Developmental Pattern of Expression and Genomic Organization of the Calponin-h1 Gene A CONTRACTILE SMOOTH MUSCLE CELL MARKER*

Calponin-h1 is a 34-kDa myofibrillar thin filament, actin-binding protein that is expressed exclusively in smooth muscle cells (SMCs) in adult animals. To exam- ine the molecular mechanisms that regulate SMC-spe-cific gene expression, we have examined the temporal, spatial, and cell cycle-regulated patterns of expression of calponin-h1 gene expression and isolated and struc- turally characterized the murine calponin-h1 gene. Cal-ponin-h1 mRNA is expressed exclusively in SMC-con- taining tissues in adult animals. During murine embryonic development, calponin-h1 gene expression is (i) detectable in E9.5 embryos in the dorsal aorta, cardiac outflow tract, and tubular heart, (ii) sequentially up-regulated in SMC-containing tissues, and (iii) down-regulated to non-detectable levels in the heart during late fetal development. In addition, the gene is expressed in resting rat aortic SMCs, but its expression is rapidly down-regulated when growth-arrested cells re-enter phase G 1 of the cell cycle and proliferate. Calpo- nin-h1 is encoded by a 10.7-kilobase single copy gene composed of seven exons, which is part of a multigene family. Transient transfection analyses demonstrated that 1.5 kilobases of calponin-h1 5 (cid:42) -flanking sequence is sufficient to program high level transcription of a lucif- erase reporter gene in cultured primary rat aortic SMCs and the smooth muscle cell line, A7r5. Taken together, these data suggest that the calponin-h1 gene will serve as an excellent model system with which to examine the molecular mechanisms

The vascular smooth muscle cell (SMC) 1 is responsible for maintaining both arterial tone via contraction-relaxation and vessel integrity by proliferation and synthesis of extracellular matrix (1,2). During postnatal development, SMCs located in the arterial tunica media are maintained in the resting, or G 0 /G 1 , stage of the cell cycle and express high levels of con-tractile protein isoforms (3). However, in response to vascular injury and the concomitant release of growth factors, vascular SMCs re-enter the cell cycle, proliferate, and modulate their phenotype to subserve a primarily synthetic function (4 -13). This phenotypic modulation has been implicated in the pathogenesis of a number of disease states, including atherosclerosis and restenosis following percutaneous transluminal coronary angioplasty (3, 14 -17). Despite the central role of SMCs in vascular biology, relatively little is currently understood about the molecular mechanisms that control SMC lineage specification, differentiation, and phenotypic modulation. This is due, in part, to the fact that relatively few SMC lineage-specific markers have been identified and to the poorly understood embryological origin(s) of the SMC lineage(s) (18 -27).
Previous studies have suggested that the myofibrillar thin filament protein calponin is expressed exclusively in SMCs (11, 28 -31). Two species of calponin mRNA, designated h1 and h2, have been identified, each of which is encoded by a separate gene (32). Interestingly, only the h1 protein product is detectable in SMC-containing tissues (32). In addition, a non-muscle acidic isoform of calponin with distinct functional properties has recently been described (33). Several findings suggest that calponin may regulate SMC contraction: (i) calponin binds to the thin filament proteins actin, tropomyosin, calmodulin, and caltropin (34 -38), (ii) calponin can reversibly inhibit the actinactivated myosin MgATPase (39,40), (iii) calponin can inhibit the Ca 2ϩ -dependent mobility of actin on immobilized myosin (41), (iv) calponin induces conformational changes in F-actin (42), and (v) calponin decreases the rate of cross-bridge cycling and increases maximum force production by smooth muscle myosin (43). Despite these in vitro findings, the molecular mechanism by which calponin regulates smooth muscle cell contraction in vivo remains unknown.
In the studies described in this report, we have used the murine calponin-h1 cDNA as a molecular probe to define the spatial and temporal pattern of calponin-h1 gene expression during development. In addition, we have isolated and structurally characterized a murine calponin-h1 genomic clone and performed a series of transient transfection analyses using calponin/luciferase reporter plasmids. These studies demonstrated that the calponin-h1 gene is expressed in a developmentally regulated, SMC-specific pattern. The 10.7-kb calponin-h1 gene is a single copy gene that is a member of a multigene family composed of proteins with an evolutionarily conserved actin-binding domain. Finally, transient transfection analyses demonstrate that the murine calponin-h1 promoter programs high level transcription of a luciferase reporter gene in primary cultures of rat aortic SMCs and the SMC line, A7r5. Taken together, these data suggest that the murine calponin-h1 gene should serve as an excellent model system in which to examine the transcriptional mechanisms that control SMC lineage specification and differentiation. * This work was supported in part by Public Health Service Grants HL48257 and AI34566 (to J. Q. S.) and 1R0 -1HL51145 (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 be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Isolation of Murine
Calponin-h1 cDNAs-The coding region (bp 98 -988) of the murine calponin-h1 cDNA was isolated by performing reverse transcriptase-polymerase chain reaction using murine uterine RNA and synthetic 5Ј-and 3Ј-oligonucleotide primers constructed from the previously published sequence of the mouse calponin-h1 cDNA (GenBank Accession No. Z19542) as described previously (44). The 891-bp reaction product (excluding the restriction enzyme sites) was subcloned into BamHI-HindIII-digested pGEM7Z (Promega) and was utilized for genomic screening, Southern blot, and Northern blot analyses. In addition, a 467-bp murine calponin-h1 cDNA fragment (bp 193-659), derived from the coding region of the cDNA, and a 746-bp cDNA fragment (bp 659-1404), derived from the coding region and 3Ј-untranslated region of the calponin-h1 cDNA were isolated using reverse transcriptase-polymerase chain reaction using murine uterine RNA and synthetic 5Ј-and 3Ј-oligonucleotide primers as described previously (44) and utilized for in situ hybridization analyses.
Isolation of Murine Calponin-h1 Genomic Clones-Approximately 1 ϫ 10 6 recombinant phage from a murine 129SV Lambda FIX II genomic library (Stratagene) were screened with the radiolabeled 891-bp murine calponin-h1 cDNA probe, and positively hybridizing clones were purified to homogeneity as described previously (44,45). Each clone was analyzed by Southern blot analysis, and one clone spanning the length of the gene (mCalp-5A) including approximately 6 kb of 5Ј-flanking sequence was used for all subsequent experiments.
DNA and RNA Blot Analyses-High molecular weight DNA was prepared from strain 129SV mice as described previously (44). Southern blot analyses were performed using the radiolabeled 891-bp murine calponin cDNA probe under the high and low stringency conditions described previously (44). For Northern blot analyses, RNA was prepared from tissues isolated from 12-week-old 129SV mice (Jackson Laboratories) as described previously (44). In addition, RNA was prepared from cultures of primary rat aortic SMCs and a variety of smooth muscle and non-muscle cell lineages as described previously (47). Northern blotting was performed as described previously (44). Probes included the 891-bp murine calponin cDNA probe and the 4.7-kb fulllength human retinoblastoma (Rb) gene product cDNA probe (which cross-hybridizes to the murine Rb mRNA) that was generously provided by William Kaelin (Dana Farber Cancer Institute). Quantitative image analyses were performed using a Molecular Dynamics PhosphorImager (Sunnyvale, CA).
In Situ Hybridizations-In situ hybridization was performed essentially as described by Eichele and co-workers (49). The murine calponin-h1 cDNA subcloned into pGEM11Z was in vitro transcribed using T7 or SP6 polymerase, respectively, in the presence of 35 S-labeled UTP to generate sense and antisense cRNA probes, respectively. Hybridizations were performed in 50% formamide, 0.3 M NaCl, 20 mM Tris, pH 8.0, 5 mM EDTA, 1 ϫ Denhardt's solution, 500 g/ml yeast tRNA, 10 mM dithiothreitol, 250 nM ␣-thio-ATP, 1 ϫ 10 5 dpm/l of the 35 S-labeled cRNA probe (0.3 mg/ml final probe concentration). Following hybridization, slides were washed, digested with RNase, dehydrated, and air dried. Finally, emulsion autoradiography was performed, and the sections were post-stained with Hoecht 33258 and visualized by epifluorescence and darkfield microscopy on a Zeiss Axiophot microscope. To detect nonspecific background, hybridization was performed with the radiolabeled sense calponin-h1 riboprobe to alternate sections under identical conditions. Primer Extension and RNase Protection Analyses-A 35-mer oligonucleotide probe constructed to include the reverse complement of base pairs ϩ80 to ϩ115 of the previously published murine calponin-h1 cDNA (GenBank Accession No. Z19542) (32) (5Ј-AAATGTGCAGAAGA-CATGCTGGCCAGGGGGCTCTG-3Ј) was 5Ј-end-labeled and hybridized to 40 g of mouse uterine RNA as described previously (46). Primer extension reactions were performed at 42, 50, and 56°C as described previously (46). RNase protection analyses were performed by subcloning a murine XbaI-BbsI calponin-h1 genomic subfragment (bp Ϫ427 to ϩ97, Fig. 6C) into pGEM7Z and performing in vitro transcription of the antisense strand of the AvrII-linearized plasmid (which cuts at bp Ϫ160) using SP6 polymerase to obtain an antisense probe of 257 bp. The 257-bp probe was labeled with [␣-32 P]UTP, and RNase protection analyses were performed as described previously (46).
Transfections and Luciferase Assays-A7r5 cells were transfected using 50 g of Lipofectin reagent, 15 g of luciferase reporter plasmid, and 5 g of the pMSV␤gal reference plasmid as reported previously (46). In addition, to optimize for expression of the calponin-h1 gene in primary rat aortic SMCs (the gene is down-regulated in late passage and/or proliferating SMCs), 2 ϫ 10 6 second passage primary rat aortic SMCs were plated on 90-mm dishes and grown to confluence. The cells were then incubated for 5 h with 100 g of Lipofectin reagent, 15 g of luciferase reporter plasmid, and 5 g of the pMSV␤gal reference plasmid. Following transfection, cell lysates were prepared as described previously (48). The cell lysates were normalized for protein content (Bio-Rad) and the expressed luciferase activities (light units) were corrected for variations in transfection efficiencies as determined by assaying cell extracts for ␤-galactosidase activities. All experiments were repeated at least three times to ensure reproducibility and permit the calculation of standard errors. Data are expressed as normalized light units Ϯ S.E.

SMC Lineage-restricted Expression of the Calponin-h1
Gene-Previous studies have suggested that the thin filament protein calponin is expressed exclusively in SMC-containing tissues (11, 28 -31). To determine the in vivo pattern of calponin-h1 gene expression in adult mice, the calponin-h1 cDNA was hybridized to Northern blots containing RNAs prepared from 12-week-old mouse tissues. As shown in Fig. 1A, the murine calponin-h1 cDNA hybridized to a single mRNA species of approximately 1.5 kb (arrow). In adult mice, the calponin-h1 gene is expressed at high levels in SMC-containing tissues including aorta, small intestine, lung, and uterus. Prolonged autoradiographic exposure demonstrated a low intensity hybridization signal in both the heart and thymus. The faint hybridization signal of approximately 1.2 kb represents crosshybridization to the structurally related SMC-specific protein SM22␣ (46).
To determine the cell specificity of calponin-h1 gene expression, the calponin-h1 cDNA probe was hybridized to Northern blots containing RNAs prepared from freshly isolated primary rat aortic SMCs, the smooth muscle cell line, A7r5, murine NIH 3T3 and C3H10T1/2 fibroblasts, the monkey SV-40 transformed kidney cell line, COS-7, murine Sol8 myoblasts and myotubes, the human hepatocellular carcinoma-derived cell line, HepG2, and the murine lymphoid cell line, EL-4. High levels of calponin-h1 mRNA were detected in primary rat aortic SMCs and the smooth muscle cell line, A7r5 (Fig. 1B, lanes 1 and 2), while the gene was not expressed in other cell lines including NIH 3T3, C3H10T1/2, COS-7, Sol8, Hep G2, and EL-4 cells even after prolonged autoradiographic exposure. Thus, calponin is expressed in a lineage-restricted fashion in primary vascular SMCs and the SMC line, A7r5.
Developmental Regulation of Calponin-h1 Gene Expression in Vivo-To determine the temporal and spatial patterns of calponin-h1 gene expression during mouse development, a series of in situ hybridization experiments were performed on staged murine embryos using radiolabeled antisense and sense (control) calponin-h1 cRNA probes derived from both the coding and 3Ј-untranslated regions of the murine calponin-h1 cDNA. In postcoital day (E) 9.5 mouse embryos, calponin-h1 mRNA was abundantly expressed in the aortic outflow tract or truncus arteriosus (open arrow), dorsal aorta (white arrow), as well as the developing heart tube ( Fig. 2A). Of note, several studies suggest that the SMCs in the aortic outflow tract are derived from the neural crest, while those in the dorsal aorta are derived from the lateral plate mesoderm (50 -52). No expres-sion could be detected in other tissues including the oropharynx, esophageal region of the foregut, midgut, hindgut, lung bud, or nephric ducts. In addition, no hybridization signal was detected following hybridization of the sense control probe (Fig. 2B).
In E14.0 embryos, calponin mRNA is detectable in the branch arteries, foregut, midgut, hindgut, developing lung bud, and urogenital ridge (Fig. 2C). Once again, the sense probe did not hybridize to the E14.0 embryo (Fig. 2D). Higher power views reveal that calponin-h1 mRNA is expressed exclusively in the lamina propria surrounding the gut epithelium (white arrows, Fig. 3B), bronchial epithelium (white arrow, Fig. 3C), the urogenital ridge surrounding the urogenital sinus (white arrow, Fig. 3D), and the medial layer surrounding large arteries such as the dorsal aorta (Fig. 2C) as well as the smaller branch arteries (white arrow, Fig. 3A).
In E18.5 mouse embryos, calponin-h1 mRNA gene expression can no longer be detected above background levels in the heart, while the gene continues to be expressed abundantly in the aorta as well as smaller branch arteries, the esophagus, stomach, upper and lower intestine, pulmonary bronchi and bronchioles, and the bladder (Fig. 2E). At this late fetal stage, low level nonspecific hybridization to the sense probe was detected in the heart, liver, and some skeletal muscles (Fig. 2F). Taken together, these analyses demonstrate that calponin-h1 gene expression is (i) initially detectable at least as early as embryonic day 9.5 in the dorsal aorta, the aortic outflow tract, and the developing heart tube, (ii) sequentially up-regulated in SMC-containing tissues including the smaller branch arteries, primitive gut, lung bud, and urinary tract where it continues to be expressed throughout adult development, and (iii) downregulated to non-detectable levels during late fetal development in the four-chambered heart.
Calponin-h1 Is a Marker of the Contractile/Arrested SMC Phenotype-Within the arterial wall the majority of SMCs are maintained in the G 0 /G 1 stage of the cell cycle and express contractile protein isoforms (3). However, in response to vascular injury, SMCs are stimulated to pass through the G 1 /S checkpoint of the cell cycle, proliferate, and modulate their phenotype by up-regulating the expression of intracellular matrix proteins (4 -13). Thus, it was of interest to determine the pattern of calponin-h1 gene expression in quiescent and proliferating SMCs. To address this question, primary cultures of third passage rat aortic SMCs were synchronized in the G 0 /G 1 stage of the cell cycle by serum starvation for 72 h. Under these conditions, 85% of SMCs are arrested in G 0 /G 1 of the cell cycle as assayed by propidium iodide staining and fluorescence-activated cell sorting analyses (Fig. 4A, left panel). The cells were then serum-stimulated, and RNA was harvested from replicate cultures at 0, 8, 12, 16, and 24 h post-stimulation. Following serum stimulation, primary rat aortic SMCs begin to pass through the G 1 /S transition point at approximately 12 h (data not shown), and by 24 h post-stimulation, greater than 50% of the cells are in the S and G 2 ϩ M phases of the cell cycle (Fig.  4A, right panel). Northern blot analyses revealed that at approximately 12 h post-stimulation (coincident with the G 1 /S transition), calponin-h1 mRNA began to be down-regulated, and by 24 h post-stimulation, a marked reduction in calponin gene expression was noted (Fig. 4B, arrow). Quantitative phosphorimage analyses of signal intensity demonstrated a 50% decrease in signal intensity within 24 h from the time of serum stimulation (Fig. 4C). In contrast, hybridization of this membrane to a cDNA encoding the retinoblastoma gene product (Fig. 4, B and C)  specific gene, SM22␣, is stable in cell cycle-arrested and proliferating SMCs (46). Thus, these data demonstrate that calponin-h1 gene expression is down-regulated when quiescent vascular SMCs re-enter the cell cycle and proliferate and suggest that calponin-h1 is a specific marker of the contractile SMC phenotype.
Calponin-h1 Is a Member of a Multigene Family-Previous investigators have reported that calponin shares structural domains in common with a number of other thin filament myofibrillar proteins including troponin T, troponin I, and caldesmon (29). In addition, we and others (18,46,53) have reported high level sequence identity with the SMC-specific protein SM22␣. To determine whether calponin-h1 was encoded by a single copy gene in the murine genome and whether this gene was part of a multigene family, we hybridized the calponin-h1 cDNA probe to Southern blots containing murine In situ hybridization analyses were performed using radiolabeled calponin-h1 antisense (panels A, C, and E) and sense (panels B, D, and F) riboprobes on adjacent sections of staged murine embryos. Following hybridization, each slide was subjected to emulsion autoradiography, and the sections were visualized and photographed through a Ziess Axiophot microscope under darkfield conditions. A and B, longitudinal sections through an E9.5 mouse embryo hybridized to the calponin-h1 antisense (panel A) and sense (panel B) probes. Specific hybridization (white) is seen in the embryonic heart tube, the cardiac outflow tract (open arrow), and the dorsal aorta (white arrow) at this early stage of development. Magnification, 25ϫ. C and D, longitudinal sections through an E14.0 mouse embryo hybridized to the calponin-h1 antisense (panel C) and sense (panel D) probes. Specific hybridization (white staining) is seen in the heart, aorta, and smaller branch arteries (see also Fig. 4A), the foregut, midgut, and hindgut (see also Fig. 4B), the developing lung bud (see also Fig. 4C), and the urogenital ridge (see also Fig.  4D). Magnification, 12.5ϫ. E and F, longitudinal sections through an E18.5 mouse embryo hybridized to the calponin-h1 antisense (panel E) and sense (panel F) probes. Specific hybridization is seen throughout the arterial tree, in the esophagus, stomach, small and large intestinal mucosa, the pulmonary bronchiole tree, and the bladder. Of note, at this late fetal stage of development, hybridization in the heart has decreased to background levels (compare panels E and F). Magnification, 12.5ϫ. genomic DNA under both high and low stringency conditions. Under high stringency conditions, the calponin-h1 cDNA probe hybridized to 1-2 BamHI, EcoRI, HindIII, PstI, and XbaI fragments, suggesting that calponin-h1 is encoded by a single copy murine gene (Fig. 5A). However, under moderate stringency conditions (2 ϫ SSC, 0.1% SDS at 50°C), between four and eight additional bands were detected in each lane (Fig. 5B). Taken together, these data demonstrate that calponin-h1 is a single copy gene that is a member of a multigene family.

Isolation and Structural Characterization of Calponin-h1
Genomic Clones-A full-length murine calponin-h1 genomic clone was isolated by screening a murine 129SV genomic library with a calponin-h1 cDNA probe under high stringency conditions. A partial restriction map of this 18-kb clone, designated mCalp-5A, is shown in Fig. 6A. The complete coding sequence and 1216 bp of the 5Ј-flanking sequence of the calponin-h1 gene is shown in Fig. 6C. Exons were identified by Southern hybridization with specific cDNA fragments, and their boundaries were confirmed by DNA sequence analyses. The murine calponin-h1 gene is composed of seven exons spanning approximately 10.7 kb of genomic DNA. Each of the splice junctions (Fig. 6C, underlined) conforms to the consensus splice donor-acceptor patterns described by Breathnach and Chambon (54). The transcriptional start site was identified by RNase protection and primer extension analyses (Fig. 7). As shown in Fig.  7A, primer extension analyses utilizing an antisense synthetic oligonucleotide corresponding to bp ϩ80 to ϩ115 of the previously published sequence of the murine calponin-h1 cDNA (GenBank Accession No. Z19542) (32) resulted in two major extended products of 93 and 95 bp, respectively (arrows), each of which was thermostable up to a temperature of 56°C. In addition, a less abundant (10% relative signal intensity) 118-bp primer extension product was also detected upon prolonged autoradiographic exposure (dashed arrow). Thus, primer extension analyses suggests that the 5Ј most transcriptional start site is located 101 bp 5Ј of the translational initiation codon (Fig. 6C, closed arrowhead) with alternative transcriptional start sites located 78 and 76 bp 5Ј of the initiation codon (Fig.  6C, black dots). To complement the primer extension analyses, RNase protection analyses were also performed using an antisense cRNA probe corresponding to bp Ϫ160 to ϩ97 of the calponin-h1 genomic sequence as deduced by DNA sequence, Southern blot, and primer extension analyses (Fig. 7B). These analyses revealed several major protected fragments of 97 (37% relative signal intensity), 73, 71, and 69 bp, respectively (Fig.  7B, arrows). Thus, RNase protection analyses suggests that the 5Ј most transcriptional start site is located 101 bp 5Ј of the initiation codon (Fig. 6C, closed arrowhead) with alternative transcriptional start sites located 78, 76, and 74 bp (Fig. 6C, closed arrowheads) 5Ј of the initiation codon. Taken together, these analyses served to identify the 5Ј most transcriptional A, high stringency Southern blot analysis of the murine calponin-h1 gene. High molecular weight murine SV129 DNA was digested with restriction endonucleases BamHI, EcoRI, HindIII, PstI, and XbaI and hybridized to the radiolabeled calponin-h1 cDNA probe under high stringency conditions. B, low stringency Southern blot analysis of the murine calponin-h1 gene. The filter described above was rehybridized to the radiolabeled calponin-h1 cDNA probe under low stringency conditions. Size markers are shown in kilobases to the left of each blot. start site 101 bp 5Ј of the initiation codon, which heretofore will be designated as ϩ1 (Fig. 6C). Additional alternative transcriptional start sites are located between bp ϩ24 and ϩ28.
As shown in Fig. 6B, the full-length calponin-h1 cDNA is composed of a 101-bp 5Ј-untranslated region, an 892-bp open reading frame, and a 494-bp 3Ј-untranslated region. The predicted protein contains three 29-amino acid direct repeats (amino acids 164 -192, 204 -232, and 243-271) (Fig. 6B, gray boxes). A sequence homology search of the protein sequence data bases demonstrated that this 29-amino acid motif is conserved across species within each of the calponin isoforms (29, 32, 33, 55) (Fig. 8). Interestingly, five direct repeats of this amino acid motif are present in the recently identified unc-87 body wall muscle protein of Caenorhabditis elegans (55) (Fig. 8). In addition, single copies of this motif are present in the chicken, rat, and murine SM22␣ proteins (13,18,46,53,56,57), the rat neuronal protein NP25 (58), and the Drosophila muscle protein mp20 (59) (Fig. 8). Comparison of the amino acid sequence of this motif across species and family members revealed a consensus sequence of (I/V)GLQMGTNKXASQAGMTXYGX(R/P/ scriptional start site as deduced by primer extension, and RNase protection analyses (see Fig. 7) is indicated by a black arrowhead above the nucleotide sequence. Alternative transcriptional start sites as deduced by primer extension (black dots) and RNase protection (open arrowheads) are also shown. The reaction products of primer extension reactions performed at 42, 50, and 56°C utilizing a 5Ј-end-labeled antisense oligonucleotide primer corresponding to bp ϩ80 to ϩ115 of the previously published murine calponin-h1 cDNA and SV129 murine uterine RNA were separated on a 8% acrylamide/urea gel, which was subjected to autoradiography. The upper band (dashed arrow) in the autoradiogram represents the 118-bp primer extension product, and the lower two bands (arrow) represent the 93-and 95-bp primer extension products. DNA size markers in base pairs are indicated to the left of the autoradiogram. B, RNase protection analysis of calponin-h1 mRNA. Murine uterine RNA subjected to RNase protection analysis using an antisense cRNA probe corresponding to bp Ϫ160 to ϩ97 of the murine calponin-h1 gene was performed, and the reaction products were run down an 8% sequencing gel. The first lane (Probe only) contains the radiolabeled 257-bp probe. The second lane (ϪRNA) contains the products from an RNase digestion reaction performed with tRNA and the radiolabeled 257-bp probe. The third lane (ϩRNA) contains the digestion products of a reaction mixture containing murine uterine RNA and the radiolabeled antisense cRNA probe. The 97-bp product, corresponding to a transcriptional start site 101 bp 5Ј of the initiation codon, is shown (arrow). In addition, the 73-, 71-, and 69-bp products that represent alternative transcriptional start sites are shown (arrows). K)R (Fig. 8, Consensus). Of note, each of the threonines in the consensus sequence are potential sites for protein kinase Cmediated phosphorylation (60). Structural analysis of the calponin-h1 gene revealed that the nucleotide sequences encoding the first two 29-amino acid repeats are each interrupted by an intervening intron (Fig. 6C). Taken together, these data suggest that the calponin multigene family consists of proteins that contain a conserved amino acid motif that has been conserved over 600 million years of evolution.
1216 bp of 5Ј-sequence flanking the cap site was searched for potential transcriptional regulatory elements using MacVector DNA sequence analysis software (Eastman Kodak Co., IBI). The immediate 5Ј-flanking sequence did not contain a consensus TATA or CAAT box. A search for previously described cis-acting sequences present in the transcriptional regulatory regions of skeletal, cardiac, and smooth muscle genes identified one consensus GATA binding site (WGATAR) (61) located at bp Ϫ273, two consensus CACCC boxes (62) located at bp Ϫ322 and Ϫ214, and seven consensus E boxes (63) located at bp Ϫ244, Ϫ441, Ϫ660, Ϫ916, Ϫ954, Ϫ961, Ϫ1150, and Ϫ1214. However, unlike each of the three previously characterized smooth muscle lineage-restricted or lineage-specific genes, including the SM22␣ (46), SM-myosin heavy chain (64), and SM-␣-actin (65,66), the immediate 5Ј-flanking sequence of the calponin-h1 gene did not contain a consensus CArG/SRF binding site (67,68). In addition, four consensus binding sites for the ubiquitously expressed transcription factor AP2 were identified at bps Ϫ39, Ϫ214, Ϫ237, and Ϫ322 and one consensus binding site for Sp1 was identified at bp Ϫ983.
Identification of the Calponin-h1 Transcriptional Promoter-To confirm that the immediate 5Ј-flanking region of the calponin-h1 gene functions as a transcriptional promoter in SMCs, a series of transient transfections was performed using calponin-h1-luciferase reporter constructs and the SMC line, A7r5, as well as primary cultures of rat aortic SMCs, both of which express the gene (see Fig. 1B). Transfection of A7r5 cells with the plasmids p-3400cal/luc, containing 3.4 kb of 5Ј-flanking sequence, or the p-1500cal/luc, containing 1.5 kb of 5Јflanking sequence, resulted 180-and 290-fold, respectively, increases in luciferase activity as compared to the control plasmid pGL2-Basic (Fig. 9A). The level of luciferase activity following transfection of the p-1500cal/luc plasmid was approximately 50% of that demonstrated after transfection of the positive control plasmid pGL2-Control, which contains the SV40 promoter and transcriptional enhancer.
To test the activity of the calponin-h1 promoter in primary SMCs, confluent cultures of low passage rat aortic SMCs (which express abundant levels of calponin mRNA) were transiently transfected with the p-1500cal/luc reporter plasmid. Under these transfection conditions, the p-1500cal/luc plasmid increased luciferase activity approximately 40-fold above levels obtained following transfection with the promoterless pGL2-Basic plasmid (Fig. 9B). Once again, this level of transcriptional activity was approximately 50% of that obtained following transfection with the positive control plasmid, pGL2-Control (Fig. 9B). Of note, transfection of subconfluent cultures of proliferating primary rat aortic SMCs with the p-1500cal/luc plasmid resulted in significantly lower levels of transcriptional activity relative to the positive control plasmid, suggesting that the down-regulation of calponin-h1 gene expression in proliferating SMCs may be transcriptionally regulated (data not FIG. 8. Evolutionary conservation of the 29-amino acid calponin repeat domain. A computer-based search of the protein data bases revealed that the calponin 29-amino acid repeat domain is conserved in other proteins including chicken calponin ␣, chicken calponin ␤, pig calponin-h1, rat acidic calponin, C. elegans unc-87, mouse SM22␣, human SM22␣, and Drosophila mp20. The location of this motif within each respective protein is indicated in the column under aa. The actual amino acid sequence of each motif is indicated. Amino acid identity or conservation is indicated by gray shading. A consensus amino acid motif derived from this analysis is shown below the protein sequences. shown). Taken together, these data demonstrate that the immediate 5Ј-flanking region of the calponin-h1 gene contains a promoter that is active in both quiescent primary cultures of rat aortic SMCs and the smooth muscle cell line, A7r5. DISCUSSION The myofibrillar thin filament actin-binding protein, calponin, is believed to regulate SMC contraction and has been proposed as a candidate marker of the SMC lineage (11, 13, 28 -31). In this report, we have examined the spatial and temporal pattern of calponin-h1 gene expression during murine development. In addition, we have defined the pattern of calponin-h1 gene expression in quiescent and proliferating cultured rat aortic SMCs. We have isolated and structurally characterized the murine calponin-h1 gene, which has provided novel insights into the evolution and function of the calponin multigene family. Finally, we have demonstrated that the immediate 5Ј-flanking region of the calponin-h1 gene directs high level transcription of the calponin-h1 gene in primary cultures of rat aortic SMCs. These data are relevant to several unresolved issues in SMC biology including (i) the developmental programs that control SMC lineage specification, (ii) the molecular mechanisms that regulate SMC phenotypic modulation, and (iii) the evolution and function of the calponin multigene family.
In contrast to the skeletal muscle lineage (for review, see Ref. 69), relatively little is currently understood about the molecular mechanisms that control SMC lineage specification and differentiation. This is due in part to the poorly defined embryological origin of SMCs (18 -26), the lack of definitive markers of the SMC lineage (27), and the phenotypic plasticity of this muscle cell lineage (4 -13). In this report, we have determined the pattern of expression of the murine calponin-h1 gene during murine embryogenesis and in adult mice to investigate its utility as a marker of the SMC lineage. As anticipated, the calponin-h1 gene is expressed solely in SMC-containing tissues in adult animals demonstrating that the pattern of expression of the protein is regulated at the level of gene expression. Calponin mRNA was detected at least as early as embryonic day 9.5 in the dorsal aorta as well as the aortic outflow tract. Thus, the calponin-h1 gene represents one of the earliest developmental markers of the SMC lineage. Second, we have shown that calponin-h1 gene expression is rapidly down-regulated when primary rat aortic SMCs begin to pass through the G 1 /S checkpoint of the cell cycle and proliferate. Taken together, these data suggest that the calponin-h1 gene will serve as an excellent model system to elucidate the molecular mechanisms that regulate the SMC specification and differentiation.
Our results demonstrated that the calponin-h1 gene is not only expressed in SMCs but is also transiently expressed in the early embryonic heart. Of note, several other genes that are expressed in a SMC-specific or lineage-restricted fashion during postnatal development including SM22␣ 2 and SM-␣-actin (70,71) are also expressed in embryonic cardiac myocytes. Similarly, expression of vascular smooth muscle ␣-actin in avian cardiogenesis correlates with the onset of cardiomyocyte differentiation (70). Given the common embryological origin of some SMCs and cardiac progenitors from the lateral plate mesoderm, it is tempting to speculate that a common developmental program might direct early cardiac and SMC lineage specification. In this regard, it will be of interest to determine whether distinct or similar sets of cis-acting sequences and trans-acting factors control transcription of the calponin-h1 gene in each of these two early muscle cell lineages. In this regard, it is noteworthy that the calponin-h1 promoter contains a GATA and CACCC box/Sp1 binding sites. Similar sites have been identified previously in several cardiac-specific transcriptional regulatory elements (72)(73)(74).
Finally, structural analyses of the calponin-h1 gene revealed some new insights into the function and evolution of the calponin multigene family. The calponin-h1 protein contains three conserved direct repeats of 29 amino acids (amino acids 164 -192, 204 -232, and 243-271). Previous investigators have demonstrated that amino acids 145-182 encode an actin-binding domain (39,75). In addition, each of these repeats contains a conserved threonine residue, which is phosphorylatable by protein kinase C in vitro (60). This motif is present in other putative actin-binding proteins including the non-muscle, acidic isoform of calponin, the human, murine, rat, and chicken SM22␣ proteins (13,18,46,53,56,57), the Drosophila muscle protein mp20 (59), and the recently described C. elegans unc-87 body wall protein (55) (Fig. 8). Interestingly, mutation of the unc-87 protein, which contains five copies of this motif, results in variable degrees of paralysis (55). As such, these data are consistent with the hypothesis that the calponin multigene family evolved from a common ancestral protein with a conserved amino acid motif that encodes an actin-binding domain, which is regulated by phosphorylation. Future studies of this conserved domain may provide insights into the function of calponin and related proteins in modulating the contractile phenotype.