was of interest to determine whether SM22 gene expression was differentially regulated during progression through the cell cycle. In order to address this question, cultures of low passage number primary rat aortic SMCs were synchronized in the G/G stage of the cell cycle by serum starvation for 72 h. Fluorescence activated cell sorter analyses revealed that under these conditions approximately 90% of cells are arrested in G/G (Ref. 31, and data not shown). The cells were then serum-stimulated and RNA was prepared from replicate cultures at the time of serum stimulation (t), and at 8, 12, 16, and 24 h post-stimulation. After serum stimulation the arrested vascular SMCs begin to pass through the G/S checkpoint of the cell cycle at approximately 12 h, and by 24 h post-stimulation greater than 50% of cells are in the S and G/M phases of the cell cycle (31) . A Northern blot analyses demonstrated no differences in SM22 gene expression in cell cycle arrested versus proliferating SMCs as assessed by quantitative PhosphorImager analysis of the hybridization signal (Fig. 4). Thus, in contrast to other smooth muscle contractile proteins, such as smooth muscle myosin heavy chain (3) , smooth muscle -actin (2) , and calponin,() SM22 appears to be constitutively expressed at high levels in both quiescent and proliferating vascular SMCs.


Figure 4: The cell cycle regulation of SM22 gene expression in vitro. The top panel shows a Northern blot analysis of RNA prepared from G/G synchronized cultures of primary rat aortic SMCs at t and 8, 12, 16, and 24 h post-serum stimulation hybridized to the radiolabeled SM22 cDNA probe. Quantitative image analysis of the hybridization signal (arrow) was performed using a Molecular Dynamics PhosphorImager. RNA size markers are shown in kb to the left of the blot. The bottom panel shows the ethidium-stained formaldehyde-containing gel prior to membrane transfer of RNA. The locations of the 28 S and 18 S ribosomal RNA bands are indicated to the left of the gel.



Isolation and Structural Characterization of a SM22 Genomic Clone

A full-length murine SM22 genomic clone was isolated by screening a murine 129SV genomic library with a SM22 cDNA probe under high stringency conditions. A partial restriction map of this 20-kb clone is shown in Fig. 5A. Exons were identified by hybridization with specific cDNA fragments and their boundaries confirmed by DNA sequence analysis (Fig. 5B). The murine SM22 gene is composed of five exons spanning 6.2 kb of genomic DNA.

The transcriptional start site of the SM22 gene was identified by RNase protection, primer extension, and 5`-RACE PCR analyses (Fig. 6). As shown in Fig. 6A, primer extension analyses utilizing an antisense synthetic oligonucleotide corresponding to bp 80-104 of the SM22 cDNA resulted in a major extended product of 104-bp (arrow) which was generated at reaction temperatures up to 56 °C. In addition, 5`-RACE PCR was performed utilizing an antisense oligonucleotide primer corresponding to bp 234-258 of the SM22 cDNA. DNA sequence analyses of eight random 5`-RACE clones revealed a transcriptional start site 76 bp 5` of the initiation codon in seven of eight clones and 72 bp 5` of the initiation codon in one of eight clones (data not shown). RNase protection analyses were also performed using an antisense cDNA probe corresponding to bp -88 to +44 of the SM22 genomic sequence as deduced by DNA sequence and Southern blot analyses (Fig. 6B). These analyses revealed a major protected fragment of 44 bp (arrow) corresponding to a transcriptional start site 76 bp 5` of the initiation codon. In addition, a second, minor (20% relative signal intensity) protected fragment of 54 bp was also demonstrated. Taken together, these data allowed the identification of the major transcriptional start site of the murine SM22 gene 76 bp 5` of the initiation codon.


Figure 6: Localization of the transcriptional start site of the murine SM22 gene. A, primer extension analysis of SM22 mRNA. The reaction products of primer extension reactions performed at 42, 50, and 56 °C utilizing a 5` end-labeled antisense oligonucleotide primer corresponding to bp 80-104 of the SM22 cDNA and SV129 murine uterine RNA were separated on a 6% acrylamide/urea gel which was subject to autoradiography. The upper band (black arrow) in the autoradiogram represents the 104-bp primer extension product. The lower band (hatched arrow) represents the nonhybridized 25-bp radiolabeled oligonucleotide probe. DNA size markers in bp are indicated to the left of the autoradiogram. B, RNase protection analysis of SM22 mRNA. Murine uterine RNA was subjected to RNase protection analysis using an antisense cRNA probe corresponding to bp -88 to +44 of the SM22 gene. The top panel shows the nucleotide sequence of the SM22 mRNA (RNA) and the SM22 cRNA probe (Probe). The arrows indicate the transcriptional start sites (see text). The bottom panel shows an autoradiogram of a 8% sequencing gel containing the RNase protection analysis reaction products. The first lane (Probe only) contains the radiolabeled 142-bp antisense cRNA probe only (dotted arrow). The second lane (-RNA) contains the products from an RNase digestion reaction performed with tRNA and the radiolabeled 142-bp antisense probe. The third lane (+RNA) contains the digestion products of a reaction mixture containing murine uterine RNA and the radiolabeled antisense cRNA probe. The 44-bp product, corresponding to a transcriptional start site 76-bp 5` of the initiation codon, is indicated by a black arrow.



The complete coding sequence and 1340 bp of 5`-flanking sequence of the SM22 gene is shown in Fig. 5B. Each of the splice junctions (Fig. 5B, underlined) conforms to the consensus splice donor-acceptor patterns as described by Breathnach and Chambon (39). In order to identify potential transcriptional regulatory elements, 1340 bp of 5` sequence flanking the cap site was searched for a variety of transcriptional regulatory elements using MacVector DNA sequencing software (Kodak/IBI). The nucleotide sequence TTTAAA, which might function as a TATA box was present 29-bp 5` of the start site (Fig. 5B, boxed). A consensus CAAT box was not identified in the immediate 5`-flanking region of the SM22 gene. A computer homology search for previously described muscle-specific and/or skeletal or cardiac muscle lineage-restricted transcriptional regulatory elements revealed five consensus E boxes/bHLH myogenic transcription factor binding sites (CANNTG (16, 17, 40) ) located at bp -535, -578, -866, -899, -911, and -1268, three consensus GATA-4 binding sites (WGATAR (41) ) located at bp -505, -829, -977, and two AT-rich, potential MEF-2/rSRF binding sites (YTAWAAATAR (42) ) located at bp -408 (TTtAAAATcG, small letters denote mismatches from the consensus MEF-2 sequence) and -771 (TTcAAAATAG). In addition, functionally important nuclear protein binding sites which have been identified in previously characterized skeletal and cardiac-specific transcriptional regulatory elements included two consensus CArG/SRF binding sites (43) located at bp -150 and -273 and one CACC box (44) located at bp -104. Finally, four AP2 (CCCMNSSS (45) ), one Sp1 (KRGGCKRRK (46) ), and two NF-IL6 (TKNNGNAAK (47) ) binding sites were located in the 5`-flanking region.

Identification of the cis-Acting Transcriptional Regulatory Elements That Control SM22 Gene Expression

In order to identify the functionally important cis-acting sequences that regulate transcription of the SM22 gene in SMCs, a series of transient transfections were performed using SM22-luciferase reporter constructs and primary rat aortic vascular SMCs and the SMC line, A7r5, both of which express high levels of SM22 mRNA (Fig. 3B). Transfection of A7r5 cells with the plasmid p-5000/I1SM22luc, containing 5 kb of 5`-flanking sequence and the entire 4-kb SM22 intron 1 sequence (the initiation codon is located in exon 2), resulted in a 250-300-fold induction in luciferase activity as compared to the promoterless control plasmid, pGL2-Basic (Fig. 7A, lanes 1 and 2). This level of transcriptional activity was comparable to that obtained following transfection of A7r5 cells with the RSV-containing luciferase reporter plasmid, pRSVL (Fig. 7A, lanes 2 and 8). In order to determine whether this transcriptional activity was due to the immediate 5`-flanking region of the SM22 gene, or alternatively, was due to a transcriptional regulatory element located within the first intron of the SM22 gene, the activities of the p-5000/IlSM22luc and p-5000SM22luc plasmid were compared (Fig. 7A, lanes 2 and 3). Transfection of A7r5 cells with the p-5000SM22luc plasmid, containing only 5 kb of 5`-flanking sequence, resulted in high level transcription of the luciferase reporter gene comparable (on a molar basis) to levels obtained with the p-5000/I1SM22luc plasmid. Thus, the 5`-flanking region of the SM22 gene contains cis-acting sequence elements required for high level transcription in A7r5 cells.


Figure 7: Identification and localization of transcriptional regulatory elements that control SM22 gene expression in A7r5 and primary rat aortic SMCs. A, transient transfection analyses of SM22/luciferase reporter plasmids in the smooth muscle cell line, A7r5. 15 µg of SM22/luciferase reporter plasmid and 5 µg of the pMSVgal reference plasmid were transiently transfected into replicate cultures of A7r5 cells. Cells were harvested 60 h after transfection, and cell extracts were assayed for both luciferase and -galactosidase activities. Luciferase activities (light units) were corrected for variations in transfection efficiencies as determined by -galactosidase activities. Data are expressed as normalized light units ± S.E. B, transient transfection analyses of SM22/luciferase reporter plasmids in primary rat aortic SMCs. Transient transfection analyses were performed using a series of SM22/luciferase reporter plasmids and primary rat aortic SMCs as described in A above. Data are expressed as normalized light units ± S.E.



To further localize the 5`-flanking elements of the SM22 gene that direct high level expression in SMCs, a series of 5` deletion mutants were transfected into both A7r5 cells (Fig. 7A) and primary cultured rat aortic vascular smooth muscle cells (Fig. 7B). In both A7r5 cells and primary vascular SMCs, the p-441SM22luc plasmid, containing 441 bp of 5`-flanking sequence, increased transcription of the luciferase reporter to levels comparable to the p-5000SM22luc plasmid and the p-1339SM22luc plasmids (Fig. 7, A lanes 3-5 and B, lanes 2 and 3). However, transfection of both A7r5 cells and primary vascular SMCs with the luciferase reporter plasmidsp-300SM22luc and p-162SM22luc containing 300 and 162 bp, respectively, of 5`-flanking sequence resulted in 50 and 90% reductions in normalized luciferase activities as compared with those obtained with the p-441SM22luc plasmid (Fig. 7, A, lanes 5-7 and B, lanes 3-5). These data demonstrated that 441 bp of SM22 5`-flanking sequence, containing the endogenous SM22 promoter, is sufficient to direct high level transcriptional activity in both A7r5 cells and primary rat aortic SMCs.

Cellular Specificity of the SM22 Promoter

In order to characterize the cellular specificity of the SM22 promoter sequence, the transcriptional activities of the 441-bp SM22 promoter containing plasmid, p-441SM22luc, was compared to the positive control plasmid containing the Rous sarcoma virus-long terminal repeat, pRSVL, in primary rat vascular SMCs, the smooth muscle cell line A7r5, NIH 3T3 fibroblasts, COS-7, and HepG2 cells. Consistent with the lineage-restricted pattern of SM22 mRNA expression demonstrated in these cell lines (see Fig. 3B), the promoter-containing plasmid, p-441SM22luc, was active in primary rat aortic SMCs and A7r5 cells, increasing transcription of the luciferase reporter gene approximately 2500- and 540-fold, respectively, over that induced by transfection with the promoterless pGL2-Basic plasmid (Fig. 8). This level of promoter activity was comparable to levels obtained following transfection of these cells with the RSV-long terminal repeat-driven positive control plasmid (Fig. 8). In contrast, the 441-bp SM22 promoter was inactive in NIH 3T3, COS-7, and HepG2 cells (Fig. 8).


Figure 8: Cellular-specificity of the 441-bp SM22 promoter. The p-441SM22luc (black bar) and pRSVL (hatched bar) plasmids were transiently transfected into primary rat aortic SMCs (VSMC), A7r5, NIH 3T3 (3T3), COS-7, and HepG2 cells and the normalized luciferase activities for each respective plasmid was determined as described in the legend to Fig. 7. Data are expressed as normalized luciferase light units ± S.E.



DNA sequence analyses (Fig. 5B, boxed) revealed that this 441-bp promoter contains two CArG/SRF boxes (43) , a CACC box (44) , and one A/T-rich potential MEF-2/rSRF binding site (42) , cis-acting elements which have each been demonstrated to be involved in the transcriptional programs that regulate skeletal and cardiac muscle-specific gene expression. However, unlike most previously described skeletal muscle-specific transcriptional regulatory elements, this sequence lacked a canonical E box binding site for the myogenic bHLH transcription factors (17, 40) . Thus, the endogenous 441-bp SM22 promoter contains all of the cis-acting sequence elements required to recapitulate the smooth muscle lineage-restricted pattern of SM22 gene expression demonstrated in vivo.


DISCUSSION

In this report, we have isolated and structurally characterized the murine SM22 cDNA and gene. Using the murine SM22 cDNA as a molecular probe, we have defined the tissue distribution and cell cycle-regulated pattern of SM22 gene expression. In addition, we have demonstrated that the immediate 5`-flanking region of the SM22 gene is necessary and sufficient to direct high level, lineage-restricted expression of the SM22 gene in both primary vascular SMCs and the SMC line, A7r5. Finally, we have demonstrated that the minimal SM22 promoter lacks a binding site for the bHLH family of myogenic transcription factors. These data are relevant to understanding the underlying transcriptional program that regulates SMC differentiation.

The unique contractile properties of SMCs and their ability to reversibly modulate their phenotype from primarily contractile to primarily synthetic, distinguishes this myogenic lineage from both the skeletal and cardiac muscle cell lineages. However, in contrast to the striated muscle lineages (for review, see Refs. 16-19), relatively little is currently understood about the cis-acting sequences and trans-acting factors that regulate gene expression in SMCs. This is due, in part, to the poorly understood lineage relationships of SMCs, which appear to develop from multiple locations throughout the embryo, as well as to the relative paucity of SMC-specific markers (48, 49, 50, 51, 52, 53, 54) . The data presented in this report demonstrate that the level of SM22 protein expression is regulated at the level of gene expression. However, in contrast to the smooth muscle myosin heavy chain, and possibly the -enteric actin gene, which are expressed exclusively in SMCs (3, 55, 56, 57, 58) , SM22 is expressed in other myogenic cell lineages including the embryonic skeletal muscle cell lineage C2C12. In this regard, it is noteworthy that the SM22 gene is expressed in undifferentiated skeletal myoblasts, which do not express myofibrillar protein isoforms, and that SM22 gene expression is not down-regulated in conjunction with other SMC contractile proteins during serum-induced SMC proliferation. Taken together, these data suggest that the SM22 gene is not regulated in a coordinated manner with other smooth muscle contractile proteins. Therefore, elucidation of the cis-acting sequences that regulate SM22 gene expression in SMCs could serve as a valuable tool for targeting gene expression to both contractile/arrested and synthetic/proliferative SMCs in the arterial wall in vivo.

This differential pattern of SM22 gene expression in several myogenic lineages suggests that distinct transcriptional programs have evolved to permit the regulated expression of a single gene in multiple cell lineages. However, it is noteworthy that Olson and co-workers (59) recently reported that a null mutation of the MADS box transcription factor D-MEF2 gene in Drosophila resulted in failure of somatic, cardiac, and visceral muscles to differentiate. These data suggest that this evolutionarily conserved family of transcription factors may play a critical role in coordinating muscle differentiation across lineages. Thus, it will be of interest to determine the functional role of the A/T-rich potential MEF-2/rSRF (8/10-bp sequence identity) binding site located within the minimal murine SM22 promoter. In this respect, the SM22 promoter may serve as a useful target with which to dissect the functional role of the four individual MEF-2/rSRF family members expressed in vertebrate species (versus the single D-MEF-2 gene in Drosophila) in the smooth muscle lineage. Similarly, two consensus CArG box/SRF binding sites were identified in the minimal SM22 promoter. This motif, which has been identified in multiple skeletal and cardiac-specific transcriptional regulatory elements (60) , is also present in the smooth muscle -actin promoter (61, 62, 63) , suggesting that it may play a role in the coordinate regulation of genes expressed in SMCs. Finally, a consensus CACC box was identified in the minimal SM22 promoter. This nuclear protein binding site is present in multiple skeletal and cardiac-specific transcriptional regulatory elements, where it has been demonstrated to function in conjunction with other lineage-specific nuclear protein binding sites (29, 30, 64, 65, 66) .

Current developmental paradigms suggest that tissue-specific gene expression is ultimately regulated by the expression of lineage-specific or lineage-restricted transcription factors (16, 17, 18, 19) . Interestingly, sequence analyses of the minimal SM22 promoter failed to reveal a consensus bHLH myogenic transcription factor/E-box binding site. Consistent with this observation, myogenic bHLH family members including MyoD, myogenin, myf-5, and MRF-4/herculin/myf-6 are not expressed in SMCs and null mutations of the MyoD, myogenin, and myf-5 genes, respectively, had no effect on smooth muscle cell specification or differentiation in vivo(67, 68) . Similarly, the minimal SM22 promoter lacked a consensus binding site for GATA-4, a transcription factor that has been demonstrated to transactivate multiple cardiac-specific transcriptional regulatory elements in non-muscle cell lines (32, 69) . Taken together, these studies suggest that potentially novel SMC-specific transcription factors may play a key role in regulating SMC-specific transcription. Future studies utilizing the SM22 promoter as a model system should provide fundamental insight into the molecular mechanisms that regulate SMC-specific transcription and differentiation.


FOOTNOTES

*
This work was supported in part by United States Public Health Service Grants R01-HL48257 and U01 AI34566 (to J. S.) and R01HL51145 (to M. S. P.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The GenBank accession number for the murine SM22 cDNA is L41154. The GenBank accession number for the murine SM22 gene is L41161.

§
To whom correspondence should be addressed: Dept. of Medicine, University of Chicago MC 6088, 5841 S. Maryland Ave., Chicago, IL 60637. Tel.: 312-702-2679; Fax: 312-702-2681.

The abbreviations used are: SMC, smooth muscle cells; bp, base pair(s); PCR, polymerase chain reaction; RSV, Rous sarcoma virus; kb, kilobase pair(s).

F. Samaha and M. Parmacek, unpublished data.

F. Samaha and M. Parmacek, unpublished observation.

F. Samaha, J. Seltzer, and M. Parmacek, unpublished observation.


ACKNOWLEDGEMENTS

We thank Jeffrey M. Leiden and Eric N. Olson for helpful discussions and suggestions. We also thank Lisa Gottschalk for expert preparation of illustrations and figures.


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