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• W2013741570 abstract "Although several genes are considered markers for vascular smooth muscle cell (SMC) differentiation, few have been rigorously tested for SMC specificity in mammals, particularly during development where considerable overlap exists between different muscle gene programs. Here we describe the temporospatial expression pattern of the SMC calponin gene (formerly h1 or basic calponin) during mouse embryogenesis and in adult mouse tissues and cell lines. Whereas SMC calponin mRNA expression is restricted exclusively to SMCs in adult tissues, during early embryogenesis, SMC calponin transcripts are expressed throughout the developing cardiac tube as well as in differentiating SMCs. Transcription of the SMC calponin gene initiates at two closely juxtaposed sites in the absence of a consensus TATAA or initiator element. Transient transfection assays in cultured SMC demonstrated that high level SMC calponin promoter activity required no more than 549 nucleotides of 5′ sequence. In contrast to the strict cell type-specificity of SMC calponin mRNA expression, the SMC calponin promoter showed activity in several cell lines that do not express the endogenous SMC calponin gene. These results demonstrate that SMC calponin responds to cardiac and smooth muscle gene regulatory programs and suggest that the cardiac and smooth muscle cell lineages may share a common gene regulatory program early in embryogenesis, which diverges as the heart matures. The finding that the isolated SMC calponin promoter is active in a wider range of cells than the endogenous SMC calponin gene also suggests that long-range repression or higher order regulatory mechanism(s) are involved in cell-specific regulation of SMC calponin expression. Although several genes are considered markers for vascular smooth muscle cell (SMC) differentiation, few have been rigorously tested for SMC specificity in mammals, particularly during development where considerable overlap exists between different muscle gene programs. Here we describe the temporospatial expression pattern of the SMC calponin gene (formerly h1 or basic calponin) during mouse embryogenesis and in adult mouse tissues and cell lines. Whereas SMC calponin mRNA expression is restricted exclusively to SMCs in adult tissues, during early embryogenesis, SMC calponin transcripts are expressed throughout the developing cardiac tube as well as in differentiating SMCs. Transcription of the SMC calponin gene initiates at two closely juxtaposed sites in the absence of a consensus TATAA or initiator element. Transient transfection assays in cultured SMC demonstrated that high level SMC calponin promoter activity required no more than 549 nucleotides of 5′ sequence. In contrast to the strict cell type-specificity of SMC calponin mRNA expression, the SMC calponin promoter showed activity in several cell lines that do not express the endogenous SMC calponin gene. These results demonstrate that SMC calponin responds to cardiac and smooth muscle gene regulatory programs and suggest that the cardiac and smooth muscle cell lineages may share a common gene regulatory program early in embryogenesis, which diverges as the heart matures. The finding that the isolated SMC calponin promoter is active in a wider range of cells than the endogenous SMC calponin gene also suggests that long-range repression or higher order regulatory mechanism(s) are involved in cell-specific regulation of SMC calponin expression. Expression of the smooth muscle cell calponin gene marks the early cardiac and smooth muscle cell lineages during mouse embryogenesis.Journal of Biological ChemistryVol. 272Issue 43PreviewThe luciferase activity reported for the construct −115 CALPLuc in Fig. 7B is incorrect due to a probable mutation in the coding region of the pGL3 basic luciferase gene. A newly cloned −115 CALPLuc reporter construct gave normalized luciferase activity approximating 207 of the pGL3 control. Investigators are encouraged to take care in the interpretation of seemingly unusual luciferase activity as spontaneous mutations in the pGL3 basic luciferase gene may occur during amplification in bacteria (Yang, N. Full-Text PDF Open Access The discovery of cell-specific transcription factors that trigger differentiation in skeletal and cardiac muscle has led to a search for similar regulatory factors in smooth muscle cells (SMCs), 1The abbreviations used are: SMCsmooth muscle cell(s)SM α-actinsmooth muscle α-actinSMMHCsmooth muscle myosin heavy chainRASMCrat aortic smooth muscle cell(s)RACErapid amplification of cDNA endPCRpolymerase chain reactionntnucleotide(s)TricineN-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. whose differentiation program is impaired during the course of intimal disease(1.Schwartz S.M. deBlois D. O'Brien E.R.M. Circ. Res. 1995; 77: 445-465Crossref PubMed Scopus (901) Google Scholar). Although several transcription factors have been documented in SMCs(2.Reilly C.F. Kindy M.S. Brown K.E. Rosenberg R.D. Sonenshein G.E. J. Biol. Chem. 1989; 264: 6990-6995Abstract Full Text PDF PubMed Google Scholar, 3.Cserjesi P. Lilly B. Bryson L. Wang Y. Sassoon D.A. Olson E.N. Development. 1992; 115: 1087-1101Crossref PubMed Google Scholar, 4.Gorski D.H. Patel C.V. Walsh K. Trends Cardiovasc. Med. 1993; 3: 184-190Crossref PubMed Scopus (29) Google Scholar, 5.Miano J.M. Vlasic N. Tota R.R. Stemerman M.B. Am. J. Pathol. 1993; 142: 715-724PubMed Google Scholar, 6.Martin J.F. Miano J.M. Hustad C.M. Copeland N.G. Jenkins N.A. Olson E.N. Mol. Cell. Biol. 1994; 14: 1647-1656Crossref PubMed Scopus (188) Google Scholar, 7.Lawrence R. Chang L.J. Siebenlist U. Bressler P. Sonenshein G.E. J. Biol. Chem. 1994; 269: 28913-28918Abstract Full Text PDF PubMed Google Scholar, 8.Morishita R. Gibbons G.H. Horiuchi M. Ellison K.E. Nakajima M. Zhang L. Kaneda Y. Ogihara T. Dzau V.J. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5855-5859Crossref PubMed Scopus (487) Google Scholar), none display the specificity commonly associated with factors that control cell identity by activating batteries of cell-specific genes(9.Olson E.N. Klein W.H. Genes & Dev. 1994; 8: 1-8Crossref PubMed Scopus (605) Google Scholar). Given the similarities between skeletal, cardiac, and smooth muscle cells, it is reasonable to anticipate that these different muscle cell types may share certain aspects of a myogenic gene regulatory program. smooth muscle cell(s) smooth muscle α-actin smooth muscle myosin heavy chain rat aortic smooth muscle cell(s) rapid amplification of cDNA end polymerase chain reaction nucleotide(s) N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine. In contrast to skeletal and cardiac muscle, which are derived from distinct populations of mesodermal precursor cells, SMCs arise throughout the embryo from diverse precursor cell types. The mechanisms that specify the SMC phenotype and the embryonic origins of the many different types of SMCs remain unclear. There have been relatively few studies that have examined the temporospatial patterns of expression of SMC-specific genes during embryogenesis. However, the few SMC genes that have been examined have been found to exhibit different expression patterns. Smooth muscle myosin heavy chain (SMMHC), for example, is expressed only in the SMC lineage, appearing initially in the dorsal aorta at 10.5 days postcoitum(10.Miano J.M. Cserjesi P. Ligon K.L. Periasamy M. Olson E.N. Circ. Res. 1994; 75: 803-812Crossref PubMed Scopus (323) Google Scholar). In contrast, smooth muscle α-actin (SM α-actin) is expressed in the cardiac, skeletal, and smooth muscle cell lineages during embryogenesis and in the adult (11.Ruzicka D.L. Schwartz R.J. J. Cell Biol. 1988; 107: 2575-2586Crossref PubMed Scopus (253) Google Scholar, 12.Lecain E. Alliot F. Laine M.C. Calas B. Pessac B. J. Neurosci. Res. 1991; 28: 601-606Crossref PubMed Scopus (29) Google Scholar, 13.Black F.M. Packer S.E. Parker T.G. Michael L.H. Roberts R. Schwartz R.J. Schneider M.D. J. Clin. Invest. 1991; 88: 1581-1588Crossref PubMed Scopus (97) Google Scholar, 14.Li L. Miano J.M. Cserjesi P. Olson E.N. Circ. Res. 1995; 78: 188-195Crossref Scopus (343) Google Scholar). SM22α is also expressed in cardiac, skeletal, and smooth muscle cells in the embryo before becoming restricted to SMCs late in embryogenesis(14.Li L. Miano J.M. Cserjesi P. Olson E.N. Circ. Res. 1995; 78: 188-195Crossref Scopus (343) Google Scholar, 15.Li, L., Miano, J. M., Mercer, B., Olson, E. N. (1995) J. Cell Biol. in pressGoogle Scholar). Dissection of the cis-acting regulatory elements associated with these and other SMC genes will be an important step toward understanding the similarities and differences in the myogenic regulatory programs in the three major muscle cell types. One approach to identify SMC-specific regulatory factors is to analyze promoters of genes that are unique to SMC lineages. The best studied SMC promoter is that of SM α-actin(16.Blank R.S. McQuinn T.C. Yin K.C. Thompson M.M. Takeyasu K. Schwartz R.J. Owens G.K. J. Biol. Chem. 1992; 267: 984-989Abstract Full Text PDF PubMed Google Scholar, 17.Foster D.N. Min B. Foster L.K. Stoflet E.S. Sun S. Getz M.J. Strauch A.R. J. Biol. Chem. 1992; 267: 11995-12003Abstract Full Text PDF PubMed Google Scholar). Defining SMC-specific transcription factors that activate the SM α-actin promoter, however, is complicated by its expression in multiple cell types during embryogenesis and in the adult(11.Ruzicka D.L. Schwartz R.J. J. Cell Biol. 1988; 107: 2575-2586Crossref PubMed Scopus (253) Google Scholar, 12.Lecain E. Alliot F. Laine M.C. Calas B. Pessac B. J. Neurosci. Res. 1991; 28: 601-606Crossref PubMed Scopus (29) Google Scholar, 13.Black F.M. Packer S.E. Parker T.G. Michael L.H. Roberts R. Schwartz R.J. Schneider M.D. J. Clin. Invest. 1991; 88: 1581-1588Crossref PubMed Scopus (97) Google Scholar). In addition to SM α-actin, other SMC gene promoters have been cloned and partially characterized including elastin(18.Kahari V.M. Fazio M.J. Chen Y.Q. Bashir M.M. Rosenbloom J. Uitto J. J. Biol. Chem. 1990; 265: 9485-9490Abstract Full Text PDF PubMed Google Scholar), SMMHC(19.Katoh Y. Loukianov E. Kopras E. Zilberman A. Periasamy M. J. Biol. Chem. 1994; 269: 30538-30545Abstract Full Text PDF PubMed Google Scholar), and SM22α(15.Li, L., Miano, J. M., Mercer, B., Olson, E. N. (1995) J. Cell Biol. in pressGoogle Scholar, 20.Solway J. Seltzer J. Samaha F.F. Kim S. Alger L.E. Niu Q. Morrisey E.E. Ip H.S. Parmacek M.S. J. Biol. Chem. 1995; 270: 13460-13469Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar). As yet, no cis elements have been shown to confer SMC-specific expression of these promoters. Calponin is a thin filament-associated protein that apparently functions as a negative regulatory element for SMC contraction(21.Winder S.J. Walsh M.P. Cell. Signal. 1993; 5: 677-686Crossref PubMed Scopus (128) Google Scholar), but may also have more broad cellular activities independent of contractility(22.Takahashi K. Takagi M. Ohgami K. Nakai M. Kojima A. Nadal-Ginard B. Shibata N. Circulation. 1993; 88: I-174Google Scholar). Three distinct mammalian calponin genes have been described based on their expression and nucleotide sequence differences (23.Takahashi K. Nadal-Ginard B. J. Biol. Chem. 1991; 266: 13284-13288Abstract Full Text PDF PubMed Google Scholar, 24.Strasser P. Gimona M. Moessler H. Herzog M. Small J.V. FEBS Lett. 1993; 330: 13-18Crossref PubMed Scopus (116) Google Scholar, 25.Applegate D. Feng W. Green R.S. Taubman M.B. J. Biol. Chem. 1994; 269: 10683-10690Abstract Full Text PDF PubMed Google Scholar). Whereas much effort has been directed toward understanding the function of different calponin proteins, relatively little is known about their specificity of mRNA expression. Based on studies conducted with antisera and cDNA probes, calponin was shown to predominate in SMCs(23.Takahashi K. Nadal-Ginard B. J. Biol. Chem. 1991; 266: 13284-13288Abstract Full Text PDF PubMed Google Scholar, 26.Gimona M. Herzog M. Vandekerchkhove J. Small J.V. FEBS Lett. 1990; 274: 159-162Crossref PubMed Scopus (172) Google Scholar, 27.Duband J.L. Gimona M. Scatena M. Sartore S. Small J.V. Differentiation. 1993; 55: 1-11Crossref PubMed Scopus (195) Google Scholar), but was also present in other cell types(28.Birukov K.G. Stepanova O.V. Nanaev A.K. Shirinsky V.P. Cell Tissue Res. 1991; 266: 579-584Crossref PubMed Scopus (37) Google Scholar, 29.Takeuchi K. Takahashi K. Abe M. Nishida W. Hiwada K. Nabeya T. Maruyama K. J. Biochem. (Tokyo). 1991; 103: 311-316Google Scholar). These studies, however, could not adequately distinguish between the three calponin genes. A similar problem was recently approached with respect to SMMHC mRNA expression using stringent assays for the unambiguous assignment of this marker to only SMC lineages(10.Miano J.M. Cserjesi P. Ligon K.L. Periasamy M. Olson E.N. Circ. Res. 1994; 75: 803-812Crossref PubMed Scopus (323) Google Scholar). In this study, we examined the temporospatial expression pattern of the basic or h1 calponin (hereafter referred as SMC calponin) during mouse embryogenesis and in adult mouse tissues. Our results demonstrate that SMC calponin is strictly specific for adult SMCs, but that during embryogenesis it is expressed throughout the early cardiac tube. While the SMC calponin gene is expressed exclusively in SMC lineages and embryonic heart, its promoter, which lacks core sequence elements typical of other muscle genes, displays activity in cell lines that do not express the endogenous transcript. These results reveal similarities between the cardiac and smooth muscle gene regulatory programs during early embryogenesis and suggest that complex mechanisms govern the cell type-specific expression of SMC calponin. The culture conditions for many of the cell lines analyzed here have been described previously(10.Miano J.M. Cserjesi P. Ligon K.L. Periasamy M. Olson E.N. Circ. Res. 1994; 75: 803-812Crossref PubMed Scopus (323) Google Scholar). Primary rat aortic SMCs (RASMC) were obtained by a modified explant protocol(30.Chamley-Campbell J. Campbell G.R. Ross R. Physiol. Rev. 1979; 59: 1-61Crossref PubMed Scopus (1266) Google Scholar). Briefly, rat aortae were rinsed in phosphate-buffered saline, carefully stripped of adherent periaortic fat and endothelium, and subjected to a 20-min digestion in 1% collagenase, 0.25% type II elastase, and 1% soybean trypsin inhibitor as described(31.Miano J.M. Tota R.R. Vlasic N. Stemerman M.B. Arterioscler. Thromb. 1993; 13: 211-219Crossref PubMed Scopus (130) Google Scholar). Vessels were then rinsed in Dulbecco's modified Eagle's medium and aseptically cut into small rings. The rings were placed under sterile coverslips in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mML-glutamine for 5-7 days. The cells were routinely split 1:5 and maintained in the same medium. The identity of our RASMC cultures was verified by virtue of their mRNA expression of SMC calponin (below) as well as SMMHC, SM22α, and SM α-actin. 2J. M. Miano and E. N. Olson, unpublished observations. Rat L6 skeletal myoblasts and A7r5 rat aortic SMC were maintained in the same medium as primary RASMC. Restriction enzyme-clamped PCR primers to the 5′ ends of mouse (24.Strasser P. Gimona M. Moessler H. Herzog M. Small J.V. FEBS Lett. 1993; 330: 13-18Crossref PubMed Scopus (116) Google Scholar) and rat (32.Nishida W. Kitami Y. Hiwada K. Gene (Amst.). 1993; 130: 297-302Crossref PubMed Scopus (68) Google Scholar) SMC calponin were created with a Beckman 1000 oligonucleotide synthesizer (Fullerton, CA) and used to amplify the corresponding calponin fragment from a previously described (10.Miano J.M. Cserjesi P. Ligon K.L. Periasamy M. Olson E.N. Circ. Res. 1994; 75: 803-812Crossref PubMed Scopus (323) Google Scholar) mouse uterine cDNA library or a similarly constructed rat aortic cDNA library. The sequence of the primers containing the underlined restriction sites were as follows: (a) mouse 5′ forward primer, 5′-gatacgaattcagagggtgcagacggaggctc-3′; (b) mouse 3′ reverse primer, 5′-gatacaagctttcaatccactctctcagctcc-3′; (c) rat 5′ forward primer, 5′-gatacgaattcatgtcttccgcacactttaac-3′; (d) rat 3′ reverse primer, 5′-gatacaagctttgaccttcttcacagatcccg-3′. The length of each calponin probe was, respectively, for mouse and rat calponin, 195 nt and 200 nt. These clones were ligated into the EcoRI/HindIII sites of the Bluescript SK+ vector (Stratagene; La Jolla, CA), sequenced on both strands with Sequenase II (U. S. Biochemical Corp., Cleveland, OH), and utilized for RNase protection, in situ hybridization, or genomic screening as described below. Tissue and cell culture RNA were harvested by the acid guanidinium thiocyanate/phenol method(33.Chomczynski P. Sacchi N. Anal. Biochem. 1987; 162: 156-159Crossref PubMed Scopus (63232) Google Scholar). RNA samples from PC12 and HepG2 cells were kindly provided by Keith Ligon. All SMC calponin clones were linearized with NotI and in vitro transcribed using T7 polymerase (Ambion, Austin, TX) in the presence of [α-32P]UTP (800 Ci/mmol; Amersham). Approximately 15 μg of total RNA was hybridized to each radiolabeled riboprobe according to the manufacturer's instructions (Ambion RPA II). In some experiments, a 157-nt 3′-untranslated region riboprobe corresponding to the murine SM α-actin cDNA (34.Min B. Strauch A.R. Foster D.N. Nucleic Acids Res. 1988; 16: 10374Crossref PubMed Scopus (23) Google Scholar) or an 18 S riboprobe (Ambion) was co-hybridized with the SMC calponin riboprobe. Protected fragments were resolved in a 5% polyacrylamide, 7 M urea gel and visualized following autoradiographic exposure (Kodak X-Omat AR; Rochester, NY). A monoclonal antibody to SMC calponin (clone hCP; Product No. C-2687) was purchased from Sigma and used to test for the presence of SMC calponin protein in a number of cell types. Briefly, cells were washed 3 times in phosphate-buffered saline, scraped in extraction buffer(35.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar), sheared 10 times through a 22-gauge needle, and boiled for 10 min. After spinning 10 min in a microcentrifuge, equivalent volumes of supernatant were loaded on a 10% polyacrylamide gel and stained with Coomassie Blue to verify protein integrity and equal loading. In a separate gel, resolved proteins were electroblotted to a nylon membrane (Zeta Probe; Bio-Rad, Hercules, CA) and blocked in buffered 5% non-fat milk (35.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) for 1 h at room temperature. The blot was then incubated with the monoclonal calponin antibody at a dilution of 1:2500 in buffered 1% non-fat milk and rocked for 1 h at room temperature. The blot was washed in buffered 1% nonfat milk and then incubated with a horseradish peroxidase-linked secondary anti-mouse IgG (Sigma) at a dilution of 1:1000 for 1 h at room temperature. After washing in buffered 1% nonfat milk, the SMC calponin was detected with an enhanced chemiluminescent kit (RPN 2108; Amersham) followed by exposure to hyperfilm-MP (Amersham). Adult heart, skeletal muscle, small intestine, and uterus as well as staged mouse embryos were fixed in 4% paraformaldehyde, dehydrated, and embedded in paraplast. Sections (8-10 μ) were mounted onto triple washed silane-coated glass slides (HCS Inc., Glen Head, NY) and processed for in situ hybridization as described previously(10.Miano J.M. Cserjesi P. Ligon K.L. Periasamy M. Olson E.N. Circ. Res. 1994; 75: 803-812Crossref PubMed Scopus (323) Google Scholar). Adjacent sections of 9.5- and 13.5-day embryos were hybridized with either a riboprobe corresponding to mouse SMMHC (10.Miano J.M. Cserjesi P. Ligon K.L. Periasamy M. Olson E.N. Circ. Res. 1994; 75: 803-812Crossref PubMed Scopus (323) Google Scholar) or mouse SMC calponin (see above). Slides were dipped in emulsion (Kodak NBT-2; Rochester, NY) and exposed for 9 days at 4°C before developing. Developed slides were subjected to darkfield microscopy for analysis and photography (Nikon SMZ-U Zoom 1:10). The XL 1-Blue MRA(P2) strain of bacteria (Stratagene) was used to generate ~4 × 104 recombinants/plate of the SV129 mouse genomic library (Stratagene). About 500,000 recombinants (~1.5 genomic equivalents) were lifted in duplicate onto nitrocellulose filters (Schleicher & Schuell), denatured, and neutralized as described (35.Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY1989Google Scholar) and then prehybridized for at least 6 h in 50% formamide, 5 × SSC, 5 × Denhardt's, 0.5% SDS, and 200 μg/ml salmon sperm DNA at 42°C. The 195-nt mouse SMC calponin probe described above was random prime-labeled (Boehringer Mannheim) in the presence of [α-32P]dCTP (3000 Ci/mmol; Amersham) and added to the prehybridization mixture for an additional 16 h. Filters were washed at high stringency (final wash 0.1 × SSC, 0.1% SDS at 60°C) to select for the SMC calponin gene. A total of 16 positive clones were detected after primary screening. Two clones of ~18 (CALP-5) and 14 kilobase pairs (CALP-8) were plaque purified by multiple rounds of screening using freshly labeled probe. Restriction digest analysis of these clones revealed they were independent but overlapping (see Fig. 4). Genomic Southern blotting of the CALP-5 and CALP-8 clones with DNA derived from the liver of an SV129 mouse verified the authenticity of both clones.2 The CALP-5 clone was cut with SacI and all fragments subcloned into Bluescript SK+ (Stratagene) for further restriction mapping and sequencing. These analyses coupled with PCR indicated that the CALP-5 clone harbored all of the SMC calponin coding sequence. Therefore, this clone was sequenced in its entirety on both strands with an ABI 373A automated DNA sequencer (Foster City, CA). The sequence has been deposited in GenBank (accession number U28932). Three independent assays were performed to map the SMC calponin transcription initiation sites. First, two negative strand-specific primers were synthesized for primer extension of total RNA from uterus, stomach, or liver. One primer was synthesized 5′ of the first intron (5′-cagacaagccgtaggcaggacc-3′) and the other 3′ of the first intron (5′-cgctgatggtcgtatttctgggccag-3′). These primers were 5′ end-labeled with [γ-32P]ATP (3000 Ci/mmol; Amersham) using T4 polynucleotide kinase (Boehringer Mannheim) and annealed (300 mM NaCl, 40 mM Tricine, pH 8.0, and 0.1 mM EDTA) to 25 μg of total RNA from the above mouse tissues at 65°C for 10 min as described(36.Cooper T.A. Cardone M.H. Ordahl C.P. Nucleic Acids Res. 1988; 16: 8553-8565Crossref Scopus (14) Google Scholar). Following annealing, the samples were transferred to a 48°C bath and extension was carried out for 1 h with SuperScript II reverse transcriptase (Bethesda Research Laboratories) in a final volume of 20 μl. Reactions were stopped by adding 60 μl of dH2O, 70 μl of 5 M NH4OAc, and 2 μl of 0.5 M EDTA and precipitated in 500 μl of ethanol. Dried samples were resuspended in 10 μl of Sequenase stop solution (U. S. Biochemical), denatured at 75°C, and resolved in a 6% polyacrylamide, 8 M urea gel. A sequencing ladder using a calponin cDNA clone with the same primer as that used to anneal the RNA was loaded adjacently to the primer extension reactions for fine mapping of the start sites. RNase protection analysis and 5′ RACE were used in conjunction with primer extension for mapping the start sites. For RNase protection, total RNA was hybridized to one of two independent riboprobes (see Fig. 5A) and the protected fragments resolved in a 5% polyacrylamide, 7 M urea gel. Sequencing reactions of a calponin cDNA were carried out using primers to the 3′ most end of each riboprobe. Total RNA from uterus or liver was also subjected to 5′ RACE according to the manufacturer's instructions (BRL). A final nested PCR was performed using the 5′ anchor primer provided in the kit (BRL) and a 3′ SMC calponin-specific primer, 5′-cagacaagccgtaggcaggacc-3′. A total of 10 RACE products were sequenced, two of which are presented in Fig. 5D. All promoter constructs were derived from a 2301-nt PCR fragment of the 5′ end of genomic clone CALP-5. BamHI sites were engineered at the 5′ and 3′ ends of this fragment for cloning into Bluescript. The 2301-nt BamHI fragment, which included 60 nt of untranslated exon one sequence and 2251 nt of 5′ promoter sequence, was then cloned into the BglII site of pGL3 basic luciferase (Promega, Madison, WI) to generate −2251 CALPLuc. A BamHI/HindIII digest of the 2301-nt PCR product generated a 549-nt deletion construct that was filled in and blunt cloned into the SmaI site of pGL3 basic to generate −549 CALPLuc. A BamHI/NcoI digest of the 2301-nt PCR product generated a 1342-nt deletion that was filled in and blunt cloned into the SmaI site of pGL3 basic to generate −1342 CALPLuc. The −3000 CALPLuc promoter construct was generated by ligating a 2.4-kilobase pair HindIII promoter fragment adjacent to the −549 CALPLuc deletion construct. Finally, the −115 CALPLuc deletion construct was generated by PCR using BamHI sites at both ends for cloning into the BglII site of pGL3 basic. All promoter constructs were sequenced to confirm their proper orientation in the pGL3 basic luciferase reporter vector as well as to verify the absence of any errors introduced by Taq polymerase. The promoterless pGL3 basic vector and the pGL3 control vector containing the SV40 promoter/enhancer were used as controls to normalize each promoter construct's activity. All transfections were done using a calcium phosphate precipitation method(37.Gorman C.M. Moffat L.F. Howard B.H. Mol. Cell. Biol. 1982; 2: 1044-1051Crossref PubMed Scopus (5292) Google Scholar). Cells (A7r5, L6 myoblasts, C2C12, 10T1/2, and F9 teratocarcinoma) were grown in 100-mm dishes (Falcon) according to the supplier's specifications (American Type Culture Collection, Bethesda, MD) and transfected at 50% confluence with 10 μg of plasmid DNA. Primary RASMC were grown in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and used between passage 15 and 30. Although the cell lines grow at different rates, every effort was made to transfect approximately equivalent cell numbers between lines. In some experiments, a plasmid carrying the β-galactosidase gene was cotransfected to correct for varying transfection efficiencies between cell lines. Transfections were typically carried out for 12-16 h followed by 48 h of recovery and growth. Cells were harvested in cold phosphate-buffered saline, spun down, and resuspended in 200 μl of 0.25 M Tris-HCl, pH 7.8. Cell lysates were then briefly sonicated, spun down and stored at −80°C before use. Neither mild sonication nor multiple freeze thawing influenced luciferase activity.2 Total protein was measured by the Bradford assay (Bio-Rad). Luciferase activity was assayed according to the manufacturer's specifications (Promega). A Turner Model 20 luminometer was used to measure the light reactivity of firefly luciferase. The relative light units were then normalized to total protein and expressed as a percent of the normalized luciferase activity obtained with the pGL3 control vector, which contains the SV40 promoter/enhancer (Promega). The data reflect the means (±S.E.) of at least four independent experiments done in duplicate. RNase protection assays were performed to accurately assess the specificity of SMC calponin mRNA expression in adult mouse tissues and a variety of cell lines. Consistent with previous reports (21.Winder S.J. Walsh M.P. Cell. Signal. 1993; 5: 677-686Crossref PubMed Scopus (128) Google Scholar, 24.Strasser P. Gimona M. Moessler H. Herzog M. Small J.V. FEBS Lett. 1993; 330: 13-18Crossref PubMed Scopus (116) Google Scholar), SMC calponin transcripts were restricted to adult mouse tissues with a SMC component (Fig. 1A). Upon overexposure, however, transcripts were detected in most tissues due to the presence of blood vessels (see below). In cultured cells, SMC calponin was expressed in proliferating BC3H1 cells, which have been reported to exhibit properties of smooth and skeletal muscle, and only weakly in the differentiated P19 embryonal carcinoma cell line, which also has the potential to differentiate into smooth muscle (38.Blank R.S. Swartz E.A. Thompson M.M. Olson E.N. Owens G.K. Circ. Res. 1995; 76: 742-749Crossref PubMed Scopus (82) Google Scholar) (Fig. 1B). Interestingly, SMC calponin mRNA expression was extinguished in differentiated BC3H1 cells (Fig. 1B). No other mouse lines examined expressed SMC calponin mRNA including 3T3, 10T1/2, C2, F9, and embryonic stem cells (Fig. 1B). These findings contrast with the expression of SM α-actin, which was expressed in most cell lines analyzed (Fig. 1B). No SMC calponin transcripts were detected in the rat L6 skeletal myoblast line, PC12 cells, or the HepG2 liver cell line (Fig. 1C). On the other hand, a prominent signal was observed in the A7r5 fetal rat aortic SMC line as well as primary RASMC (Fig. 1C). These latter cells also expressed SMC calponin protein as determined by Western blotting (Fig. 1D). Importantly, SMC calponin mRNA was expressed at high levels in rat SMC irrespective of passage number or growth state (Fig. 1C). Together, these results show SMC calponin to be a highly restricted marker for SMC lineages. They also demonstrate the utility of both the A7r5 fetal rat aortic SMC line and multiply passaged primary RASMC for analyzing SMC calponin promoter activity (see below). Several adult mouse tissues were analyzed by in situ hybridizatio" @default.
• W2013741570 created "2016-06-24" @default.
• W2013741570 creator A5080731495 @default.
• W2013741570 creator A5084182062 @default.
• W2013741570 date "1996-03-01" @default.
• W2013741570 modified "2023-10-18" @default.
• W2013741570 title "Expression of the Smooth Muscle Cell Calponin Gene Marks the Early Cardiac and Smooth Muscle Cell Lineages during Mouse Embryogenesis" @default.
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